Chemically modified RNAi constructs and their use

Abstract

This invention relates to a chemically modified RNAi construct for reducing the expression of a target gene. Specifically, this invention relates to a specific pattern of modified nucleotides incorporated into an RNAi construct to improve in vivo stability and efficacy. [Solution] A pharmaceutical composition comprising a chemically modified RNAi construct is described, as well as a method for inhibiting target gene expression in vivo to treat or improve various medical conditions, for example, by administering the chemically modified RNAi construct.

Description

[Technical Field]

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

[0002] Description of electronically submitted text files This application includes a sequence listing, electronically filed in ASCII format and incorporated in its entirety herein by reference. A computer-readable copy of the sequence listing, prepared on 9 December 2019, is named A-2327-WO-PCT_SeqList_ST25 and is 24.7 kilobytes in size.

[0003] This invention relates to chemically modified RNAi constructs for reducing the expression of target genes in vivo. Specifically, this invention relates to specific patterns of modified nucleotides that confer improved potency and stability to 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 post-transcriptional gene silencing mechanism found in almost all phyla and is considered an evolutionarily conserved cellular defense mechanism (Fire et al., Nature, Vol.391;806-811, 1998; Fire et al., Trends Genet, Vol.15: 358-363, 1999; and Hamilton and Baulcombe, Science, Vol.286,950-952, 1999). Physiologically, the RNAi mechanism is initiated by the generation of a double helix of 18-25 base pairs derived from longer non-coding RNA, mediated by the Dicer enzyme. These short RNA molecules are placed into an 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, mRNA silencing is induced via Ago2-mediated degradation or translational repression (Bobbin and Rossi, Annu. Rev. Pharmacol. Toxicol., Vol. 56: 103-122, 2016).

[0005] Advances in RNAi technology and delivery methods are leading to a growing number of positive outcomes from RNAi-based therapies. These therapies represent a promising class of treatments, particularly for targets previously considered "undruggable" by small molecules or biological modalities. While significant progress has been made in overcoming the inherent metabolic instability of natural RNA through the development of chemical modifications and improved delivery methods, there remains a need in this field 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 [Overview of the project] [Means for solving the problem]

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

[0008] Accordingly, the present invention provides an RNAi construct for inhibiting the expression of a target gene sequence, wherein the RNAi construct comprises a sense strand and an antisense strand, the antisense strand comprising a sequence complementary to the target gene sequence, and the sense strand comprising a sequence sufficiently complementary to the sequence of the antisense strand to form a double-stranded region, and the RNAi construct comprises a structure represented by one of the formulas described herein. In certain embodiments, the RNAi construct of the present invention has a chemical modification pattern selected from one of the patterns designated as P1 to P30 as described herein.

[0009] In some embodiments, the RNAi construct comprises a structure represented by formula (A): 5’-(N

[0010] , M , M , F , , L , L , L , L , , M , F , M , , F , M , L , L ) x N L N L N L N L N L N L N F N​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Each N independently represents a modified nucleotide selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), and deoxyribonucleotides. L N independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide. T x represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. When x is 1, 2, 3, or 4, N A x can be an integer from 0 to 4, provided that one or more of the nucleotides are independently modified nucleotides selected from debasalized nucleotides, inverted debasalized nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. A One or more nucleotides may be complementary to the nucleotides in the antisense strand. When y is 1, 2, 3, or 4, y can be an integer from 0 to 4, provided that one or more n nucleotides are modified or unmodified overhang nucleotides that do not base-pair with the nucleotides in the antisense strand. When z is 1, 2, 3, or 4, N B z can be an integer from 0 to 4, provided that one or more of the nucleotides are 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. BIf one or more nucleotides are present in the sense strand, then N A They may be complementary to nucleotides, or they may be overhang nucleotides that do not base-pair with nucleotides in the sense strand.

[0011] In some embodiments, the RNAi construct comprises a sense strand of 19–23 nucleotides in length and an antisense strand of 19–23 nucleotides in length, wherein the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a double-stranded region of 19–21 base pairs, the nucleotides at positions 2, 7, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoromodified nucleotides, the nucleotides in the sense strand at positions 8–11, and 13 (counting from the 5' end) in the antisense strand are 2'-fluoromodified nucleotides, and neither the sense strand nor the antisense strand has a total of more than 7 2'-fluoromodified nucleotides. The RNAi construct may have nucleotide overhangs at either the 3' end of the sense strand or the antisense strand, or both. 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 present invention, 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)

[0013] In formula (D), the upper strand listed in the 5'-to-3' direction is the sense strand, and the lower strand listed in the 3'-to-5' direction is the antisense strand. Each N F represents a 2'-fluoro-modified nucleotide, and each N M / independently represents a modified nucleotide selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, BNA, and deoxyribonucleotides. Each N L independently represents a modified nucleotide selected from 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, BNA, and deoxyribonucleotides. N T 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, BNA, and deoxyribonucleotides. When x is 1, 2, 3, or 4, N Ax can be an integer from 0 to 4, provided that one or more of the nucleotides are independently modified nucleotides selected from debasalized nucleotides, inverted debasalized nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. A One or more nucleotides may be complementary to the nucleotides in the antisense strand. When y is 1, 2, 3, or 4, y can be an integer from 0 to 4, provided that one or more n nucleotides are modified or unmodified overhang nucleotides that do not base-pair with the nucleotides in the antisense strand. When z is 1, 2, 3, or 4, N B z can be an integer from 0 to 4, provided that one or more of the nucleotides are 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. B If one or more nucleotides are present in the sense strand, then N A They may be complementary to nucleotides, or they may be overhang nucleotides that do not base-pair with nucleotides in the sense strand.

[0014] In some embodiments of the present invention, the RNAi construct comprises a sense strand of 19–23 nucleotides in length and an antisense strand of 19–23 nucleotides in length, wherein the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a double-stranded region of 19–21 base pairs, the nucleotides at positions 2, 14, and 16 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides, the nucleotides in the sense strand at positions 10–13 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides, and neither the sense strand nor the antisense strand has a total of more than 7 2'-fluoromodified 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 the 3' ends of both the sense strand and the antisense strand.

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

[0016] The RNAi construct may further include ligands that facilitate the delivery or uptake of the RNAi construct to specific tissues or cells, such as hepatocytes. In some embodiments, the ligand targets the delivery of the RNAi construct to hepatocytes. In these and other embodiments, the ligand may include galactose, galactosamine, or N-acetyl-galactosamine (GalNAc). In certain embodiments, the ligand includes a polyvalent galactose or polyvalent GalNAc moiety, such as a trivalent or tetravalent galactose or GalNAc moiety. The ligand may optionally be covalently bound to the 5' or 3' end of the sense strand of the RNAi construct via a linker. In some embodiments, the RNAi construct includes a ligand and linker having one of the structures of formulas I to IX described herein. In one embodiment, the RNAi construct includes a ligand and linker having the structure of formula VI. In another embodiment, the RNAi construct includes a ligand and linker having the structure of formula VII. In yet another embodiment, the RNAi construct includes a ligand and linker having the structure of formula IX.

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

[0018] The present invention includes methods for reducing or inhibiting the expression of a target gene in cells, tissues, or subjects. In one embodiment, the method includes contacting cells or tissues with any one of the RNAi constructs described herein. The cells or tissues may 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 constructs may be administered parenterally (e.g., intravenously or subcutaneously) to the subject. [Brief explanation of the drawing]

[0019] [Figure 1] Several representative embodiments of the chemical modification patterns of RNAi constructs are shown. In each schematic diagram, the upper strand represents the sense strand in the 5' to 3' direction, and the lower strand represents the antisense strand in the 3' to 5' direction. 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 debasalized nucleotides (invAb) or inverted deoxyribonucleotides (invdN). Light gray lines connecting the circles represent phosphodiester bonds, and black lines connecting the circles represent phosphorothioate bonds. Black squares represent presumed Ago2 cleavage sites within the RNAi construct. [Figure 2] This bar graph shows the expression levels of the human PNPLA3 mutant in mouse livers, which were injected with an AAV encoding the human PNPLA3 mutant and then treated with 5 mg / kg subcutaneous injection of the indicated RNAi construct having a P1-CM1 chemical modification pattern. Human PNPLA3 expression was measured by qPCR and is reported as an expression level compared to vehicle-treated animals. The expression levels shown are those observed 8 days after RNAi construct administration. [Figure 3] This bar graph shows the expression levels of the human PNPLA3 mutant in mouse livers, which were injected with an AAV encoding the human PNPLA3 mutant and treated with 5 mg / kg subcutaneous injection of the indicated RNAi construct having a P1, P2, P3, or P4 chemical modification pattern. Human PNPLA3 expression was measured by qPCR and is reported as an expression level compared to vehicle-treated animals. Expression levels are shown at 15 days after RNAi construct administration. [Figure 4A-4B]This line graph shows the total photon flux (photons / second) versus the number of weeks after RNAi construct injection in mice that received subcutaneous injection of a vehicle or a specified RNAi construct having a P9 chemical modification pattern at doses of 1 mg / kg (Figure 4A) or 3 mg / kg (Figure 4B). The total photon flux represents the signal from the luciferase reporter expressed by the mouse, which contains a sequence complementary to the RNAi construct sequence. A decrease in total photon flux indicates a decrease in luciferase reporter expression. [Figure 5] This bar graph shows the expression levels of human PNPLA3 mutants in mouse livers treated with 3 mg / kg subcutaneous injection of the indicated RNAi construct having the P9 (double-strand numbers 7318 and 8709), CM2 (double-strand number 8103), CM3 (double-strand number 8104), or CM4 (double-strand number 8105) chemical modification patterns, after injection with AAV encoding human PNPLA3 mutants. Human PNPLA3 expression was measured by qPCR and is reported as an expression level compared to vehicle-treated animals. Expression levels are shown 28 days after RNAi construct administration. [Figure 6] The bar graphs show the expression levels of mouse ASGR1 in the livers of mice treated with 5 mg / kg subcutaneous injection of the ASGR1 RNAi construct. Mouse ASGR1 expression was measured by qPCR and is reported as an expression level normalized by the Gapdh expression level. The expression levels shown are those at 4, 8, and 15 days after administration of the RNAi construct or buffer (phosphate-buffered saline, PBS). [Figure 7] This line graph shows the percentage change in serum Lp(a) levels compared to baseline in double transgenic mice administered 0.5 mg / kg subcutaneously with the indicated LPA-targeted RNAi construct. Both RNAi constructs had the same sequence, differing only in their chemical modification patterns: double-stranded no. 3632 had a CM1 modification pattern, and double-stranded no. 3635 had a P1 modification pattern. The percentage change in serum Lp(a) levels is shown at day 14 (D14) and day 28 (D28) after single subcutaneous injection of the RNAi construct. [Modes for carrying out the invention]

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

[0021] As used herein, the term “RNAi construct” refers to a drug comprising an RNA molecule that, when introduced into a cell, can downregulate the expression of a target gene via an RNA interference mechanism. RNA interference is the process by 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 to form a double-stranded region. “Hybridizing” typically refers to the pairing of complementary polynucleotides via hydrogen bonds between complementary bases in two polynucleotides (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds). The strand containing a region having a sequence substantially complementary to the target sequence (e.g., target mRNA) is referred to as the “antisense strand.” The "sense strand" refers to a strand containing a region substantially complementary to the antisense strand. In some embodiments, the sense strand may contain a region having a sequence substantially identical to the target sequence.

[0022] Double-stranded RNA molecules may include chemical modifications to ribonucleotides, including modifications to ribose sugars, bases, or ribonucleotide backbone components, such as those described herein or known in the art. Any such modifications used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.) are encompassed by the term “double-stranded RNA” for the purposes of this disclosure.

[0023] As used herein, a polynucleotide containing a first sequence is “complementary” to a second sequence if, under certain conditions such as physiological conditions, the first sequence can hybridize to a polynucleotide containing a second sequence to form a double-stranded region. Other such conditions may include moderate or stringent hybridization conditions known to those skilled in the art. A polynucleotide containing a first sequence is considered 100% complementary to a second sequence if it forms base pairs with the polynucleotide containing a second sequence over the entire length of one or both nucleotide sequences without mismatching. A sequence is “substantially complementary” to a target sequence if it 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 complementarity percentage can be calculated by dividing the number of bases in the first sequence that are complementary to the bases at the corresponding positions in the 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 double-stranded region, the sequences may be said to be substantially complementary to the other sequence. In general, if any nucleotide overhangs as defined herein exist, the sequences of such overhangs are not considered in determining the degree of complementarity between the two sequences. As an example, a 21-nucleotide sense strand and a 21-nucleotide antisense strand that hybridize to form a 19-base-pair double-stranded region with a 2-nucleotide overhang at the 3' end of each strand are considered perfectly complementary when this term is used herein.

[0024] In some embodiments, the antisense strand region includes a sequence that is fully complementary to the target gene sequence (e.g., target mRNA). In such embodiments, the sense strand may include a sequence that is fully complementary to the antisense strand sequence. In other embodiments, the sense strand may include a sequence that is substantially complementary to the antisense strand sequence, for example, a sequence having 1, 2, 3, 4, or 5 mismatches in the double-stranded region formed by the sense and antisense strands. In certain embodiments, it is preferable that any mismatches occur within the terminal region (e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the strand). In one embodiment, any mismatches in the double-stranded region formed by the sense and antisense strands occur within 6, 5, 4, 3, or 2 nucleotides of the 5' end of the antisense strand.

[0025] In certain embodiments, the sense and antisense strands of a double-stranded RNA may hybridize to form a double-stranded region, but may be two separate molecules that are not linked except for this region. Such double-stranded RNA molecules formed from two separate strands are referred to as "small interfering RNA" or "short interfering RNA" (siRNA). Therefore, in some embodiments, the RNAi construct of the present invention includes siRNA.

[0026] In other embodiments, the sense and antisense strands that hybridize to form a double-stranded 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 includes a double-stranded region (also called a stem region) and a loop region. The 3' end of the sense strand is joined to the 5' end of the antisense strand by an adjacent sequence of unpaired nucleotides that will form a loop region. The loop region is usually long enough to allow the RNA molecule to fold on its own, so that the antisense strand can base-pair with the sense strand to form a double-stranded 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 partially self-complementary regions are referred to as "small hairpin RNA" (shRNA). In certain embodiments, the RNAi construct of the present invention includes shRNA. The length of a single, at least partially self-complementary RNA molecule may be approximately 40 to 100 nucleotides, approximately 45 to 85 nucleotides, or approximately 50 to 60 nucleotides, and may include double-stranded regions and loop regions having the lengths listed herein, respectively.

[0027] The RNAi construct of the present invention comprises a sense strand and an antisense strand, the antisense strand comprising a region having a sequence substantially or completely complementary to the target gene sequence. The target gene sequence generally refers to a nucleic acid sequence comprising a partial or complete coding sequence of a polypeptide. The target gene sequence may also include non-coding regions such as the 5' or 3' untranslated region (UTR). In certain embodiments, the target gene sequence is a messenger RNA (mRNA) sequence. The mRNA sequence refers to any messenger RNA sequence comprising a protein, protein variant, or splice variant encoding an isoform derived 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. The target gene sequence may also be an RNA sequence other than an mRNA sequence, such as a tRNA sequence, a microRNA sequence, or a viral RNA sequence.

