Chemically modified RNAi constructs and uses thereof

Abstract

The present invention relates to chemically modified RNAi constructs for reducing the expression of target genes. Specifically, the present invention relates to specific patterns of modified nucleotides that are incorporated into the RNAi constructs to improve in vivo stability and efficacy. Pharmaceutical compositions containing the chemically modified RNAi constructs and methods for inhibiting target gene expression in vivo, for example, to treat or ameliorate various disease conditions, by administering the chemically modified RNAi constructs are also described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 777,677, filed December 10, 2018, which is incorporated herein by reference in its entirety.

[0002] Description of electronically submitted text files

[0001] This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the Sequence Listing in computer readable format, created on December 9, 2019, is titled A-2327-WO-PCT_SeqList_ST25, and is 24.7 kilobytes in size.

[0003] The present invention relates to chemically modified RNAi constructs for reducing the expression of target genes in vivo. Specifically, the present invention relates to specific patterns of modified nucleotides that confer improved efficacy and stability of RNAi constructs in vivo. Such RNAi constructs are useful for inhibiting the expression of target genes for therapeutic purposes. [Background technology]

[0004] RNA interference (RNAi) is a posttranscriptional gene silencing mechanism found in nearly all phyla and is thought to be 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 18-25 base-pair duplexes derived from longer noncoding RNAs. These short RNA molecules are loaded into the RNA-induced silencing complex (RISC), where the sense or passenger strand is discarded and the antisense or guide strand hybridizes to a fully or partially complementary mRNA sequence (Nakanishi, Wiley Interdiscip. Rev. RNA, Vol. 7:637-660, 2016), subsequently inducing mRNA silencing via Ago2-mediated degradation or translational repression (Bobbin and Rossi, Annu. Rev. Pharmacol. Toxicol., Vol. 56:103-122, 2016).

[0005] With the advancement of RNAi technology and delivery methods, the positive results of RNAi-based treatments continue to increase.These treatments represent a promising class of therapies, especially for targets that have been considered "undruggable" by small molecules or biological modalities.Although the development of chemical modifications and improved delivery methods have made great progress in overcoming the inherent metabolic instability of natural RNA, there remains a need in the art for RNAi agents with improved in vivo efficacy and stability suitable for therapeutic administration. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Fire et al.,Nature,Vol.391;806-811,1998 [Non-patent document 2] Fire et al.,Trends Genet,Vol.15:358-363,1999 [Non-patent document 3] Hamilton and Baulcombe,Science,Vol.286,950-952,1999 [Non-patent document 4] Nakanishi,Wiley Interdiscip.Rev.RNA,Vol.7:637-660,2016 [Non-Patent Document 5] Bobbin and Rossi,Annu.Rev.Pharmacol.Toxicol.,Vol.56:103-122,2016 Summary of the Invention [Means for solving the problem]

[0007] The present invention is based in part on the design of chemical modification patterns for RNAi constructs that improve the potency and / or duration of the gene silencing activity of the construct in vivo.The modification patterns described herein can be widely applied to various RNAi constructs with different sequences and targets.The RNAi constructs are useful for inhibiting the expression of target genes in vivo, for example, for therapeutic purposes.

[0008] Thus, the present invention provides RNAi constructs that inhibit 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 that of the antisense strand to form a duplex region, and the RNAi construct comprises a structure represented by one of the formulas described herein. In certain embodiments, the RNAi constructs of the present invention have a chemical modification pattern selected from one of the patterns designated as P1 to P30 as described herein.

[0009] In some embodiments, the RNAi construct comprises a structure represented by formula (A): 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)

[0010] In formula (A), the top strand, listed in the 5' to 3' direction, is the sense strand, and the bottom strand, listed in the 3' to 5' direction, is the sense strand. strand) is the antisense strand, each NF represents a 2'-fluoro modified nucleotide, each NM independently represents a modified nucleotide selected from a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), and a deoxyribonucleotide, each NL independently represents a modified nucleotide selected from a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide, and NT represents a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide. When x is 1, 2, 3, or 4, x can be an integer from 0 to 4, provided that one or more of the NA nucleotides are independently a modified nucleotide selected from abasic nucleotides, inverted abasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides. One or more of the NA nucleotides can be complementary to a nucleotide in the antisense strand. When y is 1, 2, 3, or 4, one or more n nucleotides can be base-paired with a nucleotide in the antisense strand. Ify can be an integer from 0 to 4, provided that the overhanging nucleotides are unmodified or unmodified. When z is 1, 2, 3, or 4, z can be an integer from 0 to 4, provided that one or more of the NB 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. One or more of the NB nucleotides can be complementary to an NA nucleotide when present in the sense strand, or base pair with a nucleotide in the sense strand. If The overhanging nucleotides may be non-overhanging nucleotides.

[0011] In some embodiments, the RNAi construct comprises a sense strand 19-23 nucleotides in length and an antisense strand 19-23 nucleotides in length, wherein the sequences of the antisense and sense strands are sufficiently complementary to each other to form a duplex region of 19-21 base pairs, wherein nucleotides at positions 2, 7, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides, and nucleotides in the sense strand at positions 8-11 and 13 (counting from the 5' end) paired with the antisense strand are 2'-fluoro-modified nucleotides, and neither the sense strand nor the antisense strand has more than seven total 2'-fluoro-modified nucleotides, respectively. The RNAi construct can have nucleotide overhangs on one or both of the 3' ends of the sense and antisense strands. In certain embodiments, the RNAi construct has a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0012] In other embodiments of the invention, the RNAi construct comprises a structure represented by formula (D): 5'-(N A ) x N L N L N L N L NM 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)

[0013] In formula (D), the top strand, listed in the 5' to 3' direction, is the sense strand, and the bottom strand, listed in the 3' to 5' direction, is the antisense strand; each NF represents a 2'-fluoro modified nucleotide; each NM independently represents a modified nucleotide selected from a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide; and each NL independently represents a 2'-O-methyl modified nucleotide. NT represents a modified nucleotide selected from abasic nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides, and NT represents a modified nucleotide selected from abasic nucleotides, inverted abasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides. When x is 1, 2, 3, or 4, x can be an integer from 0 to 4, provided that one or more of the NA nucleotides are independently a modified nucleotide selected from abasic nucleotides, inverted abasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides. One or more of the NA nucleotides can be complementary to a nucleotide in the antisense strand. When y is 1, 2, 3, or 4, one or more n nucleotides are base-paired with a nucleotide in the antisense strand. Ify can be an integer from 0 to 4, provided that the overhanging nucleotides are unmodified or unmodified. When z is 1, 2, 3, or 4, z can be an integer from 0 to 4, provided that one or more of the NB 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. One or more of the NB nucleotides can be complementary to an NA nucleotide when present in the sense strand, or base pair with a nucleotide in the sense strand. If The overhanging nucleotides may be non-overhanging nucleotides.

[0014] In some embodiments of the present invention, an RNAi construct comprises a sense strand 19-23 nucleotides in length and an antisense strand 19-23 nucleotides in length, wherein the sequences of the antisense and sense strands are sufficiently complementary to each other to form a duplex region of 19-21 base pairs, wherein nucleotides at positions 2, 14, and 16 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides, and nucleotides in the sense strand paired with positions 10-13 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides, and neither the sense nor the antisense strand has more than seven total 2'-fluoro-modified nucleotides. The RNAi construct may have a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. Alternatively, the RNAi construct may have nucleotide overhangs at both the 3' ends of the sense and antisense strands.

[0015] The RNAi construct of the present invention can comprise at least one backbone modification, such as modified internucleotide or internucleoside bond.In some embodiments, the RNAi construct described herein comprises at least one phosphorothioate internucleotide bond.In certain embodiments, the phosphorothioate internucleotide bond can be located at the 3' or 5' end of the sense strand and / or antisense strand.

[0016] The RNAi construct may further comprise a ligand that facilitates delivery or uptake of the RNAi construct to specific tissues or cells, such as liver cells. In some embodiments, the ligand targets delivery of the RNAi construct to liver cells. In these and other embodiments, the ligand may comprise galactose, galactosamine, or N-acetyl-galactosamine (GalNAc). In certain embodiments, the ligand comprises a multivalent galactose or multivalent GalNAc moiety, such as a trivalent or tetravalent galactose or GalNAc moiety. The ligand may be covalently attached to the 5' or 3' end of the sense strand of the RNAi construct, optionally via a linker. In some embodiments, the RNAi construct comprises a ligand and a linker having the structure of any of Formulas I-IX described herein. In one embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula VI. In another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula VII. In yet another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula IX.

[0017] The present invention also provides pharmaceutical compositions comprising any of the RNAi constructs described herein and a pharmaceutically acceptable carrier, excipient, or diluent. Such pharmaceutical compositions are particularly useful for reducing or inhibiting expression of a target gene in cells (e.g., liver cells) of a subject, particularly when overexpression of the target gene product in the subject is associated with a pathological phenotype.

[0018] The present invention includes a method for reducing or inhibiting the expression of a target gene in a cell, tissue, or subject. In one embodiment, the method includes contacting a cell or tissue with any one of the RNAi constructs described herein. The cell or tissue can be in vitro or in vivo. In another embodiment, the method includes administering any one of the RNAi constructs described herein to a subject. The RNAi construct can be administered to a subject parenterally (e.g., intravenously or subcutaneously). [Brief explanation of the drawings]

[0019] [Figure 1] Several representative embodiments of chemical modification patterns for RNAi constructs are shown. In each schematic diagram, the top strand represents the sense strand in the 5' to 3' direction, and the bottom strand represents the antisense strand in the 3' to 5' direction. Solid black circles represent 2'-O-methyl (2'-OMe) modified nucleotides, striped circles represent 2'-fluoro (2'-F) modified nucleotides, and white circles represent inverted abasic nucleotides (invAb) or inverted deoxyribonucleotides (invdN). Light gray lines connecting circles represent phosphodiester bonds, and black lines connecting circles represent phosphorothioate bonds. Black squares represent putative Ago2 cleavage sites within the RNAi constructs. [Figure 2] This is a bar graph of the expression levels of human PNPLA3 variants in the livers of mice injected with AAV encoding the human PNPLA3 variants and treated with a 5 mg / kg subcutaneous injection of the indicated RNAi constructs with the P1-CM1 chemical modification pattern. Human PNPLA3 expression was measured by qPCR and is reported as expression levels relative to vehicle-treated animals. Expression levels are shown at 8 days after administration of the RNAi constructs. [Figure 3]1 shows a bar graph of the expression levels of human PNPLA3 mutants in the livers of mice injected with AAV encoding the human PNPLA3 mutants and treated with 5 mg / kg subcutaneous injections of the indicated RNAi constructs with P1, P2, P3, or P4 chemical modification patterns. Human PNPLA3 expression was measured by qPCR and is reported as expression levels relative to vehicle-treated animals. Expression levels are shown 15 days after administration of the RNAi constructs. [Figure 4A-4B]

[0033] Figure 4B is a line graph showing total light flux (photons / second) versus the number of weeks after RNAi construct injection in mice receiving subcutaneous injections of vehicle or the indicated RNAi constructs with the P9 chemical modification pattern at doses of 1 mg / kg (Figure 4A) or 3 mg / kg (Figure 4B). Total light flux represents the signal from a luciferase reporter expressed by the mice that contains a sequence complementary to that of the RNAi construct. A reduction in total light flux indicates a reduction in expression of the luciferase reporter. [Figure 5] 1 shows a bar graph of the expression levels of human PNPLA3 variants in the livers of mice injected with AAV encoding human PNPLA3 variants and treated with 3 mg / kg subcutaneous injections of the indicated RNAi constructs with the P9 (duplex numbers 7318 and 8709), CM2 (duplex number 8103), CM3 (duplex number 8104), or CM4 (duplex number 8105) chemical modification patterns. Human PNPLA3 expression was measured by qPCR and is reported as expression levels relative to vehicle-treated animals. Expression levels are shown at 28 days after administration of the RNAi constructs. [Figure 6] This is a bar graph of mouse ASGR1 expression levels in the livers of mice treated with 5 mg / kg subcutaneous injection of the indicated ASGR1 RNAi constructs. Mouse ASGR1 expression was measured by qPCR and is reported as expression levels normalized by Gapdh expression levels. Expression levels are shown on days 4, 8, and 15 after administration of the RNAi constructs or buffer (phosphate-buffered saline, PBS). [Figure 7]1 is a line graph showing the percent change in serum Lp(a) levels compared to baseline in double transgenic mice administered a 0.5 mg / kg subcutaneous injection of the indicated LPA-targeting RNAi construct. Both RNAi constructs had the same sequence and differed only in the pattern of chemical modification: duplex number 3632 had the CM1 modification pattern, and duplex number 3635 had the P1 modification pattern. The percent change in Lp(a) serum levels is shown at days 14 (D14) and 28 (D28) after a single subcutaneous injection of the RNAi construct. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is based in part on the design of chemical modification patterns for RNAi constructs that result in potent and sustained knockdown of target gene expression in vivo across a variety of sequences and targets.The chemically modified RNAi constructs described herein have been shown to have improved potency and / or duration of gene silencing activity in vivo compared to previously described RNAi therapeutic agents with alternative chemical modification patterns.The modified RNAi constructs of the present invention are useful for inhibiting target gene expression in vivo, for example, to treat or ameliorate various disease conditions.Therefore, the present invention provides RNAi constructs that inhibit the expression of target gene sequences.

[0021] As used herein, the term "RNAi construct" refers to an agent comprising an RNA molecule that, when introduced into a cell, can downregulate the expression of a target gene via the RNA interference mechanism. RNA interference is a process in which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g., a messenger RNA or mRNA molecule) in a sequence-specific manner, for example, via the RNA-induced silencing complex (RISC) pathway. In some embodiments, an RNAi construct comprises a double-stranded RNA molecule comprising two antiparallel strands of consecutive nucleotides that are sufficiently complementary to each other to hybridize and form a duplex region. "Hybridizing" or "hybridization" typically refers to the pairing of complementary polynucleotides via hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides. The strand containing a region having a sequence substantially complementary to a target sequence (e.g., a target mRNA) is referred to as the "antisense strand." "Sense strand" refers to the strand that includes a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand can include a region that has substantial sequence identity to a target sequence.

[0022] Double-stranded RNA molecules can contain chemical modifications to ribonucleotides, including modifications to the ribose sugar, base, or backbone components of ribonucleotides, such as those described herein or known in the art. Any such modifications as used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.) are encompassed by the term "double-stranded RNA" for purposes of this disclosure.

[0023] As used herein, a first sequence is "complementary" to a second sequence if, under certain conditions, such as physiological conditions, a polynucleotide comprising the first sequence can hybridize to a polynucleotide comprising the second sequence to form a duplex region. Other such conditions can include moderate or stringent hybridization conditions known to those of skill in the art. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if the polynucleotide comprising the first sequence base-pairs with the polynucleotide comprising the second sequence without mismatches over the entire length of one or both nucleotide sequences. A sequence is "substantially complementary" to a target sequence if the sequence is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the target sequence. The percent complementarity can be calculated by dividing the number of bases in a first sequence that are complementary to the bases at corresponding positions in a second sequence or target sequence by the total length of the first sequence.When two sequences hybridize, if there are 5, 4, 3, or 2 or fewer mismatches across a 30-base pair duplex region, the sequence can be said to be substantially complementary to another sequence.Generally, if any nucleotide overhangs, as defined herein, exist, the sequence of such overhangs is not taken into account when determining the degree of complementarity between two sequences.For example, a 21-nucleotide sense strand and a 21-nucleotide antisense strand that hybridize to form a 19-base pair duplex region with a 2-nucleotide overhang at the 3' end of each strand are considered to be fully complementary as this term is used herein.

[0024] In some embodiments, the 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 such 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, for example, a sequence that has 1, 2, 3, 4, or 5 mismatches in the duplex region formed by the sense strand and the antisense strand. In certain embodiments, it is preferred that any mismatches occur within the terminal regions (e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the strands). In one embodiment, any mismatches in the duplex region formed by the sense strand and the antisense strand occur within 6, 5, 4, 3, or 2 nucleotides of the 5' end of the antisense strand.

