Formulations for oligonucleotide delivery

Abstract

This disclosure relates to compositions and methods for delivering nucleic acid therapeutics, particularly iRNA drugs, to several target sites and genes located within the central nervous system (CNS).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 631,377 filed on April 8, 2024, and U.S. Provisional Patent Application No. 63 / 495,652 filed on April 12, 2023. The entire contents of the above applications are incorporated herein by reference.

[0002] This disclosure generally relates to improved formulations for oligonucleotides, such as iRNA drugs, methods of delivery and manufacture, and methods of use thereof.

[0003] Array List This application was filed electronically in Extended Markup Language (XML) format and includes an array list incorporated herein by reference. The XML copy prepared on 5 April 2024 is named A108868_1750WO_SL.xm and has a size of 924,746 bytes. [Background technology]

[0004] Recent studies have described the efficient delivery of inhibitory RNA (iRNA) drugs to extrahepatic organs, particularly organs of the central nervous system (CNS) (see, for example, International Publication 2019 / 217459). While lipophilic modification of iRNA drugs has been identified as facilitating efficient CNS delivery, the formulation ultimately adopted for therapeutic delivery of any particular iRNA drug must also be free from chemical, manufacturing, and quality control (CMC) issues during drug product development. Issues such as unexpected granular formation and batch-to-batch variability can occur during the drug product development process for any of the selected therapeutic agents. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, when a candidate therapeutic agent faces a problem that may adversely affect the CMC of a new pharmaceutical product, there is a need for a promising alternative formulation that can be adopted and successfully implemented in pharmaceutical product development. [Means for solving the problem]

[0006] This disclosure presents, in at least part, compositions for enhancing the formulation of iRNA drugs during the pharmaceutical product development period, as well as resulting pharmaceutical product formulations, related methods, kits, and other compositions. In particular, the compositions herein may be administered parenterally (e.g., by injection).

[0007] In one embodiment, the Disclosure presents a composition comprising (a) a double-stranded ribonucleic acid (dsRNA) having a sense strand and an antisense strand, wherein one of the sense strand or antisense strand of the dsRNA contains at least one lipophilic modification and the other strand of the dsRNA does not contain a lipophilic modification, and (b) a divalent ion source, wherein (i) substantially all of the sense strand or antisense strand of the dsRNA containing at least one lipophilic modification in the composition are double-stranded with the strand not containing a lipophilic modification, or (ii) the strand containing at least one lipophilic modification is present in a molar excess of less than 1% compared to the other strand, the strand is present at molar equivalence, or the strand containing at least one lipophilic modification is present in a molar excess compared to the other strand. In certain embodiments, the composition is substantially free of inorganic phosphate, as will be described in more detail below.

[0008] In a relevant embodiment, the Disclosure presents a composition comprising (a) a dsRNA having a sense strand and an antisense strand, wherein one of the sense strand or antisense strand of the dsRNA contains at least one lipophilic modification and the other strand of the dsRNA does not contain a lipophilic modification, and (b) a divalent ion source, wherein (i) the sense strand or antisense strand of the dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to the dsRNA strand that does not contain a lipophilic modification, the dsRNA strand that does not contain a lipophilic modification is present in an equimolar amount relative to the sense strand or antisense strand of the dsRNA containing at least one lipophilic modification, or the dsRNA strand that does not contain a lipophilic modification is present in a molar excess compared to the sense strand or antisense strand of the dsRNA containing at least one lipophilic modification. In a particular embodiment, the composition is substantially free of inorganic phosphate, as will be described in more detail below.

[0009] In one embodiment, the sense strand of the dsRNA contains at least one lipophilic modification, and the antisense strand of the dsRNA does not contain any lipophilic modifications.

[0010] In another embodiment, there may be a molar excess of antisense chains compared to the sense chain. There may be at least about 0.1% molar excess of antisense chains compared to the sense chain. There may be at least about 0.2% molar excess of antisense chains compared to the sense chain. There may be at least about 0.3% molar excess of antisense chains compared to the sense chain. There may be at least about 0.4% molar excess of antisense chains compared to the sense chain. There may be at least about 0.5% molar excess of antisense chains compared to the sense chain. There may be at least about 1% molar excess of antisense chains compared to the sense chain. There may be at least about 2% molar excess of antisense chains compared to the sense chain. There may be at least about 3% molar excess of antisense chains compared to the sense chain. There may be at least about 4% molar excess of antisense chains compared to the sense chain. There may be about 5% or more molar excess of antisense chains compared to the sense chain.

[0011] In another embodiment, the antisense strand of the dsRNA contains at least one lipophilic modification, and the sense strand of the dsRNA does not contain a lipophilic modification. In a particular embodiment, the antisense strand of the dsRNA contains at least one lipophilic modification. In the relevant embodiment, there is a molar excess of sense strands compared to the antisense strand. In some embodiments, there is at least about 0.1% molar excess of sense strands compared to the antisense strand. There may be at least about 0.2% molar excess of sense strands compared to the antisense strand. There may be at least about 0.3% molar excess of sense strands compared to the antisense strand. There may be at least about 0.4% molar excess of sense strands compared to the antisense strand. There may be at least about 0.5% molar excess of sense strands compared to the antisense strand. There may be at least about 1% molar excess of sense strands compared to the antisense strand. There may be at least about 2% molar excess of sense strands compared to the antisense strand. There may be at least about 3% molar excess of sense strands compared to the antisense strand. There may be at least about 4% molar excess of sense strands compared to the antisense strand. A molar excess of sense strands of approximately 5% or more may be present compared to the antisense strands.

[0012] In one embodiment, the dsRNA composition is formulated for intravenous, subcutaneous, intramuscular, intradermal, intra-articular, and / or intrathecal administration / delivery.

[0013] In certain embodiments, lipophilic modification involves saturated or unsaturated C4-C 30 It is a hydrocarbon. Lipophilic modification is C4~C 30 It may be alkyl or alkenyl. Lipophilic modification is linear C6-C 18 It may be alkyl or alkenyl. Lipophilic modification is C 16 It may also be alkyl. In certain embodiments, the lipophilic modification is linked to the 2'-ribo position of a nucleic acid residue of dsRNA.

[0014] In some embodiments, the dsRNA includes at least one modified nucleotide that is not a 2'-deoxynucleotide.

[0015] In certain embodiments, the dsRNA comprises at least one modified nucleotide containing a 5'-phosphate group or a 5'-phosphate mimetic group.

[0016] In one embodiment, the divalent ion source is magnesium, calcium, copper, nickel, zinc, or strontium. In one embodiment, the divalent ion source is calcium.

[0017] In some embodiments, the molar ratio of divalent ions to dsRNA is greater than approximately 2:1.

[0018] In certain embodiments, the composition is substantially free of inorganic phosphoric acid and / or contains less than 100 ppm of inorganic phosphoric acid. The composition may contain less than 50 ppm of inorganic phosphoric acid. The composition may contain less than 10 ppm of inorganic phosphoric acid. The composition may contain less than 5 ppm of inorganic phosphoric acid. The composition may not contain inorganic phosphoric acid at all.

[0019] In some embodiments, at least one modified nucleotide that is not a 2'-deoxynucleotide is selected from nucleotides comprising 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified nucleotide, 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramide, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, and 2-hydroxymethyl-tetrahydrofuran-5-phosphate.

[0020] In one embodiment, the molar ratio of divalent ion source to dsRNA is greater than approximately 2.5:1. The molar ratio of divalent ion source to dsRNA may be greater than approximately 3.0:1. The molar ratio of divalent ion source to dsRNA may be greater than approximately 3.5:1. The molar ratio of divalent ion source to dsRNA may be greater than approximately 4.0:1.

[0021] In this specification, the maximum divalent ion (e.g., calcium) content of the compositions disclosed herein may be the amount of divalent ions in the composition that reaches a certain concentration, thereby causing the composition to reach its gelation point. In this specification, “gelation point” refers to the point at which it is impossible to sample the composition through a 25-gauge needle.

[0022] In certain embodiments, the molar ratio of divalent ion source to dsRNA is about 2:1 to about 10:1. The molar ratio of divalent ion source to dsRNA may be about 3:1 to about 10:1. The molar ratio of divalent ion source to dsRNA may be about 3:1 to about 9:1. The molar ratio of divalent ion source to dsRNA may be about 3:1 to about 8:1. The molar ratio of divalent ion source to dsRNA may be about 3:1 to about 7:1. The molar ratio of divalent ion source to dsRNA may be about 3:1 to about 6:1. The molar ratio of divalent ion source to dsRNA may be about 3:1 to about 5:1.

[0023] In one embodiment, the dsRNA comprises at least one phosphate group (5'-phosphate) or a 5'-phosphate mimic modification.