[0028] The antisense strand region of the RNAi construct may be substantially complementary or fully complementary to at least 15 consecutive nucleotides of the target gene sequence. In some embodiments, the target region of the gene sequence containing the complementary antisense strand may range from 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 the antisense strand such that the two strands hybridize under physiological conditions to form a double-stranded region. A “double-stranded region” refers to a region of two complementary or substantially complementary polynucleotides that form a double helix between two polynucleotides by base pairing, either through Watson-Crick base pairing or other hydrogen bonding interactions. The double-stranded region of an RNAi construct should be long enough to allow the RNAi construct to enter the RNA interference pathway, for example, by binding to a Dicer enzyme and / or RISC complex. For example, in some embodiments, the double-stranded region is about 15 to about 30 base pairs long. Other lengths of the double-stranded region within this range are also suitable, for example, about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In one embodiment, the double-stranded region is about 17 to about 24 base pairs long. In another embodiment, the double-stranded region is about 19 to about 21 base pairs long. In certain embodiments, the double-stranded region is about 19 base pairs long. In other embodiments, the double-stranded region is about 21 base pairs long.

[0030] In embodiments where the sense and antisense strands are two separate molecules (for example, the RNAi construct contains siRNA), the sense and antisense strands do not need to be the same length as the double-stranded region. For example, one or both strands may be longer than the double-stranded region and have one or more unpaired nucleotides or mismatches adjacent to the double-stranded region. Thus, in some embodiments, the RNAi construct includes 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 double-stranded 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 lengths of nucleotide overhangs are generally 1–6 nucleotides, 1–5 nucleotides, 1–4 nucleotides, 1–3 nucleotides, 2–6 nucleotides, 2–5 nucleotides, or 2–4 nucleotides. In some embodiments, the nucleotide overhang contains 1, 2, 3, 4, 5, or 6 nucleotides. In a particular embodiment, the nucleotide overhang contains 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang contains 2 nucleotides. In certain other embodiments, the nucleotide overhang contains a single nucleotide.

[0031] The nucleotide in the overhang may be a ribonucleotide or a modified nucleotide as described herein. In some embodiments, the nucleotide in the overhang may be a 2'-modified nucleotide (e.g., a 2'-fluoromodified nucleotide, a 2'-O-methylmodified nucleotide), a deoxyribonucleotide, an inverted nucleotide (e.g., an inverted debasalized nucleotide, an inverted deoxyribonucleotide), or a combination thereof. For example, in one embodiment, the nucleotide in the overhang may be a deoxyribonucleotide, e.g., deoxythymidine. In another embodiment, the nucleotide in the overhang may be a 2'-O-methylmodified nucleotide, a 2'-fluoromodified nucleotide, a 2'-methoxyethyl modified nucleotide, or a combination thereof. In yet another embodiment, the overhang may include a 5'-uridine-uridine-3'(5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide may include 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. If the nucleotide overhang is present in the antisense strand, the nucleotide in the overhang may be complementary to the target gene sequence, may form a mismatch with the target gene sequence, or may contain any other sequence (e.g., polypyrimidine or polypurine sequences, e.g., UU, TT, AA, GG).

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

[0033] An RNAi construct may include a nucleotide overhang at one end of a double-stranded RNA molecule and a blunt end at the other end. “Blunt end” means that the sense and antisense strands are fully base-paired at the ends of the molecule, and there are no unpaired nucleotides extending beyond the double-stranded region. In some embodiments, the RNAi construct includes a nucleotide overhang at the 3' end of the sense strand and blunt ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct includes a nucleotide overhang at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNAi construct includes blunt ends at both ends of a double-stranded RNA molecule. In these embodiments, the sense and antisense strands are of equal length, and the double-stranded region is of equal length to the sense and antisense strands (i.e., the molecule is double-stranded over its entire length).

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

[0035] In other embodiments, the sense strand or antisense strand is longer than the other strand, such that the RNAi construct includes at least one nucleotide overhang, and the two strands form a double-stranded region having a length equal to the length of the shorter strand. For example, in one embodiment, the RNAi construct includes (i) a sense strand that is 19 nucleotides long, (ii) an antisense strand that is 21 nucleotides long, (iii) a double-stranded 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 includes (i) a sense strand that is 21 nucleotides long, (ii) an antisense strand that is 23 nucleotides long, (iii) a double-stranded 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 constructs of the present invention preferably include modified nucleotides. “Modified nucleotide” refers to a nucleotide having one or more chemical modifications to a nucleoside, nucleic acid base, pentose ring, or phosphate group. As used herein, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate. However, RNAi constructs may include combinations 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 sensitivity to nucleases and other degradation processes. The efficacy of RNAi constructs in reducing the expression of target genes can also be enhanced by incorporating modified nucleotides (particularly when incorporated in specific patterns, as described in more detail herein).

[0037] In certain embodiments, modified nucleotides have ribose sugar modifications. Such sugar modifications may include modifications at the 2' and / or 5' positions of the pentose ring, as well as bicyclic sugar modifications. 2'-Modified nucleotides refer to nucleotides having a pentose ring with substituents other than OH at the 2' position. Such 2'-modifications include 2'-H (e.g., deoxyribonucleotides) and 2'-O-alkyl (e.g., O-C1-C) 10 or O-C1-C 10 Examples of 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 the modification of a pentose ring, in which case the bridge connects two atoms of the ring to form a second ring resulting in a bicyclic sugar structure. In some embodiments, the bicyclic sugar modification includes a bridge between the 4' and 2' carbon atoms of the pentose ring. A nucleotide containing a sugar moiety having a bicyclic sugar modification is referred to herein as a bicyclic nucleic acid or BNA. Examples of bicyclic sugar modifications include α-L-methyleneoxy(4'-CH2-O-2') bicyclic nucleic acid (BNA); β-D-methyleneoxy(4'-CH2-O-2')BNA (also called lock nucleic acid or LNA); ethyleneoxy(4'-(CH2)2-O-2')BNA; aminooxy(4'-CH2-ON(R)-2')BNA; oxyamino(4'-CH2-N(R)-O-2')BNA; methyl(methyleneoxy)(4'-CH(CH3)-O-2')BNA (constrained ethyl (constrained) Examples include, but are not limited to, ethyl (also called cEt); methylene-thio(4'-CH2-S-2')BNA; methylene-amino(4'-CH2-N(R)-2')BNA; methyl carboncyclic(4'-CH2-CH(CH3)-2')BNA; propylene carboncyclic(4'-(CH2)3-2')BNA; and methoxy(ethyleneoxy)(4'-CH(CH2OMe)-O-2')BNA (also called constrained MOE or cMOE). These and other glycosylated nucleotides that can be incorporated into the RNAi construct of the present invention are described in U.S. Patent No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated herein by reference in their entirety.

[0039] In some embodiments, the RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), deoxyribonucleotides, or combinations thereof. In certain embodiments, the RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, or combinations thereof. In certain embodiments, the RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or combinations thereof.

[0040] Both the sense and antisense strands of an RNAi construct may contain one or more modified nucleotides. For example, in some embodiments, the sense strand contains one, two, three, four, five, six, seven, eight, nine, or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand contains one, two, three, four, five, six, seven, eight, nine, or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides may be 2'-fluoromodified nucleotides, 2'-O-methylmodified nucleotides, or a combination thereof.

[0041] In certain embodiments, the modified nucleotides incorporated into one or both strands of the RNAi construct of the present invention have modifications to nucleic acid bases (also referred to herein as “bases”). “Modified nucleic acid bases” or “modified bases” refers to bases other than the naturally occurring purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases may be synthetic or naturally occurring modifications, and include 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, and 6-azo Examples 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, especially 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. A “universal base” refers to a base analog that indiscriminately forms base pairs with all of the native bases of RNA and DNA without altering the double helix structure of the resulting double-stranded region. Universal bases are known to those skilled in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl and other aromatic derivatives, azole carboxamides and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole.

[0043] Other suitable modified bases that can be incorporated into the RNAi construct 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 will be well aware that guanine, cytosine, adenine, thymine, and uracil can be substituted with other nucleic acid bases, such as the modified nucleic acid bases described above, without substantially altering the base-pair properties of polynucleotides containing nucleotides having such substituted nucleic acid bases.

[0044] In some embodiments, the sense and antisense strands of an RNAi construct may contain one or more debasalized nucleotides. A “debasalized nucleotide” or “debasalized nucleoside” is a nucleotide or nucleoside that lacks a nucleic acid base at the 1' position of a ribose sugar. In certain embodiments, the debasalized nucleotide is incorporated into the ends of the sense and / or antisense strands of the RNAi construct. In one embodiment, the sense strand contains the debasalized nucleotide as a terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. In another embodiment, the antisense strand contains the debasalized nucleotide as a terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. In these embodiments where the debasalized nucleotide is a terminal nucleotide, it may be an inverted nucleotide, meaning it binds to an adjacent nucleotide via a 3'-3' nucleotide bond (if it is on the 3' end of the strand) or a 5'-5' nucleotide bond (if it is on the 5' end of the strand), rather than a natural 3'-5' nucleotide bond. Debasic nucleotides may also include sugar modifications, such as any of the sugar modifications described above. In certain embodiments, debasic nucleotides include 2'-modifications such as 2'-fluoro modification, 2'-O-methyl modification, or 2'-H (deoxy) modification. In one embodiment, the debasic nucleotide includes a 2'-O-methyl modification. In another embodiment, the debasic nucleotide includes a 2'-H modification (i.e., a deoxy-debasic nucleotide).

[0045] The 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 containing sequences sufficiently complementary to each other to form a double-stranded region of at least 15 base pairs, where, The nucleotides at positions 2, 7, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoromodified nucleotides. The nucleotides in the sense strand at positions 8-11 and 13 (counting from the 5' end) in the antisense strand are 2'-fluoromodified nucleotides. Neither the sense strand nor the antisense strand contains more than 7 total 2'-fluoromodified nucleotides.

[0046] In other embodiments, the RNAi construct of the present invention comprises a sense strand and an antisense strand containing sequences sufficiently complementary to each other to form a double-stranded region of at least 19 base pairs, where, The nucleotides at positions 2, 7, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoromodified nucleotides, the nucleotides at positions 4, 6, 10, and 12 (counting from the 5' end) are optionally 2'-fluoromodified nucleotides, and all other nucleotides in the antisense strand are modified nucleotides other than 2'-fluoromodified nucleotides. Nucleotides in the sense strand at positions 8-11 and 13 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides, nucleotides in the sense strand at positions 3 and 5 (counting from the 5' end) of the antisense strand are optionally 2'-fluoromodified nucleotides, and all other nucleotides in the sense strand are modified nucleotides other than 2'-fluoromodified nucleotides.

[0047] In these embodiments, 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, BNA, and deoxyribonucleotides. In these and other embodiments, the terminal nucleotides at the 3' end, 5' end, or both the 3' and 5' ends of the sense strand may be debasalized nucleotides or deoxyribonucleotides. In these embodiments, the debasalized nucleotides or deoxyribonucleotides may be inverted, i.e., they may bind to adjacent nucleotides via a 3'-3' nucleotide bond (if located at the 3' end of the strand) or a 5'-5' nucleotide bond (if located at the 5' end of the strand), rather than via a natural 3'-5' nucleotide bond.

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

[0049] In all 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'-fluoromodified 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'-fluoromodified 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'-fluoromodified nucleotides.

[0050] In certain embodiments of the present invention, the RNAi construct comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to the target gene sequence, and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a double-stranded region, and 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 upper chain, arranged in the direction from 5' to 3', is the sense chain, and the lower chain, arranged in the direction from 3' to 5', is the antisense chain. each N F This represents a 2'-fluoromodified nucleotide, each N M This independently represents a modified nucleotide selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), and deoxyribonucleotides. each N L This independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide. N T This represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. When x is 1, 2, 3, or 4, N A The condition is that one or more nucleotides are independently modified nucleotides selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, where x is an integer from 0 to 4, and N AOne or more nucleotides may be complementary to the nucleotides in the antisense strand. When y is 1, 2, 3, or 4, y is an integer from 0 to 4, provided that one or more n nucleotides are modified or unmodified overhang nucleotides that do not base-pair with nucleotides in the antisense strand. When z is 1, 2, 3, or 4, N B z is an integer from 0 to 4, provided that one or more of the nucleotides are independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and N B If one or more nucleotides are present in the sense strand, then N A They may be complementary to nucleotides, or they may be overhang nucleotides that do not base-pair with nucleotides in the sense strand.

[0051] In some embodiments of the RNAi construct having 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 where 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 of the RNAi construct having a structure represented by formula (A), the RNAi construct has blunt ends at the 3' end of the sense strand and the 5' end of the antisense strand (i.e., y is 0). In these 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 embodiment where x is greater than 0, the terminal nucleotide at the 5' end of the sense strand is N AThe nucleotide may be an inverted nucleotide, such as an inverted debasalized nucleotide or an inverted deoxyribonucleotide.

[0052] In certain embodiments, the RNAi construct has a structure represented by formula (A), where the N at positions 4 and 12 counting from the 5' end of the antisense strand M These are 2'-fluoromodified nucleotides, respectively. In other embodiments, the N at positions 4, 6, and 12 counting from the 5' end of the antisense strand are M These are 2'-fluoromodified nucleotides, respectively. In other embodiments, the N at positions 4, 6, 10, and 12 counting from the 5' end of the antisense strand are also present. M These are 2'-fluoromodified nucleotides, respectively. In an alternative embodiment in which the RNAi construct has a structure represented by formula (A), the N at positions 10 and 12 counting from the 5' end of the antisense strand are M These are 2'-fluoromodified nucleotides, respectively. In related embodiments, the N at positions 4, 10, and 12 counting from the 5' end of the antisense strand. M These are 2'-fluoromodified nucleotides, respectively. In other alternative embodiments in which the RNAi construct has a structure represented by formula (A), the N at positions 4, 6, and 10 counting from the 5' end of the antisense strand are M These are 2'-O-methyl modified nucleotides, and are located at the 12th position from the 5' end of the antisense strand. M is a 2'-fluoromodified nucleotide. In some embodiments, the RNAi construct has a structure represented by formula (A), in which each N in the sense strand M is a 2'-O-methyl modified nucleotide. In other embodiments, each N in the sense strand M is a 2'-fluoromodified nucleotide. In yet another embodiment, the RNAi construct has a structure represented by formula (A), in both the sense strand and the antisense strand, each N M This is a 2'-O-methyl modified nucleotide.

