Products and Compositions

Abstract

To satisfy a clear need for more efficacious receptor specific ligand conjugated iRNA duplex agents and methods for their preparation for the in vivo delivery of oligonucleotide therapeutics, nucleic acids and double stranded siRNAs, because targeting ligands developed so far do not always translate to in vivo setting.SOLUTION: The present invention relates to products and compositions and their uses. In particular the invention relates to nucleic acid products that interfere with gene expression or inhibits its expression and therapeutic uses such as for the treatment of disease and disorders.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to products and compositions and their uses. In particular, the present invention relates to nucleic acid products that interfere with or inhibit gene expression and therapeutic uses such as for the treatment of diseases and disorders. [Background technology]

[0002] Double-stranded RNA (dsRNA) has been shown to block gene expression (Fire et al., 1998 and Elbashir et al., 2001), a process termed RNA interference (RNAi). Short dsRNAs direct gene-specific posttranscriptional silencing in many organisms, including vertebrates, providing a new tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that destroys messenger RNAs homologous to silencing triggers. Interfering RNAs (iRNAs), such as nucleic acids, antisense RNAs, and microRNAs, are oligonucleotides that prevent the formation of proteins by silencing genes, i.e., inhibit gene translation of proteins. Gene silencing agents are becoming increasingly important for therapeutic applications in medicine.

[0003] However, delivering nucleic acids such as RNA into cells while maintaining efficacy and target specificity and avoiding degradation by intracellular nucleases has proven challenging for those skilled in the art of developing nucleic acid molecules for therapeutic applications.

[0004] According to Watts and Corey, Journal of Pathology (2012, Vol. 226, pp. 365-379), there are algorithms that can be used to design nucleic acids, but none are perfect. Because algorithms do not take into account factors such as tertiary structure or the involvement of RNA-binding proteins, various experimental methods may be required to identify potent nucleic acids. Therefore, discovering potent nucleic acids with minimal off-target effects is a complex process, but the pharmaceutical development of these highly charged molecules requires that they can be synthesized economically, distributed to target tissues, enter cells, and function within acceptable limits of toxicity.

[0005] Therefore, the means for efficient delivery of oligonucleotide, especially double-stranded siRNA, to cells in vivo becomes increasingly important, which requires specific targeting and substantial protection from extracellular environment, especially serum protein.One way to achieve specific targeting is to conjugate targeting moiety to iRNA duplex agent.Targeting moiety serves to target iRNA duplex agent to required target site, and it is necessary to design the targeting moiety appropriate for desired receptor site, so that conjugated molecule is taken up into cell by endocytosis or the like. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 5,885,968 [Patent Document 2] WO2017 / 174657 [Non-patent literature]

[0007] [Non-Patent Document 1] Watts and Corey, Journal of Pathology (2012, Vol. 226, pp. 365-379) [Non-patent document 2] Takei et al., 2002, JBC, vol. 277(26):23800-06 Summary of the Invention [Problem to be solved by the invention]

[0008] However, the targeting ligands developed to date do not necessarily translate to in vivo settings, and there is a clear need for more effective receptor-specific ligand-conjugated iRNA duplexes and methods for preparing oligonucleotide therapeutics, nucleic acids, and double-stranded siRNAs for in vivo delivery. [Means for solving the problem]

[0009] The present invention is directed not only to lipid delivery systems, but also to the nucleic acid structure itself.

[0010] Thus, the present invention provides a nucleic acid for inhibiting expression of a target gene in a cell, the nucleic acid comprising at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the target gene, and the first strand comprises modified or unmodified nucleotides at multiple positions to facilitate processing of the nucleic acid by RISC.

[0011] The present invention also relates to a nucleic acid capable of inhibiting expression of a target gene, the nucleic acid comprising a first strand and a second strand, the first strand and the second strand being at least partially complementary to each other, the first strand being at least partially complementary to RNA transcribed from a portion of the target gene, and the first strand and / or the second strand comprising modified nucleotides.

[0012] The nucleotides at positions 2 and 14 from the 5' end of the first strand may be modified.

[0013] The nucleotides at positions 2 and 14 from the 5' end of the first strand may not be modified with a 2'O-methyl modification.

[0014] The nucleotides at positions 2 and 14 from the 5' end of the first strand may be unmodified with a modification selected from the group consisting of 2'-O-(2-methoxyethyl), 2'-O-allyl, 2'-O-DNP, 2'-CE, 2'-EA, 2'-AEM, 2'-APM, and 2'-GE.

[0015] The nucleotides at positions 2 and 14 from the 5' end of the first strand may be modified with a modification selected from the group consisting of 2'F, 4'-S, 2'-FANA, and UNA.

[0016] The nucleotides at positions 2 and 14 from the 5' end of the first strand may be unmodified.

[0017] The nucleotides at positions 2 and 14 from the 5' end of the second strand may be modified with a 2'O-methyl modification or a '-O-(2-methoxyethyl) modification.

[0018] The first strand and the second strand can be separate strands.

[0019] The nucleic acid may comprise a single strand comprising a first strand and a second strand.

[0020] The first strand and / or the second strand may each be 17 to 35 nucleotides in length, and at least one double-stranded region may be 10 to 25 nucleotides in length. The double-stranded region may comprise two separate strands or may comprise a single strand comprising a first strand and a second strand.

[0021] In one embodiment, the second strand can be as short as 11 nucleotides in length, for example, 11, 12, 13, 14, 15, 16, 17, 18, 19 nucleotides in length, or longer.

[0022] The nucleic acid can be a) blunt-ended on both ends, b) have an overhang on one end and a blunt end on the other, or c) have overhangs on both ends.

[0023] One or more nucleotides on the first strand and / or the second strand may be modified to form modified nucleotides. One or more of the odd-numbered nucleotides of the first strand may be modified. One or more of the even-numbered nucleotides of the first strand may be modified with at least a second modification, where the at least a second modification is different from the modification for the one or more odd-numbered nucleotides. At least one of the one or more modified even-numbered nucleotides may be adjacent to at least one of the one or more modified odd-numbered nucleotides.

[0024] In the nucleic acid of the present invention, a plurality of odd-numbered nucleotides in the first strand may be modified.A plurality of even-numbered nucleotides in the first strand may be modified by a second modification.The first strand may comprise adjacent nucleotides that are modified by a common modification.The first strand may comprise adjacent nucleotides that are modified by a second different modification.

[0025] One or more of the odd-numbered nucleotides of the second strand may be modified with a modification different from the modification of the odd-numbered nucleotides on the first strand, and / or one or more of the even-numbered nucleotides of the second strand may be modified with the same modification as the odd-numbered nucleotides of the first strand. At least one of the one or more modified even-numbered nucleotides of the second strand may be adjacent to one or more modified odd-numbered nucleotides. Multiple odd-numbered nucleotides of the second strand may be modified with a common modification and / or multiple even-numbered nucleotides may be modified with the same modification present on the odd-numbered nucleotides of the first strand. Multiple odd-numbered nucleotides on the second strand may be modified with a second modification, where the second modification is different from the modification of the odd-numbered nucleotides of the first strand.

[0026] The second strand comprises adjacent nucleotides that are modified with a common modification, which can be a second modification that is different from the modification of the odd-numbered nucleotides of the first strand.

[0027] In the nucleic acids of the invention, each odd-numbered nucleotide in the first strand and each even-numbered nucleotide in the second strand may be modified with a common modification, each even-numbered nucleotide in the first strand may be modified with a second modification, and each odd-numbered nucleotide in the second strand may be modified with a second, different modification.

[0028] In the nucleic acids of the invention, the modified nucleotides of the first strand may be shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.

[0029] The modification and / or modifications may each be independently selected from the group consisting of a 3'-terminal deoxy-thymine, a 2'-O-methyl, a 2'-deoxy modification, a 2'-amino modification, a 2'-alkyl modification, a morpholino modification, a phosphoramidate modification, a 5'-phosphorothioate group modification, a 5'-phosphate modification or a 5'-phosphate mimetic modification, and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification, and / or the modified nucleotide may be any one of a locked nucleotide, an abasic nucleotide, or a nucleotide containing an unnatural base.

[0030] At least one modification may be 2'-O-methyl and / or at least one modification may be 2'-F.

[0031] The nucleic acids of the present invention may comprise phosphorothioate bonds between the one, two, or three 3'-terminal nucleotides and / or the one, two, or three 5'-terminal nucleotides of the first strand and / or the second strand, including two phosphorothioate bonds between each of the three 3'-terminal nucleotides and each of the three 5'-terminal nucleotides on the first strand, and two phosphorothioate bonds between the three terminal nucleotides at the 3' end of the second strand.

[0032] Such nucleic acids may be conjugated to a ligand.

[0033] The present invention further provides a nucleic acid for inhibiting expression of a target gene in a cell, the nucleic acid comprising at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the target gene, the first strand comprising modified or unmodified nucleotides at multiple positions to facilitate processing of the nucleic acid by RISC, and wherein the nucleotide sequence is conjugated to a ligand.

[0034] The ligand may comprise (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, which conjugates the GalNAc moiety to the sequence defined in any of the preceding embodiments. The linker may be a bivalent, trivalent, or tetravalent branched structure. The nucleotide may be modified as defined herein.

[0035] The ligand has the formula I: [SX 1 -PX 2 ]3-AX 3 - (I) [In the formula, S represents a saccharide, where the saccharide is N-acetylgalactosamine; X 1is C3-C6 alkylene or (-CH2-CH2-O) m (—CH)—, where m is 1, 2, or 3; P is a phosphate or a modified phosphate (preferably a thiophosphate); X 2 is an alkylene or a group of formula (-CH2) n -O-CH2- [wherein n=1 to 6] alkylene ether, A is a branching unit, X 3 represents a cross-linking unit, The nucleic acids according to the invention may be linked to X via a phosphate or modified phosphate (preferably a thiophosphate). 3 conjugated to] may include:

[0036] Thus, the present invention further provides a compound having the following structure: [ka] [ka] [ka] [ka] (wherein Z represents a nucleic acid as defined above). The present invention provides a conjugated nucleic acid having one of the following:

[0037] The ligands may include: [ka]

[0038] The present invention also provides a composition comprising a nucleic acid or conjugated nucleic acid as defined herein and a physiologically acceptable excipient. i) a cationic lipid, or a pharmaceutically acceptable salt thereof; ii) steroids, iii) phosphatidylethanolamine phospholipids; iv) PEGylated lipids may include:

[0039] The content of the cationic lipid component in the composition can be about 55 mol % to about 65 mol % of the total lipid content of the lipid formulation, preferably about 59 mol % of the total lipid content of the lipid composition.

[0040] The composition comprises: [ka] a cationic lipid having the structure [ka] a steroid having the structure [ka]

[0041] and a phosphatidylethanolamine phospholipid having the structure [ka] The PEGylated lipid may have the structure:

[0042] Also provided is a nucleic acid or conjugated nucleic acid according to any aspect of the invention for use in the treatment of a disease or disorder and / or in the manufacture of a medicament for treating a disease or disorder.

[0043] The present invention provides a method of treating a disease or disorder comprising administering to an individual in need thereof a composition comprising a nucleic acid or conjugated nucleic acid according to any aspect of the present invention. The nucleic acid may be administered to a subject subcutaneously, intravenously, or using any other route of application, such as orally, rectally, or intraperitoneally.

[0044] Also included are methods for making the nucleic acids or conjugated nucleic acids of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention relates to nucleic acids and compositions thereof that are double-stranded and target the expressed RNA transcripts of target genes. These nucleic acids can be used in the treatment of various diseases and disorders in which reduced expression of the target gene product is desirable.

[0046] A first aspect of the present invention relates to a nucleic acid for inhibiting expression of a target gene in a cell, the nucleic acid comprising at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, the first strand being at least partially complementary to at least a portion of RNA transcribed from the target gene, and the first strand comprising modified nucleotides at selected positions to facilitate processing of the nucleic acid by RISC. The first strand may comprise unmodified nucleotides.

[0047] Another aspect of the invention relates to a nucleic acid capable of inhibiting expression of a target gene, the nucleic acid comprising a first strand and a second strand, the first strand and the second strand being at least partially complementary to each other, the first strand being at least partially complementary to RNA transcribed from a portion of the target gene, and the first strand and / or the second strand comprising modified nucleotides.

[0048] Nucleic acid refers to a nucleic acid that comprises two strands containing nucleotides and can interfere with gene expression. Inhibition can be complete or partial, resulting in downregulation of gene expression in a targeted manner. Nucleic acid comprises two separate polynucleotide strands: a first strand, which can be a guide strand, and a second strand, which can be a passenger strand. The first strand and the second strand can be part of the same self-complementary polynucleotide strand that "folds" to form a double-stranded molecule. Nucleic acid can be an siRNA molecule.

[0049] The nucleic acid may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogs. The nucleic acid may further comprise a double-stranded nucleic acid portion or duplex region formed by all or a portion of a first strand (also known in the art as a guide strand) and all or a portion of a second strand (also known in the art as a passenger strand). The duplex region is defined as beginning with the first base pair formed between the first and second strands and ending with the last base pair formed between the first and second strands, inclusive.

[0050] "Duplex region" refers to a region in two complementary or substantially complementary oligonucleotides that base-pair with each other by Watson-Crick base pairing or any other method that allows for duplexing between complementary or substantially complementary oligonucleotide strands. For example, an oligonucleotide strand having 21 nucleotide units can base-pair with another oligonucleotide having 21 nucleotide units, but only 19 nucleotides on each strand are complementary or substantially complementary, so that the "duplex region" consists of 19 base pairs. The remaining base pairs can exist as 5' and 3' overhangs or as single-stranded regions. Furthermore, 100% complementarity is not required within the duplex region. Substantial complementarity is acceptable within the duplex region. Substantial complementarity refers to complementarity between strands that allows annealing under biological conditions. Techniques for empirically determining whether two strands can anneal under biological conditions are well known in the art. Alternatively, the two strands may be synthesized and added together under biological conditions to determine whether they will anneal to each other.

[0051] The portions of the first strand and the second strand that form at least one duplex region can be fully complementary to one another, or at least partially complementary.

[0052] Depending on the length of the nucleic acid, a perfect match in terms of base complementarity between the first and second strands may not be necessary, but the first and second strands must be able to hybridize under physiological conditions.

[0053] The complementarity between the first and second strands in at least one double-stranded region can be perfect, in that there are no nucleotide mismatches or nucleotide additions / deletions in either strand. Alternatively, the complementarity need not be perfect. The complementarity can be at least 70%, 75%, 80%, 85%, 90%, or 95%.

[0054] The first strand and the second strand can each comprise a region of complementarity comprising at least 15 contiguous nucleotides.

[0055] The nucleic acid can comprise a second sequence comprising the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.

[0056] This nucleic acid requires the formation of a duplex region between all or a portion of the first strand and a portion of the target nucleic acid. The portion of the target nucleic acid that forms a duplex region with the first strand, defined as beginning with the first base pair formed between the first strand and the target sequence and ending with the last base pair formed between the first strand and the target sequence, inclusive, is referred to as the target nucleic acid sequence, or simply the target sequence. The duplex region formed between the first strand and the second strand need not be the same as the duplex region formed between the first strand and the target sequence. That is, the second strand can have a different sequence from the target sequence, but the first strand must be able to form a duplex structure with both the second strand and the target sequence.

[0057] The complementarity between the first strand and the target sequence may be perfect (no nucleotide mismatches or nucleotide additions / deletions in either nucleic acid).

[0058] The complementarity between the first strand and the target sequence may not be perfect: it may be at least 70%, 80%, 85%, 90%, or 95%.

[0059] The identity between the first strand and the complementary sequence of the target sequence can be at least 75%, 80%, 85%, 90%, or 95%, provided that the nucleic acid is capable of reducing or inhibiting expression of the target gene.

[0060] The nucleic acid may be capable of reducing expression of the target gene by at least 25%, 50%, or 75% of a comparison nucleic acid having complete identity to the first strand and target sequence.

[0061] The nucleic acid may comprise a first strand and a second strand, each of which is 17 to 35 or 19 to 25 nucleotides in length. The first strand and the second strand may be of different lengths.

[0062] The nucleic acid may be 15 to 25 nucleotide pairs in length. The nucleic acid may be 17 to 23 nucleotide pairs in length. The nucleic acid may be 17 to 25 nucleotide pairs in length. The nucleic acid may be 23 to 24 nucleotide pairs in length. The nucleic acid may be 19 to 21 nucleotide pairs in length. The nucleic acid may be 21 to 23 nucleotide pairs in length.

[0063] The nucleic acid may comprise a double-stranded region of 19 to 25 nucleotide base pairs. The double-stranded region may be 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs, which may be contiguous.

[0064] The nucleic acid can be blunt ended on both ends, have an overhang on one end and a blunt end on the other, or have overhangs on both ends.

[0065] As used herein, the term "overhang" has its usual and customary meaning in the art. That is, a single-stranded portion of a nucleic acid that extends beyond the terminal nucleotide of the complementary strand in a double-stranded nucleic acid. The term "blunt end" includes double-stranded nucleic acids in which both strands terminate at the same position, regardless of whether the terminal nucleotides are base-paired. The terminal nucleotides of the first and second strands at a blunt end may be base-paired. The terminal nucleotides of the first and second strands at a blunt end may be unpaired. The two terminal nucleotides of the first and second strands at a blunt end may be base-paired. The two terminal nucleotides of the first and second strands at a blunt end may be unpaired.