[0025] In some embodiments, the sense strand and antisense strand of double-stranded RNA hybridize to form a duplex region, but can be two separate molecules that are not connected except for this region. Such double-stranded RNA molecules formed from two separate strands are called "small interfering RNA" or "short interfering RNA" (siRNA). Thus, in some embodiments, the RNAi construct of the present invention comprises siRNA.

[0026] In other embodiments, the sense and antisense strands that hybridize to form a duplex region may be part of a single RNA molecule; i.e., the sense and antisense strands are part of a self-complementary region of the single RNA molecule. In such cases, the single RNA molecule comprises a duplex 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 a contiguous sequence of unpaired nucleotides, which form the loop region. The loop region is typically of sufficient length to allow the RNA molecule to fold back on itself so that the antisense strand can base pair with the sense strand to form the duplex or stem region. The loop region can 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 a partially 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 the single, at least partially self-complementary RNA molecule can be from about 40 nucleotides to about 100 nucleotides, from about 45 nucleotides to about 85 nucleotides, or from about 50 nucleotides to about 60 nucleotides, and can include a duplex region and a loop region, each having a length listed herein.

[0027] The RNAi construct of the present invention comprises a sense strand and an antisense strand, and the antisense strand comprises a region having a sequence substantially or completely complementary to the target gene sequence. A target gene sequence generally refers to a nucleic acid sequence comprising a partial or complete coding sequence of a polypeptide. A target gene sequence may also comprise non-coding regions, such as 5' or 3' untranslated regions (UTRs). In certain embodiments, the target gene sequence is a messenger RNA (mRNA) sequence. An mRNA sequence refers to any messenger RNA sequence, including splice variants encoding proteins, protein variants, or isoforms from any species (e.g., mouse, rat, non-human primate, human). In one embodiment, the target gene sequence is an mRNA sequence encoding a human protein. A 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.

[0028] The region of the antisense strand of the RNAi construct can be substantially complementary or completely complementary to at least 15 consecutive nucleotides of the target gene sequence. In some embodiments, the target region of the gene sequence to which the antisense strand comprises a region of complementarity can 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.

[0029] The sense strand of an RNAi construct typically contains a sequence sufficiently complementary to that of the antisense strand so that the two strands hybridize under physiological conditions to form a duplex region. A "duplex region" refers to a region of two complementary or substantially complementary polynucleotides that base-pair with each other through either Watson-Crick base pairing or other hydrogen-bonding interactions to generate a duplex between the two polynucleotides. The duplex region of an RNAi construct should be of sufficient length to allow, for example, binding of the Dicer enzyme and / or the RISC complex, thereby allowing the RNAi construct to enter the RNA interference pathway. For example, in some embodiments, the duplex region is about 15 to about 30 base pairs in length. Other lengths of the duplex region within this range are also suitable, such as 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 duplex region is about 17 to about 24 base pairs in length. In another embodiment, the duplex region is about 19 to about 21 base pairs in length. In certain embodiments, the duplex region is about 19 base pairs in length. In other embodiments, the duplex region is about 21 base pairs in length.

[0030] In embodiments in which the sense and antisense strands are two separate molecules (e.g., the RNAi construct comprises an siRNA), the sense and antisense strands need not be the same length as the duplex region. For example, one or both strands may be longer than the duplex region and may have one or more unpaired nucleotides or mismatches adjacent to the duplex region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, "nucleotide overhang" refers to an unpaired nucleotide at the end of a strand or a nucleotide that extends beyond the duplex region. Nucleotide overhangs are typically generated 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 a 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 particular embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. In certain other embodiments, the nucleotide overhang comprises a single nucleotide.

[0031] The nucleotides in the overhang can be ribonucleotides or modified nucleotides described herein. In some embodiments, the nucleotides in the overhang are 2'-modified nucleotides (e.g., 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides), deoxyribonucleotides, inverted nucleotides (e.g., inverted abasic nucleotides, inverted deoxyribonucleotides), or combinations thereof. For example, in one embodiment, the nucleotides in the overhang are deoxyribonucleotides, e.g., deoxythymidine. In another embodiment, the nucleotides in the overhang are 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-methoxyethyl-modified nucleotides, or combinations thereof. In other embodiments, the overhang comprises a 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide can comprise a ribonucleotide or a modified nucleotide, e.g., a 2'-modified nucleotide. In other embodiments, the overhang comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide. When a nucleotide overhang is present in the antisense strand, the nucleotides in 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., a polypyrimidine or polypurine sequence, such as UU, TT, AA, GG, etc.).

[0032] The nucleotide overhangs can be present at the 5'-end or 3'-end of one or both strands. For example, in one embodiment, the RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the antisense strand. In another embodiment, the RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the sense strand. In some embodiments, the RNAi construct comprises nucleotide overhangs at the 5'-end of the sense strand and the 5'-end of the antisense strand. In other embodiments, the RNAi construct comprises nucleotide overhangs at the 3'-end of the sense strand and the 3'-end of the antisense strand.

[0033] An RNAi construct may comprise a nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other end. "Blunt end" means that the sense and antisense strands are perfectly base-paired at the ends of the molecule, with no unpaired nucleotides extending beyond the duplex region. In some embodiments, an RNAi construct comprises a nucleotide overhang at the 3' end of the sense strand and a blunt end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, an RNAi construct comprises a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, an RNAi construct comprises blunt ends at both ends of the double-stranded RNA molecule. In such embodiments, the sense and antisense strands have the same length, and the duplex region is the same length as the sense and antisense strands (i.e., the molecule is double-stranded throughout its entire length).

[0034] The sense strand and antisense strand in the RNAi constructs of the present invention can each independently be about 15 to about 30 nucleotides in length, about 19 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 19 to about 21 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. 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 length. In some embodiments, the sense strand and antisense strand form a duplex region of the same length but shorter than the other strands, such that the RNAi construct has a two-nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand, each 21 nucleotides in length, (ii) a duplex region that is 19 base pairs in length, and (iii) a nucleotide overhang of two 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 in length, (ii) a duplex region that is 21 base pairs in length, and (iii) a nucleotide overhang of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the sense strand and the antisense strand have the same length and form a duplex region throughout their entire length, so that there are no nucleotide overhangs at either end of the double-stranded molecule. In one such embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand, each 21 nucleotides in length, and (ii) a duplex region that is 21 base pairs in length. In another such embodiment, the RNAi construct is blunt-ended and comprises (i) sense and antisense strands that are each 23 nucleotides in length, and (ii) a duplex region that is 23 base pairs in length.

[0035] In other embodiments, the sense strand or antisense strand is longer than the other strand, and the two strands form a duplex region having a length equal to that of the shorter strand, such that the RNAi construct comprises at least one nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand that is 19 nucleotides long, (ii) an antisense strand that is 21 nucleotides long, (iii) a duplex region that is 19 base pairs long, and (iv) a nucleotide overhang of two unpaired nucleotides at the 3'-end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand that is 21 nucleotides long, (ii) an antisense strand that is 23 nucleotides long, (iii) a duplex region that is 21 base pairs long, and (iv) a nucleotide overhang of two unpaired nucleotides at the 3'-end of the antisense strand.

[0036] The RNAi construct of the present invention preferably contains modified nucleotides. "Modified nucleotide" refers to a nucleotide having one or more chemical modifications to the 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, an RNAi construct may contain a combination of modified nucleotides and ribonucleotides. Incorporation of 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 an RNAi construct in reducing the expression of a target gene can also be enhanced by incorporating modified nucleotides (especially when incorporated in a specific pattern, as described in more detail herein).

[0037] In certain embodiments, modified nucleotides have modifications of the ribose sugar. Such sugar modifications can 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-C-C 10 or O-C1-C 10 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.

[0038] "Bicyclic 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, resulting in a bicyclic sugar structure. In some embodiments, a bicyclic sugar modification comprises a bridge between the 4' and 2' carbons of the pentose ring. Nucleotides containing a sugar moiety comprising a bicyclic sugar modification are referred to herein as bicyclic nucleic acids or BNAs. Exemplary bicyclic sugar modifications include α-L-methyleneoxy (4′-CH2-O-2′) bicyclic nucleic acids (BNAs); β-D-methyleneoxy (4′-CH2-O-2′) BNAs (also referred to as locked nucleic acids or LNAs); ethyleneoxy (4′-(CH2)2-O-2′) BNAs; aminooxy (4′-CH2-ON(R)-2′) BNAs; oxyamino (4′-CH2-N(R)-O-2′) BNAs; methyl(methyleneoxy) (4′-CH(CH3)-O-2′) BNAs (constrained ethyl methylene-thio (4'-CH2-S-2') BNA; methylene-amino (4'-CH2-N(R)-2') BNA; methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA; propylene carbocyclic (4'-(CH2)3-2') BNA; and methoxy(ethyleneoxy) (4'-CH(CHOMe)-O-2') BNA (also referred to as constrained MOE or cMOE). These and other sugar-modified nucleotides that can be incorporated into the RNAi constructs of the invention are described in U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entirety.

[0039] In some embodiments, an 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 (BNAs), deoxyribonucleotides, or combinations thereof. In certain embodiments, an 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, an RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0040] Both the sense strand and the antisense strand of an RNAi construct can 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 of the 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 of the nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides can be 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or a combination thereof.

[0041] In certain embodiments, modified nucleotides incorporated into one or both strands of an RNAi construct of the invention have a nucleobase (also referred to herein as a "base") modification. A "modified nucleobase" or "modified base" refers to a base 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 or naturally occurring modifications, including but not limited to the universal bases 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine (I), 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, 6-methylcytosine, 6-methyl-2 ... These include, but are not limited to, uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0042] In some embodiments, the modified base is a universal base. "Universal base" refers to a base analog that indiscriminately base pairs with all natural bases in RNA and DNA without altering the resulting double-stranded region's double helix structure. 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.

[0043] Other suitable modified bases that can be incorporated into the RNAi constructs of the present invention include those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Vol.10:297-310,2000, and Peacock et al., 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 can be substituted with other nucleobases, such as the modified nucleobases described above, without substantially changing the base pairing properties of polynucleotides that contain nucleotides with such substituted nucleobases.

[0044] In some embodiments, the sense and antisense strands of an RNAi construct may contain one or more abasic nucleotides. An "abasic nucleotide" or "abasic nucleoside" is a nucleotide or nucleoside lacking a nucleobase at the 1' position of the ribose sugar. In certain embodiments, an abasic nucleotide is incorporated at the end of the sense and / or antisense strand of an RNAi construct. In one embodiment, the sense strand contains an abasic nucleotide as the terminal nucleotide at its 3'-end, its 5'-end, or both its 3'-end and 5'-end. In another embodiment, the antisense strand contains an abasic nucleotide as the terminal nucleotide at its 3'-end, its 5'-end, or both its 3'-end and 5'-end. In such embodiments, the abasic nucleotide is a terminal nucleotide, which may be an inverted nucleotide, i.e., it is linked to an adjacent nucleotide via a 3'-3' internucleotide bond (if on the 3'-end of the strand) or a 5'-5' internucleotide bond (if on the 5'-end of the strand) rather than a natural 3'-5' internucleotide bond. The abasic nucleotide may also include a sugar modification, such as any of the sugar modifications described above. In certain embodiments, the abasic nucleotide includes a 2'-modification, such as a 2'-fluoro modification, a 2'-O-methyl modification, or a 2'-H (deoxy) modification. In one embodiment, the abasic nucleotide includes a 2'-O-methyl modification. In another embodiment, the abasic nucleotide includes a 2'-H modification (i.e., a deoxyabasic nucleotide).

[0045] The present inventors have discovered that incorporating modified nucleotides into an RNAi construct according to a specific pattern results in an RNAi construct with improved in vivo gene silencing activity. For example, in one embodiment, the RNAi construct of the present invention comprises a sense strand and an antisense strand that comprise sequences sufficiently complementary to each other to form a duplex region of at least 15 base pairs, wherein: the nucleotides at positions 2, 7, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro modified nucleotides; the nucleotides in the sense strand at positions 8 to 11 and 13 (counting from the 5' end) paired with the antisense strand are 2'-fluoro-modified nucleotides; Neither the sense nor the antisense strand has more than 7 total 2'-fluoro modified nucleotides each.

[0046] In another embodiment, an RNAi construct of the invention comprises a sense strand and an antisense strand that comprise sequences sufficiently complementary to each other to form a duplex region of at least 19 base pairs, wherein: the nucleotides at positions 2, 7, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro modified nucleotides, the nucleotides at positions 4, 6, 10, and 12 (counting from the 5' end) are optionally 2'-fluoro modified nucleotides, and all other nucleotides in the antisense strand are modified nucleotides other than 2'-fluoro modified nucleotides; The nucleotides in the sense strand at positions 8 to 11 and 13 (counting from the 5' end) in the antisense strand that are paired with positions 3 and 5 (counting from the 5' end) in the antisense strand are optionally 2'-fluoro-modified nucleotides, and all other nucleotides in the sense strand are modified nucleotides other than 2'-fluoro-modified nucleotides.

[0047] In these embodiments, the modified nucleotides other than 2'-fluoro modified nucleotides may be selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides. In these and other embodiments, the terminal nucleotide at the 3' end, the 5' end, or both the 3' and 5' ends of the sense strand may be an abasic nucleotide or a deoxyribonucleotide. In these embodiments, the abasic nucleotide or deoxyribonucleotide may be inverted, i.e., linked to the adjacent nucleotide via a 3'-3' internucleotide bond (if on the 3' end of the strand) or via a 5'-5' internucleotide bond (if on the 5' end of the strand), rather than the natural 3'-5' internucleotide bond.

[0048] In any of the above embodiments, the nucleotides at positions 2, 7, 12, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro modified nucleotides. In other embodiments, the nucleotides at positions 2, 4, 7, 12, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro modified nucleotides. In yet other embodiments, the nucleotides at positions 2, 4, 6, 7, 12, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro modified nucleotides. In yet other embodiments, the nucleotides at positions 2, 4, 6, 7, 10, 12, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro modified nucleotides. In an alternative embodiment, the nucleotides at positions 2, 7, 10, 12, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro modified nucleotides. In certain other embodiments, the nucleotides at positions 2, 4, 7, 10, 12, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro modified nucleotides.

[0049] In any of the above embodiments, the nucleotides in the sense strand at positions 3, 8-11, and 13 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides. In some embodiments, the nucleotides in the sense strand at positions 5, 8-11, and 13 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides. In other embodiments, the nucleotides in the sense strand at positions 3, 5, 8-11, and 13 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides.

[0050] In certain embodiments of the invention, an 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 that of the antisense strand to form a duplex region, wherein the RNAi construct comprises a structure represented by formula (A): 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 NM N L N M N L N F N L -5' (A) During the ceremony, The top strand, listed in 5' to 3' direction, is the sense strand, and the bottom strand, listed in 3' to 5' direction, is the antisense strand; each N F represents a 2'-fluoro modified nucleotide, each N M represents a modified nucleotide independently selected from a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), and a deoxyribonucleotide; each N L represents a modified nucleotide independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides; N T represents a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide; When x is 1, 2, 3, or 4, N A x is an integer from 0 to 4, and N is a nucleotide selected from the group consisting of an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide; Aone or more of the nucleotides can be complementary to nucleotides in the antisense strand; When y is 1, 2, 3, or 4, one or more n nucleotides are base-paired with nucleotides in the antisense strand. If y is an integer from 0 to 4, provided that the overhanging nucleotide is a modified or unmodified nucleotide that does not When z is 1, 2, 3, or 4, N B z is an integer from 0 to 4, and N is a nucleotide selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides, provided that one or more of the nucleotides are modified nucleotides independently selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides. B One or more of the nucleotides, when present in the sense strand, are N A may be complementary to or base paired with nucleotides in the sense strand If The overhanging nucleotides may be non-overhanging nucleotides.