[0053] In any of the above embodiments, the RNAi construct has a structure represented by formula (A), and each N in both the sense strand and the antisense strand L This can be a 2'-O-methyl modified nucleotide. In these embodiments, and in any of the embodiments described above, N in formula (A) T This may be an inverted debasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0054] In certain embodiments of the present invention, the RNAi construct comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to the target gene sequence, and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a double-stranded region, and the RNAi construct comprises 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 LN F N L -5' (B) During the ceremony, The upper chain, arranged in the direction from 5' to 3', is the sense chain, and the lower chain, arranged in the direction from 3' to 5', is the antisense chain. each N F This represents a 2'-fluoromodified nucleotide, each N L This independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide. N T This represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. When x is 1, 2, 3, or 4, N A The condition is that one or more nucleotides are independently modified nucleotides selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, where x is an integer from 0 to 4, and N A One or more nucleotides may be complementary to the nucleotides in the antisense strand. When y is 1, 2, 3, or 4, y is an integer from 0 to 4, provided that one or more n nucleotides are modified or unmodified overhang nucleotides that do not base-pair with nucleotides in the antisense strand. When z is 1, 2, 3, or 4, N Bz is an integer from 0 to 4, provided that one or more of the nucleotides are independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and N B If one or more nucleotides are present in the sense strand, then N A They may be complementary to nucleotides, or they may be overhang nucleotides that do not base-pair with nucleotides in the sense strand.

[0055] In some embodiments of the RNAi construct having 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 where 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 of the RNAi construct having a structure represented by formula (B), the RNAi construct has blunt ends at the 3' end of the sense strand and the 5' end of the antisense strand (i.e., y is 0). In these 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 embodiment where x is greater than 0, the terminal nucleotide at the 5' end of the sense strand is N A The nucleotide may be an inverted nucleotide, such as an inverted debasalized nucleotide or an inverted deoxyribonucleotide.

[0056] In any of the above embodiments, the RNAi construct has a structure represented by formula (B), and each N in both the sense strand and the antisense strand LThis can be a 2'-O-methyl modified nucleotide. In these embodiments, and in any of the embodiments described above, N in formula (B) T This may be an inverted debasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0057] In some embodiments of the present invention, the RNAi construct comprises a sense strand and an antisense strand, the antisense strand comprising a sequence complementary to the target gene sequence, the sense strand comprising a sequence sufficiently complementary to the sequence of the antisense strand to form a double-stranded 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 upper chain, arranged in the direction from 5' to 3', is the sense chain, and the lower chain, arranged in the direction from 3' to 5', is the antisense chain. each N F This represents a 2'-fluoromodified nucleotide, each N L This independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide. each N M This independently represents a modified nucleotide selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, BNA, and deoxyribonucleotides. N T This represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. x is either 0 or 1, and Ab is an inverted debasal nucleotide.

[0058] In certain embodiments, the RNAi construct has a structure represented by formula (C), in which the antisense strand contains N M is a 2'-fluoromodified 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 in the sense strand M is a 2'-fluoromodified nucleotide. In some embodiments, the RNAi construct has a structure represented by formula (C), in both the sense strand and the antisense strand, each N is present. M This is a 2'-O-methyl modified nucleotide.

[0059] In any of the above embodiments, the RNAi construct has a structure represented by formula (C), and each N in both the sense strand and the antisense strand L This can be a 2'-O-methyl modified nucleotide. In these embodiments, and in any of the embodiments described above, N in formula (C) T This can be an inverted debasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide. For example, in one embodiment, N T x is an inverted debasal nucleotide or an inverted deoxyribonucleotide, and x is 0. In another embodiment, N T is a 2'-O-methyl modified nucleotide, and x is 1. In another embodiment, N T x is an inverted debasal nucleotide or an inverted deoxyribonucleotide, and x is 1.

[0060] In certain embodiments, the RNAi construct of the present invention comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to the target gene sequence, and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a double-stranded region, and 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 NL 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 upper chain, arranged in the direction from 5' to 3', is the sense chain, and the lower chain, arranged in the direction from 3' to 5', is the antisense chain. each N F This represents a 2'-fluoromodified nucleotide, each N M This independently represents a modified nucleotide selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), and deoxyribonucleotides. each N L This independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide. N T This represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. When x is 1, 2, 3, or 4, N AThe condition is that one or more nucleotides are independently modified nucleotides selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, where x is an integer from 0 to 4, and N A One or more nucleotides may be complementary to the nucleotides in the antisense strand. When y is 1, 2, 3, or 4, y is an integer from 0 to 4, provided that one or more n nucleotides are modified or unmodified overhang nucleotides that do not base-pair with nucleotides in the antisense strand. When z is 1, 2, 3, or 4, N B z is an integer from 0 to 4, provided that one or more of the nucleotides are independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and N B If one or more nucleotides are present in the sense strand, then N A They may be complementary to nucleotides, or they may be overhang nucleotides that do not base-pair with nucleotides in the sense strand.

[0061] In some embodiments of the RNAi construct having 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 where 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 of the RNAi construct having a structure represented by formula (D), the RNAi construct has blunt ends at the 3' end of the sense strand and the 5' end of the antisense strand (i.e., y is 0). In these 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 embodiment where x is greater than 0, the terminal nucleotide at the 5' end of the sense strand is N A The nucleotide may be an inverted nucleotide, such as an inverted debasalized nucleotide or an inverted deoxyribonucleotide.

[0062] In certain embodiments, the RNAi construct has a structure represented by formula (D), where the N at positions 4, 6, 8, 9, and 16 counting from the 5' end of the antisense strand are M These are 2'-fluoromodified nucleotides, with the N at positions 7 and 12 counting from the 5' end of the antisense strand. M These are 2'-O-methyl modified nucleotides, respectively. In other embodiments, the N at positions 4 and 6, counting from the 5' end of the antisense strand, are present. M These are 2'-fluoromodified nucleotides, and are located at positions 7-9 from the 5' end of the antisense strand. M These are 2'-O-methyl-modified nucleotides, respectively. In yet another embodiment, the N3s at positions 4, 6, 8, 9, and 16, counting from the 5' end of the antisense strand, are also present. MThese are 2'-O-methyl modified nucleotides, and are located at positions 7 and 12 of the antisense strand, counting from the 5' end. M These are 2'-fluoromodified nucleotides, respectively. In an alternative embodiment in which the RNAi construct has the structure represented by formula (D), the N at positions 4, 6, 8, 9, and 12 counting from the 5' end of the antisense strand are M These are 2'-O-methyl modified nucleotides, and are located at positions 7 and 16 from the 5' end of the antisense strand. M These are 2'-fluoromodified nucleotides, respectively. In certain other embodiments in which the RNAi construct has a structure represented by formula (D), the N at positions 7, 8, 9, and 12 counting from the 5' end of the antisense strand are M These are 2'-O-methyl modified nucleotides, and are located at positions 4, 6, and 16 of the antisense strand, counting from the 5' end. M These are 2'-fluoromodified nucleotides, respectively. In these and other embodiments, the RNAi construct has the structure represented by formula (D), and the N in the sense strand M This is a 2'-fluoromodified nucleotide. In an alternative embodiment, N in the sense strand M This is a 2'-O-methyl modified nucleotide.

[0063] In any of the above embodiments, the RNAi construct has a structure represented by formula (D), and each N in both the sense strand and the antisense strand L This can be a 2'-O-methyl modified nucleotide. In these embodiments, and in any of the embodiments described above, N in formula (D) T This may be an inverted debasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0064] The RNAi constructs of the present invention may also include one or more modified nucleotide bonds. As used herein, the term “modified nucleotide bond” refers to nucleotide bonds other than the natural 3'-5' phosphodiester bond. In some embodiments, the modified nucleotide bond is a phosphorus-containing nucleotide bond such as phosphotriesters, aminoalkyl phosphotriesters, alkylphosphonates (e.g., methylphosphonate, 3'-alkylenephosphonate), phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), phosphorothioates (P=S), chiral phosphorothioates, phosphorodithioates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. In one embodiment, the modified nucleotide bond is a 2'-5' phosphodiester bond. In other embodiments, modified nucleotide bonds are phosphorus-free nucleotide bonds and may therefore be called modified nucleoside bonds. Such phosphorus-free bonds include, but are not limited to, morpholino bonds (partially formed from the sugar moiety of the nucleoside); siloxane bonds (-O-Si(H)2-O-); sulfide, sulfoxide, and sulfone bonds; formacetyl and thioformacetyl bonds; alkene-containing skeletons; sulfamic acid skeletons; methylene methylimino (-CH2-N(CH3)-O-CH2-) and methylene hydrazino bonds; sulfonic acid and sulfonamide bonds; amide bonds; and others having mixed N, O, S, and CH2 constituent parts. In one embodiment, the modified nucleoside bond is a peptide-based bond (e.g., aminoethylglycine) for generating peptide nucleic acids or PNAs, such as those described in U.S. Patent No. 5,539,082; No. 5,714,331; and No. 5,719,262.Other suitable modified nucleotide-nucleotide and nucleoside-nucleotide bonds that may be employed in the RNAi construct of the present invention are described in U.S. Patent No. 6,693,187, U.S. Patent No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated herein by reference.

[0065] In certain embodiments, the RNAi construct of the present invention includes one or more phosphorothioate nucleotide interbonds. These phosphorothioate nucleotide interbonds may be present on the sense strand, the antisense strand, or both strands of the RNAi construct. For example, in some embodiments, the sense strand includes one, two, three, four, five, six, seven, or eight or more phosphorothioate nucleotide interbonds. In other embodiments, the antisense strand includes one, two, three, four, five, six, seven, or eight or more phosphorothioate nucleotide interbonds. In yet another embodiment, both strands include one, two, three, four, five, six, seven, or eight or more phosphorothioate nucleotide interbonds. The RNAi construct may include one or more phosphorothioate nucleotide interbonds at the 3' end, 5' end, or both the 3' and 5' ends of the sense strand, the antisense strand, or both strands. For example, in certain embodiments, the RNAi construct includes about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, or 6 or more) consecutive phosphorothioate nucleotide links at the 3' ends of the sense strand, antisense strand, or both strands. In other embodiments, the RNAi construct includes about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, or 6 or more) consecutive phosphorothioate nucleotide links at the 5' ends of the sense strand, antisense strand, or both strands.

[0066] In some embodiments, the RNAi construct includes a single phosphorothioate nucleotide linkage between the 3' terminal nucleotides of the sense strand. In other embodiments, the RNAi construct includes two consecutive phosphorothioate nucleotide linkages between the 3' terminal nucleotides of the sense strand. In one embodiment, the RNAi construct includes a single phosphorothioate nucleotide linkage between the 3' terminal nucleotides of the sense strand and a single phosphorothioate nucleotide linkage between the 3' terminal nucleotides of the antisense strand. In another embodiment, the RNAi construct includes two consecutive phosphorothioate nucleotide linkages between the 3' terminal nucleotides of the antisense strand (i.e., phosphorothioate nucleotide linkages in the first and second nucleotide linkages at the 3' end of the antisense strand). In yet another embodiment, the RNAi construct includes two consecutive phosphorothioate nucleotide linkages between the 3' and 5' terminal nucleotides of the antisense strand. In another embodiment, the RNAi construct includes two consecutive phosphorothioate nucleotide links between both the 3' and 5' terminal nucleotides of the antisense strand, and two consecutive phosphorothioate nucleotide links at the 5' end of the sense strand. In yet another embodiment, the RNAi construct includes two consecutive phosphorothioate nucleotide links between both the 3' and 5' terminal nucleotides of the antisense strand, and two consecutive phosphorothioate nucleotide links between the terminal nucleotides at the 3' end of the sense strand. In another embodiment, the RNAi construct includes two consecutive phosphorothioate internucleotide bonds between the terminal nucleotides at both the 3' and 5' ends of the antisense strand, and two consecutive phosphorothioate internucleotide bonds between the terminal nucleotides at both the 3' and 5' ends of the sense strand (i.e., phosphorothioate internucleotide bonds in the first and second internucleotide bonds at both the 5' and 3' ends of the antisense strand, and phosphorothioate internucleotide bonds in the first and second internucleotide bonds at both the 5' and 3' ends of the sense strand).In another embodiment, the RNAi construct includes two consecutive phosphorothioate internucleotide bonds between both the 3' and 5' terminal nucleotides of the antisense strand, and a single phosphorothioate internucleotide bond between the terminal nucleotides of the 3' terminal of the sense strand. In any embodiment in which one or both strands include one or more phosphorothioate internucleotide bonds, the remaining internucleotide bonds in the strands may be native 3'-5' phosphodiester bonds. For example, in some embodiments, each internucleotide bond in the sense and antisense strands is selected from phosphodiesters and phosphorothioates, with at least one internucleotide bond being a phosphorothioate.

[0067] In embodiments where the RNAi construct includes a nucleotide overhang, two or more unpaired nucleotides in the overhang may be linked by phosphorothioate nucleotide linkages. In certain embodiments, all unpaired nucleotides in the nucleotide overhang at the 3' end of the antisense strand and / or sense strand are linked by phosphorothioate nucleotide linkages. In other embodiments, all unpaired nucleotides in the nucleotide overhang at the 5' end of the antisense strand and / or sense strand are linked by phosphorothioate nucleotide linkages. In yet another embodiment, all unpaired nucleotides in any nucleotide overhang are linked by phosphorothioate nucleotide linkages.

[0068] The RNAi construct of the present invention may have any one of the chemical modification patterns P1 to P30 shown in Figure 1. For example, in some embodiments, the RNAi construct comprises a sense strand of 19 to 23 nucleotides in length and an antisense strand of 19 to 23 nucleotides in length, wherein the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a double-stranded 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'-fluoromodified nucleotides, the nucleotides in the sense strand at positions 8 to 11, and 13 (counting from the 5' end) in the antisense strand are 2'-fluoromodified nucleotides, neither the sense strand nor the antisense strand has a total of more than 7 2'-fluoromodified nucleotides, and the RNAi construct has nucleotide overhangs at the 3' ends of both the sense strand and the antisense strand.

[0069] In one embodiment, the RNAi construct is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7 and 9-12, and 2'-O-methylmodified nucleotides at positions 1-6, 8, and 13-21 (counting from the 5' end), (iii) A sense strand having phosphorothioate internucleotide bonds between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 nucleotide overhangs containing two nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand.

[0070] In another embodiment, the RNAi construct is (a) (i) Length of 22 nucleotides, (ii) an inverted debasic nucleotide or inverted deoxyribonucleotide at position 1, a 2'-fluoromodified nucleotide at positions 8 and 10-13, and a 2'-O-methylmodified nucleotide (counting from the 5' end) at positions 2-7, 9 and 14-22, and, (iii) A sense strand having phosphorothioate internucleotide bonds between nucleotides at positions 20 and 21, and between nucleotides at positions 21 and 22 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 a nucleotide overhang containing one to two nucleotides at the 3' end of the antisense strand.