[0066] The nucleic acid may have an overhang at one end and a blunt end at the other. The nucleic acid may have overhangs at both ends. The nucleic acid may have blunt ends at both ends. The ends of the nucleic acid may be blunt at the 5' end of the first strand and the 3' end of the second strand, or at the 3' end of the first strand and the 5' end of the second strand.

[0067] The nucleic acid may include an overhang at the 3' end or the 5' end. The nucleic acid may have a 3' overhang on the first strand. The nucleic acid may have a 3' overhang on the second strand. The nucleic acid may have a 5' overhang on the first strand. The nucleic acid may have a 5' overhang on the second strand. The nucleic acid may have an overhang on both the 5' and 3' ends of the first strand. The nucleic acid may have an overhang on both the 5' and 3' ends of the second strand. The nucleic acid may have a 5' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may have a 3' overhang on the first strand and a 5' overhang on the second strand. The nucleic acid may have a 3' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may have a 5' overhang on the first strand and a 5' overhang on the second strand.

[0068] The length of the overhang at the 3' or 5' end of the second strand or the first strand may be selected from the group consisting of 1, 2, 3, 4, and 5 nucleotides. Optionally, the overhang may consist of 1 or 2 nucleotides, which may be modified or unmodified.

[0069] Unmodified polynucleotides, particularly ribonucleotides, may be susceptible to degradation by nucleases within the cell and therefore modified / modified nucleotides may be included in the nucleic acids of the invention.

[0070] One or more nucleotides in the second strand and / or the first strand of the nucleic acid of the invention may be modified.

[0071] Modification of the nucleic acids of the present invention generally provides a powerful tool for overcoming potential limitations inherent to natural RNA molecules, including, but not limited to, in vitro and in vivo stability and bioavailability. The nucleic acids of the present invention may be modified by chemical modification. Modified nucleic acids may also minimize the possibility of inducing human interferon activity. Modifications can further enhance functional delivery of the nucleic acid to target cells. The modified nucleic acids of the present invention may contain one or more chemically modified ribonucleotides in either or both of the first and second strands. The ribonucleotides may contain chemical modifications of the base, sugar, or phosphate moiety. The ribonucleic acid may be modified by substitution with or insertion of a nucleic acid or base analog.

[0072] One or more nucleotides of the nucleic acid of the present invention may be modified. The nucleic acid may contain at least one modified nucleotide. The modified nucleotide may be on the first strand. The modified nucleotide may be in the second strand. The modified nucleotide may be in the duplex region. The modified nucleotide may be outside the duplex region, i.e., in the single-strand region. The modified nucleotide may be on the first strand and outside the duplex region. The modified nucleotide may be on the second strand and outside the duplex region. The 3'-terminal nucleotide of the first strand may be a modified nucleotide. The 3'-terminal nucleotide of the second strand may be a modified nucleotide. The 5'-terminal nucleotide of the first strand may be a modified nucleotide. The 5'-terminal nucleotide of the second strand may be a modified nucleotide.

[0073] Nucleic acids of the invention may have one modified nucleotide, or about two to four modified nucleotides, or about four to six modified nucleotides, about six to eight modified nucleotides, about eight to ten modified nucleotides, about ten to twelve modified nucleotides, about twelve to fourteen modified nucleotides, about fourteen to sixteen modified nucleotides, about sixteen to eighteen modified nucleotides, about eighteen to twenty modified nucleotides, about twenty to twenty-two modified nucleotides, about twenty-two to twenty-four modified nucleotides, twenty-four to twenty-six modified nucleotides, or about twenty-six to twenty-eight modified nucleotides, In each case, the nucleic acid containing the modified nucleotides retains at least 50% of its activity compared to the same nucleic acid except that it does not contain the modified nucleotides. The nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more of its activity compared to the same nucleic acid but without the modified nucleotide.

[0074] The modified nucleotide may be a purine or a pyrimidine. At least half of the purines may be modified. At least half of the pyrimidines may be modified. All of the purines may be modified. All of the pyrimidines may be modified. The modified nucleotide may be selected from the group consisting of 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, nucleotides containing 5'-phosphorothioate groups, nucleotides containing 5' phosphates or 5' phosphate mimetics, and terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

[0075] Nucleic acids may contain bases such as 2-aminoadenosine, 2,6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridines (e.g., 5-bromouridine), 6-azapyrimidines, 6-alkylpyrimidines (e.g., 6-methyluridine), propyne, queusine, 2-thiouridine, 4-thiouridine, wybutoxosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl The nucleotides may include modified nucleotides selected from 5-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueusine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueusine, uridine-5-oxyacetic acid, and 2-thiocytidine.

[0076] The nucleic acids discussed herein include not only unmodified RNA, but also RNA modified, for example, to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to molecules in which the nucleic acid components, specifically sugar, base, and phosphate moieties, are the same as or essentially the same as those naturally occurring, for example, those naturally occurring in the human body. Modified nucleotides used herein refer to nucleotides in which one or more of the nucleic acid components, specifically sugar, base, and phosphate moieties, are different from those naturally occurring. Although these are called modified nucleotides, they naturally include non-nucleotide molecules, for example, polynucleotide molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows interstrand hybridization, i.e., the modified nucleotide mimics the ribophosphate backbone, due to the modification.

[0077] Many of the modifications that occur in nucleic acids, such as modifications of bases, phosphate moieties, or non-linked O's in phosphate moieties, described below, are repeated within a polynucleotide molecule. In some cases, modifications occur at all possible positions / nucleotides within a polynucleotide, but in many cases this is not the case. Modifications may occur only at the 3'- or 5'-terminal positions, or only within terminal regions, such as at terminal nucleotide positions or within the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of the nucleic acids of the invention, or only in single-stranded regions of the nucleic acids of the invention. Phosphorothioate modifications at non-linked O' positions may occur only at one or both ends, or only within terminal regions, such as at terminal nucleotide positions or within the last 2, 3, 4, or 5 nucleotides of the strand, or only within double-stranded and / or single-stranded regions, particularly at both ends. The 5' or 3' end may be phosphorylated.

[0078] The stability of the nucleic acids of the present invention can be increased by including specific bases in the overhangs or by including modified nucleotides in the single-stranded overhangs, for example, in the 5' or 3' overhangs, or both. Purine nucleotides may be included in the overhangs. All or some of the bases in the 3' or 5' overhangs may be modified. Modifications may include the use of modifications at the 2'OH group of the ribose sugar, the use of deoxyribonucleotides instead of ribonucleotides, and modifications at the phosphate group, such as phosphorothioate modifications. The overhangs do not need to be homologous to the target sequence.

[0079] The 5' overhang or 3' overhang in the first strand, the second strand, or both strands of the dsRNA agent of the present invention may be phosphorylated.In some embodiments, the overhang region contains two nucleotides, and there is a phosphorothioate between the two nucleotides, and these two nucleotides may be the same or different.In one embodiment, the overhang is present at the 3' end of the first strand, the second strand, or both strands.In one embodiment, this 3' overhang is present in the second strand.In one embodiment, this 3' overhang is present in the first strand.

[0080] Nucleases can hydrolyze the phosphodiester bonds of nucleic acids, however, chemical modifications of nucleic acids can confer improved properties and make oligoribonucleotides more stable to nucleases.

[0081] As used herein, a modified nucleic acid is: (i) a change, e.g., a substitution, of one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens (which shall be linked even if at the 5' and 3' ends of the nucleic acids of the invention); (ii) a change, e.g., substitution, of a component of the ribose sugar, e.g., the 2' hydroxyl of the ribose sugar; (iii) replacement of the phosphate moiety with a "dephospho" linker; (iv) modification or substitution of naturally occurring bases; (v) substitutions or modifications of the ribose-phosphate backbone; (vi) modification of the 3' or 5' end of the RNA, e.g., removal, modification, or replacement of a terminal phosphate group, or conjugation of a moiety (e.g., a fluorescently labeled moiety) to either the 3' or 5' end of the RNA; may include one or more of:

[0082] The terms substitution, modification and alteration indicate a difference from the naturally occurring molecule.

[0083] Specific modifications are discussed in more detail below.

[0084] Examples of modified phosphate groups include phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester.In phosphorodithioate, both non-bonding oxygens are replaced by sulfur.One, each, or both non-bonding oxygens in the phosphate group can independently be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0085] The phosphate linker may be modified by substituting the linking oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphonate). Substitution can occur at the terminal oxygen. Substitution of the non-linking oxygen with nitrogen is possible.

[0086] Modified nucleotides can include modifications of the sugar group. The 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.

[0087] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CHO)nCH2CH2OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is linked, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; O-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) and aminoalkoxy, O(CH2)nAMINE (e.g., AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino).

[0088] "Deoxy" modifications include hydrogen, halo; amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CHCHNH)CHCH-AMINE (AMINE = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl, which may be optionally substituted, for example, with an amino functionality. Other substituents of certain embodiments include 2'-methoxyethyl, 2'-OCH, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro.

[0089] The sugar group may contain one or more carbons that have the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified nucleotides may contain sugars such as arabinose.

[0090] Modified nucleotides can also include "abasic" sugars in which the nucleobase at C-I' is missing. Such abasic sugars can further contain modifications to one or more of the atoms that make up the sugar.

[0091] The 2' modifications may be used in combination with one or more phosphate linker modifications (eg, phosphorothioate).

[0092] The phosphate group may be replaced by a non-phosphorus-containing linking agent.

[0093] Examples of moieties that can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino. In certain embodiments, the replacement can include methylenecarbonylamino and methylenemethylimino groups.

[0094] The phosphate linkers and ribose sugars may be replaced by nuclease-resistant nucleotides.

[0095] Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

[0096] The 3' and 5' ends of the oligonucleotide may be modified. Such modifications may occur at the 3' or 5' end of the molecule, or at both ends. These may include modifying or replacing the entire terminal phosphate, or modifying or replacing one or more atoms of the phosphate group. For example, the 3' and 5' ends of the oligonucleotide may be conjugated to a labeling moiety, such as a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dye) or other functional molecular entity, such as a protecting group (e.g., sulfur-, silicon-, boron-, or ester-based). The functional molecular entity may be attached to the sugar via the phosphate group and / or a linker. The terminal atom of the linker may be linked to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S, or C group of the sugar. Alternatively, the linker may be linked to or replace the terminal atom of a nucleotide surrogate (e.g., PNA). These spacers or linkers include, for example, -(CH2) n -, -(CH2) n N-, -(CH2) n O-, -(CH2) n The 3'-terminus may be an -OH group.

[0097] Other examples of terminal modifications include dyes, intercalating agents (e.g., acridines), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(O O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocycles of Eu 3+ complexes).

[0098] Terminal modifications can be added for several reasons, including to modulate activity or resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5' end with phosphate or a phosphate analog. The nucleic acids of the present invention may be 5' phosphorylated or contain a phosphoryl analog at the 5' prime end on either the first or second strand. 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO) (O)POP(HO)(O)-O-5'; 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; ( 5'-phosphorothiolates ((HO)2(O)PS-5'); any further combination of oxygen / sulfur substituted monophosphate, diphosphate, and triphosphate (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5' vinylphosphonates, 5'-alkyl ether phosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0099] The nucleic acids of the present invention may contain one or more phosphorothioate modifications at one or more of the ends of the first strand and / or second strand. Optionally, each or either end of the first strand may contain one, two, or three phosphorothioate-modified nucleotides. Optionally, each or either end of the second strand may contain one, two, or three phosphorothioate-modified nucleotides. Optionally, both ends of the first strand and the 5' end of the second strand may contain two phosphorothioate-modified nucleotides. Phosphorothioate-modified nucleotides means that the linkage between the nucleotide and the adjacent nucleotide contains a phosphorothioate group instead of a standard phosphate group.

[0100] Terminal modifications may also be useful for monitoring distribution, in which case the added group may include a fluorophore, such as fluorescein or an Alexa dye. Terminal modifications may also be useful for enhancing uptake, and useful modifications for this purpose include cholesterol. Terminal modifications may also be useful for crosslinking an RNA agent to another moiety.

[0101] Adenine, guanine, cytosine, and uracil are the most common bases found in RNA. These bases can be modified or substituted to provide RNA with improved properties. For example, these bases, or synthetic and natural nucleic acid bases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin), and any one of the modifications described above, can be used to prepare nuclease-resistant oligoribonucleotides. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used.Examples include 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, e.g., 2-aminopropyladenine, 5-propynyl Uracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N <4> -acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine, or an O-alkylated base.

[0102] As used herein, the term "non-base pairing nucleotide analog" refers to a nucleotide analog containing a non-base pairing moiety, including, but not limited to, 6-desaminoadenosine (nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-MeriboU, N3-MeriboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, and N3-Me dC. In some embodiments, the non-base pairing nucleotide analog is a ribonucleotide. In other embodiments, it is a deoxyribonucleotide.

[0103] As used herein, the term "terminal functional group" includes, but is not limited to, a halogen group, an alcohol group, an amine group, a carboxyl group, an ester group, an amide group, an aldehyde group, a ketone group, and an ether group.

[0104] Certain moieties can be attached to the 5' end of the first or second strand, including abasic ribose moieties, abasic deoxyribose moieties, modified abasic ribose, and abasic deoxyribose moieties, including 2'O alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and their modifications, C6-imino-Pi; mirror nucleotides, including L-DNA and L-RNA; 5'OMe nucleotides; and nucleotide analogs, such as 4',5'-methylene nucleotides; 1-(β-D-erythrofuranosyl) nucleotides; 4'-thionucleotides, carbocyclic nucleotides; and 5'-amino-alkyl phosphates. ; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotides; alpha-nucleotides; threo-pentofuranosyl nucleotides; acyclic 3',4'-seconucleotides; 3,4-dihydroxybutyl nucleotides; 3,5-dihydroxypentyl nucleotides, 5'-5'-inverted abasic moieties; 1,4-butanediol phosphate; 5'-amino; and bridged or unbridged methylphosphonate and 5'-mercapto moieties.

[0105] Nucleic acids of the invention may contain one or more inverted nucleotides, such as inverted thymidines or inverted adenines (see, for example, Takei et al., 2002, JBC, 277(26):23800-06).

[0106] As used herein, the terms "inhibit," "down-regulate," or "reduce" with respect to gene expression means reducing the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule (e.g., mRNA) encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, below that observed in the absence of a nucleic acid of the invention. For example, expression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, or less of that observed in the absence of the inhibitor.

[0107] Nucleic acids of the invention can include abasic nucleotides. As used herein, the term "abasic" refers to a moiety in which the 1' base is absent or has another chemical group in its place, such as a 3',3'-linked or 5',5'-linked deoxyabasic ribose derivative.

[0108] The nucleic acid may comprise one or more nucleotides that are modified on the second strand and / or the first strand. Alternating nucleotides may be modified to form modified nucleotides.

[0109] Alternating, as used herein, means occurring in regular succession. In other words, alternating means occurring repeatedly in order. For example, if one nucleotide is modified, the next adjacent nucleotide is unmodified, the next adjacent nucleotide is modified, etc. One nucleotide may be modified with a first modification, the next adjacent nucleotide may be modified with a second modification, the next adjacent nucleotide is modified with the first modification, etc., where the first modification and the second modification are different.

[0110] One or more odd-numbered nucleotides in the first strand of the nucleic acid of the present invention may be modified, where the first strand is numbered from 5' to 3'. As used herein, the term "odd" refers to a number that is not divisible by 2. Examples of odd numbers are 1, 3, 5, 7, 9, 11, etc. One or more even-numbered nucleotides in the first strand of the nucleic acid of the present invention may be modified, where the first strand is numbered from 5' to 3'. As used herein, the term "even" refers to a number that is divisible by 2. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14, etc. One or more odd-numbered nucleotides in the second strand of the nucleic acid of the present invention may be modified, where the second strand is numbered from 3' to 5'. One or more even-numbered nucleotides in the second strand of the nucleic acid of the present invention may be modified, where the second strand is numbered from 3' to 5'.

[0111] One or more nucleotides on the first strand and / or the second strand may be modified to form modified nucleotides. One or more of the odd-numbered nucleotides of the first strand may be modified. One or more of the even-numbered nucleotides of the first strand may be modified with at least a second modification, where the at least a second modification is different from the modification for the one or more odd-numbered nucleotides. At least one of the one or more modified even-numbered nucleotides may be adjacent to at least one of the one or more modified odd-numbered nucleotides.

[0112] In the nucleic acid of the present invention, a plurality of odd-numbered nucleotides in the first strand may be modified.A plurality of even-numbered nucleotides in the first strand may be modified by a second modification.The first strand may comprise adjacent nucleotides that are modified by a common modification.The first strand may comprise adjacent nucleotides that are modified by a second different modification.

[0113] One or more of the odd-numbered nucleotides of the second strand may be modified with a modification different from the modification of the odd-numbered nucleotides on the first strand, and / or one or more of the even-numbered nucleotides of the second strand may be modified with the same modification as the odd-numbered nucleotides of the first strand. At least one of the one or more modified even-numbered nucleotides of the second strand may be adjacent to one or more modified odd-numbered nucleotides. Multiple odd-numbered nucleotides of the second strand may be modified with a common modification and / or multiple even-numbered nucleotides may be modified with the same modification present on the odd-numbered nucleotides of the first strand. Multiple odd-numbered nucleotides on the second strand may be modified with a second modification, where the second modification is different from the modification of the odd-numbered nucleotides of the first strand.

[0114] The second strand may include adjacent nucleotides that are modified with a common modification, which may be a second modification that is different from the modification of the odd-numbered nucleotides of the first strand.