[0024] In one embodiment, the 5'-phosphate mimetic variant is a phosphonic acid variant. In a related embodiment, the phosphonic acid variant is a 5'-vinylphosphonate variant. The phosphonic acid variant may also be a 5'-(E)-vinylphosphonate variant.

[0025] In one embodiment, the 5'-phosphate mimic variant is a 5'-vinyl phosphate variant [i.e., 4'-C(H)=C(H)-OP(O)(OH)2]. The phosphonic acid variant may be a 5'-(E)-vinyl phosphate variant.

[0026] In related embodiments, the phosphate mimic variant is a 5'-phosphonic acid variant of the antisense chain.

[0027] In some embodiments, the 5'-phosphate mimetic may be a 5'-phosphate prodrug or a 5'-phosphonic acid prodrug. In some embodiments, the 5'-phosphate prodrug or 5'-phosphonic acid prodrug has the structure of the formulation disclosed in International Publication No. 2022 / 147214, which is incorporated herein by reference. In some exemplary embodiments, the 5'-phosphate prodrug or 5'-phosphonic acid prodrug is Pmmds(

[0028] [ka] [(4SR,5SR)-3,3,5-trimethyl-1,2-dithiolan-4-ol)phosphodiester]; cPmmds(

[0029] [ka] [(4SR,5RS)-3,3,5-trimethyl-1,2-dithiolan-4-ol)phosphodiester (cisPmmds)];PdAr1s(

[0030] [ka] [(4SR,5RS)-5-phenyl-3,3-dimethyl-1,2-dithiolan-4-ol)phosphodiester];PdAr3s(

[0031] [ka] [(4SR,5RS)-5-(4-methylphenyl)-3,3-dimethyl-1,2-dithiolan-4-ol)phosphodiester];PdAr5s(

[0032] [ka] [(4SR,5RS)-5-(4-methoxyphenyl)-3,3-dimethyl-1,2-dithiolan-4-ol)phosphodiester];PdAr2s(

[0033] [ka] );PdAr4s(

[0034] [ka] );PdAr6s(

[0035] [ka] );Pmmd / Pmmds(

[0036] [ka] );Pmds(

[0037] [ka] );Cymd / Cymds(

[0038] [ka] , X is the OS); or Ptmd / Ptmds(

[0039] [ka] X is the OS), Pd / Pds(

[0040] [ka] (X is the OS).

[0041] In some exemplary embodiments, a 5'-phosphate prodrug or a 5'-phosphonic acid prodrug is,

[0042] [ka] Therefore, an siRNA containing one of the above list of 5'-modified phosphate prodrugs generally has activity comparable to that of an siRNA containing 5'-VP. In some exemplary embodiments, the 5'-phosphate prodrug or 5'-phosphonic acid prodrug is

[0043] [ka] Therefore, siRNAs containing one of the above-mentioned 5'-modified phosphate prodrugs generally exhibit improved stability compared to siRNAs containing 5'-VP, and also have better or comparable activity than siRNAs containing 5'-VP. In some embodiments, the antisense strand is substantially complementary to at least 15 consecutive nucleotides of the target mRNA. Optionally, the sense strand forms a double-stranded region with the antisense strand consisting of at least 15 consecutive base pairs.

[0044] In one embodiment, the sense strand and the antisense strand each independently have 15 to 40 nucleotides.

[0045] In certain embodiments, substantially all of the nucleotides in the sense strand are modified.

[0046] In some embodiments, the sense strand includes at least one phosphorothioate nucleotide interbonding. In related embodiments, the sense strand includes at least one block of two consecutive phosphorothioate nucleotide interbondings.

[0047] In an additional embodiment, the sense strand is 21 nucleotides long.

[0048] In certain embodiments, substantially all of the nucleotides in the antisense strand are modified.

[0049] In some embodiments, seven or fewer nucleotides in the antisense strand are 2'-deoxynucleotides. Six or fewer nucleotides in the antisense strand may be 2'-deoxynucleotides. Five or fewer nucleotides in the antisense strand may be 2'-deoxynucleotides. Four or fewer nucleotides in the antisense strand may be 2'-deoxynucleotides. Three or fewer nucleotides in the antisense strand may be 2'-deoxynucleotides. Two or fewer nucleotides in the antisense strand may be 2'-deoxynucleotides.

[0050] In one embodiment, the antisense chain includes a 5'-terminal or 3'-terminal debasalized nucleotide. The 5'-terminal or 3'-terminal debasalized nucleotide may be reversed. The 5'-terminal or 3'-terminal debasalized nucleotide may be linked by a 5'-5' or 3'-3' bond. Such terminal debasalized nucleotides may be linked to the rest of the oligonucleotide via phosphodiester nucleotide-nucleotide bonds or phosphorothioate nucleotide-nucleotide bonds. In certain embodiments, one or both terminal debasalized nucleotides are linked to the rest of the oligonucleotide via phosphorothioate nucleotide-nucleotide bonds.

[0051] In another embodiment, the antisense chain includes at least one phosphorothioate nucleotide interbonding. In a related embodiment, the antisense chain includes at least one block of two consecutive phosphorothioate nucleotide interbondings. The antisense chain may also include two blocks of two consecutive phosphorothioate nucleotide interbondings.

[0052] In certain embodiments, the antisense strand contains a single thermally destabilized nucleotide that is neither a terminal nucleotide nor a cleavage region nucleotide. The thermally destabilized nucleotide may be a glycol nucleic acid (GNA). The thermally destabilized nucleotide may be an unlocked nucleic acid (UNA). The thermally destabilized nucleotide may be a 2'-5' linked ribonucleotide (3'-RNA). The thermally destabilized nucleotide may be a threose nucleic acid (TNA).

[0053] As a further example of thermally destabilized nucleotides,

[0054] [Table 1] Examples include nucleotides, or their stereoisomers. In the formula, B is a modified or unmodified nuclear base, and the asterisk represents R, S, or a racemic mixture. In another embodiment, a thermally destabilized nucleotide contains a nuclear base mismatch between the antisense strand and the sense strand; for example, the sense strand may have a mismatch with respect to the antisense strand, while the antisense strand remains matched to the target mRNA at the same position.

[0055] When an antisense strand contains one thermally stabilized nucleotide, such nucleotide is neither a terminal nucleotide nor located at positions 11, 12, or 13, except that position 1 is defined as the 5' terminal nucleotide of the antisense strand. In certain embodiments, the thermally destabilized nucleotide is located at one of positions 2 through 9, for example, at position 4, 5, 6, 7, or 8 counting from the 5' end of the antisense strand. In one embodiment, the thermally destabilized nucleotide is located at position 6 or 7 counting from the 5' end of the antisense strand. In one embodiment, the thermally destabilized nucleotide is located at position 7 counting from the 5' end of the antisense strand.

[0056] In one embodiment, the antisense chain is 23 nucleotides long. In one embodiment, the antisense chain is 19 nucleotides long. In one embodiment, the antisense chain is 20 nucleotides long. In one embodiment, the antisense chain is 21 nucleotides long. In one embodiment, the antisense chain is 22 nucleotides long.

[0057] In another embodiment, both the sense strand and the antisense strand are 19 nucleotides long. In another embodiment, both the sense strand and the antisense strand are 20 nucleotides long. In another embodiment, both the sense strand and the antisense strand are 21 nucleotides long. In another embodiment, both the sense strand and the antisense strand are 22 nucleotides long. In another embodiment, both the sense strand and the antisense strand are 23 nucleotides long. In each of these embodiments, the iRNA drug may have blunt ends at both ends of the double-stranded region.

[0058] In another embodiment, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long. In yet another embodiment, the sense strand is 20 nucleotides long and the antisense strand is 22 nucleotides long. In yet another embodiment, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long. In each of these embodiments, the iRNA drug may have two nucleotide overhangs at one end of the double-stranded region (e.g., the 3' end of the antisense strand).

[0059] In some embodiments, the composition further comprises a diluent.

[0060] In the relevant embodiments, the composition is isotonic with respect to cerebrospinal fluid (CSF).

[0061] In certain embodiments, the composition further comprises a sodium source, a potassium source, a magnesium source, and a calcium source.

[0062] In additional embodiments, the composition comprises sodium chloride, magnesium chloride, potassium chloride, and calcium chloride.

[0063] In some embodiments, the composition has a pH of about 4 to about 10. The pH may be 6 to about 10. The pH may be 6.5 to about 8.0.

[0064] In certain embodiments, the composition has an osmotic pressure of approximately 200-400 mOsm / kg.

[0065] In one embodiment, the composition does not contain hydrogen phosphate or dihydrogen phosphate.

[0066] In another embodiment, the composition does not include a buffer.

[0067] In additional embodiments, the composition may include a stabilizer that is sucrose, glucose, mannitol, sorbitol, polyethylene glycol (PEG), histidine, arginine, lysine, phospholipids, or trehalose, or a combination thereof.