[0071] In another embodiment, the RNAi construct is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7 and 9-12, and 2'-O-methylmodified nucleotides at positions 1-6, 8, and 13-21 (counting from the 5' end), (iii) A sense strand having phosphorothioate internucleotide bonds between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 10, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8, 9, 11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 nucleotide overhangs containing two nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand.

[0072] In another embodiment, the RNAi construct is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7 and 9-12, and 2'-O-methylmodified nucleotides at positions 1-6, 8, and 13-21 (counting from the 5' end), (iii) A sense strand having phosphorothioate internucleotide bonds between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 7, 10, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 8, 9, 11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 nucleotide overhangs containing two nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand.

[0073] In another specific embodiment, the RNAi construct is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7 and 9-12, 2'-O-methylmodified nucleotides at positions 1-6, 8, and 13-20, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8-11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 nucleotide overhangs containing two nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand.

[0074] In certain embodiments, the RNAi construct comprises a sense strand of 19–21 nucleotides in length and an antisense strand of 21–23 nucleotides in length, wherein the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a 19–21 base pair double-stranded region, the nucleotides at positions 2, 7, and 14 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides, the nucleotides in the sense strand at positions 8–11, and 13 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides, neither the sense strand nor the antisense strand has a total of more than 7 2'-fluoromodified nucleotides, 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 is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 9 and 11-14, and 2'-O-methylmodified nucleotides at positions 1-8, 10, and 15-20, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-23 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 22 nucleotides, (ii) an inverted debasic nucleotide or inverted deoxyribonucleotide at position 1, a 2'-fluoromodified nucleotide at positions 10 and 12-15, and a 2'-O-methylmodified nucleotide at positions 2-9, 11, and 16-22 (counting from the 5' end), (iii) A sense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 21 and 20, and between the nucleotides at positions 21 and 22 (counting from the 5' end), (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-23 (counting from the 5' end), (iii) an antisense chain 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 1-2 nucleotides at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0077] In another embodiment, the RNAi construct is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 9 and 11-14, and 2'-O-methylmodified nucleotides at positions 1-8, 10, and 15-21 (counting from the 5' end), (iii) A sense strand having phosphorothioate internucleotide bonds between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-23 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 22 nucleotides, (ii) Reverse debasic nucleotides or reverse deoxyribonucleotides at positions 1-22, 2'-fluoro-modified nucleotides at positions 10 and 12-15, and 2'-O-methyl-modified nucleotides at positions 2-9, 11, and 16-21 (counting from the 5' end), (iii) A sense strand having a phosphorothioate internucleotide bond between the nucleotides at positions 21 and 22, (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-23 (counting from the 5' end), (iii) an antisense chain 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 1-2 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 is (a) (i) Length of 19 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7 and 9-12, and 2'-O-methylmodified nucleotides at positions 1-6, 8, and 13-19 (counting from the 5' end), (iii) A sense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 17 and 18, and between the nucleotides at positions 18 and 19 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 19 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7 and 9-12, 2'-O-methylmodified nucleotides at positions 1-6, 8 and 13-18, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 19, (iii) A sense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 17 and 18, and between the nucleotides at positions 18 and 19 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 8-11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 9 and 11-14, and 2'-O-methylmodified nucleotides at positions 1-8, 10, and 15-20, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8-11, 13, and 15-23 (counting from the 5' end), (iii) an antisense chain 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 another embodiment, the RNAi construct is (a) (i) Length of 22 nucleotides, (ii) an inverted debasic nucleotide or inverted deoxyribonucleotide at position 1, a 2'-fluoromodified nucleotide at positions 10 and 12-15, and a 2'-O-methylmodified nucleotide at positions 2-9, 11, and 16-22 (counting from the 5' end), (iii) A sense strand having phosphorothioate internucleotide bonds between the nucleotides at positions 20 and 21, and between the nucleotides at positions 21 and 22, (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8-11, 13, and 15-23 (counting from the 5' end), (iii) an antisense chain 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 1-2 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 is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 9 and 11-14, and 2'-O-methylmodified nucleotides at positions 1-8, 10, and 15-20, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 6, 8-11, 13, and 15-23 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 9, 11-14, 17, and 19, and 2'-O-methylmodified nucleotides at positions 1-8, 10, 15, 16, 18, and 20, and an inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 6, 8-11, 13, and 15-23 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 19 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7 and 9-12, 2'-O-methylmodified nucleotides at positions 1-6, 8 and 13-18, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 19, (iii) A sense strand having phosphorothioate internucleotide bonds between nucleotides at positions 18 and 19, and optionally between nucleotides at positions 17 and 18 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8-11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 9 and 11-14, and 2'-O-methylmodified nucleotides at positions 1-8, 10, and 15-20, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 7, 10, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 8, 9, 11, 13, and 15-23 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 9 and 11-14, and 2'-O-methylmodified nucleotides at positions 1-8, 10, and 15-20, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide linkage between the nucleotides at positions 20 and 21 (counting from the 5' end), and (b) (i) A length of 23 nucleotides, (ii) 2'-fluoro-modified nucleotides at positions 2, 7, 10, 12, and 14, and 2'-O-methyl-modified nucleotides at positions 1, 3 - 6, 8, 9, 11, 13, and 15 - 23 (counting from the 5' end), and (iii) An antisense strand having phosphorothioate internucleotide linkages 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), and The RNAi construct has a nucleotide overhang containing 2 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 (a) (i) A length of 21 nucleotides, (ii) 2'-fluoro-modified nucleotides at positions 9, 11 - 14, 17, and 19, and 2'-O-methyl-modified nucleotides at positions 1 - 8, 10, 15, 16, 18, and 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 linkage between the nucleotides at positions 20 and 21 (counting from the 5' end), and (b) (i) A length of 23 nucleotides, (ii) 2'-fluoro-modified nucleotides at positions 2, 7, 10, 12, and 14, and 2'-O-methyl-modified nucleotides at positions 1, 3 - 6, 8, 9, 11, 13, and 15 - 23 (counting from the 5' end), and (iii) An antisense strand having phosphorothioate internucleotide linkages 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 2 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 (a) (i) 21 nucleotides in length, (ii) 2'-fluoro-modified nucleotides at positions 9 and 11 - 14, 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 linkage between the nucleotides at positions 20 and 21 (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 linkages 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 2 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, the RNAi construct comprises a sense strand of 19–23 nucleotides in length and an antisense strand of 19–23 nucleotides in length, wherein the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a 19–21 base pair double-stranded region, the nucleotides at positions 2, 14, and 16 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides, the nucleotides in the sense strand at positions 10–13 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides, and neither the sense strand nor the antisense strand has a total of more than 7 2'-fluoromodified 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 alternative embodiments, the RNAi construct has nucleotide overhangs at the 3' ends of both the sense strand and the antisense strand.

[0091] In one particular embodiment, the RNAi construct is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7 and 9-12, 2'-O-methylmodified nucleotides at positions 1-6, 8, and 13-20, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 8, 9, 14, and 16, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 7, 10-13, 15, and 17-23 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7 and 9-12, 2'-O-methylmodified nucleotides at positions 1-6, 8, and 13-20, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 14, and 16, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8-13, 15, and 17-23 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7 and 9-12, 2'-O-methylmodified nucleotides at positions 1-8, 6, and 13-20, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 23 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 14, and 16, and 2'-O-methylmodified nucleotides at positions 1, 3, 5, 7-13, 15, and 17-23 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 19 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 5 and 7-10, 2'-O-methylmodified nucleotides at positions 1-4, 6, and 11-18, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 19, and (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 18 and 19 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (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 linkages between nucleotides at positions 1 and 2, between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21 (counting from the 5' end), and The RNAi construct has a nucleotide overhang containing 2 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 (a) (i) 20 nucleotides in length, (ii) an inverted abasic nucleotide or an inverted deoxyribonucleotide at position 1, 2'-fluoro-modified nucleotides at positions 8 - 11, and 2'-O-methyl-modified nucleotides at positions 2 - 7 and 12 - 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), and (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 - 6, 8 - 13, 15, and 17 - 21 (counting from the 5' end), and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotides at positions 1 and 2, between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21 (counting from the 5' end), and The RNAi construct has a nucleotide overhang containing 1-2 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 is (a) (i) Length of 22 nucleotides, (ii) an inverted debasic nucleotide or inverted deoxyribonucleotide at position 1, a 2'-fluoromodified nucleotide at positions 8-11, and a 2'-O-methylmodified nucleotide at positions 2-7 and 12-22 (counting from the 5' end), (iii) A sense strand having phosphorothioate internucleotide bonds between nucleotides at positions 20 and 21, and between nucleotides at positions 21 and 22 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 14, and 16, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8-13, 15, and 17-21 (counting from the 5' end), (iii) an antisense chain 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 a nucleotide overhang containing one to two nucleotides at the 3' end of the antisense strand.

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

[0098] For example, in one embodiment, the RNAi construct is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 7-10, and 2'-O-methylmodified nucleotides at positions 1-6 and 11-21 (counting from the 5' end), (iii) A sense strand having phosphorothioate internucleotide bonds between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8-11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand.

[0099] In another embodiment, the RNAi construct is (a) (i) Length of 22 nucleotides, (ii) an inverted debasic nucleotide or inverted deoxyribonucleotide at position 1, a 2'-fluoromodified nucleotide at positions 8-11, and a 2'-O-methylmodified nucleotide at positions 2-7 and 12-22 (counting from the 5' end), (iii) A sense strand having phosphorothioate internucleotide bonds between nucleotides at positions 20 and 21, and between nucleotides at positions 21 and 22 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8-11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 a nucleotide overhang containing one to two nucleotides at the 3' end of the antisense strand.

[0100] In certain embodiments of the present invention, the RNAi construct comprises a sense strand of 19-21 nucleotides in length and an antisense strand of 19-21 nucleotides in length, wherein the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a 19-21 base pair double-stranded region, the nucleotides at positions 2, 7, 12, and 14 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides, the nucleotides in the sense strand at positions 10, 11, and 13 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides, and neither the sense strand nor the antisense strand has a total of more than 7 2'-fluoromodified nucleotides. In such embodiments, the RNAi construct is (a) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 9 and 11-14, and 2'-O-methylmodified nucleotides at positions 1-8, 10, and 15-20, and inverted debasalized nucleotide or inverted deoxyribonucleotide (counting from the 5' end) at position 21, and, (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8-11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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 is (a) (i) Length of 21 nucleotides, (ii) A 2'-fluoromodified nucleotide at positions 9-12, and a 2'-O-methylmodified nucleotide at positions 1-8 and 13-20, and an inverted debasalized nucleotide or inverted deoxyribonucleotide at position 21 (counting from the 5' end), (iii) A sense strand having a phosphorothioate internucleotide bond between nucleotides at positions 20 and 21 (counting from the 5' end), (b) (i) Length of 21 nucleotides, (ii) 2'-fluoromodified nucleotides at positions 2, 7, 12, and 14, and 2'-O-methylmodified nucleotides at positions 1, 3-6, 8-11, 13, and 15-21 (counting from the 5' end), (iii) an antisense chain 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' ends of the sense strand, antisense strand, or both the antisense and sense strands of the RNAi construct include a phosphate moiety. As used herein, the term “phosphate moiety” refers to terminal phosphate groups including unmodified phosphates (-OP=O)(OH)OH) and modified phosphates. Modified phosphates include phosphates in which one or more of the O and OH groups are substituted with H, O, S, N(R) or alkyl, where R is H, an amino protecting group, or an unsubstituted or substituted alkyl group. Exemplary phosphate moieties include, but are not limited to, 5'-monophosphates; 5'-diphosphates; 5'-triphosphates; 5'-guanosine caps (7-methylated or unmethylated); 5'-adenosine caps or any other modified or unmodified nucleotide cap structures; 5'-monothiophosphates (phosphorothioates); 5'-monodhithiophosphates (phosphorodithioates); 5'-α-thiotriphosphates; 5'-γ-thiotriphosphates; 5'-phosphoamidates; 5'-vinyl phosphates; 5'-alkylphosphonates (e.g., alkyl=methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyletherphosphonates (e.g., alkylether=methoxymethyl, ethoxymethyl, etc.).

[0103] Modified nucleotides that can be incorporated into the RNAi construct of the present invention may have two or more chemical modifications as described herein. For example, a modified nucleotide may have modifications to a ribose sugar and modifications to a nucleic acid base. For example, a modified nucleotide may include a 2' sugar modification (e.g., 2'-fluoro or 2'-O-methyl) and a modified base (e.g., 5-methylcytosine or pseudouracil). In other embodiments, a modified nucleotide may include a sugar modification combined with a modification to a 5' phosphate, which will create modified internucleotide or internucleoside bonds when the modified nucleotide is incorporated into a polynucleotide. For example, in some embodiments, a modified nucleotide may 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 the RNAi construct of the present invention may include a combination of a 2' modified nucleotide or a BNA and a phosphorothioate nucleotide interbond. In certain embodiments, both the sense and antisense strands of the RNAi construct of the present invention include a combination of 2'-fluoromodified nucleotides, 2'-O-methylmodified nucleotides, and phosphorothioate nucleotide interbondings.

[0104] In certain embodiments, the nucleotide at position 1, counting from the 5' end of the antisense strand in the RNAi construct may be 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 double-stranded region is an AU base pair. In a particular embodiment, the first base pair from the 5' end of the antisense strand in the double-stranded region is an AU base pair.

[0105] The RNAi construct of the present invention can be readily prepared using methods known in the art, such as conventional solid-phase nucleic acid synthesis. The polynucleotides of the RNAi construct can be constructed on a suitable nucleic acid synthesizer utilizing 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 along with an acid-dissociable dimethoxytrityl (DMT) at the 5' position of a ribonucleoside. The final deprotection conditions are known to not significantly degrade the RNA product. All synthesis can be carried out on a large, medium, or small scale using some kind of automated or manual synthesizer. 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 be 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 sources of fluoride ions are tetrabutylammonium fluoride or aminohydrofluorides (e.g., triethylamine and aqueous HF in a dipolar aproton solvent, such as dimethylformamide).

[0108] The stability of tryesters with respect to fluorides can be altered by selecting the appropriate protecting group for use with phosphite tryesters and phosphotryesters. Methyl protection of phosphotryesters or phosphite tryesters can stabilize their binding to fluoride ions and improve process yields.

[0109] Because ribonucleosides have a reactive 2'-hydroxyl substituent, it is sometimes desirable to protect the reactive 2' position in RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group, such as one that is stable to acid treatment. Silyl protecting groups satisfy this condition and can be easily removed in the final fluoride deprotection step, which can 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, and p-nitrophenyltetrazole.

[0111] As can be understood by those skilled in the art, further methods for synthesizing the RNAi constructs described herein are obvious to them. In addition, various synthetic steps may be carried out in alternative order or sequence to obtain the desired compounds. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc. present in bases), and methods of protecting and deprotecting the RNAi constructs described herein that are useful in the synthesis of the RNAi constructs described herein are known in the art, including, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TW. Greene and PG. W. Mutts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof. Custom synthesis of RNAi drugs is also available from several private vendors, including Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).