[0115] In the nucleic acids of the invention, each odd-numbered nucleotide in the first strand and each even-numbered nucleotide in the second strand may be modified with a common modification, each even-numbered nucleotide in the first strand may be modified with a second modification, and each odd-numbered nucleotide in the second strand may be modified with a second modification.

[0116] In the nucleic acids of the invention, the modified nucleotides of the first strand may be shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.

[0117] In the first strand, one or more or each of the odd-numbered nucleotides may be modified, and in the second strand, one or more or each of the even-numbered nucleotides may be modified. In either or both strands, one or more or each of the alternating nucleotides may be modified with a second modification. In the first strand, one or more or each of the even-numbered nucleotides may be modified, and in the second strand, one or more or each of the even-numbered nucleotides may be modified. In either or both strands, one or more or each of the alternating nucleotides may be modified with a second modification. In the first strand, one or more or each of the odd-numbered nucleotides may be modified, and in the second strand, one or more of the odd-numbered nucleotides may be modified with a common modification. In either or both strands, one or more or each of the alternating nucleotides may be modified with a second modification. In the first strand, one or more or each of the even-numbered nucleotides may be modified, and in the second strand, one or more or each of the odd-numbered nucleotides may be modified with a common modification. In either or both strands, one or more or each of the alternating nucleotides may be modified with a second modification.

[0118] Nucleic acids of the invention may comprise single- or double-stranded constructs containing at least two regions of alternating modification on one or both strands. These alternating regions may contain up to about 12 nucleotides, but preferably contain about 3 to about 10 nucleotides. The regions of alternating nucleotides may be located at the termini of one or both strands of the nucleic acids of the invention. The nucleic acids may contain 4 to about 10 alternating nucleotides at each end (3' and 5'), and these regions may be separated by about 5 to about 12 adjacent nucleotides that are unmodified, differentially modified, or commonly modified.

[0119] The odd-numbered nucleotides of the first strand may be modified, and the even-numbered nucleotides may be modified with a second modification. The second strand may include adjacent nucleotides modified with a common modification, which may be the same as the modification of the odd-numbered nucleotides of the first strand. One or more nucleotides of the second strand may also be modified with a second modification. One or more nucleotides having a second modification may be adjacent to each other and to a nucleotide having a modification that is the same as the modification of the odd-numbered nucleotides of the first strand. The first strand may include a phosphorothioate bond between two nucleotides at the 3' and 5' ends. The second strand may include a phosphorothioate bond between two nucleotides at the 5' end. The second strand may be conjugated to a ligand at the 5' end.

[0120] The nucleic acid of the present invention may comprise a first strand comprising adjacent nucleotides modified with a common modification. One or more of such nucleotides may be adjacent to one or more nucleotides that may be modified with a second modification. The one or more nucleotides with the second modification may be adjacent. The second strand may comprise adjacent nucleotides modified with a common modification, which may be the same as one of the modifications of one or more nucleotides of the first strand. One or more nucleotides of the second strand may also be modified with a second modification. The one or more nucleotides with the second modification may be adjacent. The first strand may comprise a phosphorothioate bond between two nucleotides at the 5' and 3' ends. The second strand may comprise a phosphorothioate bond between two nucleotides at the 3' end. The second strand may be conjugated to a ligand at the 5' end.

[0121] For purposes of the modifications described herein, nucleotides (unless otherwise specified) are numbered 5' to 3' in the first strand and 3' to 5' in the second strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 may be modified with a modification in the first strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 may be modified with a second modification in the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 may be modified with a modification in the second strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 may be modified with a second modification in the second strand. In the nucleic acids of the invention, nucleotides are numbered from 5' to 3' in the first strand and from 3' to 5' in the second strand unless otherwise specified.

[0122] Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 may be modified in the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 may be modified with a second modification in the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 may be modified with a modification in the second strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 may be modified with a second modification in the second strand.

[0123] Obviously, if the first strand and / or the second strand is shorter or longer than 25 nucleotides in length, e.g., 19 nucleotides in length, then there are no nucleotides numbered 20, 21, 22, 23, 24, and 25 to be modified. Those skilled in the art will understand that the above description applies to shorter or longer strands as appropriate.

[0124] One or more modified nucleotides on the first strand may pair with modified nucleotides on the second strand that have a common modification. One or more modified nucleotides on the first strand may pair with modified nucleotides on the second strand that have a different modification. One or more modified nucleotides on the first strand may pair with unmodified nucleotides on the second strand. One or more modified nucleotides on the second strand may pair with unmodified nucleotides on the first strand. In other words, alternating nucleotides may be aligned on the two strands. For example, all modifications in the alternating region of the second strand may pair with the same modification in the first strand, or alternatively, the modifications may be offset by one nucleotide with a common modification in the alternating region of one strand that pairs with a different modification (i.e., a second or further modification) in the other strand. Another option is to have different modifications on each of the strands.

[0125] The modifications in the first strand may be shifted by one nucleotide relative to the modified nucleotides on the second strand so that common modified nucleotides do not pair with each other.

[0126] The modification and / or modifications may each be independently selected from the group consisting of a 3'-terminal deoxy-thymine, a 2'-O-methyl, a 2'-deoxy modification, a 2'-amino modification, a 2'-alkyl modification, a morpholino modification, a phosphoramidate modification, a 5'-phosphorothioate group modification, a 5'-phosphate modification or a 5'-phosphate mimetic modification, and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification, and / or the modified nucleotide may be any one of a locked nucleotide, an abasic nucleotide, or a nucleotide containing an unnatural base.

[0127] At least one modification may be 2'-O-methyl and / or at least one modification may be 2'-F. Additional modifications described herein may be present in the first strand and / or the second strand.

[0128] Throughout the present description, "same or common modification" refers to the same modification for any nucleotide, whether A, G, C, or U modified with a group such as a methyl group or a fluoro group. It is not intended to refer to the same addition in the same nucleotide. For example, 2'F-dU, 2'F-dA, 2'F-dC, and 2'F-dG are all considered to be the same or common modifications, as are 2'-OMe-rU, 2'-OMe-rA, 2'-OMe-rC, and 2'-OMe-rG. The 2'F modification is a different modification than the 2'OMe modification.

[0129] Some representative modified nucleic acid sequences of the present invention are provided in the examples, which are representative and not intended to be limiting.

[0130] Preferably, the nucleic acid may comprise a modification and a second or further modification, each independently selected from the group comprising a 2'-O-methyl modification and a 2'-F modification. The nucleic acid may comprise a modification that is 2'-O-methyl (2'OMe), which may be a first modification, and a second modification that is 2'-F. The nucleic acids of the invention may comprise phosphorothioate and / or deoxy modifications, which may be present at or between one, two, or three terminal nucleotides at each or any end of each or both strands.

[0131] The nucleic acids of the present invention may be conjugated to a ligand.

[0132] Some ligands may have endosomolytic properties. Endosomolytic ligands promote the lysis of endosomes and / or transport of the compositions of the present invention or their components from endosomes to the cytoplasm of cells. Endosomolytic ligands may be polyanionic peptides or peptidomimetics that exhibit pH-dependent membrane activity and membrane fusogenicity. Endosomolytic components may contain chemical groups that undergo a charge change or protonation in response to a change in pH. Endosomolytic components may be linear or branched.

[0133] Ligands can include therapeutic modifiers, e.g., to enhance uptake, diagnostic compounds or reporter groups, e.g., to monitor distribution, cross-linking agents, and nuclease-resistance-conferring moieties. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics. Ligands can include naturally occurring substances such as proteins, carbohydrates, or lipids. Ligands can be recombinant or synthetic molecules.

[0134] The ligand can also include a targeting group, such as a cell targeting agent or a tissue targeting agent. The targeting ligand can be a lectin, a glycoprotein, a lipid, or a protein.

[0135] Other examples of ligands include dyes, intercalating agents, cross-linking agents, porphyrins, polycyclic aromatic hydrocarbons, artificial endonucleases or chelating agents, lipophilic molecules, alkylating agents, phosphate, amino, mercapto, PEG, MPEG, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens, transport / absorption enhancers, synthetic ribonucleases, or imidazole clusters.

[0136] Ligands can be proteins, such as glycoproteins or peptides. Ligands can also be hormones or hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, or cofactors.

[0137] The ligand may be, for example, a substance such as a drug that can increase cellular uptake of nucleic acids by disrupting the cytoskeleton of the cell.

[0138] Ligands can increase cellular uptake of nucleic acids by activating the inflammatory response, such as tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, or gamma interferon.

[0139] The ligand can be a lipid or lipid-based molecule. The lipid or lipid-based molecule preferably binds to a serum protein. Preferably, the lipid-based ligand binds to human serum albumin (HSA). The lipid or lipid-based molecule can increase the conjugate's resistance to degradation, increase targeting or transport to target cells, and / or modulate binding to serum proteins. The lipid-based ligand can be used to modulate binding of the conjugate to target tissues.

[0140] The ligand may be a steroid. Preferably, the ligand is cholesterol or a cholesterol derivative.

[0141] The ligand can be a moiety that is taken up by target cells, such as a vitamin. Exemplary vitamins include vitamins A, E, K, and the B vitamins. Vitamins can be taken up by proliferating cells, which can be useful for delivering nucleic acids to cells, such as malignant or non-malignant tumor cells.

[0142] The ligand can be a cell-penetrating agent, such as a helical cell-penetrating agent. Preferably, such agents are amphipathic.

[0143] The ligand can be a peptide or peptidomimetic. Peptidomimetics are molecules capable of folding into a defined three-dimensional structure similar to natural peptides. Peptide or peptidomimetic ligands can include naturally occurring peptides, modified peptides, or both. The peptide or peptidomimetic can be a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide. The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. The peptide moiety can include a hydrophobic membrane translocation sequence. The peptide moiety can be a peptide capable of transporting large polar molecules such as peptides, oligonucleotides, and proteins across cell membranes, for example, the sequence of the HIV Tat protein (GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK). Preferably, the peptide or peptidomimetic is a cell-targeting peptide, for example, an arginine-glycine-aspartic acid (RGD)-peptide.

[0144] The ligand can be, for example, a cell-penetrating peptide capable of penetrating microbial or mammalian cells.

[0145] The ligand can be a pharmacokinetic modifier, which can be a lipophile, bile acid, steroid, phospholipid analog, peptide, protein binder, PEG, vitamin, or the like.

[0146] When two or more ligands are present, the ligands may all have the same properties, all may have different properties, or some may have the same properties and others may have different properties. For example, the ligands may have targeting properties, endosomolytic activity, or PK modulating properties. In a preferred embodiment, the ligands all have different properties.

[0147] The ligand may be attached to the nucleic acid at the 3' end, 5' end, and / or terminal position. Preferably, the ligand is attached to the nucleic acid by an intervening tether or linker.

[0148] In some embodiments, the nucleic acid is a double-stranded nucleic acid. In a double-stranded nucleic acid, the ligand may be bound to one or both strands. In some embodiments, the double-stranded nucleic acid contains a ligand conjugated to the second strand. In other embodiments, the double-stranded nucleic acid contains a ligand conjugated to the first strand.

[0149] The ligand may be conjugated to the nucleobase, sugar moiety, or internucleoside bond of the nucleic acid molecule. Conjugation to a purine nucleobase or its derivative may occur at any position, including the endocyclic and exocyclic atoms. Conjugation to a pyrimidine nucleotide or its derivative may also occur at any position. Conjugation to the sugar moiety of a nucleoside may occur at any carbon atom. Conjugation to an internucleoside bond may occur at the phosphorus atom of a phosphorus-containing bond, or at an oxygen, nitrogen, or sulfur atom bound to the phosphorus atom. For internucleoside bond containing an amine or amide, conjugation may occur at the nitrogen atom of the amine or amide, or to an adjacent carbon atom.

[0150] The ligand is typically a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, or polysaccharide. The ligand may be conjugated to the nucleic acid via a linker. The saccharide may be selected from N-acetylgalactosamine, mannose, galactose, glucose, glucosamine, and fucose. The saccharide may be N-acetylgalactosamine (GalNAc).

[0151] Thus, the ligands used in the present invention may comprise (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, which conjugates the GalNAc moiety to the sequence defined in any of the preceding aspects. The linker may be a bivalent, trivalent, or tetravalent branched structure. The nucleotides may be modified as defined herein.

[0152] The method for effectively delivering oligonucleotide, especially the double-stranded nucleic acid of the present invention, to cells in vivo is important, and requires specific targeting and substantial protection from extracellular environment, especially serum protein.One way to achieve specific targeting is to conjugate ligand to nucleic acid.Targeting moiety serves to target nucleic acid to required target site, and it is necessary to conjugate suitable ligand to desired receptor site so that conjugated molecule is taken up into cell by endocytosis or the like.Ligand can be any moiety or ligand that can target specific receptor.

[0153] For example, the asialoglycoprotein receptor (ASGP-R) is a high-capacity receptor that is highly abundant in hepatocytes. One of the first disclosures of tripantennary cluster glycosides was in U.S. Patent No. 5,885,968. Conjugates with three GalNAc ligands and containing phosphate groups are known and described in Dubber et al. (2003). ASGP-R exhibits 50-fold higher affinity for N-acetyl-D-galactosylamine (GalNAc) than for D-Gal.

[0154] Hepatocytes expressing a lectin (asialoglycoprotein receptor; ASGPR), which specifically recognizes the terminal β-galactosyl subunit of glycosylated proteins or other oligosaccharides (P.H. Weigel et al., 2002), can be used to target drugs to the liver by covalently linking galactose or galactosamine to the drug substance (S. Ishibashi et al., 1994). Furthermore, binding affinity can be significantly increased by the multivalency effect achieved by repeating the targeting unit (E.A. Biessen et al., 1995).

[0155] ASGPR is a mediator of active endosomal trafficking of glycoproteins containing terminal β-galactosyl, and therefore ASGPR is highly suitable for targeted delivery of drug candidates such as nucleic acids that need to be delivered intracellularly (Akinc et al.).

[0156] The saccharide, sometimes referred to as a ligand, can be selected to have affinity for at least one receptor on the target cell, particularly the receptor on the surface of mammalian liver cells, e.g., the hepatic asialoglycoprotein receptor (ASGP-R).

[0157] The saccharide may be selected from N-acetylgalactosamine, mannose, galactose, glucose, glucosamine, and fucose. The saccharide may be N-acetylgalactosamine (GalNAc).

[0158] "GalNAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetylgalactosamine. Reference to "GalNAc" or "N-acetylgalactosamine" includes both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. Both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose may be used interchangeably. Preferably, the compounds of the present invention contain the β-form of 2-(acetylamino)-2-deoxy-β-D-galactopyranose.

[0159] The ligand may comprise GalNAc.

[0160] The ligand is a compound of formula I: [SX 1 -PX 2 ]3-AX 3 - (I) [In the formula, S represents a saccharide, where the saccharide is N-acetylgalactosamine; X 1 is C3-C6 alkylene or (-CH2-CH2-O) m (—CH)—, where m is 1, 2, or 3; P is a phosphate or a modified phosphate (preferably a thiophosphate); X 2 is an alkylene or a group of formula (-CH2) n -O-CH2- [wherein n=1 to 6] alkylene ether, A is a branching unit, X 3 represents a cross-linking unit, The nucleic acids according to the invention may be linked to X via a phosphate or modified phosphate (preferably a thiophosphate). 3 conjugated to] may include:

[0161] In Formula I, the branching unit "A" is tri-branched to accommodate three saccharide ligands. The branching unit covalently bonds to the ligand and the nucleic acid. The branching unit may comprise a branched aliphatic group, including a group selected from an alkyl group, an amide group, a disulfide group, a polyethylene glycol group, an ether group, a thioether group, and a hydroxyamino group. The branching unit may comprise a group selected from an alkyl group and an ether group.

[0162] Branch unit A is [ka]

[0163] wherein each A1 independently represents O, S, C=O, or NH; Each n independently represents an integer from 1 to 20. The polymer may have a structure selected from:

[0164] The branch unit is [ka] wherein each A1 independently represents O, S, C=O, or NH; Each n independently represents an integer from 1 to 20. The polymer may have a structure selected from:

[0165] The branch unit is [ka] (Wherein A1 is O, S, C=O, or NH; Each n independently represents an integer from 1 to 20. The polymer may have a structure selected from:

[0166] The branch unit is [ka] The structure may be:

[0167] The branch unit is [ka] The structure may be:

[0168] The branch unit is [ka] The structure may be:

[0169] Optionally, the branching units consist only of carbon atoms.

[0170] X 3 is -C1~C 20 Alkylene-, -C2~C 20 Alkenylene-, formula -(C1-C 20 alkylene)-O-(C1-C 20 Alkylene ethers of -C(O)-C1-C 20 alkylene-, -C0-C4 alkylene(Cy)C0-C4 alkylene- [wherein Cy represents a substituted or unsubstituted 5- or 6-membered cycloalkylene ring, arylene ring, heterocyclylene ring, or heteroarylene ring], -C1-C4 alkylene-NHC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)NH-C1-C4 alkylene and -C1-C6 alkylene-SS-C1-C6 alkylene-.

[0171] X 3 is the formula -(C1~C 20 alkylene)-O-(C1-C 20 X may be an alkylene ether of alkylene. 3 is the formula -(C1~C20 Alkylene)-O-(C4-C 20 alkylene)-[wherein the (C4 to C 20 X may be an alkylene ether of the formula: 3 -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C6H 12 - and -CH2-O-CH 16 -, especially -CH2-O-C4H8-, -CH2-O-C6H 12 - and -CH2-O-CH 16 - (in each instance, the -CH2- group is attached to A).