[0068] In some embodiments, the composition contains more than 1 mg of dsRNA per ml of composition. The composition may contain more than 5 mg of dsRNA per ml of composition. The composition may contain more than 10 mg of dsRNA per ml of composition. The composition may contain more than 25 mg of dsRNA per ml of composition. The composition may contain more than 50 mg of dsRNA per ml of composition. The composition may contain more than 60 mg of dsRNA per ml of composition.

[0069] In certain embodiments, the dsRNA is AD-961583, AD-454973, AD-454843, AD-961584, AD-961585, AD-961586, AD-454844, AD-1302922, AD-1302923, or AD-1999409.

[0070] Additional aspects of the present disclosure provide compositions comprising (a) double-stranded ribonucleic acid (dsRNA) having sense strands and antisense strands, (b) about 80 mM to about 110 mM sodium chloride, and (c) about 8.0 mM to about 20.0 mM calcium chloride, wherein the composition contains about 50 mg to about 70 mg of dsRNA per 1 ml and is substantially free of inorganic phosphate.

[0071] Additional aspects of the present disclosure provide compositions comprising (a) double-stranded ribonucleic acid (dsRNA) having sense strands and antisense strands, (b) about 95 mM to about 100 mM sodium chloride, and (c) about 12.0 mM to about 14.0 mM calcium chloride, wherein the composition contains about 50 mg to about 70 mg of dsRNA per ml and is substantially free of inorganic phosphate.

[0072] In certain embodiments, the composition further comprises (d) about 1.0 mM to about 2.5 mM potassium chloride and (e) about 0.1 mM to about 1.0 mM magnesium chloride.

[0073] In certain embodiments, the composition further comprises (d) about 1.8 mM to about 2.0 mM potassium chloride and (e) about 0.4 mM to about 0.6 mM magnesium chloride.

[0074] In some embodiments, the composition contains about 60 mg of dsRNA per 1 ml of composition.

[0075] In one embodiment, the composition contains about 85 mM to about 100 mM sodium chloride.

[0076] In one embodiment, the composition contains about 10 mM to about 15 mM calcium chloride.

[0077] In one embodiment, the composition contains about 1.5 mM to about 2.2 mM potassium chloride.

[0078] In one embodiment, the composition contains about 0.3 mM to about 0.7 mM magnesium chloride.

[0079] In one embodiment, the composition contains about 97.6 mM sodium chloride.

[0080] In one embodiment, the composition contains about 97.0 mM sodium chloride.

[0081] In one embodiment, the composition contains about 89.5 mM sodium chloride.

[0082] In another embodiment, the composition contains about 13.0 mM calcium chloride.

[0083] In another embodiment, the composition contains about 12.9 mM calcium chloride.

[0084] In another embodiment, the composition contains about 13.9 mM calcium chloride.

[0085] In a particular embodiment, the composition contains about 1.9 mM potassium chloride.

[0086] In a particular embodiment, the composition contains about 1.7 mM potassium chloride.

[0087] In some embodiments, the composition contains about 0.5 mM magnesium chloride.

[0088] In one embodiment, the composition is a pharmaceutical composition for intrathecal administration targeting dsRNA. In related embodiments, the target is a mammal. The target may be a human.

[0089] In some embodiments, the dsRNA includes at least one modified nucleotide that is not a 2'-deoxynucleotide.

[0090] Another aspect of the present disclosure provides a composition for intrathecal administration comprising (a) a dsRNA which is AD-961583, AD-454973, AD-454843, AD-961584, AD-961585, AD-961586, AD-454844, AD-1302922, AD-1302923, or AD-1999409; (b) a calcium ion source; and (c) a diluent, wherein (i) the composition is substantially free of inorganic phosphate and (ii) the molar ratio of dsRNA to calcium ion source is greater than 3:1.

[0091] In one embodiment, the dsRNA is AD-454973.

[0092] In another embodiment, the dsRNA is AD-454843.

[0093] In an alternative embodiment, the dsRNA is AD-961583.

[0094] In additional embodiments, the dsRNA is AD-961584.

[0095] In a particular embodiment, the dsRNA is AD-961585.

[0096] In some embodiments, the dsRNA is AD-961586.

[0097] In some embodiments, the dsRNA is AD-454844.

[0098] In some embodiments, the dsRNA is AD-1302922.

[0099] In some embodiments, the dsRNA is AD-1302923.

[0100] In some embodiments, the dsRNA is AD-1999409.

[0101] Additional aspects of the present disclosure provide a composition for intrathecal administration comprising (a) a dsRNA which is AD-1395718, AD-1395724, AD-1395731, AD-1395738, AD-1395743, AD-1395756, AD-1395760, AD-1395762, AD-1395764, or AD-1395771; (b) a calcium ion source; and (c) a diluent, wherein (i) the composition is substantially free of inorganic phosphate and (ii) the molar ratio of dsRNA to calcium ion source is greater than 3:1.

[0102] In one embodiment, the dsRNA is AD-1395718.

[0103] In another embodiment, the dsRNA is AD-1395724.

[0104] In an alternative embodiment, the dsRNA is AD-1395731.

[0105] In an additional embodiment, the dsRNA is AD-1395738.

[0106] In a particular embodiment, the dsRNA is AD-1395743.

[0107] In some embodiments, the dsRNA is AD-1395756.

[0108] In some embodiments, the dsRNA is AD-1395760.

[0109] In some embodiments, the dsRNA is AD-1395762.

[0110] In some embodiments, the dsRNA is AD-1395764.

[0111] In some embodiments, the dsRNA is AD-1395771.

[0112] Another aspect of the present disclosure provides a composition for intrathecal administration comprising (a) dsRNA which is AD-1019448, AD-1019465, AD-1271082, AD-1271083, AD-1271084, AD-1271085, AD-1498524, AD-1498526, or AD-1498528; (b) a calcium ion source; and (c) a diluent, wherein (i) the composition is substantially free of inorganic phosphate and (ii) the molar ratio of dsRNA to calcium ion source is greater than 3:1.

[0113] In one embodiment, the dsRNA is AD-1019448.

[0114] In another embodiment, the dsRNA is AD-1019465.

[0115] In an alternative embodiment, the dsRNA is AD-1271082.

[0116] In an additional embodiment, the dsRNA is AD-1271083.

[0117] In a particular embodiment, the dsRNA is AD-1271084.

[0118] In some embodiments, the dsRNA is AD-1271085.

[0119] In some embodiments, the dsRNA is AD-1498524.

[0120] In some embodiments, the dsRNA is AD-1498526.

[0121] In some embodiments, the dsRNA is AD-1498528.

[0122] Another aspect of the present disclosure provides a composition comprising a double-stranded ribonucleic acid (dsRNA) having the ability to anneal to amyloid precursor protein (APP) mRNA and reduce its expression, wherein the dsRNA comprises a sense strand and an antisense strand, one of the sense strand or antisense strand of the dsRNA comprises at least one lipophilic modification, and the other strand of the dsRNA does not contain a lipophilic modification, and (i) substantially all of the sense strand or antisense strand of the dsRNA containing at least one lipophilic modification in the composition is doubled with a strand that does not contain a lipophilic modification, or (ii) the sense strand or antisense strand of the dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to the strand that does not contain a lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand that does not contain a lipophilic modification is present in a molar excess compared to the dsRNA strand containing at least one lipophilic modification.

[0123] In one embodiment, the composition further comprises a divalent ion source.

[0124] In some embodiments, the dsRNA is AD-961583, AD-454973, AD-454843, AD-961584, AD-961585, or AD-961586.

[0125] Additional aspects of the present disclosure provide compositions comprising double-stranded ribonucleic acid (dsRNA) having the ability to anneal to superoxide dismutase 1 (SOD1) mRNA and reduce its expression, wherein the dsRNA comprises a sense strand and an antisense strand, one of the sense strand or antisense strand of the dsRNA comprises at least one lipophilic modification, and the other strand of the dsRNA does not contain a lipophilic modification, and (i) substantially all of the sense strand or antisense strand of the dsRNA containing at least one lipophilic modification in the composition are double-stranded with a strand that does not contain a lipophilic modification, or (ii) the sense strand or antisense strand of the dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to the strand that does not contain a lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand that does not contain a lipophilic modification is present in a molar excess compared to the strand of the dsRNA containing at least one lipophilic modification.

[0126] In a particular embodiment, the dsRNA is AD-1395718, AD-1395724, AD-1395731, AD-1395738, AD-1395743, AD-1395756, AD-1395760, AD-1395762, AD-1395764, or AD-1395771.