[0112] The RNAi constructs of the present invention may include ligands. As used herein, “ligand” means any compound or molecule that can interact directly or indirectly with another compound or molecule. The interaction between another compound or molecule and a ligand may induce a biological response (e.g., triggering a signaling cascade, inducing receptor-mediated endocytosis) or may be a physical association. A ligand can modify one or more properties of the double-stranded RNA molecule it binds to, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, intracellular uptake, charge, and / or clearance properties of the RNA molecule.

[0113] Ligands include serum proteins (e.g., human serum albumin, low-density lipoprotein, globulin), cholesterol moieties, and vitamins (biotin, vitamin E, vitamin B). 12Ligands may include folic acid 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 their binding fragments (e.g., antibodies or binding fragments that target RNAi constructs against specific cell types such as liver). Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecyl It contains glycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptides (e.g., Antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, polymers (e.g., polyethylene glycol (PEG) (e.g., PEG-40K), polyamino acids and polyamines (e.g., spermine, spermidine).

[0114] In certain embodiments, the ligand has endosomal destabilizing properties. The endosomal destabilizing ligand promotes the lysis of endosomes and / or the transport of the RNAi construct or its components from endosomes to the cytoplasm of cells. The endosomal destabilizing ligand may be a polycationic peptide or peptide mimetic exhibiting pH-dependent membrane activity and membrane fusion properties. In one embodiment, the endosomal destabilizing ligand adopts its active conformation at the pH of endosomes. The "active" conformation is the conformation in which the endosomal destabilizing ligand promotes the lysis of endosomes and / or the transport of the RNAi construct or its components from endosomes to the cytoplasm of cells. Examples of 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 their derivatives (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 another steroid. Cholesterol-conjugated oligonucleotides have been reported to be more active than their unconjugated counterparts (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12: 103-228, 2002). Ligands comprising cholesterol moieties and other lipids for conjugation to nucleic acid molecules are also described in U.S. Patents No. 7,851,615; No. 7,745,608; and No. 7,833,992, all of which are incorporated herein by reference in their entirety. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated with a folate moiety can be taken up by cells via a receptor-mediated endocytosis pathway. Such folate-polynucleotide conjugates are described in U.S. Patent No. 8,188,247, which is incorporated herein by reference in its entirety.

[0116] Ligands can target RNAi constructs to specific tissues or cell types in order to selectively inhibit the expression of target genes in those tissues or cell types. In one embodiment, ligands target the specific delivery of RNAi constructs to hepatocytes (e.g., hepatocytes) using various means, as described in more detail below. In certain embodiments, RNAi constructs are targeted to hepatocytes by ligands that bind to surface-expressed asialoglycoprotein receptors (ASGRs) or their components (e.g., ASGR1, ASGR2).

[0117] In some embodiments, the RNAi construct can be specifically targeted to the liver by employing ligands that bind to or interact with proteins expressed on the surface of hepatocytes. For example, in certain embodiments, the ligand may include an antigen-binding protein (e.g., an antibody or its binding fragment (e.g., Fab, scFv)) that specifically binds to receptors expressed on hepatocytes, such as the asialoglycoprotein receptor and the LDL receptor. In a particular embodiment, the ligand includes an antibody or its binding fragment that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand includes a Fab fragment of an antibody that specifically binds to ASGR1 and / or ASGR2. The "Fab fragment" consists of one immunoglobulin light chain (i.e., the light chain variable region (VL) and constant region (CL)) and the CH1 region and variable region (VH) of one immunoglobulin heavy chain. In yet another embodiment, the ligand includes a single-chain variable antibody fragment (scFv fragment) of an antibody that specifically binds to ASGR1 and / or ASGR2. The “scFv fragment” comprises a VH region and a VL region of an antibody, which are present in a single polypeptide chain and optionally include a peptide linker between the VH and VL regions that allows Fv to form a desired structure for antigen binding. Exemplary antibodies and their conjugated fragments that specifically bind to ASGR1 and can be used as ligands for targeting the RNAi construct of the present invention to the liver are described in their entirety in International Publication No. 2017 / 058944, which is incorporated herein by reference. Other antibodies or their conjugated fragments that specifically bind to ASGR1, the LDL receptor, or other proteins expressed on the surface of the liver and are suitable for use as ligands in the RNAi construct of the present invention are commercially available.

[0118] In certain embodiments, the ligand includes carbohydrates. “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 oxygen, nitrogen, or sulfur atoms 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 carbohydrates incorporated into the ligand are monosaccharides selected from pentoses, hexoses, or heptoses, as well as disaccharides and trisaccharides containing such monosaccharide units. In other embodiments, the carbohydrates incorporated into the ligand are amino sugars 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 a particular embodiment, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose, and N-acetyl-galactosamine (GalNAc) are particularly effective in targeting the compound to hepatocytes because such ligands bind to ASGR expressed on the surface of hepatocytes. For example, see 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 construct of the present invention are described in U.S. Patent No. 7,491,805; No. 8,106,022; and No. 8,877,917; U.S. Patent Publication No. 20030130186; and International Publication No. 2013166155, all of which are incorporated herein by reference in their entirety.

[0120] In certain embodiments, the ligand includes a polyhydric carbohydrate moiety. As used herein, “polyhydric carbohydrate moiety” refers to a moiety comprising two or more carbohydrate units that can independently bind to or interact with other molecules. For example, a polyhydric carbohydrate moiety includes two or more binding domains composed of carbohydrates that can bind to two or more different molecules or 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,” “divalent,” “trivalent,” and “tetravalent” with respect to carbohydrate moieties refer to carbohydrate moieties having one, two, three, and four binding domains, respectively. A polyhydric carbohydrate moiety may include a polyhydric lactose moiety, a polyhydric galactose moiety, a polyhydric glucose moiety, a polyhydric N-acetyl-galactosamine moiety, a polyhydric N-acetyl-glucosamine moiety, a polyhydric mannose moiety, or a polyhydric fucose moiety. In some embodiments, the ligand includes a polyhydric galactose moiety. In other embodiments, the ligand includes a polyhydric N-acetyl-galactosamine moiety. In these and other embodiments, the polyhydric carbohydrate moiety may be divalent, trivalent, or tetravalent. In such embodiments, the polyvalent carbohydrate moiety may be bivalent or trivalent. In one particular embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another particular embodiment, the polyvalent galactose moiety is trivalent or tetravalent. Exemplary trivalent or tetravalent GalNAc-containing ligands for incorporation into the RNAi construct of the present invention are detailed below.

[0121] Ligands can be directly or indirectly bound to 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. Ligands can be bound to nucleic acid bases, sugar moieties, or internucleotide bonds of the polynucleotide (e.g., sense or antisense strand) of the RNAi construct of the present invention. Conjugation or binding to purine nucleic acid bases or their derivatives can occur at any position, including atoms inside and outside the ring. In certain embodiments, the ligand is bound to positions 2, 6, 7, or 8 of the purine nucleic acid base. Conjugation or binding to pyrimidine nucleic acid bases or their derivatives can also occur at any position. In some embodiments, the ligand can be bound to positions 2, 5, and 6 of the pyrimidine nucleic acid base. Conjugation or binding to the sugar moiety of a nucleotide can occur at any carbon atom. Examples of carbon atoms in the sugar moiety that can bind to a ligand include the 2', 3', and 5' carbon atoms. The 1' position can also bind to a ligand in debasalized nucleotides, etc. Internucleotide bonds can also facilitate ligand binding. For phosphorus-containing bonds (e.g., phosphodiesters, phosphorothioates, phosphorodithioates, phosphoramidates, etc.), the ligand can bind directly to the phosphorus atom or to an O, N, or S atom bonded to the phosphorus atom. For amine or amide-containing nucleoside bonds (e.g., PNA), the ligand can bind to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0122] In certain embodiments, the ligand may be bound to the 3' or 5' end of either the sense strand or the antisense strand. In certain embodiments, the ligand is covalently bound to the 5' end of the sense strand. In these embodiments, the ligand is bound to the 5' terminal nucleotide of the sense strand. In these and other embodiments, the ligand is bound at the 5' position of the 5' terminal nucleotide of the sense strand. In embodiments where the inverted debasalized nucleotide or inverted deoxyribonucleotide is the 5' terminal nucleotide of the sense strand and is bound to an adjacent nucleotide via a 5'-5' nucleotide bond, the ligand may be bound to the 3' position of the inverted debasalized nucleotide or inverted deoxyribonucleotide. In other embodiments, the ligand is covalently bound to the 3' end of the sense strand. For example, in some embodiments, the ligand is bound to the 3' terminal nucleotide of the sense strand. In these certain embodiments, the ligand is bound at the 3' position of the 3' terminal nucleotide of the sense strand. In embodiments where the inverted debasalized nucleotide or inverted deoxyribonucleotide is the 3' terminal nucleotide of the sense strand and is bound to an adjacent nucleotide via a 3'-3' nucleotide bond, the ligand may be bound to the 5' position of the inverted debasalized nucleotide or inverted deoxyribonucleotide. In alternative embodiments, the ligand is bound near the 3' end of the sense strand but before one or more terminal nucleotides (i.e., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is bound at the 2' position of the sugar of the 3' terminal nucleotide of the sense strand. In other embodiments, the ligand is bound at the 2' position of the sugar of the 5' terminal nucleotide of the sense strand.

[0123] In certain embodiments, the ligand is bound to the sense or antisense strand via a linker. The "linker" is an atom or atomic group that covalently binds the ligand to the polynucleotide component of the RNAi construct. Linkers can have atomic lengths of about 1 to about 30, about 2 to about 28, about 3 to about 26, about 4 to about 24, about 6 to about 20, about 7 to about 20, about 8 to about 20, about 8 to about 18, about 10 to about 18, and about 12 to about 18. In some embodiments, the linker may include a bifunctional binding moiety typically comprising an alkyl moiety having two functional groups. One of the functional groups is selected to bind to the compound of interest (e.g., the sense or antisense strand of the RNAi construct), and the other is further selected to bind essentially to any selected group, such as the ligand 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 typically employed in the difunctional bond portion include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, the difunctional bond portion includes amino, hydroxyl, carboxylic acid, thiol, and unsaturated (e.g., double or triple bonds).

[0124] Linkers that can be used to bind ligands to the sense or antisense strand in the RNAi construct of the present invention include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, and substituted C1-C1. 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, alkynyls. 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. The cleavable linker is sufficiently stable outside the cell but is cleaved after entering the target cell, releasing the two parts that the linker holds together. In some embodiments, the cleavable linker is cleaved at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 times faster, or at least 100 times faster, in the target cell or under a first reference condition (e.g., one that mimics or is selected to be equivalent to intracellular conditions) than under the target blood or a second reference condition (e.g., one that mimics or is selected to be equivalent to conditions found in blood or serum).

[0126] Cleavable linkers are sensitive to the presence of cleavage agents, such as pH, redox potential, or degradable molecules. Generally, cleavage agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degradable agents include oxidases or reductases such as mercaptans, or reducing agents that are selected for a specific substrate or are not substrate-specific, and can degrade redox-cleavable linkers by reduction when present inside cells; esterases; agents that can create endosomes or acidic environments, such as those that result in a pH of 5 or less; enzymes, peptidases (which may be substrate-specific), and phosphatases that can hydrolyze or degrade acid-cleavable linkers by acting as general acids.

[0127] Cleavable linkers may contain pH-sensitive regions. While human serum has a pH of 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable groups that are cleaved at a favorable pH, thereby releasing RNA molecules from ligands into the cell or into a desired compartment of the cell.

[0128] Linkers can contain cleavable groups that can be cleaved by specific enzymes. The type of cleavable group incorporated into a linker may depend on the target cell. For example, liver-targeting ligands may bind to RNA molecules via linkers containing ester groups. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in esterase-deficient cell types. Other types of cells rich in esterases include lung, renal cortex, and testicular cells. Linkers containing peptide bonds may be used to target peptidase-rich cells such as liver cells and synovial cells.

[0129] Generally, the suitability of a cleavable linker candidate can be evaluated by testing the ability of a degrading agent (or condition) to cleave the linker candidate. It is also desirable to test the cleavable linker candidate for its ability to resist cleavage in blood or in contact with non-target tissues. Therefore, the relative sensitivity to cleavage can be determined between a first condition selected to demonstrate cleavage in target cells and a second condition selected to demonstrate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform an initial evaluation in cell-free or culture conditions and confirm it with further evaluation in whole animals. In some embodiments, a useful linker candidate cleaves at least 2, 4, 10, 20, 50, 70, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0130] In other embodiments, redox-cleavable linkers are utilized. Redox-cleavable linkers are cleaved upon reduction or oxidation. An example of a reductively cleavable group is a disulfide linking group (-SS-). One or more of the methods described herein can be used to determine whether a cleavable linker candidate is a suitable “reductively cleavable linker” or suitable for use with, for example, a particular RNAi construct and a particular ligand. For example, a linker candidate 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. Linker candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In certain embodiments, linker candidates are cleaved by up to 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 other embodiments, a ligand is covalently bonded to the sense or antisense strand of the RNAi construct using a phosphate-based cleavable linker that is cleaved by an agent that decomposes or hydrolyzes the phosphate group. An example of an agent that hydrolyzes the phosphate group in a cell is an enzyme such as a phosphatase in the cell. Examples of cleavable groups in phosphate systems 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. Specific embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. Another specific embodiment is -OP(O)(OH)-O-. These 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 with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by an active agent such as an enzyme that can act as a general acid. Within cells, certain low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid-cleavable group may have the general formula -C=NN-, C(O)O, or -OC(O). In particular embodiments, 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 ester-based cleavable groups that are 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 alkylylene groups. The 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 include a cleavable group of a peptide system that is cleaved by enzymes such as peptidases and proteases in cells. The cleavable group of a peptide system is a peptide bond formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The cleavable group of a peptide system includes an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. The cleavable group of a peptide system is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that produce peptides and proteins. The cleavable linking group of a peptide system has the general formula -NHCHR A C(O)NHCHR B It contains C(O)-, in the formula, R A and R B These are the side chains of two adjacent amino acids. These candidates can be evaluated using a method similar to that described above.

[0135] Other types of linkers suitable for binding ligands to the sense or antisense strand in the RNAi construct of the present invention are known in the art and may include the linkers described in U.S. Patent 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 entirety.

[0136] In certain embodiments, the ligand covalently bound to the sense or antisense strand of the RNAi construct of the present invention includes a GalNAc moiety, for example, a polyvalent GalNAc moiety. In some embodiments, the polyvalent GalNAc moiety is a trivalent GalNAc moiety and is bound to the 3' end of the sense strand. In other embodiments, the polyvalent GalNAc moiety is a trivalent GalNAc moiety and is bound to the 5' end of the sense strand. In yet another embodiment, the polyvalent GalNAc moiety is a tetravalent GalNAc moiety and is bound to the 3' end of the sense strand. In yet another embodiment, the polyvalent GalNAc moiety is a tetravalent GalNAc moiety and is bound to the 5' end of the sense strand.