[0051] In some embodiments in which the RNAi construct comprises a structure represented by Formula (A), there is a nucleotide overhang 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 embodiments in which there is a 2-nucleotide overhang 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 other embodiments in which the RNAi construct comprises a structure represented by Formula (A), the RNAi construct comprises blunt ends at the 3'-end of the sense strand and the 5'-end of the antisense strand (i.e., y is 0). In those embodiments 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 of the embodiments where x is greater than 0, the terminal nucleotide at the 5' end of the sense strand, N A The nucleotide may be an inverted nucleotide, such as an inverted abasic nucleotide or an inverted deoxyribonucleotide.

[0052] In certain embodiments in which the RNAi construct comprises a structure represented by formula (A), the N at positions 4 and 12 from the 5' end in the antisense strand are M In another embodiment, N at positions 4, 6, and 12 from the 5' end in the antisense strand are each a 2'-fluoro modified nucleotide. M In yet another embodiment, N at positions 4, 6, 10, and 12, counting from the 5' end in the antisense strand, are each a 2'-fluoro modified nucleotide. M are each a 2'-fluoro modified nucleotide. In an alternative embodiment in which the RNAi construct has a structure represented by formula (A), N at positions 10 and 12 from the 5' end in the antisense strand are Mare each a 2'-fluoro modified nucleotide. In a related embodiment, N at positions 4, 10, and 12, counting from the 5' end in the antisense strand, are M In another alternative embodiment in which the RNAi construct has a structure represented by formula (A), N at positions 4, 6, and 10 from the 5' end in the antisense strand are each a 2'-fluoro modified nucleotide. M are 2'-O-methyl modified nucleotides, and N at the 12th position counting from the 5' end in the antisense strand M is a 2'-fluoro modified nucleotide. In some embodiments, wherein the RNAi construct comprises a structure represented by formula (A), each N M is a 2'-O-methyl modified nucleotide. In another embodiment, each N M is a 2'-fluoro modified nucleotide. In yet another embodiment in which the RNAi construct comprises a structure represented by formula (A), each N in both the sense and antisense strands M is a 2'-O-methyl modified nucleotide.

[0053] In any of the above embodiments in which the RNAi construct comprises a structure represented by formula (A), each N L In any of these and the above embodiments, N in formula (A) may be a 2'-O-methyl modified nucleotide. T can be an inverted abasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0054] In certain embodiments of the 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 that of the antisense strand to form a duplex region, and the RNAi construct comprises a structure represented by formula (B): 5'-(N A ) x N L N LN 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) During the ceremony, The top strand, listed in 5' to 3' direction, is the sense strand, and the bottom strand, listed in 3' to 5' direction, is the antisense strand; each N F represents a 2'-fluoro modified nucleotide, each N L represents a modified nucleotide independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides; N Trepresents a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide; When x is 1, 2, 3, or 4, N A x is an integer from 0 to 4, and N is a nucleotide selected from the group consisting of an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide; A one or more of the nucleotides can be complementary to nucleotides in the antisense strand; When y is 1, 2, 3, or 4, one or more n nucleotides are base-paired with nucleotides in the antisense strand. If y is an integer from 0 to 4, provided that the overhanging nucleotide is a modified or unmodified nucleotide that does not When z is 1, 2, 3, or 4, N B z is an integer from 0 to 4, and N is a nucleotide selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides, provided that one or more of the nucleotides are modified nucleotides independently selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides. B One or more of the nucleotides, when present in the sense strand, are N A may be complementary to or base paired with nucleotides in the sense strand If The overhanging nucleotides may be non-overhanging nucleotides.

[0055] In some embodiments in which the RNAi construct comprises a structure represented by formula (B), there is a nucleotide overhang 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 embodiments in which there is a 2-nucleotide overhang 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 other embodiments in which the RNAi construct comprises a structure represented by formula (B), the RNAi construct comprises blunt ends at the 3'-end of the sense strand and the 5'-end of the antisense strand (i.e., y is 0). In those embodiments 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 of the embodiments where x is greater than 0, the terminal nucleotide at the 5' end of the sense strand, N A The nucleotide may be an inverted nucleotide, such as an inverted abasic nucleotide or an inverted deoxyribonucleotide.

[0056] In any of the above embodiments in which the RNAi construct comprises a structure represented by formula (B), each N L In these and any of the above embodiments, N in formula (B) may be a 2'-O-methyl modified nucleotide. T can be an inverted abasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0057] In some embodiments of the invention, an 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 that of the antisense strand to form a duplex region, and the RNAi construct comprises a structure represented by formula (C): 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) During the ceremony, The top strand, listed in 5' to 3' direction, is the sense strand, and the bottom strand, listed in 3' to 5' direction, is the antisense strand; each N F represents a 2'-fluoro modified nucleotide, each N L represents a modified nucleotide independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides; each N Mrepresents a modified nucleotide independently selected from a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide; N T represents a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide; x is 0 or 1 and Ab is an inverted abasic nucleotide.

[0058] In certain embodiments in which the RNAi construct comprises a structure represented by formula (C), N M is a 2'-fluoro modified nucleotide. In these and other embodiments, each N in the sense strand M is a 2'-O-methyl modified nucleotide. In an alternative embodiment, each N M is a 2'-fluoro modified nucleotide. In some embodiments where the RNAi construct comprises a structure represented by formula (C), each N in both the sense and antisense strands M is a 2'-O-methyl modified nucleotide.

[0059] In any of the above embodiments in which the RNAi construct comprises a structure represented by formula (C), each N L In any of these and the above embodiments, N in formula (C) may be a 2'-O-methyl modified nucleotide. T can be an inverted abasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide. For example, in one embodiment, N T is an inverted abasic nucleotide or an inverted deoxyribonucleotide, and x is 0. In another embodiment, NT is a 2'-O-methyl modified nucleotide and x is 1. In yet another embodiment, N T is an inverted abasic nucleotide or an inverted deoxyribonucleotide, and x is 1.

[0060] In certain embodiments, an RNAi construct of the 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 that of the antisense strand to form a duplex region, wherein the RNAi construct comprises a structure represented by formula (D): 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) During the ceremony, The top strand, listed in 5' to 3' direction, is the sense strand, and the bottom strand, listed in 3' to 5' direction, is the antisense strand; each N F represents a 2'-fluoro modified nucleotide, each N M represents a modified nucleotide independently selected from a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), and a deoxyribonucleotide; each N L represents a modified nucleotide independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides; N T represents a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide; When x is 1, 2, 3, or 4, N A x is an integer from 0 to 4, and N is a nucleotide selected from the group consisting of an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a BNA, and a deoxyribonucleotide; A one or more of the nucleotides can be complementary to nucleotides in the antisense strand; When y is 1, 2, 3, or 4, one or more n nucleotides are base-paired with nucleotides in the antisense strand. If y is an integer from 0 to 4, provided that the overhanging nucleotide is a modified or unmodified nucleotide that does not When z is 1, 2, 3, or 4, N B z is an integer from 0 to 4, and N is a nucleotide selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides, provided that one or more of the nucleotides are modified nucleotides independently selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, and deoxyribonucleotides. B One or more of the nucleotides, when present in the sense strand, are N A may be complementary to or base paired with nucleotides in the sense strand If The overhanging nucleotides may be non-overhanging nucleotides.

[0061] In some embodiments in which the RNAi construct comprises a structure represented by formula (D), there is a nucleotide overhang 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 embodiments in which there is a 2-nucleotide overhang 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 other embodiments in which the RNAi construct comprises a structure represented by formula (D), the RNAi construct comprises blunt ends at the 3'-end of the sense strand and the 5'-end of the antisense strand (i.e., y is 0). In those embodiments 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 of the embodiments where x is greater than 0, the terminal nucleotide at the 5' end of the sense strand, N A The nucleotide may be an inverted nucleotide, such as an inverted abasic nucleotide or an inverted deoxyribonucleotide.

[0062] In certain embodiments in which the RNAi construct comprises a structure represented by formula (D), the Ns at positions 4, 6, 8, 9, and 16, counting from the 5' end of the antisense strand, are M are 2'-fluoro-modified nucleotides, and N at positions 7 and 12 counting from the 5' end of the antisense strand M are each a 2'-O-methyl modified nucleotide. In another embodiment, N M are 2'-fluoro-modified nucleotides, and Ns at positions 7 to 9 from the 5' end of the antisense strand are M In yet another embodiment, N at positions 4, 6, 8, 9, and 16, counting from the 5' end of the antisense strand, are each a 2'-O-methyl modified nucleotide. M are 2'-O-methyl modified nucleotides, and N is at the 7th and 12th positions counting from the 5' end of the antisense strand. M In an alternative embodiment in which the RNAi construct has a structure represented by formula (D), N at positions 4, 6, 8, 9, and 12 from the 5' end in the antisense strand are each a 2'-fluoro modified nucleotide. M are 2'-O-methyl modified nucleotides, and N is at the 7th and 16th positions counting from the 5' end in the antisense strand. M In certain other embodiments in which the RNAi construct comprises a structure represented by formula (D), Ns at positions 7, 8, 9, and 12, counting from the 5' end in the antisense strand, are each a 2'-fluoro modified nucleotide. M are 2'-O-methyl modified nucleotides, and N at positions 4, 6, and 16 from the 5' end in the antisense strand are M are each a 2'-fluoro modified nucleotide. In these and other embodiments in which the RNAi construct comprises a structure represented by formula (D), N M is a 2'-fluoro modified nucleotide. M is a 2'-O-methyl modified nucleotide.

[0063] In any of the above embodiments in which the RNAi construct comprises a structure represented by formula (D), each N L In any of these and the above embodiments, N in formula (D) may be a 2'-O-methyl modified nucleotide. T can be an inverted abasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0064] The RNAi constructs of the present invention may also contain one or more modified internucleotide linkages. As used herein, the term "modified internucleotide linkage" refers to an internucleotide linkage other than a natural 3'-5' phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing internucleotide linkage, such as a phosphotriester, an aminoalkylphosphotriester, an alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), a phosphinate, a phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), a phosphorothioate (P=S), a chiral phosphorothioate, a phosphorodithioate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, and a boranophosphate. In one embodiment, the modified internucleotide linkage is a 2'-5' phosphodiester linkage. In other embodiments, the modified internucleotide linkage is a non-phosphorus-containing internucleotide linkage and may therefore be referred to as a modified internucleoside linkage. Such non-phosphorus-containing linkages include, but are not limited to, morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane linkages (-O-Si(H)-O-); sulfide, sulfoxide, and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamic acid backbones; methylenemethylimino (-CH-N(CH)-O-CH-) and methylenehydrazino linkages; sulfonic acid and sulfonamide linkages; amide linkages; and others having mixed N, O, S, and CH component moieties. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to generate peptide nucleic acids or PNAs, such as those described in U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.Other suitable modified internucleotide and internucleoside linkages that can be employed in the RNAi constructs of the present invention are described in U.S. Pat. No. 6,693,187, U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entirety.

[0065] In certain embodiments, RNAi constructs of the present invention contain one or more phosphorothioate internucleotide linkages. The phosphorothioate internucleotide linkages can be present in the sense strand, the antisense strand, or both strands of the RNAi construct. For example, in some embodiments, the sense strand contains 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In other embodiments, the antisense strand contains 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In still other embodiments, both strands contain 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. The RNAi construct can contain one or more phosphorothioate internucleotide linkages at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand, the antisense strand, or both strands. For example, in certain embodiments, an RNAi construct comprises from about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 3'-end of the sense strand, the antisense strand, or both strands. In other embodiments, an RNAi construct comprises from about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, the antisense strand, or both strands.

[0066] In some embodiments, the RNAi construct comprises a single phosphorothioate internucleotide linkage between the terminal nucleotides at the 3' end of the sense strand. In other embodiments, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at the 3' end of the sense strand. In one embodiment, the RNAi construct comprises a single phosphorothioate internucleotide linkage between the terminal nucleotides at the 3' end of the sense strand and a single phosphorothioate internucleotide linkage between the terminal nucleotides at the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at the 3' end of the antisense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at the 3' end of the antisense strand). In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' end and the 5' end of the antisense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' and 5' ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at the 5' end of the sense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' and 5' ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at the 3' end of the sense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' and 5' ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' and 5' ends of the sense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at both the 5' and 3' ends of the antisense strand, and phosphorothioate internucleotide linkages at the first and second internucleotide linkages at both the 5' and 3' ends of the sense strand).In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' and 5' ends of the antisense strand, and a single phosphorothioate internucleotide linkage between the terminal nucleotides at the 3' end of the sense strand. In any of the embodiments in which one or both strands comprise one or more phosphorothioate internucleotide linkages, the remaining internucleotide linkages within the strands can be natural 3'-5' phosphodiester linkages. For example, in some embodiments, each internucleotide linkage in the sense strand and the antisense strand is selected from phosphodiester and phosphorothioate, and at least one internucleotide linkage is phosphorothioate.

[0067] In embodiments in which the RNAi construct comprises a nucleotide overhang, two or more of the unpaired nucleotides in the overhang may be linked by phosphorothioate internucleotide bonds. In certain embodiments, all unpaired nucleotides in the 3'-end nucleotide overhang of the antisense strand and / or sense strand are linked by phosphorothioate internucleotide bonds. In other embodiments, all unpaired nucleotides in the 5'-end nucleotide overhang of the antisense strand and / or sense strand are linked by phosphorothioate internucleotide bonds. In still other embodiments, all unpaired nucleotides in any nucleotide overhang are linked by phosphorothioate internucleotide bonds.

[0068] An RNAi construct of the present invention may have any one of chemical modification patterns P1 to P30 shown in Figure 1. For example, in some embodiments, the RNAi construct comprises a sense strand 19 to 23 nucleotides in length and an antisense strand 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 duplex region of 19 to 21 base pairs, the nucleotides at positions 2, 7, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides in the sense strand that are paired with positions 8 to 11 and 13 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides, neither the sense strand nor the antisense strand has more than 7 total 2'-fluoro-modified nucleotides, respectively, and the RNAi construct has nucleotide overhangs at the 3' ends of the sense strand and the antisense strand.

[0069] In one embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 and 9-12, and 2'-O-methyl modified nucleotides at positions 1-6, 8, and 13-21 (counting from the 5' end), and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides 19 and 20 and between nucleotides 20 and 21 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and at the 3' end of the antisense strand.

[0070] In another embodiment, the RNAi construct comprises: (a) (i) 22 nucleotides in length; (ii) an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 1, a 2'-fluoro modified nucleotide at positions 8 and 10-13, and a 2'-O-methyl modified nucleotide at positions 2-7, 9, and 14-22 (counting from the 5' end); and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 20 and 21 and between nucleotides at positions 21 and 22 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing 2 nucleotides at the 3' end of the sense strand and a nucleotide overhang containing 1 to 2 nucleotides at the 3' end of the antisense strand.

[0071] In another embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 and 9-12, and 2'-O-methyl modified nucleotides at positions 1-6, 8, and 13-21 (counting from the 5' end), and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides 19 and 20 and between nucleotides 20 and 21 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 10, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8, 9, 11, 13, and 15 to 21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and at the 3' end of the antisense strand.

[0072] In yet another embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 and 9-12, and 2'-O-methyl modified nucleotides at positions 1-6, 8, and 13-21 (counting from the 5' end), and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides 19 and 20 and between nucleotides 20 and 21 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 7, 10, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 8, 9, 11, 13, and 15-21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and at the 3' end of the antisense strand.

[0073] In another specific embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 and 9-12, and 2'-O-methyl modified nucleotides at positions 1-6, 8, and 13-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end); and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and at the 3' end of the antisense strand.