[0127] Another aspect of the present disclosure provides a composition comprising a double-stranded ribonucleic acid (dsRNA) having the ability to anneal to huntingtin (HTT) mRNA and reduce its expression, wherein the dsRNA comprises a sense strand and an antisense strand, one of the sense strand or antisense strand of the dsRNA comprises at least one lipophilic modification, and the other strand of the dsRNA does not contain a lipophilic modification, and (i) substantially all of the sense strand or antisense strand of the dsRNA containing at least one lipophilic modification in the composition is doubled with a strand that does not contain a lipophilic modification, or (ii) the sense strand or antisense strand of the dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to the strand that does not contain a lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand that does not contain a lipophilic modification is present in a molar excess compared to the dsRNA strand containing at least one lipophilic modification.

[0128] In one embodiment, the dsRNA targets a sequence within exon 1 of the huntingtin gene.

[0129] In certain embodiments, the dsRNA is AD-1019448, AD-1019465, AD-1271082, AD-1271083, AD-1271084, AD-1271085, AD-1498524, AD-1498526, or AD-1498528.

[0130] Additional aspects of the present disclosure provide compositions comprising double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, wherein one of the sense strand or antisense strand of the dsRNA comprises at least one lipophilic modification in one or more internal residues, and the other strand of the dsRNA does not contain a lipophilic modification, and (i) substantially all of the sense strand or antisense strand of the dsRNA comprising at least one lipophilic modification in the composition are double-stranded with a strand that does not contain a lipophilic modification, or (ii) the sense strand or antisense strand of the dsRNA comprising at least one lipophilic modification is present in a molar excess of less than 1% compared to the strand that does not contain a lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand that does not contain a lipophilic modification is present in a molar excess compared to the strand of the dsRNA comprising at least one lipophilic modification.

[0131] In some embodiments, the dsRNA contains at least one lipophilic modification at one or more internal residues of the sense strand. The dsRNA may also contain at least one lipophilic modification at either position 4–8 or position 13–18, counting from the 5' end of the strand. The dsRNA may also contain at least one lipophilic modification at position 6, counting from the 5' end of the strand.

[0132] In certain embodiments, the dsRNA includes at least one lipophilic modification in one or more internal residues of the antisense strand.

[0133] In one embodiment, at least one lipophilic modification is saturated or unsaturated C4-C 30 Contains hydrocarbons. At least one lipophilic modification is C4-C 30 It may contain alkyl or alkenyl compounds. At least one lipophilic modification is linear C6-C6. 18 It may contain alkyl or alkenyl compounds. At least one lipophilic modification is C 16 Alkyl compounds may be included. At least one lipophilic modification may be ligated at the 2'-ribo position of a nucleic acid residue of the dsRNA.

[0134] Another aspect of the present disclosure provides a composition comprising a double-stranded ribonucleic acid (dsRNA) having a sense strand and an antisense strand, wherein one of the sense strand or antisense strand of the dsRNA has at least one lipophilic modification at one or more terminal residues, and the other strand of the dsRNA does not have a lipophilic modification, and (i) substantially all of the sense strand or antisense strand of the dsRNA having at least one lipophilic modification in the composition is double-stranded with a strand that does not have a lipophilic modification, or (ii) the sense strand or antisense strand of the dsRNA having at least one lipophilic modification is present in a molar excess of less than 1% compared to the strand that does not have a lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand that does not have a lipophilic modification is present in a molar excess compared to the strand of the dsRNA having at least one lipophilic modification.

[0135] In certain embodiments, the dsRNA includes at least one lipophilic modification at the 5' terminal residue(s) and / or 3' terminal residue(s) of the sense strand. The dsRNA may also include at least one lipophilic modification at the 3' terminal residue of the sense strand.

[0136] In some embodiments, the dsRNA includes at least one lipophilic modification at the 5' terminal residue of the sense strand.

[0137] In one embodiment, the dsRNA includes at least one lipophilic modification at the 5' terminal residue(s) and / or 3' terminal residue(s) of the antisense strand. The dsRNA may also include at least one lipophilic modification at the 3' terminal residue(s) of the antisense strand.

[0138] In another embodiment, the dsRNA includes at least one lipophilic modification at the 5' terminal residue of the antisense strand.

[0139] In certain embodiments, at least one lipophilic modification comprises a saturated or unsaturated C4-C 30 hydrocarbon. At least one lipophilic modification may comprise a C4-C 30 alkyl or alkenyl. At least one lipophilic modification may comprise a linear C6-C 18 alkyl or alkenyl. At least one lipophilic modification may comprise a C 16 alkyl. At least one lipophilic modification may be linked at the 2'-ribo position of the nucleic acid residue of the dsRNA.

[0140] Another aspect of the disclosure provides a solid prepared by lyophilization of the compositions of the disclosure.

[0141] A further aspect of the disclosure provides a kit comprising (a) a diluent comprising a divalent cation source and substantially free of inorganic phosphate, and (b) a double-stranded ribonucleic acid (dsRNA) having a sense strand and an antisense strand, wherein the dsRNA comprises at least one modified nucleotide that is not a 2'-deoxynucleotide and the molar ratio of dsRNA to calcium ions is greater than 1:2.

[0142] Another aspect of the disclosure provides (a) a solid prepared by lyophilization of the compositions of the disclosure, and (b) a diluent substantially free of inorganic phosphate.

[0143] A further aspect of the disclosure provides a method of treating a subject having a disorder that would benefit from reduced expression of a target gene, the method comprising administering to the subject a therapeutically effective amount of the compositions of the disclosure thereby treating the subject.

[0144] In certain embodiments, the subject is human.

[0145] In some embodiments, the target gene is an amyloid precursor protein (APP), superoxide dismutase 1 (SOD1), or the huntingtin gene, and may be exon 1 of the huntingtin gene.

[0146] In one embodiment, the subject is afflicted with an APP-related disease. In certain embodiments, the APP-related disease is cerebral amyloid angiopathy (CAA), early-onset Alzheimer's disease (EOAD), familial Alzheimer's disease, early-onset familial Alzheimer's disease (EOFAD), or late-onset Alzheimer's disease. In certain embodiments, the APP-related disease is Alzheimer's disease (AD).

[0147] In certain embodiments, the expression of APP is inhibited by at least about 30%.

[0148] In another embodiment, the method further comprises administering an additional therapeutic agent to the subject.

[0149] In some embodiments, the dsRNA of the composition is administered at a dose of about 0.1 mg / kg to about 50 mg / kg.

[0150] In certain embodiments, the composition is administered intrathecally to the subject.

[0151] In one embodiment, administration of the composition to the subject causes a decrease in Aβ accumulation. Optionally, administration of the composition to the subject causes a decrease in the accumulation of Aβ(1-40) and / or Aβ(1-42).

[0152] In some embodiments, administration of the composition to the subject causes a decrease in amyloid plaque formation and / or accumulation in the subject.

[0153] In certain embodiments, the method reduces the expression of a target gene in brain or spinal tissue. In related embodiments, the brain or spinal tissue is the cortex, cerebellum, striatum, cervical, lumbar, and / or thoracic spine.

[0154] Another aspect of the disclosure provides a method of inhibiting the expression of APP in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition of the disclosure, thereby inhibiting APP expression in the subject.

[0155] Additional aspects of the present disclosure are methods for treating or preventing APP-related diseases or disorders in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition of the present disclosure, thereby treating or preventing the APP-related disease or disorder in the subject.

[0156] In certain embodiments, the APP-related disease or disorder is cerebral amyloid angiopathy (CAA), or Alzheimer's disease (AD). In one embodiment, the AD is early-onset familial Alzheimer's disease (EOFAD). In some embodiments, the APP-related disease or disorder is early-onset Alzheimer's disease (EOAD), familial Alzheimer's disease, or late-onset Alzheimer's disease.

[0157] In some embodiments, the composition is administered by intrathecal injection. The intrathecal injection may be carried out in conjunction with intravenous administration.

[0158] Alternatively, intrathecal administration is used without intravenous administration.

[0159] In some embodiments, administering the composition causes a decrease in the intensity, severity, or frequency of, or a delay in the onset of, at least one symptom or feature of the APP-related disease or disorder.

[0160] In certain embodiments, administering the composition does not cause significant side effects in the subject. Optionally, the subject does not have tremors or convulsions when the composition is administered to the subject.

[0161] In some embodiments, the composition is administered at least once every two weeks, once a month, once every two months, once every three months, once every four months, once every five months, and / or once every six months.

[0162] Another aspect of the present disclosure provides a method for administering dsRNA to a subject in need thereof, comprising intrathecal administration of a composition of the present disclosure to the subject, thereby administering dsRNA to the subject.

[0163] In one embodiment, the subject is a human.

[0164] In another embodiment, the dsRNA targets one or more of the following genes / mRNAs: amyloid precursor protein (APP); superoxide dismutase 1 (SOD1); and / or huntingtin (HTT). In a particular embodiment, exon 1 of the huntingtin gene mRNA is targeted.

[0165] Another aspect of the present disclosure provides a method for inhibiting the expression of SOD1 in cells or tissues of interest, comprising administering a composition of the present disclosure to a target in an amount sufficient to reduce the expression of SOD1 in the cells or tissues of interest, thereby inhibiting the expression of SOD1 in the cells or tissues of interest.