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

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

[0139] In one embodiment, the RNAi construct comprises a ligand and a linker having the structure of the following formula I, where each n is independently 1 to 3, k is 1 to 3, m is 1 or 2, j is 1 or 2, and the ligand is bound 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, where each n is independently 1 to 3, k is 1 to 3, m is 1 or 2, j is 1 or 2, and the ligand is bound to the 3' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0141] In another embodiment, the RNAi construct comprises a ligand and a linker having the structure of formula III below, wherein the ligand is bound to the 3' end of the sense strand of a 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 the following formula IV, wherein the ligand is bound to the 3' end of the sense strand of a 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 bound 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 bound 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 the following formula VII, where X = O or S, and the ligand is bound 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 n is independently 1 to 3, and the ligand is bound 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, where the ligand is bound to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0148] The phosphorothioate bond can be replaced with a phosphodiester bond, as shown in any one of formulas I to IX, in order to covalently bond the ligand and linker to the nucleic acid chain.

[0149] The present invention also includes pharmaceutical compositions and formulations comprising RNAi constructs described herein and pharmaceutically acceptable carriers, excipients, or diluents. Such compositions and formulations are useful for reducing the expression of target genes in subjects where this is desired. When considering clinical applications, pharmaceutical compositions and formulations will be prepared in a form appropriate for the intended use. Generally, this will inevitably involve preparing compositions that are essentially free of pyrogens and other impurities that may be harmful to humans or animals.

[0150] The terms “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not cause adverse allergic or other undesirable reactions when administered to animals or humans. As used herein, “pharmaceutically acceptable carriers, excipients or diluents” include solvents, buffers, solutions, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are acceptable for use in the formulation of pharmaceuticals, such as drugs suitable for administration to humans. The use of such media and drugs for pharmaceutically active substances is known in the art. Their use in therapeutic compositions is envisioned unless any conventional media or drug is incompatible with the RNAi construct of the present invention. Auxiliary active ingredients may also be incorporated into the composition, provided they do not inactivate the RNAi construct of the composition.

[0151] The composition and method of formulation of a pharmaceutical composition depend on several conditions, including but not limited to the route of administration, the type and degree of the disease or disorder being treated, or the dose administered. In some embodiments, the pharmaceutical composition is formulated based on the intended route of delivery. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral delivery forms include intravenous, intra-arterial, 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 comprise a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such embodiments, the pharmaceutical composition may comprise a targeted ligand (e.g., a GalNAc-containing or antibody-containing ligand as described herein).

[0152] In some embodiments, the pharmaceutical composition comprises an effective amount of the RNAi construct described herein. “Effective amount” is an amount sufficient to produce a favorable or desired clinical outcome. In some embodiments, the effective amount is sufficient to reduce the expression of a target gene in a particular tissue or cell type of interest (e.g., liver or hepatocytes).

[0153] The pharmaceutical composition of the present invention may be administered via any common route, as long as the target tissue is available through 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 composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0154] Colloidal dispersion systems such as oil-in-water emulsions, micelles, mixed micelles and liposomes, polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems can be used as delivery vehicles for the RNAi constructs of the present invention. Suitable commercially available lipid emulsions 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 liposomes (i.e., artificial membrane vesicles). The RNAi constructs of the present invention can be encapsulated within liposomes or can form complexes with liposomes, particularly cationic liposomes. Alternatively, the RNAi constructs of the present invention can form complexes with lipids, particularly cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersions are well known in the art. Exemplary formulations are also disclosed in U.S. Patent 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 in International Publication No. 03 / 093449.

[0155] In some embodiments, the RNAi constructs of the present invention are completely encapsulated, for example, in a lipid formulation to form SNALPs 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 are extremely useful for systemic administration because they exhibit a long circulating lifetime after intravenous injection and accumulate at distal sites (e.g., sites physically distant from the administration site). Nucleic acid-lipid particles typically have an average diameter of about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm and are substantially non-toxic. In addition, nucleic acids present in nucleic acid-lipid particles are resistant to degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, and 6,815,432, and in International Publication No. 96 / 40964.

[0156] Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile solutions or dispersions for injection. Generally, these preparations are sterile and fluid enough to be easily injected. The preparations should be stable under manufacturing and storage conditions and should be protected from microbial contamination such as bacteria and fungi. Suitable solvents or dispersion media may include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coating materials such as lecithin, maintaining the particle size required in the case of dispersions, and using surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injection composition can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0157] Sterile solutions for injection can be prepared by incorporating an active compound in an appropriate amount with any other desired components (e.g., as listed above) into a solvent, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating various sterilized active components into a sterile vehicle containing a basic dispersion medium and other desired components, e.g., the components listed above. For sterile powders for the preparation of sterile solutions for injection, preferred preparation methods include vacuum drying and freeze-drying techniques, from which the powders of the active component and any additional desired components are obtained from a pre-sterilized filtered solution.

[0158] The compositions of the present invention can generally be formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed by free amino groups) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed by free carboxyl groups 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 aqueous solutions, for example, the solution is usually appropriately buffered, and the liquid diluent is initially isotonic with, for example, sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, a sterile aqueous medium known to those skilled in the art is employed, especially in consideration of this disclosure. For example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution, or injected into the proposed injection site (see, for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). With regard to administration to humans, the preparation should meet the sterility, pyrogenicity, overall safety, and purity standards required by FDA standards. In certain embodiments, the pharmaceutical composition of the present invention comprises or consists of sterile saline and the RNAi construct described herein. In other embodiments, the pharmaceutical composition of the present invention comprises or consists of the RNAi construct described herein and sterile water (e.g., water for injection, WFI). In yet another embodiment, the pharmaceutical composition of the present invention comprises or consists of the RNAi construct described herein and phosphate-buffered saline (PBS).

[0160] In some embodiments, the pharmaceutical compositions of the present invention are packaged in or stored within an administration device. Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, autoinjectors, injection pumps, wearable syringes, and injection pens. Devices for aerosolized or powder formulations include, but are not limited to, inhalers, inhalers, and aspirators. Accordingly, the present invention includes an administration device containing a 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 the expression of a target gene in cells by contacting the cells with any one of the RNAi constructs described herein. The cells may 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 the expression of the target gene. The reduction in target gene expression in cells or animals treated with the RNAi construct of the present invention can be determined by comparing it to the target gene expression in cells or animals that have not been treated with the RNAi construct or that have been treated with a control RNAi construct. For example, in some embodiments, reduction or inhibition of target gene expression is evaluated by (a) measuring the amount or level of target mRNA in cells treated with the RNAi construct of the present invention, (b) measuring the amount or level of target mRNA in cells treated with a control RNAi construct (e.g., an RNAi agent targeting RNA molecules that are not expressed in cells or RNAi constructs having nonsense or scrambled sequences) or without the construct, and (c) comparing the target mRNA level measured from cells treated in (a) with the target mRNA level measured from control cells in (b). Before comparison, the target mRNA levels in treated and control cells can be normalized to RNA levels relative to a control gene (e.g., 18S ribosomal RNA or housekeeping gene). Target mRNA levels can be measured by a variety of methods, including Northern blotting, nuclease protection assays, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, and droplet digital PCR.

[0162] In other embodiments, reduction or inhibition of target gene expression is evaluated by (a) measuring the amount or level of the target protein in cells treated with the RNAi construct of the present invention; (b) measuring the amount or level of the target protein in cells treated with a control RNAi construct (e.g., an RNAi agent targeting RNA molecules that are not expressed in cells or RNAi constructs having nonsense or scrambled sequences) or without the construct; and (c) comparing the target protein level measured from cells treated in (a) with the target protein level measured from control cells in (b). Methods for measuring target protein levels are known to those skilled in the art and include Western blotting, immunoassays (e.g., ELISA), and flow cytometry.

[0163] The present invention also provides a method for reducing or inhibiting the expression of a target gene in a subject where such reduction is needed, the method comprising administering one of the RNAi constructs described herein to the subject. The RNAi constructs of the present invention can be used, for example, to treat or improve diseases, disorders, or disabilities associated with abnormal target gene expression or activity, such as when overexpression of a 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, and herpesvirus genes. In some embodiments, the target gene is a gene that codes for human microRNA (miRNA).

[0164] In certain embodiments, the RNAi construct of the present invention reduces the expression of a target gene by at least 50% in cells or subjects. In some embodiments, the RNAi construct of the present invention reduces the expression of a target gene by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% in cells or subjects. In other embodiments, the RNAi construct of the present invention reduces the expression of a target gene by about 90% or more in liver cells, for example, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more. The percentage 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 the results obtained, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims. [Examples]

[0166] Example 1. In vivo activity of PNPLA3RNAi constructs using different chemical modification patterns To evaluate the effect of different chemical modification patterns on the in vivo efficacy of RNAi constructs, RNAi constructs targeting the patatin-like phospholipase domain 3 (PNPLA3) gene were synthesized using various patterns of 2'-fluoro-modified and 2'-O-methyl-modified nucleotides, as described in more detail below, and evaluated in a humanized mouse model expressing PNPLA3.

[0167] RNAi constructs were synthesized using solid-phase phosphoramidite chemistry. The synthesis was performed on a MerMade12 (Bioautomation) instrument.

[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) Activating agent 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 (70:20:10 (v / v / v) tetrahydrofuran / pyridine / 0.02 M iodine in water, BIO420 / 4000, EMD) Diethylamine solution (20% DEA in acetonitrile, NC0017-0505, EMD) Thiolation reagent (40:60 (v / v) pyridine / acetonitrile 0.05M 5-N-[(dimethylamino)methylene]amino-3H-1,2,4-dithiazol-3-thion (BIOSULII / 160K)) Adenosine, guanosine, cytosine, and uridine 5'-aminohexyl linker phosphoramidites, phosphorylated phosphoramidites, 2'-deoxythymidine phosphoramidites, and 2'-methoxy and 2'-fluorophosphoramidites (Thermo Fisher Scientific), approximately 10 mL of acetonitrile at 0.10 M on a molecular sieve (3 Å, JTBaker). 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 MerMade12 instrument. Solid supports were added to each column (4 mL SPE tube with upper and lower frits), and the columns were attached to the instrument. The columns were washed twice with acetonitrile. The phosphoramidite and reagent solution lines were purged. Synthesis was started using Poseidon software. Synthesis was carried out by repeating the deprotection / coupling / oxidation / capping synthetic cycle. Specifically, the 5'-dimethoxytrityl (DMT) protecting group was removed by adding a detritylation reagent to the solid support. The solid support was washed with acetonitrile. Phosphoramidite and activator solution were added to the support and subsequently incubated to bond the incoming nucleotides to the free 5'-hydroxyl groups. The support was washed with acetonitrile. Oxidation or thiolation reagents were added to the support to convert the phosphite triester to 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. The support was washed with acetonitrile and dried under vacuum.

[0170] GalNAc conjugation A sense chain for conjugation to the trivalent N-acetyl-galactosamine (GalNAc) moiety (structure shown in formula VII below) was prepared using a 5'-aminohexyl linker. 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 vacuum filtration with 2 mL aliquots of 1% trifluoroacetic acid (TFA) in dichloromethane (DCM). Once no further orange / yellow coloration was observed in the eluate, the resin was washed with dichloromethane. The resin was then washed with 2% diisopropylethylamine in 5 mL of N,N-dimethylformamide (DMF). In a separate vial, a solution of GalNAc3-Lys2-Ahx (67 mg, 40 μmol) in DMF (0.5 mL) (its structure and synthesis are described below) 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, the column was capped, and incubated overnight at room temperature. The resin was washed with DMF and DCM and dried under vacuum.

[0171] Cutting The synthesis column was removed from the synthesis apparatus or vacuum manifold. The solid support from each column was transferred to a 10 mL vial. 4 mL of high-concentration 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 the freezer and cooled in the 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 fraction was desalted by size exclusion chromatography and analyzed by ion-pair reverse-phase high-performance liquid chromatography-mass spectrometry (HPLC-MS). The pooled fraction was freeze-dried to obtain a white amorphous powder.

[0173] Analytical anion exchange chromatography (AEX): Column: Thermo DNAPac PA200RS (4.6 × 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 over 6.2 minutes Preparative anion exchange chromatography (AEX): Column: Tosoh TSK Gel SuperQ-5PW21×150mm, 13μm 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% over 20 minutes Preparative size exclusion chromatography (SEC): Column: GE Hi-Prep 26 / 10 Equipment: GE AKTA Pure Buffer solution: 20% ethanol aqueous solution Flow rate: 10mL / min Injection volume: 15 mL using a sample load 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 in water, 50 mM hexafluoroisopropanol (HFIP) Buffer B: 15.7 mM DIEA and 50 mM HFIP in 50:50 water / acetonitrile Flow rate: 0.5mL / min Gradient: 10-30% over 6 minutes

[0174] annealing Small amounts of sense and antisense strands were weighed into individual vials. siRNA reconstitution buffer (Qiagen) or phosphate-buffered saline (PBS) was added to the vials to a concentration of approximately 2 mM by dry weight. The actual sample concentration was measured on a NanoDrop One (ssDNA, extinction coefficient = 33 μg / OD260). Next, the two strands were mixed in equimolar ratios, and the sample was heated in a 90°C water bath for 5 minutes and slowly cooled to room temperature. The sample was analyzed by AEX. The double helices were registered and subjected to in vivo testing as described in more detail below.

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

[0176] Fmoc-Ahx-OH (1.13 g, 3.19 mmol) in DCM (30 mL) was added to a 50 mL Falcon tube, 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 immersed in a shaker for 2 hours. The solvent was drained, and the resin was washed with 17:2:1 DCM / MeOH / DIEA (30 mL x 2) and DCM (30 mL x 4) and then dried. UV spectrophotometric detection at 290 nm determined the added amount to be 0.76 mmol / g.

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

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

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

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

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

[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), TATU (3.22 g, 10 mmol) was added, and the solution was stirred for 5 minutes. DIEA (2.96 mL, 17 mmol) was added to this solution, and the mixture was then added to the 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 the solvent was drained after 5 minutes. This process was repeated three more times, and the combined filtrate was concentrated under reduced pressure. The residue was pulverized with diethyl ether (50 mL), the suspension was filtered, and the product was dried to obtain the crude product. The crude product was purified by reverse-phase chromatography and eluted with 0-20% MeCN in water. The fractions were combined and freeze-dried to obtain the product as a white solid.