[0074] In certain embodiments, the RNAi construct comprises a sense strand 19-21 nucleotides in length and an antisense strand 21-23 nucleotides in length, wherein the sequences of the antisense and sense strands are sufficiently complementary to each other to form a duplex region of 19-21 base pairs, wherein the nucleotides at positions 2, 7, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides in the sense strand at positions 8-11 and 13 (counting from the 5' end) paired with the antisense strand are 2'-fluoro-modified nucleotides, and wherein neither the sense strand nor the antisense strand has more than 7 total 2'-fluoro-modified nucleotides, respectively, and the RNAi construct has a nucleotide overhang at the 3' end of the sense strand and a blunt end at the 5' end of the antisense strand / 3' end of the sense strand.

[0075] In one embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9 and 11-14, and 2'-O-methyl modified nucleotides at positions 1-8, 10, and 15-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end), and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0076] In another embodiment, the RNAi construct comprises: (a) (i) 22 nucleotides in length; (ii) an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 1, a 2'-fluoro modified nucleotide at positions 10 and 12-15, and a 2'-O-methyl modified nucleotide at positions 2-9, 11, and 16-22 (counting from the 5' end); and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 21 and 20 and between nucleotides at positions 21 and 22 (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing 1 to 2 nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0077] In yet another embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9 and 11-14, and 2'-O-methyl modified nucleotides at positions 1-8, 10, and 15-21 (counting from the 5' end), and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides 19 and 20 and between nucleotides 20 and 21 (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0078] In yet another embodiment, the RNAi construct comprises: (a) (i) 22 nucleotides in length; (ii) an inverted abasic nucleotide or an inverted deoxyribonucleotide at positions 1 to 22, a 2'-fluoro modified nucleotide at positions 10 and 12 to 15, and a 2'-O-methyl modified nucleotide at positions 2 to 9, 11, and 16 to 21 (counting from the 5' end); and (iii) a sense strand having a phosphorothioate internucleotide linkage between nucleotides at positions 21 and 22; (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing 1 to 2 nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0079] In one particular embodiment, the RNAi construct comprises: (a) (i) 19 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 and 9-12, and 2'-O-methyl modified nucleotides at positions 1-6, 8, and 13-19 (counting from the 5' end), and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 17 and 18 and between nucleotides at positions 18 and 19 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0080] In another specific embodiment, the RNAi construct comprises: (a) (i) 19 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 and 9-12, and 2'-O-methyl modified nucleotides at positions 1-6, 8, and 13-18, and an inverted abasic nucleotide or an inverted deoxyribonucleotide (counting from the 5' end) at position 19; and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 17 and 18 and between nucleotides at positions 18 and 19 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0081] In another specific embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9 and 11-14, and 2'-O-methyl modified nucleotides at positions 1-8, 10, and 15-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end), and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0082] In yet another embodiment, the RNAi construct comprises: (a) (i) 22 nucleotides in length; (ii) an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 1, a 2'-fluoro modified nucleotide at positions 10 and 12-15, and a 2'-O-methyl modified nucleotide at positions 2-9, 11, and 16-22 (counting from the 5' end); and (iii) a sense strand having phosphorothioate internucleotide linkages between the nucleotides at positions 20 and 21 and between the nucleotides at positions 21 and 22; (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing 1 to 2 nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0083] In yet another embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9 and 11-14, and 2'-O-methyl modified nucleotides at positions 1-8, 10, and 15-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end), and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 6, 8-11, 13, and 15-23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0084] In another specific embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9, 11 to 14, 17, and 19, and 2'-O-methyl modified nucleotides at positions 1 to 8, 10, 15, 16, 18, and 20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide (counting from the 5' end) at position 21; and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 6, 8-11, 13, and 15-23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0085] In another specific embodiment, the RNAi construct comprises: (a) (i) 19 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 and 9-12, and 2'-O-methyl modified nucleotides at positions 1-6, 8, and 13-18, and an inverted abasic nucleotide or an inverted deoxyribonucleotide (counting from the 5' end) at position 19; and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 18 and 19, and optionally between nucleotides at positions 17 and 18 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0086] In another specific embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9 and 11-14, and 2'-O-methyl modified nucleotides at positions 1-8, 10, and 15-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end), and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 7, 10, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 8, 9, 11, 13, and 15-23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0087] In another specific embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9 and 11-14, and 2'-O-methyl modified nucleotides at positions 1-8, 10, and 15-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end), and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 10, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8, 9, 11, 13, and 15 to 23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0088] In another specific embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9, 11 to 14, 17, and 19, and 2'-O-methyl modified nucleotides at positions 1 to 8, 10, 15, 16, 18, and 20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide (counting from the 5' end) at position 21; and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 10, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8, 9, 11, 13, and 15 to 23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0089] In another specific embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9 and 11-14, and 2'-O-methyl modified nucleotides at positions 1-8, 10, and 15-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end), and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 7, 10, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 6, 8, 9, 11, 13, and 15-23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0090] In some embodiments of the present invention, an RNAi construct comprises a sense strand 19-23 nucleotides in length and an antisense strand 19-23 nucleotides in length, the sequences of the antisense and sense strands being sufficiently complementary to each other to form a duplex region of 19-21 base pairs, the nucleotides at positions 2, 14, and 16 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides in the sense strand paired with positions 10-13 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides, and neither the sense strand nor the antisense strand has more than seven total 2'-fluoro-modified nucleotides. In these embodiments, the RNAi construct has a nucleotide overhang at the 3' end of the sense strand and a blunt 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 sense strand and the 3' end of the antisense strand.

[0091] In one particular embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 and 9-12, and 2'-O-methyl modified nucleotides at positions 1-6, 8, and 13-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end); and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 9, 14, and 16, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 7, 10-13, 15, and 17-23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0092] In another specific embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 and 9-12, and 2'-O-methyl modified nucleotides at positions 1-6, 8, and 13-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end); and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 14, and 16, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 13, 15, and 17 to 23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0093] In another specific embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 and 9-12, and 2'-O-methyl modified nucleotides at positions 1-8, 6, and 13-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end); and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 23 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 14, and 16, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 7-13, 15, and 17-23 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0094] In another specific embodiment, the RNAi construct comprises: (a) (i) 19 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 5 and 7-10, and 2'-O-methyl modified nucleotides at positions 1-4, 6, and 11-18, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 19 (counting from the 5' end); and (iii) a sense strand having a phosphorothioate internucleotide bond between the nucleotides at positions 18 and 19 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 9, 14, and 16, and 2'-O-methyl modified nucleotides at positions 1, 3, 5, 7, 10-13, 15, and 17-21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0095] In another specific embodiment, the RNAi construct comprises: (a) (i) 20 nucleotides in length; (ii) an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 1, a 2'-fluoro modified nucleotide at positions 8 to 11, and a 2'-O-methyl modified nucleotide at positions 2 to 7 and 12 to 20 (counting from the 5' end); and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 18 and 19 and between nucleotides at positions 19 and 20 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 14, and 16, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 13, 15, and 17 to 21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing 1 to 2 nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0096] In another embodiment, the RNAi construct comprises: (a) (i) 22 nucleotides in length; (ii) an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 1, a 2'-fluoro modified nucleotide at positions 8 to 11, and a 2'-O-methyl modified nucleotide at positions 2 to 7 and 12 to 22 (counting from the 5' end); and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 20 and 21 and between nucleotides at positions 21 and 22 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 14, and 16, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 13, 15, and 17 to 21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing 2 nucleotides at the 3' end of the sense strand and a nucleotide overhang containing 1 to 2 nucleotides at the 3' end of the antisense strand.

[0097] In certain embodiments of the invention, an RNAi construct comprises a sense strand 19-23 nucleotides in length and an antisense strand 19-23 nucleotides in length, wherein the sequences of the antisense and sense strands are sufficiently complementary to each other to form a duplex region of 19-21 base pairs, wherein nucleotides at positions 2, 7, 12, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides, and nucleotides in the sense strand paired with positions 10-13 (counting from the 5' end) in the antisense strand are 2'-fluoro-modified nucleotides, wherein neither the sense strand nor the antisense strand has more than 7 total 2'-fluoro-modified nucleotides, respectively, and the RNAi construct has nucleotide overhangs at the 3' ends of the sense strand and the antisense strand.

[0098] For example, in one embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 7 to 10 and 2'-O-methyl modified nucleotides at positions 1 to 6 and 11 to 21 (counting from the 5' end), and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides 19 and 20 and between nucleotides 20 and 21 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang comprising two nucleotides at the 3' end of the sense strand and a nucleotide overhang comprising two nucleotides at the 3' end of the antisense strand.

[0099] In another embodiment, the RNAi construct comprises: (a) (i) 22 nucleotides in length; (ii) an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 1, a 2'-fluoro modified nucleotide at positions 8 to 11, and a 2'-O-methyl modified nucleotide at positions 2 to 7 and 12 to 22 (counting from the 5' end); and (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 20 and 21 and between nucleotides at positions 21 and 22 (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has a nucleotide overhang containing 2 nucleotides at the 3' end of the sense strand and a nucleotide overhang containing 1 to 2 nucleotides at the 3' end of the antisense strand.

[0100] In certain embodiments of the invention, an RNAi construct comprises a sense strand 19-21 nucleotides in length and an antisense strand 19-21 nucleotides in length, wherein the sequences of the antisense and sense strands are sufficiently complementary to each other to form a duplex region of 19-21 base pairs, wherein the nucleotides at positions 2, 7, 12, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoro modified nucleotides, and the nucleotides in the sense strand at positions paired with positions 10, 11, and 13 (counting from the 5' end) in the antisense strand are 2'-fluoro modified nucleotides, and wherein neither the sense strand nor the antisense strand has more than 7 total 2'-fluoro modified nucleotides, respectively. (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9 and 11-14, and 2'-O-methyl modified nucleotides at positions 1-8, 10, and 15-20, and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end), and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has two blunt ends.

[0101] In another such embodiment, the RNAi construct comprises: (a) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 9 to 12 and 2'-O-methyl modified nucleotides at positions 1 to 8 and 13 to 20 and an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 21 (counting from the 5' end); and (iii) a sense strand having a phosphorothioate internucleotide bond between the 20th and 21st nucleotides (counting from the 5' end); (b) (i) 21 nucleotides in length; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methyl modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 19 and 20, and between the nucleotides at positions 20 and 21 (counting from the 5' end); The RNAi construct has two blunt ends.

[0102] In some embodiments of the present invention, the 5'-end of the sense strand, the 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, including unmodified phosphate (-OP=O)(OH)OH) and modified phosphate. Modified phosphates include phosphates in which one or more of the O and OH groups are replaced with H, O, S, N(R), or alkyl, where R is H, an amino-protecting group, or unsubstituted or substituted alkyl. Exemplary phosphate moieties include, but are not limited to, 5'-monophosphate; 5'-diphosphate; 5'-triphosphate; 5'-guanosine cap (7-methylated or unmethylated); 5'-adenosine cap or any other modified or unmodified nucleotide cap structure; 5'-monothiophosphate (phosphorothioate); 5'-monodithiophosphate (phosphorodithioate); 5'-α-thiotriphosphate; 5'-γ-thiotriphosphate; 5'-phosphoramidate; 5'-vinyl phosphate; 5'-alkylphosphonates (e.g., alkyl = methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyl ether phosphonates (e.g., alkyl ether = methoxymethyl, ethoxymethyl, etc.).

[0103] Modified nucleotides that can be incorporated into RNAi constructs of the present invention can have two or more chemical modifications described herein. For example, modified nucleotides can have a modification to the ribose sugar and a modification to the nucleobase. By way of example, modified nucleotides can 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 can include a sugar modification combined with a modification to the 5' phosphate, which will generate a modified internucleotide or internucleoside linkage when the modified nucleotide is incorporated into a polynucleotide. For example, in some embodiments, modified nucleotides can include sugar modifications such as a 2'-fluoro modification, a 2'-O-methyl modification, or a bicyclic sugar modification and a 5' phosphorothioate group. Thus, in some embodiments, one or both strands of an RNAi construct of the present invention include a combination of 2'-modified nucleotides or BNAs and phosphorothioate internucleotide linkages. In certain embodiments, both the sense and antisense strands of the RNAi constructs of the invention comprise a combination of 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, and phosphorothioate internucleotide linkages.

[0104] In certain embodiments, the first nucleotide from the 5' end of the antisense strand in the RNAi construct can comprise A, dA, dU, U, or dT. In some embodiments, at least one of the first three base pairs from the 5' end of the antisense strand in the duplex region is an AU base pair. In one particular embodiment, the first base pair from the 5' end of the antisense strand in the duplex region is an AU base pair.

[0105] The RNAi construct of the present invention can be easily produced using techniques known in the art, for example, conventional solid-phase nucleic acid synthesis. The polynucleotide of the RNAi construct can be assembled on a suitable nucleic acid synthesizer using standard nucleotide or nucleoside precursors (e.g., phosphoramidites). Automated nucleic acid synthesizers are commercially available from several vendors, including the DNA / RNA synthesizer from Applied Biosystems (Foster City, CA), the MerMade synthesizer from BioAutomation (Irving, TX), and the OligoPilot synthesizer from GE Healthcare Life Sciences (Pittsburgh, PA). An exemplary method for synthesizing the RNAi construct of the present invention is described in Example 1.

[0106] Oligonucleotides can be synthesized via phosphoramidite chemistry using a 2' silyl protecting group with acid-labile dimethoxytrityl (DMT) at the 5' position of the ribonucleoside. Final deprotection conditions are known not to significantly degrade the RNA product. All syntheses can be performed on any automated or manual synthesizer on a large, medium, or small scale. Synthesis can also be performed in multi-well plates, columns, or glass slides.

[0107] The 2'-O-silyl group can be removed by exposure to fluoride ions, which can include any source of fluoride ions, such as salts containing fluoride ions paired with inorganic counterions, such as cesium fluoride and potassium fluoride, or salts containing fluoride ions paired with organic counterions, such as tetraalkylammonium fluoride. Crown ether catalysts can be used in combination with inorganic fluorides in the deprotection reaction. Preferred fluoride ion sources are tetrabutylammonium fluoride or aminohydrofluorides (e.g., triethylamine and aqueous HF combined in a dipolar aprotic solvent, such as dimethylformamide).

[0108] The choice of protecting groups for use on the phosphite triesters and phosphotriesters can alter the stability of the triesters to fluoride. Methyl protection of the phosphotriester or phosphite triester can stabilize the bond to fluoride ions and improve process yields.

[0109] Because ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position in RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group, e.g., one that is stable to acid treatment. Silyl protecting groups meet this requirement and can be easily removed in a final fluoride deprotection step that may result in minimal RNA degradation.

[0110] Tetrazole catalysts can be used in standard phosphoramidite coupling reactions. Preferred catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, p-nitrophenyltetrazole.

[0111] As can 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. In addition, various synthetic steps may be performed in an alternative order or sequence to obtain the desired compound. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc. present in bases), and protecting group techniques (protection and deprotection) useful in synthesizing the RNAi constructs described herein are known in the art, including, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Buts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley & Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley & Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley & Sons (1995), and subsequent editions thereof. Custom synthesis of RNAi agents is also available from several commercial vendors, including Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).

[0112] The RNAi construct of the present invention may contain a ligand. As used herein, "ligand" refers to any compound or molecule that can directly or indirectly interact with another compound or molecule. The interaction between another compound or molecule and a ligand can induce a biological response (e.g., trigger a signal transduction cascade, induce receptor-mediated endocytosis), or it can simply be a physical association. A ligand can modify one or more properties of the double-stranded RNA molecule to which it is bound, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties of the RNA molecule.

[0113] Ligands include serum proteins (e.g., human serum albumin, low-density lipoproteins, globulins), cholesterol moieties, vitamins (biotin, vitamin E, vitamin B 12), folate moieties, steroids, bile acids (e.g., cholic 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 to specific cell types, such as the liver). Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl groups, hexadecyl Examples of suitable glycerols include glycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine, peptides (e.g., antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, polymers such as polyethylene glycol (PEG) (e.g., PEG-40K), polyamino acids, and polyamines (e.g., spermine, spermidine).