[0172] The ligand is a compound of formula (II): [SX 1 -PX 2 ]3-AX 3 - (II) [In the formula, S represents a sugar, X 1 is C3-C6 alkylene or (-CH2-CH2-O) m (—CH)—, where m is 1, 2, or 3; P is a phosphate or a modified phosphate (preferably a thiophosphate); X 2 is a C1-C8 alkylene, A is, [ka] a branching unit selected from X 3 is a bridging unit, The nucleic acids according to the invention may be linked to X via a phosphate or modified phosphate (preferably a thiophosphate). 3 conjugated to] may include:

[0173] Branch unit A is [ka] It may have the structure:

[0174] Branch unit A is [ka] (In the formula, X 3 is attached to the nitrogen atom) It may have the structure:

[0175] X 3 is C1~C 20 Preferably, X 3 -C3H6-, -C4H8-, -C6H 12 - and -C8H 16 -, especially -C4H8-, -C6H 12 - and -C8H 16 - is selected from the group consisting of

[0176] The ligand is a compound of formula (III): [SX 1 -PX 2 ]3-AX 3 - (III) [In the formula, S represents a sugar, X 1 is C3-C6 alkylene or (-CH2-CH2-O) m (—CH)—, where m is 1, 2, or 3; P is a phosphate or a modified phosphate (preferably a thiophosphate); X 2 is an alkylene ether of formula -C3H6-O-CH2-, A is a branching unit, X 3 -CH2-O-CH2-, -CH2-O-C2H4-, -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C5H 10 -, -CH2-O-CH 12 -, -CH2-O-C7H 14 - and -CH2-O-CH 16- (in each instance, the -CH2- group is attached to A), The nucleic acids according to the invention may be linked to X via a phosphate or modified phosphate (preferably a thiophosphate). 3 conjugated to] may include:

[0177] The branching units may comprise carbon. Preferably, the carbon units are carbon.

[0178] X 3 -CH2-O-C4H8-, -CH2-O-C5H 10 -, -CH2-O-CH 12 -, -CH2-O-C7H 14 - and -CH2-O-CH 16 Preferably, X 3 -CH2-O-C4H8-, -CH2-O-C6H 12 - and -CH2-O-CH 16 may be selected from the group consisting of:

[0179] In any of the above embodiments, P represents a modified phosphate group. [ka] where Y 1 and Y 2 are independently =O, =S, -O - , -OH, -SH, -BH3, -OCH2CO2, -OCH2CO2R x , -OCH2C(S)OR x , and -OR X where R x represents C1-C6 alkyl, and [ka] indicates the bond to the rest of the compound.

[0180] For example, Y1 may represent -OH, and Y 2 may represent =O or =S, or Y 1 -O - It may represent Y 2 may represent =O or =S, Y 1 may represent =O, Y 2 -CH3, -SH, -OR X , or -BH3, Y 1 may represent =S, Y 2 -CH3, OR X , or -SH.

[0181] Those skilled in the art will recognize that in certain cases, Y 1 and Y 2 It will be appreciated that there is delocalization between

[0182] Preferably, the modified phosphate group is a thiophosphate group. The thiophosphate group includes bithiophosphate (i.e., Y 1 represents =S, and Y 2 Ga-S - represents), and monothiophosphates (i.e., when Y 1 -O - represents Y 2 If =S, or Y 1 represents =O, and Y 2 Ga-S - Preferably, P is a monothiophosphate. The present inventors have found that conjugates having a thiophosphate group instead of a phosphate group have improved potency and duration of action in vivo.

[0183] P is ethyl phosphate (i.e., Y 1 represents =O, and Y 2 represents OCH2CH3).

[0184] The saccharide, sometimes referred to as a ligand, can be selected to have affinity for at least one receptor on the target cell, particularly the receptor on the surface of mammalian liver cells, e.g., the hepatic asialoglycoprotein receptor (ASGP-R).

[0185] For any of the above aspects, the saccharide may be selected from N-acetyl galactosamine, mannose, galactose, glucose, glucosamine, and fructose. Preferably, the saccharide is two molecules of N-acetylgalactosamine (GalNAc). The compound of the invention may have three ligands, each preferably an N-acetylgalactosamine.

[0186] "GalNAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetylgalactosamine. Reference to "GalNAc" or "N-acetylgalactosamine" includes both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. In certain embodiments, both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose may be used interchangeably. Preferably, compounds of the present invention contain the β-form of 2-(acetylamino)-2-deoxy-β-D-galactopyranose.

[0187] [ka] 2-(Acetylamino)-2-deoxy-D-galactopyranose

[0188] [ka] 2-(Acetylamino)-2-deoxy-β-D-galactopyranose

[0189] [ka] 2-(acetylamino)-2-deoxy-α-D-galactopyranose

[0190] For any of the compounds of formula (III) above, X 1 is (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2, or 3. X 1 may be (-CH-CH-O)(-CH)-. 1 may be (-CH-CH-O)(-CH)-. 1 may be (-CH2-CH2-O)3(-CH2)2-. Preferably, X 1 is (-CH2-CH2-O)2(-CH2)2-. Alternatively, X 1 represents a C3-C6 alkylene. X 1 may be propylene. 1 may be butylene. 1 may be pentylene. 1 Preferably, the alkyl is a linear alkylene. In particular, X 1 may be butylene.

[0191] For compounds of formula (III), X 2 represents an alkylene ether of the formula -C3H6-O-CH2-, i.e., a C3 alkoxymethylene, or -CH2CH2CH2OCH2-.

[0192] Thus, the present invention provides a compound having the following structure: [ka] [ka] [ka] [ka] (wherein Z represents a nucleic acid as defined above). Further provided is a conjugated nucleic acid having one of:

[0193] In another aspect, the present invention provides a nucleic acid or conjugated nucleic acid for inhibiting expression of a target gene in a cell, the nucleic acid comprising at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the first strand, the first strand being at least partially complementary to at least a portion of RNA transcribed from the target gene, the first strand comprising modified nucleotides at selected positions to facilitate processing of the nucleic acid by RISC, and the nucleic acid being conjugated to a ligand either directly or indirectly via a linker. The nucleic acid may be conjugated to a ligand as described herein. The nucleotides of the first strand and / or the second strand may be modified as described herein.

[0194] The ligand may be conjugated to the nucleic acid via a linker as shown in Formula I, wherein the first strand has odd-numbered nucleotides modified with 2'OMe modifications and even-numbered nucleotides modified with 2'F, and the second strand has even-numbered nucleotides modified with 2'OMe modifications and odd-numbered nucleotides modified with 2'F.

[0195] The ligand may be GalNac and may be attached via a linker.

[0196] The nucleic acids described herein can be formulated with lipids in the form of liposomes. Such formulations are sometimes described in the art as lipoplexes. Lipid / liposome-containing formulations can be used to assist in the delivery of the nucleic acids of the present invention to target cells. The lipid delivery systems described herein can be used as an alternative to conjugated ligands. When the nucleic acids of the present invention are used with lipid delivery systems or ligand-conjugated delivery systems, the modifications described herein can be present.

[0197] Such lipoplexes are i) a cationic lipid, or a pharmaceutically acceptable salt thereof; ii) steroids, iii) phosphatidylethanolamine phospholipids; iv) PEGylated lipids The lipid formulation may include a lipid formulation comprising:

[0198] The cationic lipid can be an amino cationic lipid.

[0199] The cationic lipid has the formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: X represents O, S, or NH; R 1 and R 2 are independently C4 to C 22 Linear or branched alkyl chain or C4-C with one or more double bonds 22 represents a straight or branched alkenyl chain, the alkyl or alkenyl chain optionally containing an intervening ester, amide, or disulfide; When X represents S or NH, R 3 and R 4 each independently represents hydrogen, methyl, ethyl, a monoamine or polyamine moiety, or R 3 and R 4 together form a heterocyclyl ring, If X represents O, then R 3 and R 4 each independently represents hydrogen, methyl, ethyl, a monoamine or polyamine moiety, or R 3 and R 4 together form a heterocyclyl ring, or R 3 represents hydrogen, and R 4 represents C(NH)(NH2).

[0200] The cationic lipid has the formula (IA): [ka] or a pharmaceutically acceptable salt thereof.

[0201] The cationic lipid has the formula (IB): [ka] or a pharmaceutically acceptable salt thereof.

[0202] The content of the cationic lipid component can be about 55 mol % to about 65 mol % of the total lipid content of the formulation. In particular, the cationic lipid component is about 59 mol % of the total lipid content of the formulation.

[0203] The formulation further comprises a steroid. The steroid may be cholesterol. The steroid content may be about 26 mol % to about 35 mol % of the total lipid content of the lipid formulation. More specifically, the steroid content may be about 30 mol % of the total lipid content of the lipid formulation.

[0204] Phosphatidylethanolamine phospholipids include 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DMPE), and 1,2-dimethicone-3-phosphate dehydrogenase (DMPE). The phospholipid may be selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2-disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE), and 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (SLPE). The phospholipid content may be about 10 mol % of the total lipid content of the formulation.

[0205] The PEGylated lipid may be selected from the group consisting of 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG), and C16-ceramide-PEG. The PEGylated lipid content may be about 1-5 mol % of the total lipid content of the formulation.

[0206] The content of the cationic lipid component in the formulation can be about 55 mol % to about 65 mol % of the total lipid content of the lipid formulation, preferably about 59 mol % of the total lipid content of the lipid formulation.

[0207] The molar ratio of components i):ii):iii):iv) of the formulation may be selected from 55:34:10:1, 56:33:10:1, 57:32:10:1, 58:31:10:1, 59:30:10:1, 60:29:10:1, 61:28:10:1, 62:27:10:1, 63:26:10:1, 64:25:10:1, and 65:24:10:1.

[0208] The composition comprises: [ka] a cationic lipid having the structure [ka] a steroid having the structure [ka] and a phosphatidylethanolamine phospholipid having the structure [ka] The PEGylated lipid may have the structure:

[0209] Neutral liposome compositions may be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions may be formed from dimyristoyl phosphatidylglycerol, and anionic fusogenic liposomes may be formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition may be formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0210] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids in the plasma membrane of tissue culture cells. DOTMA analogs can also be used to form liposomes.

[0211] Derivatives and analogs of the lipids described herein may also be used to form liposomes.

[0212] Liposomes containing nucleic acids can be prepared by various methods. In one example, the lipid components of the liposome are dissolved in a detergent to form micelles containing the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The nucleic acid preparation is then added to the micelles containing the lipid components. The cationic groups of the lipids interact with the nucleic acid and aggregate around the nucleic acid to form liposomes. After aggregation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of nucleic acid.

[0213] If necessary, a carrier compound to aid in aggregation may be added during the aggregation reaction, e.g., by controlled addition. For example, the carrier compound may be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH may be adjusted to favor aggregation.

[0214] The nucleic acid formulation may include a surfactant. In one embodiment, the nucleic acid is formulated as an emulsion that includes a surfactant.

[0215] Non-ionized surfactants are nonionic surfactants. Examples include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, nonionic alkanolamides, and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers.

[0216] Surfactants that acquire a negative charge when dissolved or dispersed in water are anionic surfactants. Examples include carboxylates, such as soaps, acyl acrylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyltaurates, and sulfosuccinates, and phosphates.

[0217] Surfactants that acquire a positive charge when dissolved or dispersed in water are cationic surfactants. Examples include quaternary ammonium salts and ethoxylated amines.

[0218] Surfactants that have the ability to carry a positive or negative charge are amphoteric surfactants. Examples include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0219] As used herein, a "micelle" is defined as a specific type of molecular aggregate in which amphiphilic molecules are arranged in a spherical structure such that all of the hydrophobic portions of the molecules face inward and the hydrophilic portions are in contact with the surrounding aqueous phase. The opposite arrangement exists if the environment is hydrophobic. Micelles can be formed by mixing aqueous solutions of nucleic acids, alkali metal alkyl sulfates, and at least one micelle-forming compound.

[0220] Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogs, polidocanol alkyl ethers and their analogs, chenodeoxycholate, deoxycholate, and mixtures thereof.

[0221] Phenol and / or m-cresol may be added to the mixed micelle composition to act as a stabilizer and preservative. A tonicity agent such as glycerin may also be added.

[0222] The nucleic acid preparation may be incorporated into particles, such as microparticles, which can be produced by spray drying, freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these methods.

[0223] The present invention also provides pharmaceutical compositions comprising the nucleic acids or conjugated nucleic acids of the present invention. The pharmaceutical compositions can be used as pharmaceuticals or diagnostic agents, either alone or in combination with other agents. For example, the nucleic acids or conjugated nucleic acids of the present invention can be combined with delivery vehicles (e.g., liposomes), and excipients such as carriers and diluents. Other agents, such as preservatives and stabilizers, can also be added. Methods for delivery of nucleic acids are known in the art and within the knowledge of those skilled in the art.

[0224] The nucleic acids or conjugated nucleic acids of the invention can also be administered in combination with other therapeutic compounds, either separately or simultaneously, e.g., administered as a combined unit dose. The invention also includes pharmaceutical compositions comprising the nucleic acids or conjugated nucleic acids of the invention in a physiologically / pharmaceutically acceptable excipient, e.g., stabilizers, preservatives, diluents, buffers, etc.

[0225] The pharmaceutical compositions may be specially formulated for administration in solid or liquid form. The compositions may be formulated for oral administration, parenteral administration (including, for example, subcutaneous, intramuscular, intravenous, or epidural injection), topical application, intravaginal or rectal administration, sublingual administration, ophthalmic administration, transdermal administration, or nasal administration. Delivery using subcutaneous or intravenous methods is preferred.

[0226] Dosage levels of the medicaments and pharmaceutical compositions of the present invention can be determined by one of ordinary skill in the art through routine experimentation. In one embodiment, a unit dose can contain between about 0.01 mg / kg and about 100 mg / kg of nucleic acid. Alternatively, the dose can be 10 mg / kg to 25 mg / kg, or 1 mg / kg to 10 mg / kg, or 0.05 mg / kg to 5 mg / kg, or 0.1 mg / kg to 5 mg / kg, or 0.1 mg / kg to 1 mg / kg, or 0.1 mg / kg to 0.5 mg / kg, or 0.5 mg / kg to 1 mg / kg. Dosage levels can also be calculated based on other parameters, such as body surface area.

[0227] The pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in lyophilized form. In one embodiment, the pharmaceutical composition may comprise a lyophilized lipoplex or an aqueous suspension of lipoplexes. The lipoplex preferably comprises a nucleic acid of the present invention. Such lipoplexes may be used to deliver the nucleic acid of the present invention to target cells either in vitro or in vivo.

[0228] The pharmaceutical compositions and medicaments of the present invention may be administered in a pharmaceutically effective amount to a mammalian subject, which may be selected from humans, dogs, cats, horses, cows, pigs, goats, sheep, mice, rats, hamsters, and guinea pigs.

[0229] A further aspect of the invention relates to a nucleic acid or conjugated nucleic acid of the invention or a pharmaceutical composition comprising the nucleic acid or conjugated nucleic acid of the invention for use in the treatment of a disease or disorder. The invention includes pharmaceutical compositions comprising a nucleic acid or conjugated nucleic acid according to the invention in a physiologically / pharmaceutically acceptable excipient, such as a stabilizer, preservative, diluent, buffer, etc.

[0230] The pharmaceutical compositions may be in the form of a sterile injectable aqueous suspension or solution, or in lyophilized form.

[0231] A pharmaceutically acceptable composition may comprise a therapeutically effective amount of a nucleic acid or conjugated nucleic acid of any embodiment of the present invention, alone or formulated with one or more pharmaceutically acceptable carriers, excipients, and / or diluents.

[0232] Examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) glycols such as propylene glycol. (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; and (24) other non-toxic, compatible substances used in pharmaceutical formulations.

[0233] The stabilizer can be an agent that stabilizes the nucleic acid or conjugated nucleic acid, such as a protein capable of forming a complex with the nucleic acid, a chelating agent (e.g., EDTA), a salt, an RNAse inhibitor, and a DNAse inhibitor.

[0234] In some cases, in order to prolong the effect of a drug, it is desirable to slow down the absorption of the drug from subcutaneous or intramuscular injection.This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility.The absorption rate of the drug then depends on its dissolution rate, which may depend on crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0235] The nucleic acids described herein may be capable of inhibiting expression of a target gene in a cell. The nucleic acids described herein may be capable of partially inhibiting expression of a target gene in a cell. Inhibition may be complete, i.e., 0% of the expression level of target gene expression in the absence of the nucleic acid of the invention. Inhibition of target gene expression may be partial, i.e., 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of target gene expression in the absence of the nucleic acid of the invention. Inhibition may last for 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or up to 3 months when used in a subject, such as a human subject. The nucleic acid or conjugated nucleic acid, or a composition comprising the same, may be for use once, weekly, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, or every 8 weeks. The nucleic acid or conjugated nucleic acid may be for subcutaneous or intravenous use.

[0236] In cells and / or subjects treated or receiving a nucleic acid or conjugated nucleic acid of the invention, target gene expression may be inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% compared to untreated cells and / or subjects. The level of inhibition may allow for treatment of a disease associated with target gene expression or overexpression or may allow further investigation into the function of the target gene gene product.