[0184] Table 1 below shows the location of modifications in each sense and antisense sequence of the modified PNPLA3RNAi construct. The nucleotide sequences are listed according to the following notation. dT, dA, dG, dC = corresponding deoxyribonucleotides; a, u, g, and c = corresponding 2'-O-methylribonucleotides; Af, Uf, Gf, and Cf = corresponding 2'-deoxy-2'-fluoro("2'-fluoro")ribonucleotides; Phos = terminal nucleotide having a monophosphate group at its 5' end; invAb = inverted debasalized nucleotide (i.e., debasalized nucleotide that, when on the 3' end of a chain, is bound to an adjacent nucleotide via its 3' substituent (3'-3' bond), or when on the 5' end of a chain, is bound to an adjacent nucleotide via its 5' substituent (5'-5' nucleotide bond)); and invdX = inverted deoxyribonucleotide (i.e., deoxyribonucleotide that, when on the 3' end of a chain, is bound to an adjacent nucleotide via its 3' substituent (3'-3' bond), or when on the 5' end of a chain, is bound to an adjacent nucleotide via its 5' substituent (5'-5' nucleotide bond)). The insertion of "s" in a sequence indicates that two adjacent nucleotides are joined by a phosphorothiodiester group (e.g., a phosphorothioate nucleotide bond). Unless otherwise specified, all other nucleotides are joined by a 3'-5' phosphodiester group. All RNAi constructs are conjugated via the 5' end of the sense strand to the GalNAc portion shown in formula VII. Table 1 also lists the pattern designation and sequence family designation for each RNAi construct. The pattern designation is schematically shown in Figure 1. If two RNAi constructs have 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 to have either a P1 chemical modification pattern or a CM1 control chemical modification pattern. siRNA molecules with the CM1 control chemical modification pattern have been reported to have potency and long-term gene silencing effects in vivo. See Nair et al., J.Am.Chem.Soc., Vol.136:16958-16961, 2014. The potency of chemically modified RNAi constructs in inhibiting PNPLA3 gene expression in humanized mouse models expressing wild-type human PNPLA3 or mutant human PNPLA3 was evaluated. To create the mouse models, 1 × 10⁶ cells per animal were used in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). 12 Associated adenovirus (AAV; serotype AAV8 or AAV7; endotoxin-free), diluted with viral particles, was intravenously injected into the tail vein of C57BL / 6NCrl male mice (Charles River Laboratories Inc.) to detect human PNPLA3, PNPLA3 rs738409 , or PNPLA3 rs738409-rs738408 Gene expression was promoted. The mice were generally 10-12 weeks old, and each treatment group contained 4-6 animals (n).

[0187] All RNAi constructs are AAV-PNPLA3, PNPLA3 rs738409 , and / or PNPLA3 rs738409-rs738408 The study was conducted in mice injected with the following: at least two vehicle-treated control groups: AAV empty vector treated with a vehicle, and AAV-PNPLA3, PNPLA3 rs738409 , or PNPLA3 rs738409-rs738408This was also included. Two weeks after AAV injection, mice were treated by subcutaneous injection of a single dose of RNAi construct (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) at a dose of 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, rapidly frozen in liquid nitrogen, and processed for purified RNA using the QIACube HT instrument (9001793) and the Qiagen RNeasy 96 QIACube HT kit (74171) according to the manufacturer's instructions. Samples were analyzed using the QIAxpert system (9002340). RNA was treated with Promgea RQ1 RNase-Free DNase (M6101) and prepared for real-time qPCR using the Applied Biosystem TaqMan™ RNA-to-CT™ 1-Step Kit (4392653). Real-time qPCR was performed on a QuantStudio real-time PCR instrument. Results are shown as relative knockdown of human PNPLA3 mRNA expression compared to vehicle-treated control animals, with human PNPLA3 gene expression normalized to mouse Gapdh (TaqMan™ assay from Invitrogen, hs00228747_m1 and 4352932E, respectively).

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

[0189] Variations of the P1 modification pattern were created to modify the chain length, the properties of the RNAi construct's ends (i.e., overhang vs. blunt end), and / or to include inverted debasal nucleotides at the 5' or 3' end of the sense strand, and applied to RNAi constructs having the same core sequence. The RNAi constructs with the 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 (double-strand numbers 3540, 5241, 5614, and 5615) were applied to human PNPLA3. rs738409 The mutant gene was subcutaneously administered at a dose of 5 mg / kg to mice expressing the mutant gene. Human PNPLA3 expression levels in the liver were evaluated 15 days after administration of the RNAi construct. The results are shown in Figure 3. RNAi constructs with P2, P3, or P4 patterns resulted in a higher mean reduction in PNPLA3 expression compared to RNAi constructs with the P1 pattern.

[0190] Further variants of the P3 pattern were created to increase the efficacy and duration of in vivo mRNA knockdown. In the P3 pattern (double-strand number 6191), the 2'-fluoromodified nucleotides at positions 4 and 6 from the 5' end of the antisense strand were replaced with 2'-O-methylmodified nucleotides to generate the P9 pattern (double-strand number 6267). RNAi constructs with a P9 pattern (double-strand number 7320) having an inverted adenosine deoxyribonucleotide instead of an inverted debasalized nucleotide at the 3' end of the sense strand were also synthesized. All three constructs were evaluated in the humanized mouse model described above. In animals treated with 5 mg / kg of double-strand number 6267, human PNPLA3 liver expression decreased by 97% at day 22 after administration, while animals treated with 5 mg / kg of double-strand number 6191 showed a 92% reduction in human PNPLA3 liver expression levels at the same time. Animals treated with 3 mg / kg of double-stranded gene number 7320 showed a 95% reduction in human PNPLA3 liver expression levels at 28 days post-administration, indicating that double-stranded gene number 7320 is more potent and resulted in longer-lasting gene knockdown than double-stranded genes 6191 and 6267.

[0191] The P9 pattern was applied to PNPLA3RNAi constructs with two different core sequences (double-strand numbers 7318, 7320, 7062, 8513, and 8709), and in vivo efficacy was evaluated at doses of 1 mg / kg and 3 mg / kg in bioluminescence imaging assays. For the bioluminescence imaging assays, an associated adenovirus (AAV) vector was designed to contain a synthetic strand of the murine cytomegalovirus promoter, the entire sequence of firefly luciferase, and subsequently, immediately downstream from the firefly luciferase stop codon, a mRNA sequence specific to the RNAi construct under test. Ten additional nucleotides were flanked at each end of the mRNA sequence. The vector "PP3A(DM)" was packaged in AAV serotype AAVDJ8 (endotoxin-free). Before injection, administer PP3A(DM) in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) at a rate of 5 × 10⁴ per animal. 11The virus was diluted into virus particles and intravenously injected into the tail vein of BALB / c male mice (Charles River Laboratories Inc.). The mice were generally 10–12 weeks old, and each group consisted of n=5 animals.

[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, the mice were imaged on the IVIS Spectrum In Vivo Imaging System (PerkinElmer). Subsequently, mice were randomly selected into groups according to baseline total luminosity scores from a defined target region including the liver. Once randomized, mice were treated subcutaneously with a single dose of RNAi construct (0.5 mM) at 1.0 or 3.0 milligrams per kilogram of body weight, diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136), or with phosphate-buffered saline alone (designated as "vehicle"). Mice were imaged weekly according to the same protocol, applying the same gating constraints to the total luminosity score. The data is expressed as total luminous flux (photons per second, y-axis) versus weeks after RNAi construct injection (x-axis). A decrease in total luminous flux indicates a decrease in luciferase reporter expression.

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

[0194] These RNAi constructs (double-strand numbers 7318, 7320, 7062, 8513, and 8709) were also evaluated in the humanized mouse model described above. Specifically, the RNAi constructs were evaluated at 0.5, 1, or 3 mg / kg in humanized PNPLA3 rs738409-rs738408 The mutant gene was subcutaneously administered to mice expressing the RNAi construct. Human PNPLA3 expression levels in the liver were evaluated by qPCR 28 or 42 days after administration of the RNAi construct. The results are expressed 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 a P9 modification pattern are more potent and result in longer-lasting gene knockdown than previously tested patterns. Administration of a single dose of 0.5 mg / kg of the RNAi construct reduced human PNPLA3 hepatic expression by approximately 50% four weeks after single-dose administration, while administration of a 1 mg / kg construct reduced human PNPLA3 hepatic expression by approximately 70% four weeks after single-dose administration. The 1 mg / kg dose was sufficient to maintain a reduction of over 55% in PNPLA3 expression over six weeks from the single-dose dose. Administration of a single dose of 3 mg / kg of the RNAi construct reduced human PNPLA3 hepatic expression by more than 90% four weeks after single-dose administration. Human PNPLA3 hepatic expression was still reduced by more than 75% six weeks after administration of the 3 mg / kg dose. The improved potency and duration of gene knockdown were observed in RNAi constructs with two different sequences, indicating that the P9 chemical modification pattern is effective in stabilizing RNAi constructs at least partially independently of the nucleic acid base sequence.

[0197] Next, the in vivo efficacy of PNPLA3RNAi constructs with a P9 chemical modification pattern was compared to PNPLA3RNAi constructs with one of three different control modification patterns. CM2, CM3, and CM4 modification patterns have been previously reported to improve the metabolic stability of siRNA molecules, leading to improved efficacy 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 both the sense and antisense strands, differing only in their chemical modification patterns. Two different constructs with P9 modification patterns were synthesized: one with an inverted debase at the 3' end of the sense strand (double-strand number 7318), and the other with an inverted deoxythymidine at the 3' end of the sense strand (double-strand number 8709). RNAi constructs having one of the CM2, CM3, or CM4 modification patterns were also synthesized (double-strand numbers 8103, 8104, and 8105, respectively). Subsequently, each RNAi construct was 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. Human PNPLA3 expression levels in the liver were evaluated by qPCR 28 days after administration of the RNAi constructs. The results are shown in Figure 5. The RNAi construct with a P9 modification pattern (double-strand number 7318) having an inverted debase at the 3' end of the sense strand resulted in the highest reduction in hepatic PNPLA3 expression among all the constructs tested. The RNAi construct with a P9 modification pattern (double-strand number 8709) having an inverted deoxythymidine at the 3' end of the sense strand resulted in a higher reduction in hepatic PNPLA3 expression than the construct with the CM4 pattern (double-strand number 8105), and a reduction in hepatic PNPLA3 expression comparable to that of the constructs with the CM2 and CM3 patterns (double-strand numbers 8103 and 8104, respectively).

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

[0199] [Table 3]

[0200] Example 2. In vivo activity of ASGR1RNAi constructs using different chemical modification patterns As shown in Example 1, the P1 chemical modification pattern applied to 13 different RNAi constructs having 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, RNAi constructs targeting asialoglycoprotein receptor 1 (ASGR1) mRNA were synthesized using the P1 chemical modification pattern according to the method described in Example 1. RNAi constructs with the same sequence were synthesized using the CM1 control chemical modification pattern. The sequences of the RNAi constructs are provided in Table 4 below, using the same notation as shown in Table 1 above. The GalNAc moiety having the structure shown in formula VII was conjugated to the 5' end of the sense strand of the RNAi construct designated as double-stranded number 1520, and the GalNAc moiety having the structure shown in formula IX was conjugated to the 5' end of the sense strand of the RNAi construct designated as double-stranded number 1421. The conjugation of the GalNAc moiety to the sense strand of the RNAi construct was carried out as described in Example 1, except that the GalNAc moiety having the structure shown in formula IX was prepared as follows. TATU (3.22 g, 10 mmol) was added 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)ethoxy)acetic acid (5.37 g, 10 mmol) in DMF (40 mL), and the solution was stirred for 5 minutes. DIEA (2.96 mL, 17 mmol) was added to this solution, and the mixture was subsequently added to the resin described in Example 1 above. The suspension was kept at room temperature overnight, and the solvent was drained. The resin was washed with DMF (3 x 30 mL) and DCM (3 x 30 mL).

[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 mice (Charles River) aged 10–12 weeks were fed a standard solid diet (2020×Teklad global soy protein-free extruded rodent diet; Harlan). On day 0, mice received subcutaneous injection of either buffer or 0.25 ml buffer at a dose of 5 mg of the specified RNAi construct per kg of body weight (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 the 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 constructs to the amount of ASGR1 mRNA in the liver tissue of animals injected with the buffer. The results show that animals treated with the RNAi construct having the P1 modification pattern (double-strand number 1520) exhibited a greater reduction in hepatic ASGR1 expression than animals treated with the RNAi construct having the CM1 control modification pattern at all measurement time points (Figure 6). Similar to the results described in Example 1 using the RNAi construct targeting human PNPLA3 mRNA, the P1 chemical modification pattern enhances the potency of the RNAi construct.

[0203] Example 3. In vivo activity of LPA RNAi constructs using different chemical modification patterns To further evaluate the ability of the chemical modification patterns described herein to enhance the in vivo potency of RNAi constructs, RNAi constructs targeting the LPA gene, a third liver 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 notation as in Table 1 above. Table 5 also lists the pattern designation and sequence family designation for each RNAi construct. The pattern designation is schematically shown in Figure 1. If two RNAi constructs have 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 to have either a CM1 control chemical modification pattern (double-strand number 3632) or a P1 chemical modification pattern (double-strand number 3635). The in vivo potency of the two constructs was evaluated in a dual-transgenic mouse model expressing fully functional human Lp(a) particles with average serum baseline Lp(a) levels of approximately 50–60 mg / dL. Lp(a) is a low-concentration lipoprotein consisting of LDL particles and apolipoprotein (a) (apo(a)), a glycoprotein bound to apolipoprotein B of the LDL particles by disulfide bonds. Apo(a) is encoded by the LPA gene and alters serum Lp(a) levels in response to changes in LPA gene expression. Dual transgenic mice were created by crossing transgenic mice expressing human apo(a) from yeast artificial chromosomes (YACs) containing the fully 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, and subsequently on days 14 and 28 after injection. Serum Lp(a) concentrations were measured using an Lp(a) ELISA assay (catalog number 10-1106-01, Mercodia AB, Uppsala, Sweden). Based on the baseline Lp(a) levels of the animals, the percentage change in Lp(a) levels at specific time points was calculated for each animal. The results are shown in Figure 7. Two weeks after injection, although not statistically significant, administration of double-stranded cell 3635 with a P1 modification pattern resulted in a higher mean reduction in serum Lp(a) levels (-49%) compared to double-stranded cell 3632 with a control CM1 modification pattern (-35%).

[0206] In the second series of experiments, LPA RNAi constructs were synthesized that targeted different regions of LPA mRNA than those used in the first set of experiments, using P1 chemical modification patterns or variants thereof. The RNAi constructs with novel patterns were evaluated in a double transgenic mouse model for both the magnitude and duration of in vivo suppression of LPA gene expression. Specifically, LPA RNAi constructs derived from three different sequence families, each possessing one of the P1 modification patterns or a variant pattern (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 weeks 1, 2, and 4 after administration of the LPA RNAi constructs. The results for this set of experiments are shown in Table 6 below. Across all three sequence families, RNAi constructs with P2, P4, P6, or P7 modification patterns resulted in a greater reduction and duration of Lp(a) serum levels compared to RNAi constructs with P1 modification patterns. RNAi constructs with P6 or P7 chemical modification patterns resulted in a reduction of over 80% 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 dual transgenic mouse model. The variant chemical modification patterns were applied to RNAi constructs containing sequences from five different sequence families. The sense and antisense strand sequences 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 dual transgenic mice expressing human Lp(a) particles. Serum Lp(a) levels in the animals were measured before injection to obtain baseline levels, and at weeks 2, 3, and 4 after administration of the LPA RNAi construct. The results are shown in Table 7 below. Some of the pattern variants, including P9, P19, P22, P24, P27, P28, and P29, resulted in a reduction of over 50% in serum Lp(a) levels at 4 weeks after a single subcutaneous injection of 1 mg / kg. RNAi constructs with a P27 chemical modification pattern were particularly effective in suppressing Lp(a) serum levels, resulting in a sustained reduction of approximately 75% in Lp(a) levels within four 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. The disclosed invention is not limited to the specific methodologies, protocols, and materials described herein, and it is understood that these may change. It is also understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit the scope of the appended claims.