[0114] In certain embodiments, the ligand has endosome-destabilizing properties. The endosome-destabilizing ligand promotes endosomal lysis and / or transport of the RNAi construct of the present invention or its components from the endosome to the cytoplasm of the cell. The endosome-destabilizing ligand can be a polycationic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosome-destabilizing ligand adopts its active conformation at endosomal pH. The "active" conformation is one in which the endosome-destabilizing ligand promotes endosomal lysis and / or transport of the RNAi construct of the present invention or its components from the endosome to the cytoplasm of the cell. Exemplary endosome-destabilizing ligands include 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 derivatives thereof (Turk et al., Biochem. Biophys. Acta, Vol. 1559:56-68, 2002). In one embodiment, the endosome-destabilizing component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosome-destabilizing component may be linear or branched.

[0115] In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or other 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 cholesterol moieties and other lipids for conjugation to nucleic acid molecules are also described in U.S. Pat. Nos. 7,851,615; 7,745,608; and 7,833,992, all of which are incorporated herein by reference in their entireties. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated to a folate moiety can be taken up into cells via receptor-mediated endocytosis. Such folate-polynucleotide conjugates are described in U.S. Pat. No. 8,188,247, which is incorporated herein by reference in its entirety.

[0116] The ligand can target the RNAi construct to a specific tissue or cell type to selectively inhibit expression of the target gene in that tissue or cell type. In one embodiment, the ligand targets the specific delivery of the RNAi construct to liver cells (e.g., hepatocytes) using various means, as described in more detail below. In certain embodiments, the RNAi construct is targeted to liver cells by a ligand that binds to the surface-expressed asialoglycoprotein receptor (ASGR) or its components (e.g., ASGR1, ASGR2).

[0117] In some embodiments, the RNAi construct can be specifically targeted to the liver by employing a ligand that binds to or interacts with a protein expressed on the surface of liver cells. For example, in certain embodiments, the ligand can comprise an antigen-binding protein (e.g., an antibody or a binding fragment thereof (e.g., Fab, scFv)) that specifically binds to a receptor expressed on liver cells, such as the asialoglycoprotein receptor and the LDL receptor. In a particular 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. A "Fab fragment" is composed of one immunoglobulin light chain (i.e., the light chain variable region (VL) and constant region (CL)) and the CH1 region and variable region (VH) of one immunoglobulin heavy chain. 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. An "scFv fragment" comprises the VH and VL regions of an antibody, these regions being present in a single polypeptide chain, and optionally including a peptide linker between the VH and VL regions that enables the Fv to form the desired structure for antigen binding. Exemplary antibodies and binding fragments thereof that specifically bind to ASGR1 and can be used as ligands for targeting the RNAi constructs of the invention to the liver are described in WO 2017 / 058944, the entire contents of which are incorporated herein by reference. Other antibodies or binding fragments thereof that specifically bind to ASGR1, LDL receptor, or other proteins expressed on the surface of the liver and that are suitable for use as ligands in the RNAi constructs of the invention are commercially available.

[0118] 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 linear, branched, or cyclic) with 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 incorporated into the ligand is a monosaccharide selected from pentose, hexose, or heptose, as well as 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.

[0119] In some embodiments, the ligand comprises a hexose or hexosamine. The hexose may be selected from glucose, galactose, mannose, fucose, or fructose. The 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 particular embodiments, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose, and N-acetyl-galactosamine (GalNAc) are particularly effective for targeting compounds to liver cells, as such ligands bind to ASGR, which is expressed on the surface of liver cells. See, e.g., D'Souza and Devarajan, J. Control Release, Vol. 203:126-139, 2015. Examples of GalNAc- or galactose-containing ligands that can be incorporated into the RNAi constructs of the invention are described in U.S. Patent Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Application Publication No. 20030130186; and WO 2013166155, all of which are incorporated herein by reference in their entireties.

[0120] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety containing two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate moiety contains two or more binding domains composed of carbohydrates that can bind to two or more different molecules or to two or more different sites on the same molecule. The valency of a carbohydrate moiety refers to the number of individual binding domains within the carbohydrate moiety. For example, the terms "monovalent," "bivalent," "trivalent," and "tetravalent" with respect to a carbohydrate moiety refer to carbohydrate moieties having one, two, three, and four binding domains, respectively. A multivalent carbohydrate moiety can comprise a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety can be bivalent, trivalent, or tetravalent. In such embodiments, the polyvalent carbohydrate moiety can be biantennary or triantennary. In a specific embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another specific embodiment, the polyvalent galactose moiety is trivalent or tetravalent. Exemplary trivalent or tetravalent GalNAc-containing ligands for incorporation into the RNAi constructs of the invention are described in detail below.

[0121] The ligand can be directly or indirectly bound or conjugated to the RNA molecule of the RNAi construct. For example, in some embodiments, the ligand is covalently bound directly to the sense or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently bound to the sense or antisense strand of the RNAi construct via a linker. The ligand can be bound to the nucleobase, sugar moiety, or internucleotide linkage of the polynucleotide (e.g., the sense or antisense strand) of the RNAi construct of the present invention. Conjugation or binding to a purine nucleobase or a derivative thereof can occur at any position, including endocyclic and exocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is bound to the ligand. Conjugation or binding to a pyrimidine nucleobase or a derivative thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase can be bound to the ligand. Conjugation or binding to the sugar moiety of the nucleotide can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that can be attached to a ligand include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to a ligand, such as in abasic nucleotides. Internucleotide linkages can also facilitate ligand attachment. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the ligand can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the ligand can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0122] In certain embodiments, the ligand can be attached to the 3' or 5' end of either the sense strand or the antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. In such embodiments, the ligand is attached to the 5'-terminal nucleotide of the sense strand. In these and other embodiments, the ligand is attached at the 5' position of the 5'-terminal nucleotide of the sense strand. In embodiments in which an inverted abasic nucleotide or inverted deoxyribonucleotide is the 5'-terminal nucleotide of the sense strand and is attached to the adjacent nucleotide via a 5'-5' internucleotide bond, the ligand can be attached to the 3' position of the inverted abasic nucleotide or 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 certain such embodiments, the ligand is attached at the 3' position of the 3'-terminal nucleotide of the sense strand. In embodiments in which the inverted abasic nucleotide or inverted deoxyribonucleotide is the 3'-terminal nucleotide of the sense strand and is attached to the adjacent nucleotide via a 3'-3' internucleotide linkage, the ligand can be attached to the 5'-position of the inverted abasic nucleotide or inverted deoxyribonucleotide. In alternative embodiments, the ligand is attached near the 3'-end of the sense strand but before one or more terminal nucleotides (i.e., before one, two, three, or four terminal nucleotides). In some embodiments, the ligand is attached at the 2'-position of the sugar of the 3'-terminal nucleotide of the sense strand. In other embodiments, the ligand is attached at the 2'-position of the sugar of the 5'-terminal nucleotide of the sense strand.

[0123] In certain embodiments, the ligand is attached to the sense or antisense strand via a linker. A "linker" is an atom or group of atoms that covalently attaches the ligand to the polynucleotide component of the RNAi construct. Linkers can be about 1 to about 30 atoms in length, about 2 to about 28 atoms in length, about 3 to about 26 atoms in length, about 4 to about 24 atoms in length, about 6 to about 20 atoms in length, about 7 to about 20 atoms in length, about 8 to about 20 atoms in length, about 8 to about 18 atoms in length, about 10 to about 18 atoms in length, and about 12 to about 18 atoms in length. In some embodiments, the linker may comprise a bifunctional linking moiety, typically comprising an alkyl moiety bearing two functional groups. One of the functional groups is selected to attach to a compound of interest (e.g., the sense or antisense strand of the RNAi construct), and the other is selected to attach to essentially any selected group, such as a ligand, as described herein. In certain embodiments, the linker comprises a chain structure or oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups commonly employed in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, the bifunctional linking moiety comprises amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bond), etc.

[0124] Linkers that can be used to attach a ligand to the sense or antisense strand in the RNAi constructs of the invention include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10 Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0125] In certain embodiments, the linker is cleavable. A cleavable linker is one that is sufficiently stable outside the cell but is cleaved after entry into the target cell to release the two moieties that the linker holds together. In some embodiments, the cleavable linker is cleaved at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold or more, or at least 100-fold faster in the target cell or under a first reference condition (e.g., which may be selected to mimic or correspond to intracellular conditions) than in the subject's blood or under a second reference condition (e.g., which may be selected to mimic or correspond to conditions found in blood or serum).

[0126] Cleavable linkers are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradative molecules. Generally, cleaving agents are found to be more prevalent or at higher levels or activity inside cells than in serum or blood. Examples of such degradative agents include oxidizing or reducing agents that are selective for specific substrates or have no substrate specificity, including oxidizing or reducing enzymes or reducing agents such as mercaptans, which are present inside cells and can degrade redox-cleavable linkers by reduction; esterases; agents that can create endosomes or acidic environments, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linkers by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0127] The cleavable linker may contain a moiety that is sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand into the cell interior or desired compartment of the cell.

[0128] The linker can include a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the linker can depend on the target cell. For example, a liver targeting ligand can be linked to an RNA molecule via a linker containing an ester group. Liver cells are rich in esterase, so the linker will be cleaved more efficiently in liver cells than in cell types that are not rich in esterase. Other types of cells that are rich in esterase include lung, renal cortex, and testicular cells. Linkers containing peptide bonds can be used when targeting peptidase-rich cells such as liver cells and synovial cells.

[0129] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or when in contact with non-target tissues. Thus, the relative susceptibility to cleavage can be determined between first conditions selected to be indicative of cleavage in target cells and second conditions selected to be indicative of cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell culture, organ or tissue culture, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in whole animals. In some embodiments, useful candidate linkers are cleaved at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 70-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0130] In other embodiments, a redox-cleavable linker is utilized. A redox-cleavable linker is cleaved upon reduction or oxidation. One example of a reductively cleavable group is a disulfide linking group (-SS-). One or more methods described herein can 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 particular RNAi construct and a particular ligand. For example, a candidate linker can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate that would be observed in cells, such as target cells. The candidate linker can also be evaluated under conditions selected to mimic blood or serum conditions. In certain embodiments, the candidate linker is cleaved at a maximum of 10% in blood. In other embodiments, useful linker candidates are degraded at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 70-fold, or 100-fold 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).

[0131] In yet other embodiments, the ligand is covalently attached to the sense or antisense strand of the RNAi construct using a phosphate-based cleavable linker that is cleaved by an agent that degrades or hydrolyzes phosphate groups. One example of an agent that hydrolyzes phosphate groups within a cell is an enzyme such as an intracellular phosphatase. 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(S)(Rk)-O-, -SP(O)(Rk)-S- and -OP(S)(Rk)-S-, where Rk can be hydrogen or alkyl. Particular 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 particular embodiment is -OP(O)(OH)-O-. These linker candidates can be evaluated using methods similar to those described above.

[0132] In other embodiments, the linker may include an acid-cleavable group, which is a group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable group is cleaved in an acidic environment of about pH 6.5 or below (e.g., about 6.0, 5.5, 5.0 or below) or by an agent, such as an enzyme, that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, may provide a cleavage environment for the acid-cleavable group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). A particular embodiment is where the carbon bonded to the oxygen (alkoxy group) of the ester is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethyl, pentyl, or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0133] In other embodiments, the linker may include an ester-based cleavable group that 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. Ester cleavable groups have the general formula -C(O)O- or -OC(O)-. These linker candidates can be evaluated using methods similar to those described above.

[0134] In further embodiments, the linker may comprise a peptidic cleavable group that is cleaved by enzymes such as peptidases and proteases in cells. Peptidic cleavable groups are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptidic cleavable groups include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptidic cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins. Peptidic cleavable linking groups have the general formula -NHCHR A C(O)NHCHR B C(O)—, where R A and R B are the side chains of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0135] Other types of linkers suitable for attaching a ligand to the sense or antisense strand in the RNAi constructs of the invention are known in the art and can include those described in U.S. Pat. Nos. 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551, all of which are incorporated herein by reference in their entireties.

[0136] In certain embodiments, the ligand covalently attached to the sense or antisense strand of an RNAi construct of the invention comprises a GalNAc moiety, e.g., 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 other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3'-end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5'-end of the sense strand.

[0137] In certain embodiments, an RNAi construct of the invention comprises a ligand having the following structure: [ka] In a preferred embodiment, a ligand having this structure is covalently attached to the 5' end of the sense strand via a linker, such as those described herein. In one embodiment, the linker is an aminohexyl linker.

[0138] Examples of trivalent and tetravalent GalNAc moieties and linkers that can be attached to double-stranded RNA molecules in the RNAi constructs of the present invention are provided below in structural formulas I-IX, where "Ac" in the formulas listed herein represents an acetyl group.

[0139] In one embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula I below, 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 (represented by a solid wavy line). [ka]

[0140] In another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula II below, 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 (represented by a solid wavy line). [ka]

[0141] In yet another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula III below, wherein the ligand is attached to the 3' end of the sense strand of the double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0142] In yet another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula IV below, where the ligand is attached to the 3' end of the sense strand of the double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0143] In certain embodiments, the RNAi construct comprises a ligand and a linker having the structure of Formula V below, where 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 (represented by a solid wavy line). [ka]

[0144] In other embodiments, the RNAi construct comprises a ligand and a linker having the structure of Formula VI below, where 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 (represented by a solid wavy line). [ka]

[0145] In one particular embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula VII below, where X=O or S, and the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a wavy line). [ka]

[0146] In some embodiments, the RNAi construct comprises a ligand and a linker having the structure of Formula VIII below, where 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 (represented by a solid wavy line). [ka]

[0147] In certain embodiments, the RNAi construct comprises a ligand and a linker having the structure of Formula IX below, where the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0148] Phosphorothioate bonds can be substituted for the phosphodiester bonds shown in any one of Formulas I-IX to covalently attach the ligand and linker to the nucleic acid strand.

[0149] The present invention also includes pharmaceutical compositions and preparations comprising the RNAi constructs described herein and pharmaceutically acceptable carriers, excipients or diluents.Such compositions and preparations are useful for reducing the expression of target genes in subjects that require it.When considering clinical use, pharmaceutical compositions and preparations will be prepared in a form that is suitable for the intended use.In general, this will entail preparing compositions that are essentially free from pyrogens and other impurities that may be harmful to humans or animals.

[0150] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carriers, excipients, or diluents" include solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for human administration. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the RNAi construct of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the composition, provided that they do not inactivate the RNAi construct of the composition.

[0151] The composition and method of pharmaceutical composition formulation depend on several factors, including, but not limited to, the route of administration, the type and severity of the disease or disorder being treated, or the dose to be administered. In some embodiments, the pharmaceutical composition is formulated based on the intended delivery route. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral delivery modes include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal, or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such embodiments, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such embodiments, the pharmaceutical composition may include a targeting ligand (e.g., a GalNAc-containing or antibody-containing ligand described herein).

[0152] In some embodiments, the pharmaceutical composition comprises an effective amount of an RNAi construct described herein. An "effective amount" is an amount sufficient to produce a beneficial or desired clinical result. In some embodiments, an effective amount is an amount sufficient to reduce expression of a target gene in a particular tissue or cell type (e.g., liver or hepatocytes) of a subject.

[0153] Administration of the pharmaceutical compositions of the present invention can be via any common route, as long as the target tissue is available via that 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 hepatic portal vein. In some embodiments, the pharmaceutical compositions are administered parenterally. For example, in certain embodiments, the pharmaceutical compositions are administered intravenously. In other embodiments, the pharmaceutical compositions are administered subcutaneously.

[0154] Colloidal dispersion systems, such as oil-in-water emulsions, micelles, mixed micelles, and macromolecular complexes, including liposomes, nanocapsules, microspheres, beads, and lipid-based systems, can be used as delivery vehicles for the RNAi constructs of the present invention. Commercially available lipid emulsions suitable for delivering the nucleic acids of the present invention include Intralipid® (Baxter International Inc.), Liposyn® (Abbott Pharmaceuticals), Liposyn® II (Hospira), Liposyn® III (Hospira), Nutrilipid (B. Braun Medical Inc.), and other similar lipid emulsions. A preferred colloidal system for use as an in vivo delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The RNAi constructs of the present invention can be encapsulated within liposomes or complexed with liposomes, particularly cationic liposomes. Alternatively, the RNAi constructs of the present invention can be complexed with lipids, particularly cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC)), distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos. 5,981,505, 6,217,900, 6,383,512, 5,783,565, 7,202,227, 6,379,965, 6,127,170, 5,837,533, 6,747,014, and WO 03 / 093449.