[0237] The target genes are factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JU gene, FOS gene, BCL-2 gene, hepcidin, activated protein C, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase II alpha gene, p73 gene mutation, p21 (WAF I / CIPI) gene mutation, p27 (KIPI) gene mutation, and PPM. The target gene may be a mutation in the ID gene, a mutation in the RAS gene, a mutation in the caveolin I gene, a mutation in the MIB I gene, a mutation in the MTAI gene, a mutation in the M68 gene, a mutation in a tumor suppressor gene, or a mutation in the p53 tumor suppressor gene. In particular, the target gene may be TMPRSS6 or ALDH2.

[0238] A further aspect of the invention relates to the use of a nucleic acid of the invention in the manufacture of a medicament for treating a disease or disorder.

[0239] The present invention also includes methods of treating a disease or disorder, comprising administering to an individual in need thereof a pharmaceutical composition comprising a nucleic acid described herein. The nucleic acid composition can be administered twice weekly, once weekly, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks. The nucleic acid can be administered to a subject subcutaneously or intravenously.

[0240] In one embodiment, the subject is administered an initial dose and one or more maintenance doses of nucleic acid or conjugated nucleic acid.The maintenance dose(s) may be the same as the initial dose or less, for example, less than half of the initial dose.The maintenance dose is administered, for example, once every 2, 5, 10, or 30 days or less.The treatment regimen may last for different periods depending on the nature of the specific disease, its severity, and the overall condition of the patient.

[0241] In one embodiment, the composition comprises multiple nucleic acid agent species. In another embodiment, the nucleic acid agent species have sequences that are non-overlapping and non-adjacent to the naturally occurring target sequence of another species. In another embodiment, the multiple nucleic acid agent species are specific for different naturally occurring target genes. In another embodiment, the nucleic acid agent is allele-specific.

[0242] The nucleic acids or conjugated nucleic acids of the invention may be administered or intended for use in combination with other therapeutic compounds, either separately or simultaneously, e.g., administered as a combined unit dose.

[0243] The nucleic acids or conjugated nucleic acids of the present invention can be produced using methods routine in the art, including chemical synthesis or expression of the nucleic acid in vitro (e.g., run-off transcription) or in vivo. For example, using solid-phase chemical synthesis or using an expression vector. In one embodiment, the expression vector can produce the nucleic acid of the present invention in a target cell. Methods for synthesizing the nucleic acids described herein are known to those of skill in the art.

[0244] In one aspect, the present invention relates to a nucleic acid capable of inhibiting expression of a target gene, the nucleic acid comprising a first strand and a second strand, the first strand and the second strand being at least partially complementary to each other, the first strand being at least partially complementary to RNA transcribed from a portion of the target gene, and the first strand and / or the second strand comprising modified nucleotides. Further preferred features of the nucleic acids disclosed herein are as follows:

[0245] 1. A nucleic acid for inhibiting expression of a target gene in a cell, the nucleic acid comprising at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the target gene, and the first strand comprises modified or unmodified nucleotides at multiple positions to facilitate processing of the nucleic acid by RISC.

[0246] In one embodiment, "facilitates processing by RISC" means that the nucleic acid can be processed by RISC, e.g., any modifications present allow the nucleic acid to be processed by RISC, preferably so that siRNA activity can occur.

[0247] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotide on the second strand corresponding to position 13 of the first strand is not modified with a 2'O-methyl modification.

[0248] A nucleotide on the second strand that "corresponds" to a position on the first strand is preferably a nucleotide that base pairs with that nucleotide on the first strand.

[0249] In one embodiment, the nucleotide on the second strand corresponding to position 13 of the first strand is the nucleotide that base pairs with position 13 of the first strand.

[0250] In one embodiment, the nucleotide on the second strand corresponding to position 11 of the first strand is the nucleotide that base pairs with position 11 of the first strand.

[0251] In one embodiment, the nucleotide on the second strand corresponding to position 12 of the first strand is the nucleotide that base pairs with position 12 of the first strand.

[0252] This nomenclature can be applied to other positions on the second strand.

[0253] For example, in a double-stranded, blunt-ended 19-mer nucleic acid, position 13 of the first strand will pair with position 7 of the second strand. Position 11 of the first strand will pair with position 9 of the second strand. This nomenclature can be applied to other positions on the second strand.

[0254] The nucleotide corresponding to position 13 of the first strand is preferably position 13 of the second strand, counting from 3' of the second strand, starting from the first nucleotide of the double-stranded region. Similarly, position 11 of the second strand is preferably the 11th nucleotide from 3' of the second strand, starting from the first nucleotide of the double-stranded region. This nomenclature can be applied to other positions in the second strand.

[0255] In one aspect, when the first and second strands are partially complementary, the nucleotide on the second strand that "corresponds" to a position on the first strand may not necessarily form a base pair if that position is one where there is a mismatch, although the nomenclature principles still apply.

[0256] Preferably, the first and second strands are completely complementary across the duplex region (disregarding any overhang regions) and have no mismatches within the double-stranded region of the nucleic acid.

[0257] The following are also preferred:

[0258] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotide on the second strand corresponding to position 11 of the first strand is not modified with a 2'O-methyl modification.

[0259] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotides on the second strand corresponding to positions 11 and 13 of the first strand are not modified with a 2'O-methyl modification.

[0260] In one embodiment, the nucleotide on the second strand corresponding to position 12 of the first strand is not modified with a 2'O-methyl modification. This restriction on nucleic acids may be viewed together with any other restrictions described herein.

[0261] Thus, another aspect of the invention is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotides on the second strand corresponding to positions 11-13 of the first strand are not modified with a 2'O-methyl modification.

[0262] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2' O-methyl modification, and the nucleotides on the second strand corresponding to positions 11, or 13, or positions 11 and 13, or positions 11-13 of the first strand are modified with a 2' fluoro modification.

[0263] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides on the second strand corresponding to positions 11, or 13, or positions 11 and 13, or positions 11-13 of the first strand are not modified with a 2' O-methyl modification.

[0264] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2' fluoro modification.

[0265] The nucleic acids disclosed herein preferably have more than 50% of the nucleotides in the first strand and / or the second strand comprising a 2'O-methyl modification, e.g., more than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first strand and / or the second strand comprising a 2'O-methyl modification, measured as a percentage of total nucleotides in both the first strand and the second strand.

[0266] The nucleic acids disclosed herein preferably have more than 50% of the nucleotides in the first and / or second strands, measured as a percentage of total nucleotides in both the first and second strands, that comprise a naturally occurring RNA modification, e.g., more than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and / or second strands comprise such a modification. Suitable naturally occurring modifications include, in addition to 20' methyl, other 2' sugar modifications, particularly 2' H modifications that result in DNA nucleotides.

[0267] The nucleic acids disclosed herein preferably comprise no more than 20%, such as no more than 15%, such as no more than 10%, of the nucleotides in the first and / or second strand, as a percentage of the total nucleotides in both the first and second strand, that have a 2' modification that is not a 2'O-methyl modification.

[0268] The nucleic acids disclosed herein preferably contain 20% or less (e.g., 15% or less or 10% or less) 2' fluoro modifications in the first strand and / or the second strand as a percentage of total nucleotides in both strands.

[0269] A nucleic acid as disclosed herein, wherein all nucleotides on the second strand except for nucleotides corresponding to positions 2 and 14 from the 5' end of the first strand and positions 11, 13, 11 and 13, or 11-13 of the first strand are modified with a 2'O-methyl modification. Preferably, the nucleotides that are not modified with 2'O-methyl are modified with a fluoro at the 2' position.

[0270] Preferred are nucleic acids disclosed herein in which all nucleotides of the nucleic acid are modified at the 2' position of the sugar. Preferably, these nucleotides are modified with a 2'-fluoro modification if the modification is not a 2'O-methyl modification.

[0271] The nucleic acids of the invention may contain one or more nucleotides modified with 2'H at the 2' position, thus having DNA nucleotides within the nucleic acid. The nucleic acids of the invention may contain DNA nucleotides at positions 2 and / or 14 of the first strand, counting from the 5' end of the first strand. The nucleic acids may contain DNA nucleotides in the second strand corresponding to positions 11, 13, 11 and 13, or 11-13 of the first strand.

[0272] In one embodiment, there is no more than one DNA per nucleic acid of the invention.

[0273] The nucleic acids of the present invention may contain one or more LNA nucleotides. The nucleic acids of the present invention may contain an LNA nucleotide at positions 2 and / or 14 of the first strand, counting from the 5'-end of the first strand. The nucleic acids may contain an LNA in the second strand corresponding to positions 11, 13, 11 and 13, or 11-13 of the first strand.

[0274] Preferably, the nucleic acids disclosed herein are siRNAs.

[0275] In one embodiment, the nucleic acid has a first strand modified with alternating 2-O-methyl and 2-fluoro modifications, with positions 2 and 14 (starting from the 5' end) modified with 2'fluoro. Preferably, the second strand has 2'fluoro modifications at nucleotides on the second strand corresponding to positions 11, or 13, or positions 11 and 13, or positions 11-13 of the first strand. Preferably, the second strand has 2'fluoro modifications at positions 11-13, starting from position 1 of the complementary (double-stranded) region and counting from the 3' end, with the remaining modifications being naturally occurring modifications, preferably 2'O-methyl.

[0276] In one embodiment, the nucleic acid of the invention comprises one or more inverted ribonucleotides, preferably inverted adenines, using a 5'-5' or 3'-3' linkage, preferably a 3'-3' linkage at the 3' end of the second strand.

[0277] In one embodiment, the nucleic acid comprises one or more phosphorodithioate linkages, for example, 1, 2, 3, or 4 phosphorodithioate linkages. Preferably, there are up to four phosphorodithioate linkages, one at the 5' end and one at the 3' end of the first strand and the second strand.

[0278] All features of the nucleic acids can be combined with all other aspects of the invention disclosed herein.

[0279] In particular, preferred nucleic acids are siRNA molecules in which the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with 2'O-methyl modifications, and which comprise one or more or all of the following: (i) an inverted nucleotide, preferably a 3'-3' linkage at the 3' end of the second strand; (ii) one or more phosphorodithioate linkages; (iii) the nucleotides of the second strand corresponding to positions 11 or 13 of the first strand are not modified with a 2' O-methyl modification, and preferably one or both of these positions contain a 2' fluoro modification. (iv) the nucleic acid contains at least 80% of its nucleotides having 2'-O-methyl modifications; (v) the nucleic acid contains 20% or less nucleotides with 2' fluoro modifications;

[0280] The present invention also provides a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand and the nucleotides at positions 7 and / or 9, or 7-9 from the 5' end of the second strand are modified with a 2' fluoro modification, and at least 90% of the remaining nucleotides are 2'-O methyl modified or contain another naturally occurring 2' modification.

[0281] With respect to double-stranded 19-base nucleic acids with no overhangs and blunt ends, a particularly preferred example is:

[0282] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotide at position 7 from the 5' end of the second strand is not modified with a 2'O-methyl modification.

[0283] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotide at position 9 from the 5' end of the second strand is not modified with a 2'O-methyl modification.

[0284] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotides at positions 7 and 9 from the 5' end of the second strand are not modified with a 2'O-methyl modification.

[0285] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotides at positions 7 to 9 from the 5' end of the second strand are not modified with a 2'O-methyl modification.

[0286] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2' O-methyl modification, and the nucleotides at positions 7 and / or 9, or 7 to 9 from the 5' end of the second strand are modified with a 2' fluoro modification.

[0287] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides at positions 7 and / or 9, or positions 7 to 9 from the 5' end of the second strand are not modified with a 2' O-methyl modification.

[0288] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides at positions 7 and / or 9, or 7 to 9 from the 5' end of the second strand are modified with a 2' fluoro modification.

[0289] Preferably, the nucleic acids disclosed herein comprise more than 50% of the nucleotides in the first strand and / or the second strand, when measured as a percentage of total nucleotides in both the first strand and the second strand, comprising a 2'O-methyl modification, e.g., more than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first strand and / or the second strand comprise a 2'O-methyl modification.

[0290] Preferably, more than 50% of the nucleotides in the first and / or second strand, measured as a percentage of total nucleotides in both the first and second strands, comprise a naturally occurring RNA modification, e.g., more than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and / or second strands comprise such a modification. Suitable naturally occurring modifications include, in addition to 20' methyl, other 2' sugar modifications, particularly 2' H modifications that result in DNA nucleotides.

[0291] A nucleic acid as disclosed herein, which comprises 20% or less, for example 15% or less, for example more than 10% of the nucleotides in the first strand and / or the second strand, preferably as a percentage of the total nucleotides in both the first strand and the second strand, having a 2' modification that is not a 2'O-methyl modification.

[0292] A nucleic acid as disclosed herein, comprising 20% ​​or less (e.g., 15% or less or 10% or less) 2' fluoro modifications in the first strand and / or the second strand, preferably as a percentage of total nucleotides in both strands.

[0293] A nucleic acid as disclosed herein, wherein all nucleotides except for the nucleotides at positions 2 and 14 from the 5' end of the first strand and positions 7 and / or 9 from the 5' end of the second strand are modified with a 2'O-methyl modification. Preferably, the nucleotides that are not modified with 2'O-methyl are modified with a fluoro at the 2' position.

[0294] A nucleic acid as disclosed herein, wherein all nucleotides except for the nucleotides at positions 2 and 14 from the 5' end of the first strand and positions 7 to 9 from the 5' end of the second strand are modified with a 2'O-methyl modification. Preferably, the nucleotides that are not modified with 2'O-methyl are modified with a fluoro at the 2' position.

[0295] For nucleic acids comprising a 20 base pair duplex region, the second strand preferably does not have a 2'O-methyl group at nucleotides 8, 9, or 10 from the 5' end of the duplex, which correspond to positions 13, 12, and 11, respectively, of the first strand.

[0296] For nucleic acids comprising a 21 base pair duplex region, the second strand preferably does not have a 2'O-methyl group at nucleotides 9, 10 or 11 counting from the 5' end of the duplex, which correspond to positions 13, 12, and 11, respectively, of the first strand.

[0297] In one aspect, the nucleic acid is not any one or more or all of Patisiran, Revusiran, Fitusiran, Cemdisiran, Givosiran, Inclisiran, Lumasiran, Votrisiran, Cosdosiran, and Teprasiran, which have the following sequence:

[0298] Patisiran 3'CAUUGGUUCUCAUAAGGUA 5' 5'GUAACCAAGAGUAUUCCAU 3' Revusiran 3'-CUACCCUAAAGUACAUUGGUUCU-5' 5'-UGGGAUUUCAUGUAACCAAGA 3' Physiollan 3'-GACCAAUUGUGGUAAAUGAAGUU-5' 5'-GGUUAACACCAUUUACUUCAA 3' Semudisilane 3'-TTUUUUCGUUCUAUAAAAAUAUUAU-5' 5'-AAGCAAGAUAUUUUUAUAAUA 3' Gibosilan 3'-UGGUCUUUCUCACAGAGUAGAAU 5' 5'-CAGAAAGAGUGUCUCAUCUUA 3' Inclisiran 3'-AAGAUCUGGACAAAACGAAAACA 5' 5'-CUAGACCUGUTUUGCUUUUGU 3'

[0299] The sequences of these molecules are also available on the WHO website http: / / www.who.int / medicines / services / inn / en / .

[0300] For example, Cemdisian is It is a double strand of

[0301] Patissiran is Guanylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-adenylyl-(3'→5')-adenylyl-(3'→5')-2'-O-methylcytidylyl-(3'→5')-2'-O-methylcytidylyl-(3'→5')-adenylyl-(3'→5')-adenylyl-(3'→5')-guanylyl-(3'→5')-adenylyl-(3'→5')- Guanylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-adenylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-2'-O-methylcytidylyl-(3'→5')-2'-O-methylcytidylyl-(3'→5')-adenylyl-(3'→5')-2'-O-methyluridylyl -(3'→5')-thymidylyl-(3'→5')-thymidine thymidylyl-(5'→3')-thymidylyl-(5'→3')-cytidylyl-(5'→3')-adenylyl-(5'→3')-2'-O-methyluridylyl-(5'→3')-uridylyl-(5'→3')-guanylyl-(5'→3')-guanylyl-(5'→3')-uridylyl-(5'→3')-uridylyl lyl-(5'→3')-cytidylyl-(5'→3')-uridylyl-(5'→3')-cytidylyl-(5'→3')-adenylyl-(5'→3')-2'-O-methyluridylyl-(5'→3')-adenylyl-(5'→3')-adenylyl-(5'→3')-guanylyl-(5'→3')-guanylyl-(5'→3')-uridylyl-(5'→3')-adenosine and is an RNA duplex.

[0302] Inclisiran is It is a double strand of

[0303] Gibosiran is It is a double strand of

[0304] Revsiran is is.

[0305] The fish lily is It is a double strand of

[0306] Lumaciran is is.

[0307] [ka]

[0308] Botrysilane is is.