[0166] In one embodiment, the composition is administered intrathecally to the subject.

[0167] In certain embodiments, administration of dsRNA reduces the level of SOD1 mRNA in the target cells or tissues by at least 50%, and optionally at least 80%, compared to a suitable control.

[0168] In some embodiments, the dsRNA of the composition is administered in doses ranging from approximately 0.1 mg / kg to approximately 50 mg / kg.

[0169] Another aspect of the present disclosure provides a method for inhibiting HTT expression in target cells or tissues, comprising administering a composition of the present disclosure in an amount sufficient to reduce HTT expression in target cells or tissues, thereby inhibiting HTT expression in target cells or tissues.

[0170] In one embodiment, the composition comprises a dsRNA that targets exon 1 of HTT.

[0171] In some embodiments, the dsRNA of the composition is administered at a dose of about 0.1 mg / kg to about 50 mg / kg.

[0172] Another aspect of the disclosure provides a kit for performing the methods of the disclosure, the kit comprising: a) a composition comprising a dsRNA; b) instructions for use; and c) optionally, means for administering the composition to a subject.

[0173] An additional aspect of the disclosure is a method for reducing or preventing particle formation in a solution comprising a divalent ion source and a double-stranded ribonucleic acid (dsRNA) having a sense strand and an antisense strand, wherein the dsRNA comprises at least one lipophilic modification on either the sense strand or the antisense strand and wherein the complementary strand does not comprise a lipophilic modification, the method comprising maintaining the sense strand or antisense strand of the dsRNA comprising the lipophilic modification at a molar excess of less than 1% compared to the strand not comprising the lipophilic modification (the complementary strand), thereby reducing or preventing particle formation in a solution comprising a divalent ion source and a double-stranded ribonucleic acid (dsRNA).

[0174] In one embodiment, the sense strand and the antisense strand are present in equimolar amounts.

[0175] In another embodiment, the strand not comprising a lipophilic modification is present in a molar excess compared to the strand of the dsRNA comprising at least one lipophilic modification.

[0176] In a further embodiment, the sense strand has at least one lipophilic modification and the antisense strand does not have a lipophilic modification.

[0177] In an alternative embodiment, the antisense strand has a lipophilic modification and the sense strand does not have a lipophilic modification.

[0178] In certain embodiments, the divalent ion source is calcium, magnesium, copper, nickel, zinc, or strontium. In one embodiment, the divalent ion source is calcium.

[0179] In another embodiment, at least one lipophilic modification is C16 or a longer lipophilic modification.

[0180] Another aspect of the present disclosure provides a method for preparing a formulation, comprising annealing a sense strand and an antisense strand to form a double-stranded solution containing double-stranded RNA (dsRNA), vacuum freeze-drying the double-stranded solution to provide a double-stranded composition, and dissolving the double-stranded composition in an injection solution, wherein one of the sense strand and the antisense strand contains a lipophilic modification, the injection solution contains a divalent cation source (e.g., calcium) and does not contain phosphate buffer, and the double-stranded composition contains an antisense strand in a molar excess of 0 to 5% relative to the sense strand (e.g., about 1 to 2% molar excess).

[0181] In certain embodiments, the divalent ion source is calcium, magnesium, copper, nickel, zinc, or strontium. In one embodiment, the divalent ion source is calcium.

[0182] In one embodiment, the double-stranded composition contains an antisense strand in an antisense strand in an excess of about 1-2% molars relative to the sense strand.

[0183] In another embodiment, the sense chain includes at least one lipophilic modification, and the antisense chain does not include any lipophilic modifications.

[0184] In another embodiment, at least one lipophilic modification is C16 or a longer lipophilic modification.

[0185] In one embodiment, the dsRNA is selected from the group consisting of AD-961583, AD-454973, AD-454843, AD-961584, AD-961585, and AD-961586. [Brief explanation of the drawing]