[0155] In some embodiments, the RNAi constructs of the present invention are fully encapsulated in, for example, a lipid formulation to form SNALP or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles. SNALPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs exhibit long circulatory life after intravenous injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them extremely useful for systemic administration. 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 nontoxic. Additionally, nucleic acids present in nucleic acid-lipid particles are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, 6,815,432, and WO 96 / 40964.

[0156] Pharmaceutical compositions suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy syringability exists. Preparations should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. Suitable solvents or dispersion media may contain, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0157] Sterile injectable solution can be prepared by incorporating active compound in appropriate amount with any other desired components (for example, as listed above) into a solvent, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other desired components, for example, the components listed above.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method includes vacuum drying and freeze-drying, which produces powder of active ingredient and any other desired components from its solution that has been previously sterilized and filtered.

[0158] The compositions of the present invention can generally be formulated in a neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with 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 with free carboxyl groups can 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.).

[0159] For parenteral administration in an aqueous solution, for example, the solution is usually suitably buffered and the liquid diluent first rendered isotonic, for example, with sufficient saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, a sterile aqueous medium is employed, as known to those skilled in the art, particularly in light of the present disclosure. By way of example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards required by FDA standards. In certain embodiments, the pharmaceutical compositions of the invention comprise or consist of sterile saline and the RNAi constructs described herein. In other embodiments, pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and sterile water (e.g., water for injection, WFI). In yet other embodiments, pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and phosphate buffered saline (PBS).

[0160] In some embodiments, the pharmaceutical composition of the present invention is packaged in or stored in an administration device.Devices for injection preparations include, but are not limited to, injection ports, pre-filled syringes, automatic injection devices, injection pumps, wearable syringes, and injection pens.Devices for aerosolized or powdered preparations include, but are not limited to, inhalers, inhalers, inhalers, etc.Therefore, the present invention includes an administration device comprising the pharmaceutical composition of the present invention for treating or preventing one or more diseases or disorders.

[0161] The present invention provides a method for reducing or inhibiting target gene expression in a cell by contacting the cell with any one of the RNAi constructs described herein. The cell can be in vitro or in vivo. Target gene expression can be assessed by measuring the amount or level of target mRNA, target protein, or another biomarker associated with target gene expression. The reduction in target gene expression in a cell or animal treated with an RNAi construct of the present invention can be determined compared to target gene expression in a cell or animal not treated with the RNAi construct or treated with a control RNAi construct. For example, in some embodiments, reduction or inhibition of target gene expression is assessed by (a) measuring the amount or level of target mRNA in cells treated with an 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 directed at an RNA molecule not expressed in the cell or an RNAi construct having a nonsense or scrambled sequence) or 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). Prior to comparison, the target mRNA levels in the treated and control cells can be normalized to RNA levels for a control gene (e.g., 18S ribosomal RNA or a housekeeping gene). Target mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assay, fluorescent in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, droplet digital PCR, etc.

[0162] In other embodiments, reduction or inhibition of target gene expression is assessed by (a) measuring the amount or level of target protein in cells treated with an RNAi construct of the present invention, (b) measuring the amount or level of target protein in cells treated with a control RNAi construct (e.g., an RNAi agent directed at an RNA molecule that is not expressed in cells or an RNAi construct having a nonsense or scrambled sequence) or 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, immunoassays (e.g., ELISA), and flow cytometry.

[0163] The present invention also provides a method for reducing or inhibiting target gene expression in a subject in need thereof, the method comprising administering any one of the RNAi constructs described herein to the subject. The RNAi constructs of the present invention can be used to treat or ameliorate a disease, condition, or disorder associated with aberrant target gene expression or activity, for example, when overexpression of the gene product results in a pathological phenotype. Exemplary target genes include, but are not limited to, 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. Target genes can also include viral genes such as hepatitis B and C virus genes, human immunodeficiency virus genes, herpes virus genes, etc. In some embodiments, the target gene is a gene encoding a human microRNA (miRNA).

[0164] In certain embodiments, the RNAi constructs of the invention reduce target gene expression in a cell or subject by at least 50%. In some embodiments, the RNAi constructs of the invention reduce target gene expression in a cell or subject by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%. In other embodiments, the RNAi constructs of the invention reduce target gene expression in liver cells by about 90% or more, e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. The percent reduction in target gene expression can be measured by any of the methods described herein and other methods known in the art.

[0165] The following examples, including the experiments conducted and results obtained, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims. [Example]

[0166] Example 1. In vivo activity of PNPLA3 RNAi constructs with different chemical modification patterns To assess the effect 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- and 2'-O-methyl-modified nucleotides and evaluated in a humanized mouse model expressing PNPLA3, as described in more detail below.

[0167] RNAi constructs were synthesized using solid-phase phosphoramidite chemistry on a MerMade12 (Bioautomation) machine.

[0168] material Acetonitrile (DNA synthesis grade, AXO152-2505, EMD) Capping Reagent A (80:10:10 (v / v / v) tetrahydrofuran / lutidine / acetic anhydride, BIO221 / 4000, EMD) Capping Reagent B (16% 1-methylimidazole / tetrahydrofuran, BIO345 / 4000, EMD) Activator solution (0.25 M 5-(ethylthio)-1H-tetrazole (ETT) in acetonitrile, BIO152 / 0960, EMD) Detritylation reagent (3% dichloroacetic acid in dichloromethane, BIO830 / 4000, EMD) Oxidizing reagent (0.02 M iodine in 70:20:10 (v / v / v) tetrahydrofuran / pyridine / water, BIO420 / 4000, EMD) Diethylamine solution (20% DEA in acetonitrile, NC0017-0505, EMD) Thiolating reagent (0.05 M 5-N-[(dimethylamino)methylene]amino-3H-1,2,4-dithiazole-3-thione (BIOSULII / 160K) in 40:60 (v / v) pyridine / acetonitrile) 5'-aminohexyl linker phosphoramidites of adenosine, guanosine, cytosine, and uridine, phosphorylated phosphoramidites, 2'-deoxythymidine phosphoramidite, and 2'-methoxy and 2'-fluorophosphoramidites (Thermo Fisher Scientific), 0.10 M in acetonitrile over approximately 10 mL of molecular sieves (3 Å, JT Baker). CPG support (high-load general-purpose support, 500A (BH5-3500-G1), 79.6μmol / g, 0.126g (10μmol)) Ammonium hydroxide (high concentration, JTBaker)

[0169] synthesis The reagent solution, phosphoramidite solution, and solvent were connected to the MerMade 12 instrument. A solid support was added to each column (4 mL SPE tubing with upper and lower frits), and the column was attached to the instrument. The column was washed twice with acetonitrile. The phosphoramidite and reagent solution lines were purged. Synthesis was initiated using Poseidon software. Synthesis was performed by repeated deprotection / coupling / oxidation / capping synthesis cycles. 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. The phosphoramidite and activator solutions were added to the support, followed by incubation to couple the incoming nucleotide to the free 5'-hydroxyl group. The support was washed with acetonitrile. An oxidation or thiolation reagent was added to the support to convert the phosphite triester to a phosphate triester or phosphorothioate. Capping reagents A and B were added to the support to terminate any unreacted oligonucleotide chains. The support was washed with acetonitrile. After the final reaction cycle, the resin was washed with diethylamine solution to remove the 2-cyanoethyl protecting group, and the support was washed with acetonitrile and dried under vacuum.

[0170] GalNAc conjugation The sense strand was prepared using a 5'-aminohexyl linker for conjugation to a trivalent N-acetyl-galactosamine (GalNAc) moiety (structure shown in Formula VII below). After automated synthesis, the column was removed from the instrument and transferred to a vacuum manifold in a hood. The 5'-monomethoxytrityl (MMT) protecting group was removed from the solid support by successive treatment with 2 mL aliquots of 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) using vacuum filtration. Once no further orange / yellow color was observed in the eluate, the resin was washed with dichloromethane. The resin was then washed with 5 mL of 2% diisopropylethylamine in N,N-dimethylformamide (DMF). In a separate vial, a solution of GalNAc3-Lys2-Ahx (67 mg, 40 μmol) (the structure and synthesis of which are described below) in DMF (0.5 mL) was prepared using 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, and the column was capped and incubated overnight at room temperature. The resin was washed with DMF and DCM and dried under vacuum.

[0171] Disconnect The synthesis columns were removed from the synthesizer or vacuum manifold. The solid support from each column was transferred to a 10 mL vial. 4 mL of concentrated ammonium hydroxide was added to the solid support. The cap was tightly attached to the bottle, and the mixture was heated at 55°C for 4 hours. The bottle was moved to a freezer and cooled in a hood for 20 minutes before being opened. The mixture was filtered through an 8 mL SPE tube to remove the solid support. The vial and solid support were rinsed with 1 mL of 50:50 ethanol / water.

[0172] Analysis and Purification A portion of the combined filtrate was analyzed and purified by anion exchange chromatography. The pooled fractions were desalted by size exclusion chromatography and analyzed by ion-pair reversed-phase high-performance liquid chromatography-mass spectrometry (HPLC-MS). The pooled fractions were lyophilized to yield a white amorphous powder.

[0173] Analytical Anion Exchange Chromatography (AEX): Column: Thermo DNAPac PA200RS (4.6 x 50 mm, 4 μm) Equipment: Agilent 1100 HPLC Buffer A: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5 Buffer B: 20 ​​mM sodium phosphate, 10% acetonitrile, pH 8.5, 1 M sodium bromide Flow rate: 1 mL / min at 40°C Gradient: 20-65% B in 6.2 min Preparative Anion Exchange Chromatography (AEX): Column: Tosoh TSK Gel SuperQ-5PW21 x 150 mm, 13 μm Equipment: Agilent1200 HPLC Buffer A: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5 Buffer B: 20 ​​mM sodium phosphate, 10% acetonitrile, pH 8.5, 1 M sodium bromide Flow rate: 8mL / min Injection volume: 5mL Gradient: 35-55% B over 20 min Preparative Size Exclusion Chromatography (SEC): Column: GE Hi-Prep 26 / 10 Equipment: GE AKTA Pure Buffer solution: 20% ethanol in water Flow rate: 10mL / min Injection volume: 15 mL using a sample loading pump Ion-pair reversed phase (IP-RP) HPLC: Column: Water Xbridge BEH OST C18, 2.5 μm, 2.1 × 50 mm Equipment: Agilent 1100 HPLC Buffer A: 15.7 mM DIEA, 50 mM hexafluoroisopropanol (HFIP) in water Buffer B: 15.7 mM DIEA, 50 mM HFIP in 50:50 water / acetonitrile Flow rate: 0.5mL / min Gradient: 10-30% B over 6 min

[0174] annealing Small amounts of the 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 based on dry weight. Actual sample concentrations were measured on a NanoDrop One (ssDNA, extinction coefficient = 33 μg / OD260). The two strands were then mixed in an equimolar ratio, and the samples were heated in a 90°C water bath for 5 minutes and slowly cooled to room temperature. Samples were analyzed by AEX. Duplexes were registered and subjected to in vivo testing as described in more detail below.

[0175] Preparation of GalNAc3-Lys2-Ahx Formula VII [ka] where X=O or S. The wavy line represents the point of attachment to the 5'-terminal nucleotide of the sense strand of the RNAi construct.

[0176] To a 50 mL falcon tube was added Fmoc-Ahx-OH (1.13 g, 3.19 mmol) in DCM (30 mL), followed by DIEA (2.23 mL, 12.78 mmol). The solution was added to 2-Cl trityl chloride resin (3.03 g, 4.79 mmol) in a 50 mL centrifuge tube and placed on a shaker for 2 hours. The solvent was drained, and the resin was washed with 17:2:1 DCM / MeOH / DIEA (2 x 30 mL), DCM (4 x 30 mL), and then dried. The loading was determined to be 0.76 mmol / g by UV spectrophotometric detection at 290 nm.

[0177] 3 g of the loaded 2-Cl trityl resin was suspended in 20% 4-methylpiperidine in DMF (20 mL), and after 30 min the solvent was drained. This process was repeated once more, and the resin was washed with DMF (30 mL x 3) and DCM (30 mL x 3).

[0178] To a solution of Fmoc-Lys(ivDde)-OH (3.45 g, 6 mmol) in DMF (20 mL) was added TATU (1.94 g, 6 mmol), followed by DIEA (1.83 mL, 10.5 mmol). The solution was then added to the deprotected resin, and the suspension was placed on a shaker overnight. The solvent was drained, and the resin was washed with DMF (30 mL × 3) and DCM (30 mL × 3).

[0179] The resin was treated with 20% 4-methylpiperidine in DMF (15 mL) and the solvent was drained after 10 min. This process was repeated once more, and the resin was washed with DMF (15 mL x 4) and DCM (15 mL x 4).

[0180] To a solution of Fmoc-Lys(Fmoc)-OH (3.54 g, 6 mmol) in DMF (20 mL) was added TATU (1.94 g, 6 mmol), followed by DIEA (1.83 mL, 10.5 mmol). The solution was then added to the deprotected resin, and the suspension was placed on a shaker overnight. The solvent was drained, and the resin was washed with DMF (30 mL × 3) and DCM (30 mL × 3).

[0181] The resin was treated with 5% hydrazine in DMF (20 mL) and the solvent was drained after 5 min. This process was repeated four more times, and the resin was washed with DMF (30 mL × 4) and DCM (30 mL × 4).

[0182] 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) was added TATU (3.22 g, 10 mmol), and the solution was stirred for 5 min. DIEA (2.96 mL, 17 mmol) was added to this solution, and then the mixture was added to the above resin. The suspension was kept at room temperature overnight, and the solvent was drained. The resin was washed with DMF (3 × 30 mL) and DCM (3 × 30 mL).

[0183] The resin was treated with 1% TFA in DCM (30 mL, containing 3% triisopropylsilane), and after 5 min the solvent was drained. This process was repeated three more times, and the combined filtrates were concentrated under reduced pressure. The residue was triturated with diethyl ether (50 mL), and the suspension was filtered and dried to give the crude product. The crude product was purified by reverse-phase chromatography, eluting with 0–20% MeCN in water. The fractions were combined and lyophilized to give the product as a white solid.

[0184] Table 1 below shows the location of the modifications in the sense and antisense sequences of each of the modified PNPLA3 RNAi constructs. Nucleotide sequences are listed according to the following notation: dT, dA, dG, dC = the corresponding deoxyribonucleotides; a, u, g, and c = the corresponding 2'-O-methylribonucleotides; Af, Uf, Gf, and Cf = the corresponding 2'-deoxy-2'-fluoro ("2'-fluoro") ribonucleotides; Phos = the terminal nucleotide having a monophosphate group at its 5' end; invAb = an inverted abasic nucleotide (i.e., an abasic nucleotide that is attached to an adjacent nucleotide through its 3'-substituent when at the 3' end of the strand (3'-3' linkage) or through its 5'-substituent when at the 5' end of the strand (5'-5' internucleotide linkage)); and invdX = an inverted deoxyribonucleotide (i.e., a deoxyribonucleotide that is attached to an adjacent nucleotide through its 3'-substituent when at the 3' end of the strand (3'-3' linkage) or through its 5'-substituent when at the 5' end of the strand (5'-5' internucleotide linkage)). The insertion of an "s" in a sequence indicates that two adjacent nucleotides are linked by a phosphorothiodiester group (e.g., a phosphorothioate internucleotide linkage). Unless otherwise specified, all other nucleotides are linked by a 3'-5' phosphodiester group. All RNAi constructs are conjugated to the GalNAc moiety shown in Formula VII via the 5' end of the sense strand. Table 1 also lists the pattern and sequence family designations of each RNAi construct. The pattern designations are displayed diagrammatically in Figure 1. When an RNAi construct has the same sequence family designation as another RNAi construct, the two constructs have the same core sequence but different chemical modification patterns.