[0309] [ka]

[0310] Kosudosiran is Adenylyl-(3'→5')-2'-O-methylguanylyl-(3'→5')-guanylyl-(3'→5')-2'-O-methyladenylyl-(3'→5')-guanylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-uridylyl-(3'→5')-2'-O-methylcytidylyl-(3'→5')-cytidylyl-(3'→5')- Adenylyl-(3'→5')-2'-O-methylcytidylyl-(3'→5')-adenylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-uridylyl-(3'→5')-2'-O-methylcytidylyl-(3'→5')-uridylyl-(3'→5')-2'-O-methylguanylyl-(3'→5')-guanylyl-(3'→5')-2 '-O-Methylcytidine [(2R,3S)-3-hydroxyoxolan-2-yl]methyl hydrogen uridylyl-(5'→3')-2'-deoxycytidylyl-(5'→3')-cytidylyl-(5'→3')-uridylyl-(5'→3')-cytidylyl-(5'→3')-adenylyl-(5'→3')-adenylyl-(5'→3')-guanylyl-(5' →3')-guanylyl-(5'→3')-uridylyl-(5'→3')-guanylyl-(5'→3')-uridylyl-(5'→3')-adenylyl-(5'→3')-adenylyl-(5'→3')-guanylyl-(5'→3')-adenylyl-(5'→3')-cytidylyl-(5'→3')-cytidylyl-(5'→3')-5'-guanylate duplex is.

[0311] Teprasiran is Guanylyl-(3'→5')-2'-O-methyladenylyl-(3'→5')-guanylyl-(3'→5')-2'-O-methyladenylyl-(3'→5')-adenylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-adenylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-uridylyl-(3'→5')-2'-O-methyluridylyl 2'-O-lysyl-(3'→5')-cytidylyl-(3'→5')-2'-O-methyladenylyl-(3'→5')-cytidylyl-(3'→5')-2'-O-methylcytidylyl-(3'→5')-cytidylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-uridylyl-(3'→5')-2'-O-methylcytidylyl-(3'→5')-adenosine Methyluridylyl-(3'→5')-guanylyl-(3'→5')-2'-O-methyladenylyl-(3'→5')-adenylyl-(3'→5')-2'-O-methylguanylyl-(3'→5')-guanylyl-(3'→5')-2'-O-methylguanylyl-(3'→5')-uridylyl-(3'→5')-2'-O-methylguanylyl-(3'→5')-adenylyl-( 3'→5')-2'-O-methyladenylyl-(3'→5')-adenylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-adenylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-uridylyl-(3'→5')-2'-O-methylcytidylyl-(3'→5')-uridylyl-(3'→5')-2'-O-methylcytidine duplex is.

[0312] Screening Method The present invention provides a method for selecting siRNA molecules having RNAi (RNA interference) activity, comprising: (1) modifying a nucleotide on the second (sense) strand corresponding to position 11 or 13 of the first (antisense) strand to introduce a 2'O-methyl modification in the nucleotide sugar; (2) determining whether the 2'O-methyl modification reduces the activity of the siRNA compared to an siRNA that differs only by the absence of the modification at its 2' position, or an siRNA that has a 2'-fluoro modification at the same nucleotide; and (3) If the modification affects siRNA activity, select molecules that do not have a 2'O-methyl modification at a position that reduces the activity of the siRNA, and / or modify the 2' position of the nucleotide with 2 fluoro or use an unmodified nucleotide at that position. The present invention relates to a method, including:

[0313] A method for selecting siRNA molecules having RNAi (RNA interference) activity, comprising: (1) modifying a nucleotide on the second (sense) strand corresponding to position 11 or 13 of the first (antisense) strand to introduce a nucleotide having a modification on the nucleotide sugar other than a 2' fluoro modification; (2) determining whether the modification reduces the activity of the siRNA compared to an siRNA that differs only by not having the modification at its 2' position, or an siRNA that has a 2' fluoro modification at the same nucleotide; and (3) if the modification affects siRNA activity, selecting a molecule that does not have the modification that reduces the activity of the siRNA, and / or modifying the 2' position of the nucleotide with a 2' fluoro modification or using an unmodified nucleotide at that position in the siRNA. A method comprising:

[0314] In another embodiment, the method uses the above method, but the modification of the nucleotide on the second (sense) strand corresponding to position 11 or 13 of the first (antisense) strand introduces a bulky modification, such as a fluoro or a modification that is more bulky than an unmodified RNA nucleotide, at the 2' position, and siRNA activity is evaluated with respect to the 2' bulky modification. O-methyl modifications are considered bulky, and in one embodiment, the modification is at least as bulky, i.e., large, as 2O-methyl.

[0315] The modifications can be modifications containing groups that are 2'-O-(2-methoxyethyl), 2'-O-allyl, 2'-O-DNP, 2'-CE, 2'-EA, 2'-AEM, 2'-APM, and 2'-GE.

[0316] The present invention also provides a method for selecting an siRNA molecule having RNAi (RNA interference) activity, comprising the steps of: 1. modifying the siRNA molecule by changing the 2' position of the sugar moiety of each base to a 2'O-methyl residue; assessing the activity of the siRNA to identify positions at which a 2'O-methyl modification reduces the activity of the siRNA compared to the same siRNA having a 2'fluoro modification at the same position, or compared to an siRNA without the modification at the same position; and 3. Selecting 2'O-methyl modified siRNA molecules at all positions that do not exhibit reduced activity in step 2. The present invention relates to a method, including:

[0317] In another embodiment, the method uses the above method, but the modification at the 2' nucleotide position in step (1) is a bulky modification, such as a fluoro or a modification that is bulkier than an unmodified RNA nucleotide, at the 2' position, and the siRNA activity is evaluated with respect to the bulky 2' modification. The modification can be a modification containing a group such as 2'-O-(2-methoxyethyl), 2'-O-allyl, 2'-O-DNP, 2'-CE, 2'-EA, 2'-AEM, 2'-APM, or 2'-GE.

[0318] RNAi activity can be assessed by any method disclosed herein or other methods known in the art.

[0319] The present invention also relates to a method for preparing siRNA molecules, comprising formulating into a pharmaceutical composition siRNA molecules containing 2'O-methyl modifications at all positions that do not exhibit reduced siRNA activity compared to 2'-fluoro modifications at the same positions. Preferably, these siRNA molecules have been identified using one of the methods described above. For example, the siRNA may be linked to GalNac or other targeting ligands described herein.

[0320] The siRNA is preferably a nucleic acid of the invention as described above.

[0321] In the preceding disclosure, 2'-NH2 modifications may be used as an alternative to 2' fluoro modifications in any embodiment of the invention, particularly in siRNA modifications. However, 2' fluoro modifications are more preferred.

[0322] In any aspect or embodiment of the invention described herein, the nucleic acid (or use, method, composition, or any other teachings involving nucleic acids) comprises one DNA nucleotide at position 2 or 14, counting from the 5' end of the first strand, and additionally and / or alternatively comprises one, two, or three DNA nucleotides on the second strand at positions corresponding to any one, two, or three of positions 11, 12, and 13 of the first strand.

[0323] In any aspect or embodiment of the invention described herein, the nucleic acid (or use, method, composition, or any other teachings involving nucleic acids) comprises a DNA nucleotide or 2' fluoro modification at one or more positions on the second strand corresponding to positions 11-13 of the first strand. More than one modification may be present.

[0324] In any aspect or embodiment of the invention described herein, the nucleic acid (or any use, method, composition, or any other teachings involving nucleic acids herein) does not comprise a bulky modification group, such as a 2'-O methyl group, at either or both of positions 2 or 14, counting from the 5' end of the first strand, and / or at any position on the second strand corresponding to positions 11, 12, or 13 on the first strand. A bulky modification can be, for example, any modification larger than an 'OH group at the 2' position of an RNA sugar moiety.

[0325] In further embodiments of the invention, the invention relates to any nucleic acid, conjugated nucleic acid, nucleic acid for use, method, composition, or use according to any disclosure herein, wherein the terminal nucleotide at the 3' end of at least one of the first strand and the second strand is an inverted nucleotide and is joined to the adjacent nucleotide via the 3' carbon of the terminal nucleotide and the 3' carbon of the adjacent nucleotide, and / or the terminal nucleotide at the 5' end of at least one of the first strand and the second strand is an inverted nucleotide and is joined to the adjacent nucleotide via the 5' carbon of the terminal nucleotide and the 5' carbon of the adjacent nucleotide; Optionally, a. The 3' and / or 5' inverted nucleotides of the first strand and / or the second strand are linked to adjacent nucleotides via phosphate groups by means of phosphodiester bonds, or b. the 3' and / or 5' inverted nucleotides of the first strand and / or the second strand are linked to adjacent nucleotides via phosphorothioate groups; or c. The 3' and / or 5' inverted nucleotides of the first strand and / or the second strand are linked to adjacent nucleotides via phosphorodithioate groups.

[0326] In further embodiments of the invention, the invention relates to any nucleic acid, conjugated nucleic acid, nucleic acid for use, method, composition, or use according to any disclosure herein, wherein the nucleic acid comprises phosphorodithioate linkages, optionally, the linkages are between the two 5'-most nucleosides and / or the two 3'-most nucleosides of the second strand, and / or optionally, the nucleic acid further does not comprise any internal phosphorothioate linkages.

[0327] The present invention also relates to any first strand or any second strand of the nucleic acids disclosed herein that contain no more than two base changes when compared to the specific SEQ ID NOs provided. For example, one base may be changed in either sequence.

[0328] In one embodiment, the change may be made to the 5'-most nucleotide of the antisense (first) strand. In one embodiment, the change may be made to the 3'-most nucleotide of the antisense (first) strand. In one embodiment, the change may be made to the 5'-most nucleotide of the sense (second) strand. In one embodiment, the change may be made to the 3'-most nucleotide of the sense (second) strand.

[0329] In one embodiment, this change occurs at the 5'-most nucleotide of the antisense (first) strand. The base of the 5' nucleotide may be changed to any other nucleotide. A or U is preferred at the 5' end, and A or U is taught herein as a possible 5'-end base in all of the antisense sequences disclosed herein.

[0330] The invention will now be described with reference to the following non-limiting figures and examples. [Brief explanation of the drawings]

[0331] [Figure 1A] FIG. 1 shows the in vitro knockdown activity of siRNAs in which position 14 of the first strand is modified with 2′-OMe or 2′-OH. [Figure 1B] FIG. 1 shows the in vitro knockdown activity of siRNAs in which position 14 of the first strand is modified with 2′-OMe or 2′-OH. [Figure 2A] FIG. 1 shows the in vitro knockdown activity of siRNAs having 2′-OMe or 2′-OH at position 14 of the first strand. [Figure 2B] FIG. 1 shows the in vitro knockdown activity of siRNAs having 2′-OMe or 2′-OH at position 14 of the first strand. [Figure 3A] FIG. 1 shows the in vitro knockdown activity of siRNAs with 2′-OMe or 2′-OH at positions 2, 3, and 4 of the first strand. [Figure 3B]FIG. 1 shows the in vitro knockdown activity of siRNAs with 2′-OMe or 2′-OH at positions 2, 3, and 4 of the first strand. [Figure 4A] FIG. 1 shows the in vitro knockdown activity of siRNAs with 2′-OMe and 2′-OH at positions 2, 3, and 4 of the first strand. [Figure 4B] FIG. 1 shows the in vitro knockdown activity of siRNAs with 2′-OMe and 2′-OH at positions 2, 3, and 4 of the first strand. [Figure 5A] FIG. 1 shows the in vitro knockdown activity of siRNAs with 2′-OMe and 2′-F at position 2 of the first strand. [Figure 5B] FIG. 1 shows the in vitro knockdown activity of siRNAs with 2′-OMe and 2′-F at position 2 of the first strand. [Figure 6A-1] FIG. 1 shows the knockdown activity of differently modified ALDH2 mutants derived from one sequence. [Figure 6A-2] This is a continuation of Figure 6A-1. [Figure 6B] FIG. 1 shows the knockdown activity of differently modified ALDH2 mutants derived from one sequence. [Figure 6C] FIG. 1 shows the knockdown activity of differently modified ALDH2 mutants derived from one sequence. [Figure 7A] FIG. 1 shows the knockdown activity of differently modified ALDH2 sequences. [Figure 7B] FIG. 1 shows the knockdown activity of differently modified ALDH2 sequences. [Figure 8A] FIG. 1 shows the knockdown activity of differently modified ALDH2 sequences. [Figure 8B] FIG. 1 shows the knockdown activity of differently modified ALDH2 sequences. [Figure 9A] FIG. 1 shows the knockdown activity of differently modified DGAT2 sequences. [Figure 9B] FIG. 1 shows the knockdown activity of differently modified DGAT2 sequences. [Figure 10A] FIG. 1 shows the effect of DNA modifications at certain positions in the TMPRSS6 siRNA sequence. [Figure 10B] FIG. 1 shows the effect of DNA modifications at certain positions in the TMPRSS6 siRNA sequence. [Figure 11A] FIG. 1 shows the effect of LNA modifications at certain positions in the TMPRSS6 siRNA sequence. [Figure 11B] FIG. 1 shows the effect of LNA modifications at certain positions in the TMPRSS6 siRNA sequence. [Figure 12A] FIG. 1 shows the knockdown activity of GalNAc conjugates with various modification patterns in both liposomal transfection and receptor-mediated uptake. [Figure 12B] FIG. 1 shows the knockdown activity of GalNAc conjugates with various modification patterns in both liposomal transfection and receptor-mediated uptake. [Figure 12C] FIG. 1 shows the knockdown activity of GalNAc conjugates with various modification patterns in both liposomal transfection and receptor-mediated uptake. [Figure 12D] FIG. 1 shows the knockdown activity of GalNAc conjugates with various modification patterns in both liposomal transfection and receptor-mediated uptake. [Figure 13A] FIG. 1 shows tolerance to DNA modifications at two or more positions in the TMPRSS6 siRNA sequence. [Figure 13B] FIG. 1 shows tolerance to DNA modifications at two or more positions in the TMPRSS6 siRNA sequence. [Figure 14A] FIG. 1 discloses the resistance to DNA in ALDH2-targeting siRNA. [Figure 14B] FIG. 1 discloses the resistance to DNA in ALDH2-targeting siRNA. [Figure 15A]FIG. 10 discloses the resistance to DNA in a second siRNA targeting ALDH2. [Figure 15B] FIG. 10 discloses the resistance to DNA in a second siRNA targeting ALDH2. [Figure 16A] FIG. 1 discloses the resistance to DNA in siRNA targeting DGAT2. [Figure 16B] FIG. 1 discloses the resistance to DNA in siRNA targeting DGAT2. [Figure 17A] FIG. 1 discloses the effect of 2-O-MOE at certain positions. [Figure 17B] FIG. 1 discloses the effect of 2-O-MOE at certain positions. [Figure 18A] FIG. 1 discloses tolerance to 2′-OMe in siRNAs targeting the GHR. [Figure 18B] FIG. 1 discloses tolerance to 2′-OMe in siRNAs targeting the GHR. [Example]

[0332] Example 1 All oligonucleotides were obtained from commercial oligonucleotide suppliers (Eurogentec, Belgium) or synthesized on a 10 μmol scale using phosphoramidite chemistry on an AKTA oligopilot 10 synthesizer (GE Healthcare). Commercially available base-loaded CPG solid support (500A, 50 μmol / g), 2'O-methyl RNA phosphoramidites, and 2'fluoro DNA phosphoramidites (ChemGenes and LinkTech) were used according to the manufacturer's recommended procedures. Amidite coupling was performed using a 0.1 M solution of the phosphoramidites in acetonitrile in the presence of 0.3 M benzylthiotetrazole (BTT) activator. The auxiliary reagents used were 0.05 M I in pyridine / HO (9 / 1, v / v) as the oxidant, 40% AcO in acetonitrile as CapA, 20% N-methylimidazole in acetonitrile as CapB, 3% dichloroacetic acid in toluene as DMT removal, and 20% diethylamine in acetonitrile as the final wash (EMP Biotech). EDITH (LinkTech) was used as the thiolation reagent. Acetonitrile (<20 ppm HO) was purchased from EMP Biotech. All other reagents and solvents were commercially available and used at standard reagent quality.

[0333] ST23 is a GalNac C4 phosphoramidite (structural elements below as described in WO2017 / 174657).

[0334] [ka]

[0335] ST41 is as follows (and as described in WO2017 / 174657):

[0336] [ka]

[0337] Phosphorothioates were introduced using 50 mM EDITH in acetonitrile. All oligonucleotides were synthesized in DMT-off mode. Upon completion of assembly of the oligonucleotide strand on the solid support, a diethylamine wash was performed.

[0338] Single strands were cleaved from the CPG using 40% aqueous methylamine, and all remaining protecting groups were removed (90 min, RT). The crude product was concentrated and purified by ion-exchange chromatography (Resource Q, 6 mL, GE Healthcare) on an AKTA Pure HPLC System (GE Healthcare) using a sodium chloride gradient (10 mM Tris buffer pH = 7.5, 10% acetonitrile). Product-containing fractions were analyzed, pooled, and concentrated. Salt removal was achieved by size-exclusion chromatography (Zetadex, EMP Biotech). Finally, the individual single strands were lyophilized.

[0339] For duplex formation, the single strands were reconstituted in water at a concentration of approximately 2 mg / mL. Equimolar amounts of each single strand were added, mixed, and heated to 80°C for 5 minutes. After cooling, the resulting siRNA was analyzed for complete duplex formation by native IP-RP HPLC. The product solution was stored at -20°C until further use.

[0340] This example uses 19-mer siRNAs unless otherwise clear from the description and figures.

[0341] Example 2 The effect of 2'-OMe at position 14 of the first strand on siRNA activity was tested using a sequence targeting mouse CLIC4. CLCO1 is modified with alternating 2'-OMe / 2'-OH. CLC15 is modified with 2'-OMe at position 14 of the first strand, while this position in CLC16 is not modified with 2'-OMe. All other positions in CLC15 and CLC16 are similarly modified. "UT" indicates an untreated sample to which the siRNA-treated sample was normalized. "Luc" was used as a non-targeting control.