[0186] [Figure 1] Figures 1A and 1B show, respectively, the modified double-stranded structures of the present disclosure and components of exemplary formulations, including both diluents and pharmaceutical product formulations. Figure 1A shows graphic representations of four related modified APP-targeted double-stranded structures AD-454844, AD-1302922, AD-1302923, and AD-1999409. The double-stranded structures shown include the following sequences: AD-454844: sense chain SEQ ID NO: 3, antisense chain SEQ ID NO: 14; AD-1302922: sense chain SEQ ID NO: 9, antisense chain SEQ ID NO: 20; AD-1302922: sense chain SEQ ID NO: 10, antisense chain SEQ ID NO: 21; and AD-1999409: sense chain SEQ ID NO: 11, antisense chain SEQ ID NO: 22. The baseline sequences for each of these four double-stranded sequences are identical, and all four double-stranded sequences contain the same antisense strand (A-882382; Sequence IDs 14 and 20-22 are identical). These four double-stranded sequences are distinguished as follows: (1) The AD-454844 double-stranded sequence has a 2'-O-C16 modification at the 6th residue from the 5' end of the sense strand (A-882381; Sequence ID 3); (2) The AD-1302922 double-stranded sequence has the 2'-O-C16 modification shifted to the 5' end residue of the sense strand of the AD-1302922 double-stranded sequence (A-2364988; Sequence ID 9); (3) AD-13 The 02923 double-stranded compound has a 2'-O-docosanyl-cytidine-3' phosphate (C22) modification at the 6th residue from the 5' end of the sense strand (A-2365995; SEQ ID NO: 10); (4) The AD-1999409 double-stranded compound has a 2'-O-decyl-cytidine-3' phosphate (C10) modification at the 6th residue from the 5' end of the sense strand (A-3724055; SEQ ID NO: 11). Figure 1B shows a diagram illustrating the components of such formulations. [Figure 2]Figure 2 shows that the APP-targeted siRNA formulation "APP F3DP," containing a 0.1% molar excess of a non-lipophilic modified antisense strand compared to a lipophilic modified sense strand, did not form granules (right panel), and a novel iRNA formulation ("APP F8DP," center panel) with a 5% molar excess of a non-lipophilic modified antisense strand compared to a lipophilic modified sense strand also did not form granules. In contrast, a different novel siRNA formulation ("APP F7DP," left panel) characterized by a 5% molar excess of a lipophilic modified sense strand compared to a non-lipophilic modified antisense strand formed granules. Therefore, such pellets were observed only when there was an excess of the sense strand modified at the C16 lipophilic moiety. [Figure 3] Figure 3 shows that in the calcium exchange test, granular formation was observed for the "APP F4DP" siRNA formulation (right panel) having a 0.85% molar excess of the lipophilic modified sense chain compared to the non-lipophilic modified antisense chain. However, this granular formation can be effectively eliminated by preparing a formulation containing an excess of the non-lipophilic modified antisense chain instead. No granular formation was observed for either the novel formulation "APP F5DP," which has a 1.5% molar excess of the non-lipophilic modified antisense chain compared to the lipophilic modified sense chain, or the novel formulation "APP F6DP," which has a 7.4% molar excess of the non-lipophilic modified antisense chain compared to the lipophilic modified sense chain. Similar to Figure 2 above, granular formation was observed only when the sense chain modified at the C16 lipophilic portion was in a molar excess compared to the non-lipophilic modified antisense chain. [Figure 4]Figures 4A-4E show that increased turbidity was observed in double-stranded samples prepared in a solution containing divalent cations and a molar excess of sense strands relative to the antisense strands, and that the resolution of such turbidity was evaluated under various conditions. Figure 4A shows the turbidity observed by visual inspection of the double-stranded samples in solution, where each double-stranded sample was prepared in a sodium solution containing a 5% molar excess of sense strands compared to the antisense strands ("before counterion exchange," upper vial). Next, counterion exchange was performed on the samples (calcium exchange was performed on all double-stranded samples, while magnesium exchange was performed on one of the C16-containing double-stranded samples, shown on the left). Turbid suspensions were observed in all double-chain solutions except for C10-containing double-chain solutions after counterion exchange [Ultrafiltration (UF) was performed on high-salt solutions (20 mM CaCl2 or 50 mM MgCl2) followed by low-salt solutions (1 mM CaCl2 or MgCl2) for the double-chain solutions shown in the lower panel "after counterion exchange"]. Figure 4B shows that the location of the lipophilic modification significantly affects the turbidity observed in the main-chain solution. Terminal C16-containing double-chains were extremely turbid when formulated with an excess of sense chains, but even with terminal C16-containing double-chains, turbidity could be eliminated by adjusting the dose to have an excess of antisense chains. Figure 4C shows that under conditions of an excess of sense chains and the presence of counterions, the turbidity level increases for lipophilic portions with chain lengths longer than C10. Both the internally C16-containing and internally C22-containing double-stranded polymers showed increased turbidity levels compared to the internally C10-containing double-stranded polymer at all sense-to-antisense ratios tested. However, the internally C22-containing double-stranded polymers showed only a slight increase in turbidity compared to the corresponding internally C16-containing double-stranded polymers. Figure 4D shows the effect of counterions on precipitate formation / turbidity. Both the calcium-exchanged and magnesium-exchanged C16 double-stranded polymers showed similar turbidity behavior and resolved in a similar manner (by removing the excess sense strand).Figure 4E shows that sequence variations between double strands with corresponding parallel nucleotide modification patterns can also significantly affect precipitate formation and associated turbidity levels. The turbidity of terminally modified AD-1302922 APP-targeted double strands and internally modified AD-454844 APP-targeted double strands (sharing a common sequence and identical antisense strands) was compared with a different internally modified APP-targeted double strand, AD-961583 (described in more detail below and simply referred to here as "APP"). Compared with precipitate resolution data for the AD-961583 double strand, both the internal and terminally modified double strands AD-1302922 and AD-454844 showed significantly lower turbidity than the AD-961583 double strand. [Figure 5] Figures 5A and 5B show the effect of excess antisense chains on resolving turbidity in counterion-containing solutions when an excess antisense chain is present in the APP-targeted double-stranded molecule "AD-961583" containing internal C16 lipophilicity. Figure 5A shows a schematic diagram of antisense chain dose adjustment in the APP-targeted AD-961583 double-stranded molecule (sense chain: SEQ ID NO: 4; antisense chain: SEQ ID NO: 15) and a suspension of the AD-961583 double-stranded molecule [which formed particulate matter under conditions where the sense chain was in excess and counterions (divalent cations) were present]. In all cases, turbidity (upper vial) was resolved by adding the antisense chain (lower vial). Figure 5B shows the quantification of the sample after dose adjustment and the turbidity level after plotting. [Figure 6]Figures 6A and 6B show the effect of excess antisense chain on turbidity in a counterion-containing solution of the internally C16 lipophilic "AD-1395762" superoxide dismutase 1 (SOD1) target duplex. Figure 6A shows a schematic diagram of the SOD1 target AD-1395762 duplex (sense chain: SEQ ID NO: 74; antisense chain: SEQ ID NO: 93), and the dose adjustment of the antisense chain in a suspension consisting of AD-1395762 duplexes that, unlike the APP target duplexes examined herein, did not form significant granular material even under conditions where the sense chain was 5.8% excess and counterions (divalent cations) were present. In all cases, turbidity was not observed by visual inspection. Figure 6B shows the quantification of the sample after dose adjustment and the turbidity level after plotting. [Figure 7] Figures 7A and 7B show the effect of an excess antisense strand on resolving turbidity in a counterion-containing solution when an antisense strand is present in the internally C16 lipophilic-containing "AD-1498524" huntingtin gene exon 1 (HTTex1) double-stranded molecule. Figure 7A shows a schematic diagram of the HTT exon 1-targeted AD-1498524 double-stranded molecule (sense strand: SEQ ID NO: 83; antisense strand: SEQ ID NO: 102), and the adjustment of the antisense strand dose of a suspension consisting of AD-1498524 double-stranded molecules that formed granular material under conditions of excess sense strand and the presence of counterions (divalent cations). In all cases, the observed turbidity (upper vial) dissipated upon addition of the antisense strand (lower vial). Figure 7B shows the quantification of the sample after dose adjustment and the turbidity level after plotting. [Figure 8]Figure 8 shows graphic representations of five related modified APP target double-stranded structures: AD-454844, AD-1302922, AD-1302923, AD-1999409 (also shown in Figure 1A above), and AD-960500. The double-stranded structures shown contain the following sequences: AD-454844: sense chain sequence number 3, antisense chain sequence number 14; AD-1302922: sense chain sequence number 9, antisense chain sequence number 20; AD-1302922: sense chain sequence number 10, antisense chain sequence number 21; AD-1999409: sense chain sequence number 11, antisense chain sequence number 22; and AD-960500: sense chain sequence number 162, antisense chain sequence number 163. The baseline sequences for each of these five double-stranded sequences are identical, and all five double-stranded sequences contain the same antisense strand (A-882382; Sequence IDs 14, 20-22, and 163 are identical). These five double-stranded sequences are distinguished as follows: (1) The AD-454844 double-stranded sequence has a 2'-O-C16 modification at the 6th residue from the 5' end of the sense strand (A-882381; Sequence ID 3); (2) The AD-1302922 double-stranded sequence has a 2'-O-C16 modification shifted to the 5' terminal residue of the sense strand (A-2364988; Sequence ID 9) of the AD-1302922 double-stranded sequence; (3) The AD-1302923 double-stranded sequence is (4) The sense strand (A-2365995; SEQ ID NO: 10) has a 2'-O-docosanyl-cytidine-3' phosphate (C22) modification at the 6th residue from the 5' end; (5) The AD-1999409 double strand has a 2'-O-decyl-cytidine-3' phosphate (C10) modification at the 6th residue from the 5' end of the sense strand (A-3724055; SEQ ID NO: 11); and (6) The AD-960500 double strand does not have lipophilic modifications. [Figure 9]Figures 9A-9C show that in double-stranded samples prepared in a solution containing either Ca2+ or Mg2+ ions and a molar excess of sense chains relative to the antisense chains, an increase in turbidity was observed with respect to such double-stranded samples, and that such turbidity could be reversed by adding antisense chains to the samples. Figure 9A shows the turbidity observed by visual inspection of double-stranded samples in solution, where each double-stranded sample was prepared in a sodium solution containing a 5% molar excess of sense chains compared to the antisense chains. Next, counterion exchange was performed on the samples (calcium exchange was performed on double-stranded AD-960500 (no lipophilic modification), AD-454844 (internal C16 sense chain lipophilic modification), AD-1302922 (terminal C16 sense chain lipophilic modification), AD-1302923 (internal C22 sense chain lipophilic modification), and AD-1999409 (internal C10 sense chain lipophilic modification), while magnesium exchange was performed on the corresponding solutions for double-stranded AD-454844 (internal C16 sense chain lipophilic modification) and AD-1302922 (terminal C16 sense chain lipophilic modification)). After the indicated counterion exchange, slight turbidity was observed in all double-stranded solutions except for the AD-960500 control double-stranded (no lipophilic modification) and the C10-containing AD-1999409 double-stranded. Figure 9B shows that turbidity was eliminated by adding a larger amount of antisense chains to each calcium-exchanged double-chain solution that generated turbidity under conditions of excess sense chains (lipophilic modified). For all double-chain solutions, when a molar excess of antisense chains was generated compared to the sense chains (lipophilic modified), and calcium exchange was performed on them, virtually no turbidity was observed. Figure 9C shows that turbidity was eliminated by adding a larger amount of antisense chains to each calcium-exchanged or magnesium-exchanged double-chain solution that generated turbidity under conditions of excess sense chains (note that, in contrast to the significant decrease in turbidity levels when using magnesium-exchanged C16 solution in the presence of excess sense chains, the calcium-exchanged terminal C16 solution produced the highest turbidity level when sense chains were in excess).As described above, for all the double-chain solutions tested, a molar excess of antisense chains was generated compared to the sense chain (lipophilic modified type). When divalent cation exchange (regardless of whether it was calcium exchange or magnesium exchange) was performed on these antisense chains, virtually no turbidity was observed. [Figure 10] Figure 10A shows a typical ion-pair reversed-phase (IPRP) HPLC for the annealing of APP double-stranded ALN-961583. Figure 10B shows a typical ion-pair reversed-phase (IPRP) HPLC for the annealing of SOD double-stranded ALN-1395762. Figure 10C shows a typical ion-pair reversed-phase (IPRP) HPLC for the annealing of HTT double-stranded ALN-1498524. [Figure 11] Figure 11A shows the sodium salt form of the formulation of APP double-stranded AD-961583, including its structure and molecular formula. Figure 11B shows the sodium salt form of the formulation of HTT double-stranded AD-1498524, including its structure and molecular formula. Figure 11C shows the sodium salt form of the formulation of SOD1 double-stranded AD-1395762, including its structure and molecular formula.

[0187] This disclosure is further illustrated by the detailed description below. [Modes for carrying out the invention]

[0188] This disclosure presents, at least in part, compositions for enhanced formulations of iRNA drugs for CNS-directed delivery (e.g., via intrathecal injection), as well as the resulting pharmaceutical product formulations, related methods, kits, and other compositions. For target administration of dsRNA via parenteral administration, the dsRNA must be formulated in a suitable aqueous solution. In the course of research efforts on the preparation of such formulations, it has been surprisingly discovered that, in order to avoid long-term stability problems, dsRNA having at least one lipophilic modification must be formulated by special methods. In particular, formulation of dsRNA in the presence of divalent cations (e.g., calcium, magnesium, copper, nickel, zinc, or strontium) may result in difficult-to-resolve precipitation problems. In such cases, the stoichiometry of individual strands of dsRNA in the formulation should be controlled to prevent precipitation of lipophilic molecules in aqueous solution.