[0185] [Table 1]

[0186] In the initial experimental setup, 13 different PNPLA3 RNAi constructs with different sequences were synthesized with either the P1 chemical modification pattern or the CM1 control chemical modification pattern. siRNA molecules with the CM1 control chemical modification pattern have been reported to have potent and long-lasting gene silencing effects in vivo. See Nair et al., J. Am. Chem. Soc., Vol. 136:16958-16961, 2014. The efficacy of the chemically modified RNAi constructs in inhibiting PNPLA3 gene expression in humanized mouse models expressing wild-type human PNPLA3 or a mutant form of human PNPLA3 was evaluated. To generate the mouse models, 1 × 10 RNAi constructs were cultured per animal in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). 12 Adenovirus (AAV; serotype AAV8 or AAV7; endotoxin-free) diluted into viral particles was intravenously injected into the tail vein of C57BL / 6NCrl male mice (Charles River Laboratories Inc.), and human PNPLA3, PNPLA3 rs738409 , or PNPLA3 rs738409-rs738408 Mice were generally 10-12 weeks old, with an n of 4-6 animals per treatment group.

[0187] All RNAi constructs were AAV-PNPLA3, PNPLA3 rs738409 , and / or PNPLA3 rs738409-rs738408 At least two vehicle-treated control groups were tested: AAV empty vector treated with vehicle, and AAV-PNPLA3, PNPLA3 rs738409 , or PNPLA3 rs738409-rs738408Two weeks after AAV injection, mice were treated with a single dose of RNAi construct (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) via subcutaneous injection at 0.5, 1.0, 3.0, or 5.0 milligrams per kilogram of animal. On days 8, 15, 22, 28, or 42 after RNAi construct injection, livers were collected from the animals, snap-frozen in liquid nitrogen, and processed for purified RNA using a QIACube HT instrument (9001793) and a Qiagen RNeasy 96 QIACube HT kit (74171) according to the manufacturer's instructions. Samples were analyzed using a QIAxpert system (9002340). RNA was treated with Promgea RQ1 RNase-Free DNase (M6101) and prepared for real-time qPCR using the Applied Biosystems TaqMan™ RNA-to-CT™ 1-Step Kit (4392653). Real-time qPCR was performed on a QuantStudio real-time PCR instrument. Results are presented as the relative knockdown of human PNPLA3 mRNA expression compared to vehicle-treated control animals, based on human PNPLA3 gene expression normalized to mouse Gapdh (TaqMan™ assays from Invitrogen, hs00228747_m1 and 4352932E, respectively).

[0188] Results from the initial set of experiments comparing RNAi constructs with the P1 chemical modification pattern (duplex numbers 4544, 3552, 2393, 3464, 3918, 2390, 2391, 2392, 3465, 3467, 2394, 3539, and 3916) with RNAi constructs with the CM1 control modification pattern (duplex numbers 2118, 2119, 2125, 2120, 2121, 2124, 2370, 2371, 2122, 2368, 2369, 2123, and 3558) are shown in Figure 2. rs738409When administered subcutaneously at 5 mg / kg to mice expressing the mutant gene, constructs with the P1 pattern generally significantly reduced PNPLA3 expression, measured 8 days after injection, compared with constructs with the CM1 pattern, regardless of sequence.

[0189] Variations in P1 modification patterns were created and applied to RNAi constructs with the same core sequence to modify strand length, the nature of the ends (i.e., overhangs vs. blunt ends) of the RNAi constructs, and / or to include inverted abasic nucleotides at the 5' or 3' end of the sense strand. RNAi constructs with novel patterns were evaluated for improved in vivo efficacy in a humanized mouse model. Specifically, RNAi constructs with P1, P2, P3, or P4 chemical modification patterns (duplex numbers 3540, 5241, 5614, and 5615) were used to transfect human PNPLA3. rs738409 Mice expressing the mutant gene were subcutaneously administered a dose of 5 mg / kg. 15 days after administration of the RNAi construct, the expression level of human PNPLA3 in the liver was assessed. The results are shown in Figure 3. RNAi constructs with the P2, P3, or P4 pattern resulted in a higher average reduction in PNPLA3 expression compared to the RNAi construct with the P1 pattern.

[0190] Further variations of the P3 pattern were generated to increase the potency and duration of mRNA knockdown in vivo. In the P3 pattern (duplex #6191), the 2'-fluoro-modified nucleotides at positions 4 and 6 from the 5' end of the antisense strand were changed to 2'-O-methyl-modified nucleotides to generate the P9 pattern (duplex #6267). An RNAi construct was also synthesized with the P9 pattern (duplex #7320), which features an inverted adenosine deoxyribonucleotide at the 3' end of the sense strand instead of an inverted abasic nucleotide. All three constructs were evaluated in the humanized mouse model described above. In animals treated with 5 mg / kg of duplex #6267, hepatic expression of human PNPLA3 was reduced by 97% at 22 days post-administration, while animals treated with 5 mg / kg of duplex #6191 showed a 92% reduction in hepatic expression levels of human PNPLA3 at the same time point. Animals treated with 3 mg / kg duplex #7320 showed a 95% reduction in hepatic expression levels of human PNPLA3 at 28 days post-administration, making duplex #7320 more potent and resulting in longer-lasting gene knockdown than duplexes #6191 and #6267.

[0191] The P9 pattern was applied to PNPLA3 RNAi constructs with two different core sequences (duplex numbers 7318, 7320, 7062, 8513, and 8709), and in vivo efficacy was evaluated at doses of 1 mg / kg and 3 mg / kg in an in vivo bioluminescence imaging assay. For the bioluminescence imaging assay, a companion adenovirus (AAV) vector was designed to contain a murine cytomegalovirus promoter, the entire sequence of firefly luciferase, followed by a synthetic strand of mRNA sequence specific to the RNAi construct being tested, immediately downstream from the firefly luciferase stop codon. Each end of the mRNA sequence was flanked by 10 additional nucleotides. The vector "PP3A(DM)" was packaged into the AAV serotype AAVDJ8 (endotoxin-free). Before injection, PP3A(DM) was injected at 5 × 10 per animal in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). 11The virus particles were diluted and injected intravenously into the tail vein of BALB / c male mice (Charles River Laboratories Inc.). Mice were generally 10-12 weeks old, with n = 5 animals per group.

[0192] Two weeks after AAV injection, mice were injected with RediJect D-luciferin bioluminescent substrate (PerkinElmer, 770504) according to the manufacturer's instructions. After a 10-minute pulse, mice were imaged on an IVIS Spectrum In Vivo Imaging System (PerkinElmer). Subsequently, mice were randomized into groups according to baseline total flux scores from defined regions of interest, including the liver. Once randomized, mice were treated via subcutaneous injection with a single dose of 1.0 or 3.0 milligrams per kilogram of body weight of RNAi constructs (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136), or with phosphate-buffered saline alone (designated "vehicle"). Mice were imaged weekly following the same protocol, applying the same gating constraints to the total flux score. Data are presented as total light flux (photons per second, y-axis) versus weeks after RNAi construct injection (x-axis). A reduction in total light flux indicates a reduction in expression of the luciferase reporter.

[0193] The results of this experiment are shown in Figures 4A and 4B. The signal from the luciferase reporter from animals treated with various RNAi constructs with the P9 pattern was significantly reduced compared to the signal from vehicle-treated animals for at least 3 weeks after a single 1 mg / kg dose of the RNAi construct (Figure 4A), and for at least 5 weeks after a single 3 mg / kg dose (Figure 4B). For many of the RNAi constructs, a single 3 mg / kg dose was sufficient to inhibit luciferase reporter expression for up to 6 weeks.

[0194] These RNAi constructs (duplex numbers 7318, 7320, 7062, 8513, and 8709) were also evaluated in the humanized mouse model described above. Specifically, the RNAi constructs were injected into humanized PNPLA3 at 0.5, 1, or 3 mg / kg. rs738409-rs738408 Mice expressing the mutant gene were subcutaneously administered the RNAi construct. Human PNPLA3 expression levels in the liver were assessed by qPCR 28 or 42 days after administration of the RNAi construct. Results are presented as relative knockdown of human PNPLA3 mRNA expression compared to vehicle-treated control animals and are shown in Table 2 below.

[0195] [Table 2]

[0196] RNAi constructs with the P9 modification pattern are more potent and result in longer-lasting gene knockdown than previously tested patterns. Administration of the RNAi construct at a single dose of 0.5 mg / kg reduced liver expression of human PNPLA3 by approximately 50% four weeks after administration, while administration of the construct at a dose of 1 mg / kg reduced liver expression of human PNPLA3 by approximately 70% four weeks after administration. The 1 mg / kg dose was sufficient to maintain a greater than 55% reduction in PNPLA3 expression for six weeks after administration of a single dose. Administration of a single dose of the RNAi construct at 3 mg / kg reduced liver expression of human PNPLA3 by more than 90% four weeks after administration of a single dose. Liver expression of human PNPLA3 was still reduced by more than approximately 75% six weeks after administration of the 3 mg / kg dose. Improved potency and duration of gene knockdown was observed with RNAi constructs with two different sequences, indicating that the P9 chemical modification pattern is effective in stabilizing RNAi constructs at least partially independent of the nucleobase sequence.

[0197] 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 metabolic stability of siRNA molecules, resulting in improved potency and duration of gene silencing. See Foster et al., Molecular Therapy, Vol. 26:708-717, 2018. All RNAi constructs had the same core nucleotide sequence in the sense and antisense strands; only the chemical modification pattern differed. Two different constructs with the P9 modification pattern were synthesized: one with an inverted abasic residue at the 3' end of the sense strand (duplex number 7318) and one with an inverted deoxythymidine residue at the 3' end of the sense strand (duplex number 8709). RNAi constructs with one of the CM2, CM3, or CM4 modification patterns were also synthesized (duplex numbers 8103, 8104, and 8105, respectively). Each of the RNAi constructs was then injected into humanized PNPLA3 at a dose of 3 mg / kg. rs738409-rs738408 The RNAi constructs were subcutaneously administered to mice expressing the mutant gene. 28 days after administration, human PNPLA3 expression levels in the liver were assessed by qPCR. The results are shown in Figure 5. The RNAi construct with a P9 modification pattern (duplex number 7318) with an inverted abasic residue at the 3' end of the sense strand resulted in the greatest reduction in hepatic PNPLA3 expression among all the constructs tested. The RNAi construct with a P9 modification pattern (duplex number 8709) with an inverted deoxythymidine at the 3' end of the sense strand resulted in a greater reduction in hepatic PNPLA3 expression than the construct with the CM4 pattern (duplex number 8105) and comparable to the constructs with the CM2 and CM3 patterns (duplex numbers 8103 and 8104, respectively).

[0198] In another experimental setting, alternative variants of the P3 modification pattern were designed and their in vivo efficacy was evaluated in a humanized PNPLA3 mouse model. The variants of the P3 pattern were applied to RNAi constructs with two different sequences. The sequences of the sense and antisense strands of each of the RNAi constructs are shown in Table 1, and the modification patterns are shown schematically in Figure 1. The RNAi constructs were administered at a dose of 3 mg / kg to humanized PNPLA3. rs738409-rs738408 The RNAi constructs were subcutaneously administered to mice expressing the mutant gene. 28 days after administration, human PNPLA3 expression levels in the liver were assessed by qPCR. The results are shown in Table 3 below. All RNAi constructs reduced hepatic expression of human PNPLA3 by approximately 90% or more 4 weeks after a single subcutaneous injection of 3 mg / kg.

[0199] [Table 3]

[0200] Example 2. In vivo activity of ASGR1 RNAi constructs with different chemical modification patterns As shown in Example 1, the P1 chemical modification pattern applied to 13 different RNAi constructs with different sequences targeting human PNPLA3 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, an RNAi construct targeting asialoglycoprotein receptor 1 (ASGR1) mRNA was synthesized using the P1 chemical modification pattern according to the method described in Example 1. An RNAi construct with the same sequence was synthesized using the CM1 control chemical modification pattern. The sequences of the RNAi constructs are provided in Table 4 below using the same notation as described in Table 1 above. A GalNAc moiety having the structure shown in Formula VII was conjugated to the 5' end of the sense strand of the RNAi construct designated duplex number 1520, and a GalNAc moiety having the structure shown in Formula IX was conjugated to the 5' end of the sense strand of the RNAi construct designated duplex number 1421. Conjugation of a GalNAc moiety to the sense strand of an RNAi construct was carried out as described in Example 1, except that for the GalNAc moiety having the structure shown in Formula IX, the GalNAc moiety was prepared as follows: To a solution of 2-(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) was added TATU (3.22 g, 10 mmol), 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 (3×30 mL) and DCM (3×30 mL).

[0201] [Table 4]

[0202] 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 10–12 weeks were fed a standard chow diet (2020× Teklad global soy protein-free extruded rodent diet; Harlan). On day 0, mice received a subcutaneous injection of buffer or 5 mg / kg body weight of the indicated RNAi construct in 0.25 ml buffer (n = 9 per group). Three animals were captured on day 4, three on day 8, and three on day 15 for further analysis. Total RNA from the livers of captured 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 construct with the amount of ASGR1 mRNA in the liver tissue of animals injected with buffer. The results show that animals receiving an RNAi construct with the P1 modification pattern (duplex number 1520) showed a greater reduction in hepatic ASGR1 expression at all time points measured than animals receiving an RNAi construct with the CM1 control modification pattern (Figure 6). Similar to the results described in Example 1 using an RNAi construct targeting human PNPLA3 mRNA, the P1 chemical modification pattern increases the potency of the RNAi construct.

[0203] Example 3. In vivo activity of LPA RNAi constructs with different chemical modification patterns To further evaluate the ability of the chemical modification patterns described herein to increase the in vivo efficacy of RNAi constructs, RNAi constructs targeting a third liver gene, the LPA gene, were synthesized according to the method described in Example 1 and conjugated to a GalNAc moiety having the structure shown in Formula VII. The sequences of the RNAi constructs are provided in Table 5 below using the same designations as those described in Table 1 above. Table 5 also lists the pattern designation and sequence family designation of each RNAi construct. The pattern designations are shown diagrammatically in Figure 1. When an RNAi construct has the same sequence family designation as another RNAi construct, the two constructs have the same core sequence but different chemical modification patterns.

[0204] [Table 5]

[0205] In initial experiments, RNAi constructs with the same nucleotide sequence were synthesized with either the CM1 control chemical modification pattern (duplex no. 3632) or the P1 chemical modification pattern (duplex no. 3635). The in vivo efficacy of the two constructs was evaluated in a double transgenic mouse model expressing fully functional human Lp(a) particles with an average serum baseline Lp(a) level of approximately 50-60 mg / dL. Lp(a) is a low-density lipoprotein composed of LDL particles and the glycoprotein apolipoprotein(a) (apo(a)) linked to the LDL particle's apolipoprotein B by a disulfide bond. Apo(a) is encoded by the LPA gene, and changes in LPA gene expression alter serum Lp(a) levels. Double transgenic mice were generated by crossing transgenic mice expressing human apo(a) from a yeast artificial chromosome (YAC) containing the entire human LPA gene (Frazer et al., Nature Genetics, Vol. 9:424-431, 1995) with transgenic mice expressing human apoB-100 (Linton et al., 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 collected before injection, followed by 14 and 28 days after injection. Serum Lp(a) concentrations were measured using an Lp(a) ELISA assay (catalog number 10-1106-01, Mercodia AB, Uppsala, Sweden). The percent change in Lp(a) levels for each animal at a particular time point was calculated based on the animal's baseline Lp(a) level. The results are shown in Figure 7. At two weeks post-injection, administration of duplex 3635 with the P1 modification pattern resulted in a greater mean reduction in serum Lp(a) levels (-49%) compared to duplex number 3632 with the control CM1 modification pattern (-35%), although this was not statistically significant.