[0342] This experiment was performed in MS1. 24 hours prior to transfection, cells were plated at a density of 40,000 cells per 6 wells, transfected with 5 nM siRNA and 1 μg / ml Atufect, and lysed 48 hours later. Total RNA was extracted, and CLIC4 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0343] The data are shown in Figures 1a and 1b.

[0344] Example 3 The effect of 2'-OMe at position 14 of the first strand on siRNA activity was tested using a sequence targeting mouse CLIC4. CLCO1 is modified with alternating 2'-OMe / 2'-OH. CLC22 is modified with 2'-OMe at positions 4, 9, and 14 of the first strand, while CLC28 is modified with 2'-OMe at positions 4, 9, and 15 of the first strand. The second strands of CLC22 and CLC28 are similarly modified. "UT" indicates an untreated sample normalized to the siRNA-treated sample. "Luc" was used as a non-targeting control.

[0345] This experiment was performed in MS1. 24 hours prior to transfection, cells were plated at a density of 40,000 cells per 6 wells, transfected with 5 nM siRNA and 1 μg / ml Atufect, and lysed 48 hours later. Total RNA was extracted, and CLIC4 and actin mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0346] The data are shown in Figures 2a and 2b.

[0347] Example 4 Using a sequence targeting mouse CLIC4, we tested the effect of 2'-OMe at position 2 of the first strand on siRNA activity. CLC56 is modified with 2'-OMe at positions 2 and 4 of the first strand and with 2'-OH at position 3. In contrast, CLC57 has 2'-OH at positions 2 and 4 and 2'-OMe at position 3. All other positions on the first and second strands are similarly modified. "UT" indicates an untreated sample normalized to the siRNA-treated sample. "Luc" was used as a non-targeting control.

[0348] This experiment was performed in MS1. 24 hours prior to transfection, cells were plated at a density of 40,000 cells per 6 wells, transfected with 5 and 1 nM siRNA and 1 μg / ml Atufect, and lysed 48 hours later. Total RNA was extracted, and CLIC4 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0349] The data are shown in Figures 3a and 3b(A).

[0350] Example 5 Using a sequence targeting mouse CLIC4, we tested the effect of 2'-OMe at position 2 of the first strand on siRNA activity. CLC56 is modified with 2'-OMe at positions 2 and 4 of the first strand and with 2'-OH at position 3. In contrast, CLC57 has 2'-OH at positions 2 and 4 and 2'-OMe at position 3. All other positions on the first and second strands are similarly modified. "UT" indicates an untreated sample normalized to the siRNA-treated sample. "Luc" was used as a non-targeting control.

[0351] This experiment was performed in MS1. 24 hours prior to transfection, cells were plated at a density of 40,000 cells per 6 wells, transfected with 1-0.008 nM siRNA and 1 μg / ml Atufect, and lysed 48 hours later. Total RNA was extracted, and CLIC4 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0352] The data are shown in Figure 3a and Figure 3b(B).

[0353] Example 6 The effect of 2'-OMe at position 2 of the first strand on siRNA activity was tested using a sequence targeting mouse CLIC4. CLCO1 is modified with alternating 2'-OMe / 2'-OH. CLC28 has 2'-OMe at position 4 of the first strand, while CLC59 has 2'-OMe at position 2, and CLC60 has 2'-OMe at position 3 of the first strand. All other positions on the first and second strands are similarly modified. "UT" indicates an untreated sample to which the siRNA-treated sample was normalized. "Luc" was used as a non-targeting control.

[0354] This experiment was performed in MS1. 24 hours prior to transfection, cells were plated at a density of 40,000 cells per 6-well plate, transfected with 5 and 1 nM siRNA (A) or 1 to 0.008 nM siRNA (B) and 1 μg / ml Atufect, and lysed 48 hours later. Total RNA was extracted, and CLIC4 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0355] The data are shown in Figures 4a and 4b.

[0356] Example 7 Using sequences targeting human HFE2, the effect of 2'-OMe at position 2 of the first strand on siRNA activity was tested. HFE04 is modified with 2'-F at position 2 and 2'-OMe at position 3 of the first strand, while HFE06 is modified with 2'-OMe at position 2 and 2'-F at position 3. All other positions on the first and second strands are similarly modified. "UT" indicates an untreated sample normalized to the siRNA-treated sample. "Luc" was used as a non-targeting control.

[0357] This experiment was performed in Hep3B. 24 hours prior to transfection, cells were plated at a density of 120,000 cells per 6 well, transfected with 1 nM siRNA and 1 μg / ml Atufect, and lysed 72 hours later. Total RNA was extracted, and HFE2 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0358] The data are shown in Figures 5a and 5b.

[0359] Example 8 Examples 8a and 8b represent biological replicates of the same experiment.

[0360] Example 8a Tolerance to 2'-OMe was investigated by targeting one position at a time in the context of an alternating pattern (2'-OMe to 2'-F change and vice versa): ALD01 is perfectly alternating, ALD13-ALD21 contain 2'-F to 2'-Me changes at every even position on the first strand, ALD22-ALD31 contain 2'-OMe to 2'-F changes at every odd position on the first strand, ALD32-ALD41 contain 2'-F to 2'-OMe changes at every odd position on the second strand, and ALD42-ALD50 contain 2'-OMe to 2'-F changes at every even position on the second strand. ALD13 contains a 2'-OMe at position 2 of the first strand, ALD19 contains a 2'-OMe at position 14 of the first strand, ALD35 contains a 2'-OMe at position 7 of the second strand, and ALD36 contains a 2'-OMe at position 9 of the second strand.

[0361] This experiment was performed in Hep3B. 24 hours prior to transfection, cells were plated at a density of 150,000 cells per 6 wells, transfected with 0.1 nM siRNA and 1 μg / ml Atufect, and lysed 48 hours later. Total RNA was extracted, and ALDH2 and actin mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0362] The sequences are listed in Figure 6a and the results are shown in Figure 6b.

[0363] Example 8b Tolerance to 2'-OMe was investigated by targeting one position at a time in the context of an alternating pattern (2'-OMe to 2'-F change and vice versa): ALD01 is perfectly alternating, ALD13-ALD21 contain 2'-F to 2'-Me changes at every even position on the first strand, ALD22-ALD31 contain 2'-OMe to 2'-F changes at every odd position on the first strand, ALD32-ALD41 contain 2'-F to 2'-OMe changes at every odd position on the second strand, and ALD42-ALD50 contain 2'-OMe to 2'-F changes at every even position on the second strand. ALD13 contains a 2'-OMe at position 2 of the first strand, ALD19 contains a 2'-OMe at position 14 of the first strand, ALD35 contains a 2'-OMe at position 7 of the second strand, and ALD36 contains a 2'-OMe at position 9 of the second strand.

[0364] This experiment was performed in Hep3B. 24 hours prior to transfection, cells were plated at a density of 150,000 cells per 6 wells, transfected with 0.1 nM siRNA and 1 μg / ml Atufect, and lysed 48 hours later. Total RNA was extracted, and ALDH2 and actin mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0365] The sequences are listed in Figure 6a and the results are shown in Figure 6c.

[0366] Example 9 Effect of modifications on the activity of two different ALDH2 siRNA sequences

[0367] (Example 9-1) We analyzed the tolerance of ALDH2-directed siRNAs for 2'-OMe at positions 2 and 14 in the first strand and at positions 7 and 9 in the second strand. ALD58 contains alternating 2'-OMe / 2'-F in both strands. ALD59-ALD61 all contain 2'-F at positions 2 and / or 14 in the first strand with an alternating second strand, whereas ALD62-ALD64 all contain 2'-F at positions 7 and / or 9 in the second strand with an alternating first strand. Positions 2 (ALD60) and 14 (ALD59) lose activity when modified with 2'-OMe, but activity is restored without 2'-OMe at positions 2 and 14 (ALD61). In the second strand, positions 7 (ALD63) and 9 (ALD62) lose activity when modified with 2'OMe, but activity is restored when positions 7 and 9 lack 2'OMe (ALD64).

[0368] This experiment was performed in Hep3B cells. Cells were plated at a density of 150,000 cells per 6 well, transfected with 0.1 nM siRNA and 1 μg / ml Atufect after 24 hours, and lysed after 48 hours. Total RNA was extracted, and ALDH2 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD from three technical replicates.

[0369] The results are shown in Figures 7a and 7b.

[0370] (Example 9-2) Different siRNAs against ALDH2 were analyzed for tolerance of 2'-OMe at positions 2 and 14 in the first strand and at positions 7 and 9 in the second strand. ALD72 contains alternating 2'-OMe / 2'-F in both strands. ALD73 to ALD75 all contain 2'-F at positions 2 and / or 14 in the first strand with an alternating second strand, whereas ALD76 to ALD78 all contain 2'-F at positions 7 and / or 9 in the second strand with an alternating first strand. Positions 2 (ALD74) and 14 (ALD73) lose activity when modified with 2'-OMe, but activity is restored without 2'-OMe at positions 2 and 14 (ALD75). In the second strand, positions 7 (ALD77) and 9 (ALD76) lose activity when modified with 2'OMe, but activity is restored when positions 7 and 9 lack 2'OMe (ALD78).

[0371] This experiment was performed in Hep3B cells. Cells were plated at a density of 150,000 cells per 6 well, transfected with 0.1 nM siRNA and 1 μg / ml Atufect after 24 hours, and lysed after 48 hours. Total RNA was extracted, and ALDH2 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD from three technical replicates.

[0372] The results are shown in Figures 8a and 8b.

[0373] Example 10 Effect of modifications on the activity of siRNA targeting DGAT2 siRNAs against DGAT2 were analyzed for tolerance of 2'-OMe at positions 2 and 14 in the first strand and at positions 7 and 9 in the second strand. DGT01 contains alternating 2'-OMe / 2'-F in both strands. DGT02-DGT04 all contain 2'-F at positions 2 and / or 14 in the first strand with an alternating second strand, whereas DGT05-DGT07 all contain 2'-F at positions 7 and / or 9 in the second strand with an alternating first strand. Positions 2 (DGT03) and 14 (DGT02) lose activity when modified with 2'-OMe, but activity is at least partially restored without 2'-OMe at positions 2 and 14 (DGT04). In the second strand, positions 7 (DGT06) and 9 (DGT05) lose activity when modified with 2'OMe, but activity is restored when positions 7 and 9 lack 2'OMe (DGT07).

[0374] This experiment was performed in Huh-7 cells. Cells were plated at a density of 80,000 cells per 6 well, transfected with 1 nM siRNA and 1 μg / ml Atufect after 24 hours, and lysed after 48 hours. Total RNA was extracted, and ALDH2 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0375] The results are shown in Figures 9a and 9b.

[0376] Example 11 Effect of DNA modifications on siRNA activity The tolerance of TMPRSS6-directed siRNAs to DNA modifications at positions 2 and 14 in the first strand and at positions 7 and 9 in the second strand was analyzed. TMP01 contains alternating 2'-OMe / 2'-F in both strands. TMP93 contains a 2'-OMe at position 14 in the first strand, while TMP113 contains a 2'-H at the same position. TMP94 contains a 2'-OMe at position 2 in the first strand, while TMP112 contains a 2'-H at the same position. TMP97 contains a 2'-OMe at position 9 in the second strand, while TMP117 contains a 2'-H at the same position. TMP98 contains a 2'-OMe at position 7 in the second strand, while TMP116 contains a 2'-H at the same position.

[0377] This experiment was performed in Hep3B. Cells were plated at a density of 150,000 cells per 6 well, transfected with 0.1 nM siRNA and 1 μg / ml Atufect after 24 hours, and lysed after 48 hours. Total RNA was extracted, and ALDH2 and actin mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD from three technical replicates.

[0378] The results are shown in Figures 10a and 10b.

[0379] Example 12 Effect of LNA modification on siRNA activity The tolerance of TMPRSS6-directed siRNAs to LNA modification at positions 2 and 14 in the first strand and at positions 7 and 9 in the second strand was analyzed. TMP01 contains alternating 2'-OMe / 2'-F in both strands. TMP93 contains 2'-OMe at position 14 in the first strand, while TMP111 contains an LNA at the same position. TMP94 contains 2'-OMe at position 2 in the first strand, while TMP110 contains an LNA at the same position. TMP97 contains 2'-OMe at position 9 in the second strand, while TMP115 contains an LNA at the same position. TMP98 contains 2'-OMe at position 7 in the second strand, while TMP114 contains an LNA at the same position.

[0380] This experiment was performed in Hep3B. Cells were plated at a density of 150,000 cells per 6 well, transfected with 0.1 nM siRNA and 1 μg / ml Atufect after 24 hours, and lysed after 48 hours. Total RNA was extracted, and ALDH2 and actin mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD from three technical replicates.

[0381] The results are shown in Figures 11a and 11b.

[0382] Example 13 Knockdown activity of various GalNAc-siRNA conjugates targeting TMPRSS6

[0383] Example 13A The effect of 2'-O-methylation at certain strand positions of the second strand was investigated in the context of GalNAc-siRNA conjugates. All conjugates contain the same first strand. STS12009V23 contains all 2'-O-methylated second strands, STS12009V25 has one 2'-F modification at position 9 of the second strand, STS12009V26 has one 2'-F modification at position 7 of the second strand, and STS12009V27 has three 2'-F modifications at positions 7 to 9 of the second strand.

[0384] This experiment was performed in Hep3B cells. Cells were plated at a density of 150,000 cells per 6 wells, transfected 24 hours later with 5-0.005 nM siRNA and 1 μg / ml Atufect, and lysed 72 hours later. Total RNA was extracted, and TMPRSS6 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD from three technical replicates.

[0385] Example 13B The effect of 2'-O-methylation at certain positions of the second strand was investigated in the context of GalNAc-siRNA conjugates. All conjugates contain the same first strand. STS12009V41L4 contains a second strand with alternating 2'-F / 2'-OMe, STS12009V23 contains a second strand that is entirely 2'-O-methylated, STS12009V25 has one 2'-F modification at position 9 of the second strand, STS12009V26 has one 2'-F modification at position 7 of the second strand, and STS12009V27 has three 2'-F modifications at positions 7-9 of the second strand.

[0386] This experiment was performed in Hep3B cells. Cells were plated at a density of 150,000 cells per 6 wells, transfected 24 hours later with 10-0.001 nM siRNA and 1 μg / ml Atufect, and lysed 72 hours later. Total RNA was extracted, and TMPRSS6 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD from three technical replicates.

[0387] Example 13C The effect of 2'-O-methylation at certain positions of the second strand was investigated in the context of GalNAc-siRNA conjugates. All conjugates contain the same first strand. STS12009V23 contains all 2'-O-methylated second strands, STS12009V25 has one 2'-F modification at position 9 of the second strand, STS12009V26 has one 2'-F modification at position 7 of the second strand, and STS12009V27 has three 2'-F modifications at positions 7 to 9 of the second strand.

[0388] This experiment was performed on primary mouse hepatocytes. Cells were plated at a density of 250,000 cells per 6-well plate and treated with 100–0.25 nM GalNAc-siRNA. Transfection with 10 nM GalNAc-siRNA and 1 μg / ml Atufect served as a control.

[0389] After 24 hours, cells were lysed, total RNA was extracted, and TMPRSS6 and actin mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD from three technical replicates.

[0390] The results are shown in Figures 12a-12d.

[0391] Example 14 Effects of DNA modifications at multiple positions TMPRSS6-directed siRNAs were analyzed for DNA tolerance at positions 2 and 14 in the first strand and at positions 7 to 9 in the second strand. TMP70 contains alternating 2'-OMe / 2'-F in both strands, whereas TMP119 contains 2'-OMe at all positions except positions 2 and 14 in the first strand and positions 7 to 9 in the second strand. TMP120 to TMP126 contain different numbers of DNA substitutions at the 2'-F position.

[0392] This experiment was performed in Hep3B cells. Cells were plated at a density of 150,000 cells per 6 well, transfected with 1 nM and 0.1 nM siRNA and 1 μg / ml Atufect after 24 hours, and lysed after 48 hours. Total RNA was extracted, and TMPRSS6 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD from three technical replicates.

[0393] The results are shown in Figures 13a and 13b.

[0394] Example 15 DNA integration at key locations. Using siRNA targeting human ALDH2, we analyzed the tolerance of positions 2 and 14 of the first strand to DNA and positions 7–9 of the second strand to DNA. ALD58 contains alternating 2'-OMe / 2'-F residues on both strands, whereas ALD61 and ALD90–ALD92 contain a reduced 2'-F pattern on the first strand, with DNA at position 2 (ALD90), DNA at position 14 (ALD91), and DNA at positions 2 and 14 (ALD92). ALD93–ALD96 contain a reduced 2'-F pattern on the second strand, with DNA at position 7 (ALD94), DNA at position 9 (ALD95), and DNA at positions 7 and 9 (ALD96). ALD97 contains 2'-F residues at positions 7, 8, and 9 of the second strand, whereas ALD98 contains DNA at these positions.

[0395] This experiment was performed in Hep3B cells. Cells were plated at a density of 150,000 cells per 6 wells, transfected with 0.1 nM and 0.01 nM siRNA and 1 μg / ml Atufect after 24 hours, and lysed after 48 hours. Total RNA was extracted, and ALDH2 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0396] The data are shown in Figures 14a and 14b.