[0189] The oligonucleotides used in the compositions and methods of this disclosure are double-stranded RNAs and are referred to herein as “double-stranded RNAi agents,” “double-stranded RNA (dsRNA) molecules,” “dsRNA agents,” “dsRNA,” “RNAi,” “iRNA,” or “iRNA agents.” The term “dsRNA” refers to a complex of one or more (e.g., two) ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid portions (e.g., strands) that are said to have a “sense” direction and an “antisense” direction with respect to the target RNA, i.e., the APP gene. In some embodiments of this disclosure, the double-stranded RNA (dsRNA) induces the degradation of the target RNA, i.e., mRNA, through a post-transcriptional gene expression arrest mechanism referred herein as RNA interference or RNAi. In one embodiment, the dsRNA contains two distinct strands that form a double-stranded structure. In another embodiment, the dsRNA is a single oligonucleotide having two distinct portions that form a double-stranded structure, where the two portions form part of a hairpin or dumbbell-shaped structure. Hairpin and dumbbell-shaped oligomeric compounds may have a double-stranded region of at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs or equal thereto. In some embodiments, the double-stranded region may be 200, 100, or 50 or less in length. In some embodiments, the range of the double-stranded region is 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length. In some embodiments, the hairpin oligomeric compound may have a single-stranded overhang or terminal unpaired region at 3' in some embodiments and on the antisense side of the hairpin in some embodiments. In some embodiments, the overhang is 1-4, more commonly 2-3 nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference may also be referred to herein as “shRNA”.

[0190] In addition to stoichiometric control of sense and antisense double strands, certain solutions and formulations presented herein may be substantially free of inorganic phosphate. It has been further discovered herein that nucleic acid formulations substantially free of inorganic phosphate, which are also formulated for intrathecal delivery by the addition of divalent cations (e.g., calcium), exhibit a significant reduction in granular formation in solution compared to the corresponding solutions and formulations having a substantial amount of inorganic phosphate source.

[0191] While not intending to dwell on theory, granular formation was observed in certain dsRNA formulations prepared for delivery via intrathecal injection and containing a calcium ion source. Granular formation was identified as being due to the interaction between calcium ions and excess lipophilic partial-containing strands of dsRNA (in the presence of an inorganic phosphate source as appropriate), which promotes granular / crystal formation. Since including a calcium ion source in intrathecal formulations has been shown to reduce the incidence and severity of certain adverse events (e.g., tremors, convulsions, etc.) in subjects administered nucleic acid formulations via intrathecal injection, attempts were made herein to remove the inorganic phosphate source from such nucleic acid formulations. Notably, granular-free nucleic acid formulations were discovered that reliably delivered nucleic acids when administered to subjects via intrathecal injection.

[0192] Accordingly, certain compositions of this disclosure carry an excess of non-lipophilic modified dsRNA strands compared to lipophilic modified dsRNA strands. Furthermore, some compositions substantially do not contain a source of inorganic phosphate. In certain embodiments, it is assumed that reducing granular formation in nucleic acid formulations of this disclosure can be achieved by reducing the source of inorganic phosphate in such compositions to, for example, 100 ppm or less, 50 ppm or less, or 10 ppm or less, but the compositions particularly exemplified herein do not contain a source of inorganic phosphate.

[0193] In several embodiments, the compositions of this disclosure are used for the delivery of nucleic acid drugs, such as iRNA drugs, including dsRNA, as particularly exemplified herein. The level of nucleic acid drug contained in the compositions of this disclosure may be, for example, in the range of about 5 mg / mL to about 300 mg / mL. In relevant embodiments, the composition contains about 10 mg / mL to about 200 mg / mL of nucleic acid drug. In further embodiments, the composition contains about 20 mg / mL to about 100 mg / mL of nucleic acid drug. In one embodiment, the composition contains about 40 mg / mL to about 80 mg / mL of nucleic acid drug. The composition may also contain about 60 mg / mL of nucleic acid drug.

[0194] The compositions of this disclosure include physiologically significant ionic species, such as Na + , K + Mg 2+ Cl - , or Ca 2+ The composition may include several salts that provide sources such as sodium chloride, potassium chloride, magnesium chloride, and calcium chloride. The composition may further include, but is not limited to, other trace elements and their salts, including, selenium, copper, chromium, iodine, fluoride, zinc, manganese, molybdenum, and iron.

[0195] Sodium ions are included in the formulations of this disclosure at relatively high concentrations, taking into account at least part of their role in normal physiological functions. + ¹⁴ is the primary cation in extracellular fluid. This ion plays a crucial role in many physiological processes, including blood volume control, blood pressure, osmotic equilibrium, pH, and nerve impulse generation.

[0196] Potassium ions are the main cations in intracellular fluid, and together with sodium ions in extracellular fluid, K + It is the primary source of electrical potentials that cross cell membranes. Therefore, it plays a crucial role in normal function and is involved in bodily functions such as neurotransmission, muscle contraction, and cardiac function.

[0197] Calcium ions are equally important for many physiological processes. In particular, Ca 2+ Ions are one of the most widespread secondary signaling molecules used in signal transduction. Within endothelial cells, Ca 2+ Ions may control several signaling pathways that cause relaxation of the smooth muscle surrounding blood vessels. 2+ Dysfunction within the activation pathway may lead to increased tension caused by uncontrolled smooth muscle contraction. This type of dysfunction may be observed in cardiovascular disease, hypertension, and diabetes. The compositions of this disclosure contain Ca 2+ It has also been identified that including ions can mitigate certain adverse events (e.g., tremors, seizures, and other neurological problems) observed in subjects who receive calcium-free or low-calcium nucleic acid preparations via intrathecal injection.

[0198] Magnesium ions are used in relatively high concentrations during normal metabolism. Magnesium deficiency is considered rare, except when accompanied by severe loss of other electrolytes, such as vomiting and diarrhea. However, deficiency of this ion is frequently recognized in modern diets and is associated with symptoms such as muscle tremors and weakness. This inorganic substance is important in many enzymatic reactions and stabilizes excitable membranes. When administered intravenously, magnesium may have an anesthetic effect, which indirectly provides evidence of its action on vascular wall endothelial components to stabilize and normalize the surface of blood vessel walls.

[0199] In some embodiments, the compositions of the present disclosure contain sodium ions (Na) at concentrations of 0.1 mM to 1 M. + ) includes a source (for example, provided as sodium chloride). In related embodiments, Na + The source exists at a concentration of approximately 40 mM to 300 mM. + The source may be present at a concentration of approximately 70 mM to approximately 200 mM. In related embodiments, Na +The source is present at a concentration of approximately 80 mM to approximately 120 mM. In related embodiments, Na + The source is present at a concentration of approximately 80 mM to 110 mM. In related embodiments, Na + The source is present at a concentration of approximately 85 mM to approximately 100 mM. In related embodiments, Na + The source is present at a concentration of approximately 90 mM to 100 mM. In an alternative embodiment, Na + The source is present at a concentration of approximately 90 mM to 105 mM, and optionally approximately 95 mM to 100 mM. In related embodiments, Na + The source is present at a concentration of approximately 97 mM to 98 mM. In one embodiment, Na + The source is present at a concentration of approximately 97.6 mM. In one embodiment, Na + The source is present at a concentration of approximately 97.0 mM. In one embodiment, Na + The source is present at a concentration of approximately 89.5 mM. In one embodiment, Na + The source is present at a concentration of 97.6 mM. In one embodiment, Na + The source exists at a concentration of 97.56 mM.

[0200] In certain embodiments, the composition of the Disclosure contains calcium ions (Ca) at concentrations of 0.1 mM to 1 M. 2+ ) includes a source (for example, provided as calcium chloride). In some embodiments, Ca 2+ The source exists at concentrations ranging from approximately 0.1 mM to approximately 200 mM. In further embodiments, Ca 2+ The source exists at a concentration of approximately 0.5 mM to 100 mM. Ca 2+ The source may be present at a concentration of approximately 0.8 mM to approximately 50 mM. In some embodiments, Ca 2+ The source is present at a concentration of approximately 1 mM to approximately 25 mM. In some embodiments, Ca 2+ The source is present at a concentration of approximately 2 mM to approximately 20 mM. In some embodiments, Ca 2+ The source is present at a concentration of approximately 8 mM to approximately 20 mM. In some embodiments, Ca 2+ The source exists at a concentration of approximately 5 mM to 15 mM. Alternatively, Ca 2+The source is present at a concentration of approximately 10 mM to 15 mM. In some embodiments, Ca 2+ The source is present at a concentration of approximately 12 mM to 15 mM. In some embodiments, Ca 2+ The source is present at a concentration of approximately 12 mM to 14 mM. In some embodiments, Ca 2+ The source exists at a concentration of approximately 12.5 mM to 13.5 mM. Ca 2+ The source may be present at concentrations of approximately 12.5 mM, 12.75 mM, 12.9 mM, 13.0 mM, 13.25 mM, 13.5 mM, or 13.9 mM. In certain embodiments, Ca 2+ The source is present at a concentration of approximately 13.0 mM. In a particular embodiment, Ca 2+ The source exists at a concentration of 13.0 mM.