[0206] In a second series of experiments, LPA RNAi constructs were synthesized that targeted regions of LPA mRNA different from those in the first set of experiments using the P1 chemical modification pattern or a variant of that pattern. The RNAi constructs with the novel patterns were evaluated in a double transgenic mouse model for both increased magnitude and duration of in vivo suppression of LPA gene expression. Specifically, LPA RNAi constructs from three different sequence families with the P1 modification pattern or one of the pattern variants (e.g., P2, P4, P6, or P7 chemical modification patterns) were subcutaneously administered to the double transgenic mice at a dose of 2 mg / kg. Serum Lp(a) levels in the animals were measured before injection to obtain baseline levels and at 1, 2, and 4 weeks after administration of the LPA RNAi constructs. The results of this set of experiments are shown in Table 6 below. Across the three sequence families, RNAi constructs with P2, P4, P6, or P7 modification patterns resulted in greater and longer-lasting reductions in serum Lp(a) levels compared to RNAi constructs with P1 modification patterns. RNAi constructs with P6 or P7 chemical modification patterns resulted in greater than 80% reductions in serum Lp(a) levels for up to 4 weeks after a single subcutaneous injection of 2 mg / kg.

[0207] [Table 6]

[0208] Next, alternative variants of the chemical modification pattern were designed and evaluated for in vivo efficacy in a double transgenic mouse model. The variants of the chemical modification pattern were applied to RNAi constructs with sequences from five different sequence families. The sequences of the sense and antisense strands of each RNAi construct are shown in Table 5, and the modification patterns are schematically shown in Figure 1. The RNAi constructs were subcutaneously administered at a dose of 1 mg / kg to double transgenic mice expressing human Lp(a) particles. Serum Lp(a) levels in the animals were measured before injection to obtain baseline levels and at 2, 3, and 4 weeks after administration of the LPA RNAi construct. The results are shown in Table 7 below. Several of the pattern variants, such as P9, P19, P22, P24, P27, P28, and P29, resulted in a greater than 50% reduction in Lp(a) serum levels 4 weeks after a single subcutaneous injection of 1 mg / kg. The RNAi construct with the P27 chemical modification pattern was particularly effective in suppressing Lp(a) serum levels, as it resulted in a sustained reduction of Lp(a) levels by approximately 75% 4 weeks after a single injection.

[0209] [Table 7]

[0210] 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 particular methodology, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the appended claims.

[0211] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. An RNAi construct for inhibiting 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 duplex region, the RNAi construct comprising a structure represented by formula (A): 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) During the ceremony, the top strand, listed in 5' to 3' direction, is the sense strand, and the bottom strand, listed in 3' to 5' direction, is the antisense strand; Each N Ｆ represents a 2'-fluoro modified nucleotide, Each N Ｍ represents a modified nucleotide independently selected from a 2'-fluoro modified nucleotide, and a 2'-O-methyl modified nucleotide; Each N Ｌ represents a 2'-O-methyl modified nucleotide, N Ｔ represents a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, and a deoxyribonucleotide; x is an integer from 0 to 4, provided that when x is 1, 2, 3, or 4, N Ａ each of the nucleotides is independently a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, and a deoxyribonucleotide; y is an integer from 0 to 4, with the proviso that when y is 1, 2, 3, or 4, n nucleotides are independently modified or unmodified overhanging nucleotides that do not base pair with nucleotides in the antisense strand; z is an integer from 0 to 4, provided that when z is 1, 2, 3, or 4, N Ｂ An RNAi construct, wherein each of the nucleotides is independently a modified nucleotide selected from 2'-O-methyl modified nucleotides and deoxyribonucleotides.

2. The RNAi construct of claim 1, wherein one or more of the N A nucleotides are complementary to nucleotides in the antisense strand, and one or more of the N B nucleotides are complementary to N A nucleotides when present in the sense strand or are overhanging nucleotides that do not base pair with nucleotides in the sense strand.

3. The RNAi construct of claim 1, wherein the sense strand and the antisense strand are each independently 19 to 23 nucleotides in length.

4. (i) x is 0, y is 2, and z is 2; (ii) x is 2, y is 0, and z is 4; (iii) x is 2, y is 0, and z is 2; or (iv) x is 0, y is 0, and z is 2; The RNAi construct of claim 1.

5. (i) x is 1 and N Ａ is an inverted abasic nucleotide, y is 2, and z is 2; (ii) x is 3, and the 5'-terminal N Ａ is an inverted abasic nucleotide, y is 0, and z is 4; (iii) x is 1 and N Ａ is an inverted abasic nucleotide, y is 0, and z is 2; The RNAi construct of claim 1.

6. x is 2, and each N Ａ The nucleotides are 2'-O-methyl modified nucleotides, y is 0, z is 4, and each N Ｂ The RNAi construct of claim 1, wherein the nucleotides are 2'-O-methyl modified nucleotides.

7. N Ｔ The RNAi construct according to any one of claims 1 to 6, wherein is an inverted abasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

8. (i) N at positions 4 and 12 from the 5' end of the antisense strand Ｍ are each a 2'-fluoro modified nucleotide, (ii) N at positions 4, 6, and 12 from the 5' end of the antisense strand Ｍ are each a 2'-fluoro modified nucleotide, (iii) Ns at positions 4, 6, 10, and 12 from the 5' end of the antisense strand Ｍ are each a 2'-fluoro modified nucleotide, (iv) N at positions 10 and 12 from the 5' end of the antisense strand Ｍ are each a 2'-fluoro modified nucleotide, or (v) Ns at positions 4, 10, and 12 from the 5' end of the antisense strand Ｍ are each a 2'-fluoro modified nucleotide, The RNAi construct according to any one of claims 1 to 7.

9. N at positions 4, 6, and 10 counting from the 5' end of the antisense strand Ｍ are 2'-O-methyl modified nucleotides, and N at the 12th position counting from the 5' end of the antisense strand Ｍ The RNAi construct according to any one of claims 1 to 7, wherein is a 2'-fluoro modified nucleotide.

10. Each N in both the sense and antisense strands Ｍ The RNAi construct according to any one of claims 1 to 7, wherein is a 2'-O-methyl modified nucleotide.

11. Each N in the sense strand Ｍ The RNAi construct according to any one of claims 1 to 9, wherein is a 2'-O-methyl modified nucleotide.

12. Each N in the sense strand Ｍ The RNAi construct according to any one of claims 1 to 9, wherein is a 2'-fluoro modified nucleotide.

13. An RNAi construct for inhibiting 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 duplex region, the RNAi construct comprising a structure represented by formula (B): 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) During the ceremony, the top strand, listed in 5' to 3' direction, is the sense strand, and the bottom strand, listed in 3' to 5' direction, is the antisense strand; Each N Ｆ represents a 2'-fluoro modified nucleotide, Each N Ｌ represents a 2'-O-methyl modified nucleotide, N Ｔ represents a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, and a deoxyribonucleotide; x is an integer from 0 to 4, provided that when x is 1, 2, 3, or 4, N Ａ each of the nucleotides is independently a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, and a deoxyribonucleotide; y is an integer from 0 to 4, with the proviso that when y is 1, 2, 3, or 4, n nucleotides are independently modified or unmodified overhanging nucleotides that do not base pair with nucleotides in the antisense strand; z is an integer from 0 to 4, provided that when z is 1, 2, 3, or 4, N Ｂ Each nucleotide is independently a modified nucleotide selected from 2'-O-methyl modified nucleotides and deoxyribonucleotides, and the RNAi construct.

14. The RNAi construct of claim 13, wherein one or more of the N A nucleotides are complementary to nucleotides in the antisense strand, and one or more of the N B nucleotides are complementary to N A nucleotides when present in the sense strand or are overhanging nucleotides that do not base pair with nucleotides in the sense strand.

15. (i) x is 0, y is 2, and z is 2; (ii) x is 0, y is 0, and z is 2; (iii) x is 1 and N Ａ is an inverted abasic nucleotide, y is 2, and z is 2; (iv) x is 2, y is 0, and z is 4, or (v) x is 3, and N at the 5' end Ａ is an inverted abasic nucleotide, y is 0, and z is 4; The RNAi construct of claim 13.

16. x is 2, and each N Ａ The nucleotides are 2'-O-methyl modified nucleotides, y is 0, z is 4, and each N Ｂ The RNAi construct of claim 13, wherein the nucleotides are 2'-O-methyl modified nucleotides.

17. N Ｔ The RNAi construct according to any one of claims 13 to 16, wherein is an inverted abasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

18. An RNAi construct that inhibits 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 duplex region, the RNAi construct comprising a structure represented by formula (C): 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) During the ceremony, the top strand, listed in 5' to 3' direction, is the sense strand, and the bottom strand, listed in 3' to 5' direction, is the antisense strand; Each N Ｆ represents a 2'-fluoro modified nucleotide, Each N Ｌ represents a 2'-O-methyl modified nucleotide, Each N Ｍ represents a modified nucleotide independently selected from a 2'-fluoro modified nucleotide, and a 2'-O-methyl modified nucleotide; N Ｔ represents a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, and a deoxyribonucleotide; An RNAi construct wherein x is 0 or 1 and Ab is an inverted abasic nucleotide.

19. Each N in both the sense and antisense strands Ｍ The RNAi construct of claim 18, wherein is a 2'-O-methyl modified nucleotide.

20. N Ｔ 20. The RNAi construct of claim 19, wherein x is an inverted abasic nucleotide or an inverted deoxyribonucleotide, and x is 0.

21. N Ｔ is a 2'-O-methyl modified nucleotide and x is 1.

22. N in the antisense strand Ｍ The RNAi construct of claim 18, wherein is a 2'-fluoro modified nucleotide.

23. Each N in the sense strand Ｍ The RNAi construct of claim 22, wherein is a 2'-O-methyl modified nucleotide.

24. Each N in the sense strand Ｍ The RNAi construct of claim 22, wherein is a 2'-fluoro modified nucleotide.

25. N Ｔ The RNAi construct of any one of claims 22 to 24, wherein x is an inverted abasic nucleotide or an inverted deoxyribonucleotide, and x is 0.

26. An RNAi construct for inhibiting 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 duplex region, the RNAi construct comprising a structure represented by formula (D): 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) During the ceremony, the top strand, listed in 5' to 3' direction, is the sense strand, and the bottom strand, listed in 3' to 5' direction, is the antisense strand; Each N Ｆ represents a 2'-fluoro modified nucleotide, Each N Ｍ represents a modified nucleotide independently selected from a 2'-fluoro modified nucleotide, and a 2'-O-methyl modified nucleotide; Each N Ｌ represents a 2'-O-methyl modified nucleotide, N Ｔ represents a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, and a deoxyribonucleotide; x is an integer from 0 to 4, provided that when x is 1, 2, 3, or 4, N Ａ each of the nucleotides is independently a modified nucleotide selected from an abasic nucleotide, an inverted abasic nucleotide, an inverted deoxyribonucleotide, a 2'-O-methyl modified nucleotide, and a deoxyribonucleotide; y is an integer from 0 to 4, with the proviso that when y is 1, 2, 3, or 4, n nucleotides are independently modified or unmodified overhanging nucleotides that do not base pair with nucleotides in the antisense strand; z is an integer from 0 to 4, provided that when z is 1, 2, 3, or 4, N Ｂ An RNAi construct, wherein each of the nucleotides is independently a modified nucleotide selected from 2'-O-methyl modified nucleotides and deoxyribonucleotides.

27. The RNAi construct of claim 26, wherein one or more of the N A nucleotides are complementary to nucleotides in the antisense strand, and one or more of the N B nucleotides are complementary to N A nucleotides when present in the sense strand or are overhanging nucleotides that do not base pair with nucleotides in the sense strand.

28. 27. The RNAi construct of claim 26, wherein the sense strand and the antisense strand are each independently 19 to 23 nucleotides in length.

29. (i) x is 2, y is 0, and z is 4; (ii) x is 1 and N Ａ is an inverted abasic nucleotide, y is 2, and z is 2; (iii) x is 1 and N Ａ is an inverted abasic nucleotide, y is 0, and z is 2; (iv) x is 0, y is 0, and z is 2; or (v) x is 2, y is 0, and z is 2; The RNAi construct of claim 26.

30. x is 2, and each N Ａ The nucleotides are 2'-O-methyl modified nucleotides, y is 0, z is 4, and each N Ｂ 27. The RNAi construct of claim 26, wherein the nucleotides are 2'-O-methyl modified nucleotides.

31. N Ｔ The RNAi construct of any one of claims 26 to 30, wherein is an inverted abasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

32. N at positions 4, 6, 8, 9, and 16 counting from the 5' end of the antisense strand Ｍ are 2'-fluoro-modified nucleotides, and Ns at positions 7 and 12 counting from the 5' end of the antisense strand are Ｍ The RNAi construct according to any one of claims 26 to 31, wherein each of

33. N at positions 4, 6, 8, 9, and 16 counting from the 5' end of the antisense strand Ｍ are 2'-O-methyl modified nucleotides, and N is at the 7th and 12th positions counting from the 5' end of the antisense strand. Ｍ The RNAi construct of any one of claims 26 to 31, wherein each of

34. N at positions 4, 6, 8, 9, and 12 counting from the 5' end of the antisense strand Ｍ are 2'-O-methyl modified nucleotides, and N is at the 7th and 16th positions counting from the 5' end of the antisense strand. Ｍ The RNAi construct of any one of claims 26 to 31, wherein each of

35. N at positions 7, 8, 9, and 12 counting from the 5' end of the antisense strand Ｍ are 2'-O-methyl modified nucleotides, and Ns at positions 4, 6, and 16 counting from the 5' end of the antisense strand are Ｍ The RNAi construct of any one of claims 26 to 31, wherein each of

36. N in the sense strand Ｍ The RNAi construct of any one of claims 26 to 35, wherein is a 2'-fluoro modified nucleotide.

37. N in the sense strand Ｍ The RNAi construct of any one of claims 26 to 35, wherein is a 2'-O-methyl modified nucleotide.

38. 38. The RNAi construct of any one of claims 1 to 37, wherein the sense strand, the antisense strand, or both the sense and antisense strands comprise one or more phosphorothioate internucleotide linkages.

39. 39. The RNAi construct of claim 38, wherein the antisense strand comprises two consecutive phosphorothioate internucleotide bonds between the terminal nucleotides at both the 3' and 5' ends.

40. 40. The RNAi construct of claim 38 or 39, wherein the sense strand comprises a single phosphorothioate internucleotide linkage between the terminal nucleotides at the 3' end.

41. 40. The RNAi construct of claim 38 or 39, wherein the sense strand comprises two consecutive phosphorothioate internucleotide bonds between the terminal nucleotides at the 3' end.

42. The RNAi construct of any one of claims 1 to 41, further comprising a ligand.

43. 43. The RNAi construct of claim 42, wherein the ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folate moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or antigen-binding fragment thereof.

44. 43. The RNAi construct of claim 42, wherein the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine.

45. 45. The RNAi construct of claim 44, wherein the ligand comprises a multivalent galactose moiety or a multivalent N-acetyl-galactosamine moiety.

46. 46. ​​The RNAi construct of claim 45, wherein the multivalent galactose moiety or the multivalent N-acetyl-galactosamine moiety is trivalent or tetravalent.

47. 47. The RNAi construct of any one of claims 42 to 46, wherein the ligand is covalently bound to the sense strand, optionally via a linker.

48. 48. The RNAi construct of claim 47, wherein the ligand is covalently attached to the 5' end of the sense strand.

49. A pharmaceutical composition comprising the RNAi construct of any one of claims 1 to 48 and a pharmaceutically acceptable carrier or excipient.

50. 49. An in vitro method for inhibiting expression of a target gene in a cell, comprising contacting the cell with an RNAi construct of any one of claims 1 to 48.

51. 50. The pharmaceutical composition of claim 49, wherein the composition is administered to a subject to inhibit expression of a target gene in the subject.

52. 52. The pharmaceutical composition of claim 51, wherein the composition is administered to the subject via a parenteral route of administration.

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