[0397] Example 16 DNA integration at key locations. Using a second siRNA targeting human ALDH2, we analyzed the tolerance of positions 2 and 14 of the first strand to DNA and positions 7–9 of the second strand to DNA. While ALD72 contains alternating 2'-OMe / 2'-F on both strands, ALD75 and ALD99–ALD101 contain a reduced 2'-F pattern on the first strand, with DNA at position 2 (ALD99), DNA at position 14 (ALD100), and DNA at positions 2 and 14 (ALD101). ALD102–ALD105 contain a reduced 2'-F pattern on the second strand, with DNA at position 7 (ALD103), DNA at position 9 (ALD104), and DNA at positions 7 and 9 (ALD105). ALD106 contains 2'-F at positions 7, 8, and 9 of the second strand, while ALD107 contains DNA at these positions.

[0398] This experiment was performed in Hep3B cells. Cells were plated at a density of 150,000 cells per 6 wells, transfected with 0.1 nM and 0.01 nM siRNA and 1 μg / ml Atufect after 24 hours, and lysed after 48 hours. Total RNA was extracted, and ALDH2 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0399] The data are shown in Figures 15a and 15b.

[0400] Example 17 DNA integration at key locations. Using siRNA targeting human DGAT2, we analyzed the tolerance of positions 2 and 14 of the first strand to DNA and positions 7–9 of the second strand to DNA. DGT01 contains alternating 2'-OMe / 2'-F on both strands, whereas DGT04 and DGT11–DGT13 contain a reduced 2'-F pattern on the first strand, with DNA at position 2 (DGT11), DNA at position 14 (DGT12), and DNA at positions 2 and 14 (DGT13). DGT14–DGT17 contain a reduced 2'-F pattern on the second strand, with DNA at position 7 (DGT15), DNA at position 9 (DGT16), and DNA at positions 7 and 9 (DGT17). DGT18 contains 2'-F at positions 7, 8, and 9 of the second strand, while DGT19 contains DNA at these positions.

[0401] This experiment was performed in Huh7. Cells were plated at a density of 80,000 cells per 6 well, transfected with 10 nM and 1 nM siRNA and 1 μg / ml Atufect 24 hours later, and lysed 72 hours later. Total RNA was extracted, and DGAT2 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0402] The data are shown in Figures 16a and 16b.

[0403] Example 18 Incorporation of 2'-O-methoxyethyl (MOE) at key positions. Using siRNA targeting ALDH2, we analyzed the tolerance of 2'-O-MOE at positions 2 and 14 of the first strand and at positions 7 and 9 of the second strand. ALD108 contains a reduced number of 2'-Fs in both strands. In this context, 2'-O-MOEs are placed at positions 2 (ALD115), 14 (ALD116), or both positions 2 and 14 (ALD117) of the first strand. Similarly, 2'-O-MOEs are placed at positions 7 (ALD118), 9 (ALD119), or both positions 7 and 9 (ALD120) of the second strand. siRNA against luciferase was used as a non-targeting control ("Luc").

[0404] This experiment was performed in Hep3B. Cells were plated at a density of 150,000 cells per 6 well, transfected with 0.1 nM siRNA and 1 μg / ml Atufect after 24 hours, and lysed after 48 hours. Total RNA was extracted, and ALDH2 and actin mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0405] The data are shown in Figures 17a and 17b.

[0406] Example 19 Identification of key positions in the first strand. 2'-OMe tolerance was investigated in siRNAs targeting GHR by targeting one position at a time in an alternating pattern (2'-OMe to 2'-F and vice versa). GHR03 contains a perfect alternation of 2'-OMe / 2'-F, GHR07-GHR15 contain 2'-F to 2'-OMe changes at every even position on the first strand, and GHR16-GHR25 contain 2'-OMe to 2'-F changes at every odd position on the first strand. GHR07 contains 2'-OMe at position 2 on the first strand, and GHR13 contains 2'-OMe at position 14 on the first strand. An siRNA targeting luciferase ("Luc") was used as a control.

[0407] This experiment was performed in MCF-7 cells. 24 hours prior to transfection, cells were plated at a density of 120,000 cells per 6-well plate, transfected with 1 nM siRNA and 1 μg / ml Atufect, and lysed 48 hours later. Total RNA was extracted, and GHR and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents the mean ± SD of three technical replicates.

[0408] The data are shown in Figures 18a and 18b.

[0409] [Table 1A]

[0410] [Table 1B]

[0411] [Table 1C]

[0412] [Table 1D]

[0413] [Table 1E]

[0414] [Table 1F]

[0415] [Table 1G]

[0416] [Table 1H]

[0417]

Table 1I

[0418]

Table 1J

[0419] Table 1K

[0420]

Table 1L

[0421]

Table 1M

[0422] Table 2A

[0423] Table 2B

[0424] Table 2C

[0425]

Table 2D

[0426] Table 2E

[0427] Description of the Invention 1. A nucleic acid for inhibiting expression of a target gene in a cell, the nucleic acid comprising at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, the first strand being at least partially complementary to at least a portion of RNA transcribed from the target gene, and the first strand comprising modified or unmodified nucleotides at multiple positions to facilitate processing of the nucleic acid by RISC.

[0428] 2. A nucleic acid according to statement 1, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified.

[0429] 3. A nucleic acid according to statement 1 or statement 2, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification.

[0430] 4. The nucleic acid according to any one of statements 1 to 3, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a modification selected from the group consisting of 2'-O-(2-methoxyethyl), 2'-O-allyl, 2'-O-DNP, 2'-CE, 2'-EA, 2'-AEM, 2'-APM, and 2'-GE.

[0431] 5. The nucleic acid according to any one of statements 1 to 3, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a modification selected from the group consisting of 2'F, 4'-S, 2'-FANA, and UNA.

[0432] 6. A nucleic acid according to statement 1, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are unmodified.

[0433] 7. The nucleic acid according to any one of statements 1 to 6, wherein in the second strand, the nucleotides at positions 2 and 14 from the 5' end of the second strand are modified with a 2'O-methyl modification or an '-O-(2-methoxyethyl) modification.

[0434] 8. The nucleic acid according to any one of statements 1 to 7, wherein the first strand and the second strand are separate strands.

[0435] 9. The nucleic acid according to any one of statements 1 to 7, comprising a single strand comprising a first strand and a second strand.

[0436] 10. The nucleic acid according to any one of statements 1 to 9, wherein the first strand and / or the second strand are each 17 to 35 nucleotides in length.

[0437] 11. The nucleic acid according to any one of statements 1 to 10, wherein at least one duplex region consists of 19 to 25 nucleotide base pairs.

[0438] 12. a) both ends are blunt, or b) have an overhang at one end and a blunt end at the other, or c) having overhangs at both ends; A nucleic acid according to any of the preceding statements.

[0439] 13. A nucleic acid according to any of the preceding statements, wherein one or more nucleotides on the first strand and / or the second strand are modified to form modified nucleotides.

[0440] 14. The nucleic acid according to statement 13, wherein one or more of the odd-numbered nucleotides of the first strand are modified.

[0441] 15. The nucleic acid according to statement 14, wherein one or more of the even numbered nucleotides of the first strand are modified with at least a second modification, and wherein the at least second modification is different from the modification of statement 14.

[0442] 16. The nucleic acid according to statement 15, wherein at least one of the one or more modified even-numbered nucleotides is adjacent to at least one of the one or more modified odd-numbered nucleotides.

[0443] 17. The nucleic acid according to any of statements 14 to 16, wherein a plurality of odd number of nucleotides is modified.

[0444] 18. The nucleic acid according to statement 15 or 17, wherein a plurality of even-numbered nucleotides are modified by a second modification.

[0445] 19. A nucleic acid according to any of statements 13 to 18, wherein the first strand comprises adjacent nucleotides that are modified with a common modification.

[0446] 20. The nucleic acid according to any of statements 14 to 19, wherein the first strand comprises adjacent nucleotides modified with a second modification different from the modification of statement 14.

[0447] 21. The nucleic acid according to any of statements 14 to 20, wherein one or more of the odd-numbered nucleotides of the second strand are modified with a modification different from the modification of statement 14.

[0448] 22. The nucleic acid according to any of statements 14 to 21, wherein one or more of the even-numbered nucleotides of the second strand are modified by the modification of statement 14.

[0449] 23. The nucleic acid according to statement 21 or 22, wherein at least one of the one or more modified even nucleotides of the second strand is adjacent to one or more modified odd nucleotides.

[0450] 24. The nucleic acid according to any of statements 21 to 23, wherein a plurality of odd-numbered nucleotides of the second strand are modified with a common modification.

[0451] 25. The nucleic acid according to any of statements 21 to 24, wherein an even number of nucleotides are modified by a modification according to statement 14.

[0452] 26. The nucleic acid according to any of statements 21 to 25, wherein a plurality of odd numbered nucleotides are modified with a second modification, and the second modification is different from the modification of statement 14.

[0453] 27. The nucleic acid according to any of statements 21 to 26, wherein the second strand comprises adjacent nucleotides that are modified with a common modification.

[0454] 28. The nucleic acid according to any of statements 21 to 27, wherein the second strand comprises adjacent nucleotides modified with a second modification different from the modification of statement 14.

[0455] 29. The nucleic acid according to statements 13 to 28, wherein each odd-numbered nucleotide in the first strand and each even-numbered nucleotide in the second strand is modified with a common modification.

[0456] 30. The nucleic acid according to statement 29, wherein each even-numbered nucleotide in the first strand is modified with a second modification, and each odd-numbered nucleotide in the second strand is modified with a second modification, provided that positions 2 and 14 are not modified with 2'OMe.

[0457] 31. The nucleic acid according to any of statements 13 to 30, wherein the modified nucleotides of the first strand are shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.

[0458] 32. The nucleic acid according to any one of statements 13 to 31, wherein the first modification and the second modification are each independently selected from the group consisting of a 3'-terminal deoxy-thymine, a 2'-O-methyl, a 2'-deoxy modification, a locked nucleotide, an abasic nucleotide, a 2'-amino modification, a 2'-alkyl modification, a morpholino modification, a phosphoramidate modification, a nucleotide containing an unnatural base, a 5'-phosphorothioate group modification, a 5' phosphate modification or a 5' phosphate mimetic modification, and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification.

[0459] 33. A nucleic acid according to any of the preceding statements, wherein the modified nucleotide is any one of a locked nucleotide, an abasic nucleotide, or a nucleotide containing an unnatural base.

[0460] 34. The nucleic acid according to any one of statements 13 to 33, wherein the first modification is 2'-O-methyl.

[0461] 35. The nucleic acid according to any one of statements 13 to 34, wherein the second modification is 2'-F.

[0462] 36. A nucleic acid according to any one of statements 1 to 35 conjugated to a ligand.

[0463] 37. The nucleic acid according to any one of statements 1 to 36, wherein the linkages between the one, two, or three 3'-terminal nucleotides and / or the 5'-terminal nucleotides in the first strand and / or the second strand of the nucleic acid comprise phosphorothioate linkages.

[0464] 38. The nucleic acid according to any one of statements 1 to 37, wherein both the 5'-end and the 3'-end of the first strand and the 3'-end of the second strand comprise two phosphorothioate linkages.

[0465] 39. A nucleic acid for inhibiting expression of a target gene in a cell, the nucleic acid comprising at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the target gene, the first strand comprising modified nucleotides or modified nucleotides at multiple positions to facilitate processing of the nucleic acid by RISC, and the nucleic acid is conjugated to a ligand.

[0466] 40. The nucleic acid according to any of statements 36 to 39, wherein the ligand comprises one or more GalNac ligands or derivatives thereof.

[0467] 41. The nucleic acid according to any of statements 36 to 40, wherein the ligand is conjugated to the nucleic acid defined in any of the preceding statements by a bivalent or trivalent branched linker.

[0468] 42. The nucleic acid according to statements 39 to 41, wherein the nucleotides are modified as defined in any of the preceding statements.

[0469] 43. The ligand has the formula I: [SX 1 -PX 2 ]3-AX 3 - (I) [In the formula, S represents a saccharide, where the saccharide is N-acetylgalactosamine; X 1 is C3-C6 alkylene or (-CH2-CH2-O) m (—CH)—, where m is 1, 2, or 3; P is a phosphate or a modified phosphate (preferably a thiophosphate); X 2 is an alkylene or a group of formula (-CH2) n -O-CH2- [wherein n=1 to 6] alkylene ether, A is a branching unit, X 3 represents a cross-linking unit, The nucleic acids according to the invention may be linked to X via a phosphate or modified phosphate (preferably a thiophosphate). 3 conjugated to] 43. The nucleic acid according to any of statements 36 to 42, comprising

[0470] 44. The following structure: [ka] [ka] [ka] [ka] wherein Z is a nucleic acid according to any of statements 1 to 35. A conjugated nucleic acid having one of:

[0471] 45. The ligand is [ka] 45. The nucleic acid according to any one of statements 39 to 44, comprising

[0472] 46. ​​A nucleic acid or conjugated nucleic acid according to any of the preceding statements, wherein the duplex comprises separate strands.

[0473] 47. A nucleic acid or conjugated nucleic acid according to any of the preceding statements, wherein the duplex comprises a single strand comprising a first strand and a second strand.

[0474] 48. A nucleic acid or conjugated nucleic acid as defined in any of statements 1 to 47, i) a cationic lipid, or a pharmaceutically acceptable salt thereof; ii) steroids, iii) phosphatidylethanolamine phospholipids; iv) PEGylated lipids and a formulation comprising A composition comprising:

[0475] 49. The composition of claim 48, wherein the content of the cationic lipid component in the formulation is about 55 mol % to about 65 mol % of the total lipid content of the lipid formulation, preferably about 59 mol % of the total lipid content of the lipid formulation.

[0476] 50. The formulation is [ka] The cationic lipid has the structure Steroids are [ka] having the structure Phosphatidylethanolamine phospholipids [ka] having the structure PEGylated lipids, [ka] having the structure 49. The composition of claim 48 or 49.

[0477] 51. A composition comprising a nucleic acid or conjugated nucleic acid according to any of statements 1 to 47 and a physiologically acceptable excipient.

[0478] 52. A nucleic acid or conjugated nucleic acid according to any of statements 1 to 47 for use in the treatment of a disease or disorder.

[0479] 53. Use of a nucleic acid or conjugated nucleic acid according to any of statements 1 to 47 in the manufacture of a medicament for treating a disease or disorder.

[0480] 54. A method of treating a disease or disorder comprising administering to an individual in need thereof a composition comprising a nucleic acid or conjugated nucleic acid according to any one of statements 1 to 47.

[0481] 55. The method of statement 54, wherein the nucleic acid or conjugated nucleic acid is administered subcutaneously or intravenously to the subject.

[0482] 56. A process for producing a nucleic acid or a conjugated nucleic acid according to any one of statements 1 to 47.

Claims

1. 1. A conjugated nucleic acid for inhibiting expression of a target gene in a cell, comprising: a nucleic acid comprising a double-stranded region, the double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand; and a ligand; the first strand is at least partially complementary to at least a portion of the RNA transcribed from the target gene; the double-stranded region consists of 19 nucleotide base pairs, the first strand and the second strand are fully complementary to each other across the double-stranded region, the first strand comprises modified nucleotides at multiple positions, and the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification; the nucleic acid is modified on a first strand with alternating 2'-O methyl and 2' fluoro modifications; the second strand is modified with 2' fluoro modifications at positions 11 to 13, counting from the 3' end, starting at position 1 of the double-stranded region, and the remaining modifications are 2' O-methyl modifications; The ligand has Formula I: [S-X 1 -P-X 2 ] 3 -A-X 3 - (I) [In the formula, S represents a saccharide, where the saccharide is N-acetylgalactosamine; X 1 is C 3 ~C 6 Alkylene or (-CH 2 -CH 2 -O) m (-CH 2 ) 2 wherein m is 1, 2, or 3; P is a phosphate or a modified phosphate; X 2 is an alkylene or a group of the formula (-CH 2 ) n -O-CH 2 - alkylene ethers of the formula: wherein n=1 to 6; A is a branching unit, X 3 represents a cross-linking unit, The nucleic acid may be linked to X via a phosphate or modified phosphate. 3 conjugated to] A conjugated nucleic acid comprising:

2. 2. The conjugated nucleic acid of claim 1, wherein the terminal nucleotide at the 3' end of at least one of the first strand and the second strand is an inverted nucleotide and is linked to an adjacent nucleotide via the 3' carbon of the terminal nucleotide and the 3' carbon of the adjacent nucleotide, and / or the terminal nucleotide at the 5' end of at least one of the first strand and the second strand is an inverted nucleotide and is linked to an adjacent nucleotide via the 5' carbon of the terminal nucleotide and the 5' carbon of the adjacent nucleotide, or the nucleic acid comprises a phosphorodithioate linkage.

3. 3. The conjugated nucleic acid of claim 1 or 2, wherein P in formula (I) is a thiophosphate.

4. The nucleic acid is linked to X via a thiophosphate 3 4. The conjugated nucleic acid of claim 1, wherein the conjugated nucleic acid is conjugated to

5. The following structure: 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 (wherein Z is the nucleic acid).

5. The conjugated nucleic acid of any one of claims 1 to 4, having a structure selected from:

6. The ligand is 【Transformation 5】 5. The conjugated nucleic acid of claim 1, comprising the structure:

7. 7. A composition comprising a conjugated nucleic acid according to any one of claims 1 to 6 and a physiologically acceptable excipient.

8. 8. A pharmaceutical composition comprising a conjugated nucleic acid according to any one of claims 1 to 6 or a composition according to claim 7 for use in the treatment of a disease or disorder.

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