[0211] Those skilled in the art will recognize or be able to verify, through mere routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents shall be covered by the following claims.

Claims

1. An RNAi construct comprising a sense strand and an antisense strand that inhibits the expression of a target gene sequence, wherein the antisense strand comprises a sequence complementary to the target gene sequence, the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a double-stranded region, and 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) During the ceremony, The upper chain, arranged in the direction from 5' to 3', is the sense chain, and the lower chain, arranged in the direction from 3' to 5', is the antisense chain. Each N Ｆ This represents a 2'-fluoromodified nucleotide, Each N Ｍ This independently represents a modified nucleotide selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), and deoxyribonucleotides. Each N Ｌ This independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide. N Ｔ This represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. When x is 1, 2, 3, or 4, N Ａ The condition is that one or more of the nucleotides are independently modified nucleotides selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, where x is an integer from 0 to 4, and the N Ａ One or more nucleotides may be complementary to the nucleotides in the antisense strand. When y is 1, 2, 3, or 4, y is an integer from 0 to 4, provided that one or more n nucleotides are modified or unmodified overhang nucleotides that do not base-pair with the nucleotides in the antisense strand. When z is 1, 2, 3, or 4, N Ｂ The condition is that one or more of the nucleotides are 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, then z is an integer from 0 to 4, and the N Ｂ When one or more nucleotides are present in the sense strand, N Ａ An RNAi construct that may be complementary to a nucleotide, or may be an overhanging nucleotide that does not base-pair with the nucleotide in the sense strand.

2. The RNAi construct according to claim 1, wherein the sense strand and the antisense strand are each independently 19 to 30 nucleotides long.

3. The RNAi construct according to claim 1, wherein the sense strand and the antisense strand are each independently 19 to 25 nucleotides long.

4. The RNAi construct according to claim 1, wherein x is 0, y is 2, and z is 2.

5. x is 1, N Ａ The RNAi construct according to claim 1, wherein is an inverted debasal nucleotide, y is 2, and z is 2.

6. The RNAi construct according to claim 1, wherein x is 2, y is 0, and z is 4.

7. The RNAi construct according to claim 1, wherein x is 2, y is 0, and z is 2.

8. x is 3, and the 5' end is N Ａ The RNAi construct according to claim 1, wherein is an inverted debasal nucleotide, y is 0, and z is 4.

9. The RNAi construct according to claim 1, wherein x is 0, y is 0, and z is 2.

10. x is 1, N Ａ The RNAi construct according to claim 1, wherein is an inverted debasal nucleotide, y is 0, and z is 2.

11. N Ｔ The RNAi construct according to any one of claims 1 to 10, wherein is an inverted debasalized nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

12. Each N in both the sense chain and the antisense chain Ｌ The RNAi construct according to any one of claims 1 to 11, wherein is a 2'-O-methyl modified nucleotide.

13. The N atoms at positions 4 and 12, counting from the 5' end of the antisense chain. Ｍ The RNAi construct according to any one of claims 1 to 12, wherein each of them is a 2'-fluoromodified nucleotide.

14. The nitrogen atom at position 6, counting from the 5' end of the aforementioned antisense chain. Ｍ The RNAi construct according to claim 13, wherein is a 2'-fluoromodified nucleotide.

15. The nitrogen atom at position 10, counting from the 5' end of the aforementioned antisense chain. Ｍ The RNAi construct according to claim 14, wherein is a 2'-fluoromodified nucleotide.

16. The N10 and N12 positions, counting from the 5' end of the antisense chain. Ｍ The RNAi construct according to any one of claims 1 to 12, wherein each of them is a 2'-fluoromodified nucleotide.

17. The nitrogen atom at position 4, counting from the 5' end of the aforementioned antisense chain. Ｍ The RNAi construct according to claim 16, wherein is a 2'-fluoromodified nucleotide.

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

19. Each N in both the sense chain and the antisense chain Ｍ The RNAi construct according to any one of claims 1 to 12, wherein is a 2'-O-methyl modified nucleotide.

20. Each N in the sense chain Ｍ The RNAi construct according to any one of claims 1 to 18, wherein is a 2'-O-methyl modified nucleotide.

21. Each N in the sense chain Ｍ The RNAi construct according to any one of claims 1 to 18, wherein is a 2'-fluoromodified nucleotide.

22. An RNAi construct comprising a sense strand and an antisense strand that inhibits the expression of a target gene sequence, wherein the antisense strand comprises a sequence complementary to the target gene sequence, the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a double-stranded region, and the RNAi construct comprises 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 upper chain, arranged in the direction from 5' to 3', is the sense chain, and the lower chain, arranged in the direction from 3' to 5', is the antisense chain. Each N Ｆ This represents a 2'-fluoromodified nucleotide, Each N Ｌ This independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide. N Ｔ This represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. When x is 1, 2, 3, or 4, N Ａ The condition is that one or more of the nucleotides are independently modified nucleotides selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, where x is an integer from 0 to 4, and the N Ａ One or more nucleotides may be complementary to the nucleotides in the antisense strand. When y is 1, 2, 3, or 4, y is an integer from 0 to 4, provided that one or more n nucleotides are modified or unmodified overhang nucleotides that do not base-pair with the nucleotides in the antisense strand. When z is 1, 2, 3, or 4, N Ｂ The condition is that one or more of the nucleotides are 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, then z is an integer from 0 to 4, and the N Ｂ When one or more nucleotides are present in the sense strand, N Ａ An RNAi construct that may be complementary to a nucleotide, or may be an overhanging nucleotide that does not base-pair with the nucleotide in the sense strand.

23. The RNAi construct according to claim 22, wherein x is 0, y is 2, and z is 2.

24. The RNAi construct according to claim 22, wherein x is 0, y is 0, and z is 2.

25. x is 1, N Ａ The RNAi construct according to claim 22, wherein is an inverted debasal nucleotide, y is 2, and z is 2.

26. The RNAi construct according to claim 22, wherein x is 2, y is 0, and z is 4.

27. x is 3, and the 5' end is N Ａ The RNAi construct according to claim 22, wherein is an inverted debasal nucleotide, y is 0, and z is 4.

28. N Ｔ The RNAi construct according to any one of claims 22 to 27, wherein is an inverted debasalized nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

29. Each N in both the sense chain and the antisense chain Ｌ The RNAi construct according to any one of claims 22 to 28, wherein is a 2'-O-methyl modified nucleotide.

30. An RNAi construct comprising a sense strand and an antisense strand that inhibits the expression of a target gene sequence, wherein the antisense strand comprises a sequence complementary to the target gene sequence, the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a double-stranded 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 upper chain, arranged in the direction from 5' to 3', is the sense chain, and the lower chain, arranged in the direction from 3' to 5', is the antisense chain. Each N Ｆ This represents a 2'-fluoromodified nucleotide, Each N Ｌ This independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide. Each N Ｍ This independently represents a modified nucleotide selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, BNA, and deoxyribonucleotides. N Ｔ This represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. An RNAi construct where x is 0 or 1 and Ab is an inverted debasal nucleotide.

31. Each N in both the sense chain and the antisense chain Ｍ The RNAi construct according to claim 30, wherein is a 2'-O-methyl modified nucleotide.

32. N Ｔ The RNAi construct according to claim 31, wherein x is an inverted debasalized nucleotide or an inverted deoxyribonucleotide, and x is 0.

33. N Ｔ The RNAi construct according to claim 31, wherein is a 2'-O-methyl modified nucleotide and x is 1.

34. N in the antisense chain Ｍ The RNAi construct according to claim 30, wherein is a 2'-fluoromodified nucleotide.

35. Each N in the sense chain Ｍ The RNAi construct according to claim 34, wherein is a 2'-O-methyl modified nucleotide.

36. Each N in the sense chain Ｍ The RNAi construct according to claim 34, wherein is a 2'-fluoromodified nucleotide.

37. N Ｔ The RNAi construct according to any one of claims 34 to 36, wherein x is an inverted debasalized nucleotide or an inverted deoxyribonucleotide, and x is 0.

38. Each N in both the sense chain and the antisense chain Ｌ The RNAi construct according to any one of claims 30 to 37, wherein is a 2'-O-methyl modified nucleotide.

39. An RNAi construct comprising a sense strand and an antisense strand that inhibits the expression of a target gene sequence, wherein the antisense strand comprises a sequence complementary to the target gene sequence, the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a double-stranded region, and 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 upper chain, arranged in the direction from 5' to 3', is the sense chain, and the lower chain, arranged in the direction from 3' to 5', is the antisense chain. Each N Ｆ This represents a 2'-fluoromodified nucleotide, Each N Ｍ This independently represents a modified nucleotide selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), and deoxyribonucleotides. Each N Ｌ This independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide. N Ｔ This represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. When x is 1, 2, 3, or 4, N Ａ The condition is that one or more of the nucleotides are independently modified nucleotides selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, where x is an integer from 0 to 4, and the N Ａ One or more nucleotides may be complementary to the nucleotides in the antisense strand. When y is 1, 2, 3, or 4, y is an integer from 0 to 4, provided that one or more n nucleotides are modified or unmodified overhang nucleotides that do not base-pair with the nucleotides in the antisense strand. When z is 1, 2, 3, or 4, N Ｂ The condition is that one or more of the nucleotides are 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, then z is an integer from 0 to 4, and the N Ｂ When one or more nucleotides are present in the sense strand, N Ａ An RNAi construct that may be complementary to a nucleotide, or may be an overhanging nucleotide that does not base-pair with the nucleotide in the sense strand.

40. The RNAi construct according to claim 39, wherein the sense strand and the antisense strand are each independently 19 to 30 nucleotides long.

41. The RNAi construct according to claim 39, wherein the sense strand and the antisense strand are each independently 19 to 25 nucleotides long.

42. The RNAi construct according to claim 39, wherein x is 2, y is 0, and z is 4.

43. x is 1, N Ａ The RNAi construct according to claim 39, wherein is an inverted debasal nucleotide, y is 2, and z is 2.

44. x is 1, N Ａ The RNAi construct according to claim 39, wherein is an inverted debasal nucleotide, y is 0, and z is 2.

45. The RNAi construct according to claim 39, wherein x is 0, y is 0, and z is 2.

46. The RNAi construct according to claim 39, wherein x is 2, y is 0, and z is 2.

47. N Ｔ The RNAi construct according to any one of claims 39 to 46, wherein is an inverted debasalized nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

48. Each N in both the sense chain and the antisense chain Ｌ The RNAi construct according to any one of claims 39 to 47, wherein is a 2'-O-methyl modified nucleotide.

49. The N atoms at positions 4, 6, 8, 9, and 16, counting from the 5' end of the antisense chain. Ｍ These are 2'-fluoromodified nucleotides, and the N at positions 7 and 12 counting from the 5' end of the antisense strand. Ｍ The RNAi construct according to any one of claims 39 to 48, wherein each of them is a 2'-O-methyl modified nucleotide.

50. The N atoms at positions 4, 6, 8, 9, and 16, counting from the 5' end of the antisense chain. Ｍ These are 2'-O-methyl modified nucleotides, and the N at positions 7 and 12 counting from the 5' end of the antisense strand. Ｍ The RNAi construct according to any one of claims 39 to 48, wherein each of them is a 2'-fluoromodified nucleotide.

51. The N atoms at positions 4, 6, 8, 9, and 12, counting from the 5' end of the antisense chain. Ｍ These are 2'-O-methyl modified nucleotides, and the N at positions 7 and 16 counting from the 5' end of the antisense strand. Ｍ The RNAi construct according to any one of claims 39 to 48, wherein each of them is a 2'-fluoromodified nucleotide.

52. The N atoms at positions 7, 8, 9, and 12, counting from the 5' end of the antisense chain. Ｍ These are 2'-O-methyl modified nucleotides, and the N at positions 4, 6, and 16 counting from the 5' end of the antisense strand. Ｍ The RNAi construct according to any one of claims 39 to 48, wherein each of them is a 2'-fluoromodified nucleotide.

53. N in the sense chain Ｍ The RNAi construct according to any one of claims 39 to 52, wherein is a 2'-fluoromodified nucleotide.

54. N in the sense chain Ｍ The RNAi construct according to any one of claims 39 to 52, wherein is a 2'-O-methyl modified nucleotide.

55. The RNAi construct according to any one of claims 1 to 54, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand include one or more phosphorothioate nucleotide interlinks.

56. The RNAi construct according to claim 55, wherein the antisense strand includes two consecutive phosphorothioate nucleotide interbonds between both the 3' and 5' terminal nucleotides.

57. The RNAi construct according to claim 55 or 56, wherein the sense strand includes a single phosphorothioate nucleotide bond between the terminal nucleotides at the 3' end.

58. The RNAi construct according to claim 55 or 56, wherein the sense strand includes two consecutive phosphorothioate nucleotide interlinks between the terminal nucleotides at the 3' end.

59. An RNAi construct according to any one of claims 1 to 58, further comprising a ligand.

60. The RNAi construct according to claim 59, wherein the ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folic acid moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or an antigen-binding fragment thereof.

61. The RNAi construct according to claim 59, wherein the ligand targets the delivery of the RNAi construct to hepatocytes.

62. The RNAi construct according to claim 59, wherein the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine.

63. The RNAi construct according to claim 62, wherein the ligand comprises a polyvalent galactose moiety or a polyvalent N-acetyl-galactosamine moiety.

64. The RNAi construct according to claim 63, wherein the polyvalent galactose moiety or the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent.

65. The RNAi construct according to any one of claims 59 to 64, wherein the ligand is optionally covalently bound to the sense strand via a linker.

66. The RNAi construct according to claim 65, wherein the ligand is covalently bound to the 5' end of the sense strand.

67. A pharmaceutical composition comprising an RNAi construct according to any one of claims 1 to 66 and a pharmaceutically acceptable carrier or excipient.

68. A method for inhibiting the expression of a target gene in a cell, comprising contacting the cell with an RNAi construct according to any one of claims 1 to 66.

69. The method according to claim 68, wherein the cells are in vivo.

70. A method for inhibiting the expression of a target gene in a subject, comprising administering an RNAi construct according to any one of claims 1 to 66 to the subject.

71. The method according to claim 70, wherein the RNAi construct is administered to the subject via a parenteral administration route.

Images