[0201] In some embodiments, the compositions of the present disclosure contain potassium ions (K) at a concentration of 0.0 mM to 1 M. + ) includes a source (for example, provided as potassium chloride). In some embodiments, K + The source is present at a concentration of approximately 0.1 mM to approximately 100 mM. In further embodiments, K + The source is present at a concentration of approximately 0.2 mM to approximately 40 mM. In further embodiments, K + The source is present at a concentration of approximately 0.5 mM to approximately 20 mM. In some embodiments, K + The source is present at a concentration of approximately 1 mM to approximately 5 mM. In some embodiments, K + The source is present at a concentration of approximately 1 mM to approximately 4 mM. In some embodiments, K + The source is present at a concentration of approximately 1 mM to approximately 3 mM. In some embodiments, K + The source is present at a concentration of approximately 1.0 mM to approximately 2.5 mM. In some embodiments, K + The source is present at a concentration of approximately 1.5 mM to approximately 2.5 mM. In some embodiments, K + The source exists at a concentration of approximately 1.5 mM to 2.2 mM. +The source may be present at concentrations of approximately 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, or 3.0 mM. In certain embodiments, K + The source is present at a concentration of approximately 1.9 mM. In a particular embodiment, K + The source exists at a concentration of 1.9 mM.

[0202] In certain embodiments, the composition of the Disclosure contains magnesium ions (Mg) at a concentration of 0.0 mM to 1 M. 2+ ) Includes a source (for example, provided as magnesium chloride). In some embodiments, Mg 2+ The source is present at concentrations ranging from approximately 0.01 mM to approximately 100 mM. In further embodiments, Mg 2+ The source is present at concentrations ranging from approximately 0.1 mM to approximately 40 mM. In further embodiments, Mg 2+ The source is present at concentrations of approximately 0.2 mM to approximately 20 mM. In some embodiments, Mg 2+ The source is present at a concentration of approximately 0.3 mM to approximately 5 mM. In some embodiments, Mg 2+ The source is present at a concentration of approximately 0.3 mM to approximately 4 mM. In some embodiments, Mg 2+ The source is present at a concentration of approximately 0.3 mM to approximately 3 mM. In some embodiments, Mg 2+ The source is present at a concentration of approximately 0.3 mM to approximately 2 mM. In some embodiments, Mg 2+ The source is present at a concentration of approximately 0.3 mM to approximately 1 mM. In some embodiments, Mg 2+ The source is present at a concentration of approximately 0.1 mM to approximately 1 mM. In some embodiments, Mg 2+ The source is present at a concentration of approximately 0.3 mM to approximately 0.7 mM. In some embodiments, Mg 2+ The source is present at a concentration of approximately 0.3 mM to approximately 0.6 mM. In some embodiments, Mg 2+ The source exists at a concentration of approximately 0.4 mM to 0.6 mM. (Mg) 2+The source may be present at concentrations of approximately 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, or 0.9 mM. In certain embodiments, Mg 2+ The source is present at a concentration of approximately 0.5 mM. In further embodiments, Mg 2+ The source exists at a concentration of 0.5 mM. Alternatively, Mg 2+ The source exists at a concentration of 0.52 mM.

[0203] In some embodiments, the compositions of the Disclosure have a molar ratio of divalent cation source to nucleic acid agent greater than about 2:1. In certain embodiments, the compositions of the Disclosure have a molar ratio of divalent cation source to nucleic acid agent greater than about 2.5:1. In further embodiments, the compositions of the Disclosure have a molar ratio of divalent cation source to nucleic acid agent greater than about 3:1. In other embodiments, the compositions of the Disclosure have a molar ratio of divalent cation source to nucleic acid agent greater than about 3.5:1. In related further embodiments, the compositions of the Disclosure have a molar ratio of divalent cation source to nucleic acid agent greater than about 4:1. In certain related embodiments, the compositions of the Disclosure have a molar ratio of divalent cation source to nucleic acid agent between about 2:1 and about 10:1. The compositions of the Disclosure may have a molar ratio of divalent cation source to nucleic acid agent between about 3:1 and about 10:1. The compositions of the Disclosure may have a molar ratio of divalent cation source to nucleic acid agent between about 3:1 and about 9:1. The compositions of this disclosure may have a molar ratio of divalent cation source to nucleic acid agent of about 3:1 to about 8:1. The compositions of this disclosure may have a molar ratio of divalent cation source to nucleic acid agent of about 3:1 to about 7:1. The compositions of this disclosure may have a molar ratio of divalent cation source to nucleic acid agent of about 3:1 to about 6:1. The compositions of this disclosure may have a molar ratio of divalent cation source to nucleic acid agent of about 3:1 to about 5:1.

[0204] In some embodiments of the solutions presented herein, the pH of the solution is 4 to 10, and optionally 6 to 10. In related embodiments, the pH of the solution is about 6.5 to about 8.0. In further embodiments, the pH of the solution is 6.5 to 7.8. In certain embodiments of the solutions presented herein, the pH of the solution is 6.7 to 7.5. In some embodiments, the pH of the solution is 6.8 to 7.2. In further embodiments, the pH of the solution is about 6.9.

[0205] In some embodiments of the compositions presented herein, the aqueous solutions of the disclosure have an osmotic pressure of about 100 to 500 mOsm / kg. In further embodiments, the aqueous solutions of the disclosure have an osmotic pressure of about 200 to 400 mOsm / kg.

[0206] In certain embodiments, the compositions presented herein (i.e., aqueous compositions) can be stored at 25°C for at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month without a measurable precipitation of the solute and / or a measurable loss of the ability to induce knockdown of a target gene in a subject administered such a solution via intrathecal injection. In some embodiments, the compositions presented herein can be stored at 2–8°C for at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month without measurable precipitation of solutes and / or measurable loss of ability to induce knockdown of target genes in subjects administered such solutions via intrathecal injection.

[0207] Pharmaceutical compositions of the present disclosure This disclosure presents pharmaceutical compositions and formulations comprising RNAi agents described herein (e.g., AD-454973, AD-454843, AD-961583, AD-961584, AD-961585, AD-961586, AD-454844, AD-1302922, AD-1302923, or AD-1999409, etc.). However, it is expressly assumed that the formulations of this disclosure may be used for the delivery of any RNAi agent. In one embodiment, a pharmaceutical composition comprising an RNAi agent as described herein and a pharmaceutically acceptable carrier is presented herein. Pharmaceutical compositions containing RNAi agents are useful for treating diseases or disorders associated with the expression or activity of genes (e.g., APP, etc.), such as disorders such as Alzheimer's disease.

[0208] The pharmaceutical compositions of this disclosure can be administered in doses sufficient to inhibit the expression of the APP gene. Generally, preferred doses of the RNAi agents of this disclosure range from about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient per day, and generally range from about 1 to 50 mg per kilogram of body weight per day. For example, dsRNA can be administered in doses of about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg.

[0209] For example, the dsRNAs disclosed herein are approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, It may be administered in doses of 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or approximately 10 mg / kg. Intermediate values ​​and ranges between the listed values ​​are also intended to be part of this disclosure.

[0210] In another embodiment, dsRNA is approximately 0.1 to 50 mg / kg, approximately 0.25 to 50 mg / kg, approximately 0.5 to 50 mg / kg, approximately 0.75 to 50 mg / kg, approximately 1 to 50 mg / mg, approximately 1.5 to 50 mg / kb, approximately 2 to 50 mg / kg, approximately 2.5 to 50 mg / kg, approximately 3 to 50 mg / kg, approximately 3.5 to 50 mg / kg, approximately 4 to 50 mg / kg, approximately 4.5 to 50 mg / kg, approximately 5 to 50 mg / kg, approximately 7.5 to 50 mg / kg, approximately 10 to 50 mg / kg, approximately 15 to 50 mg / kg, approximately 20 to 50 mg / kg, approximately 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.1 to about 45 mg / kg, about 0.25 to about 45 mg / kg, about 0. 5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / mg, about 1.5 to about 45 mg / kb, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / mg, about 1.5 to about 40 mg / kb, about 2 to about 40 mg / kg kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30mg / kg, about 5 to about 30mg / kg, about 7.5 to about 30mg / kg, about 10 to about 30mg / kg, about 15 to about 30mg / kg, about 20 to about 30mg / kg, about 20 to about 30mg / kg, about 25 to about 30mg / kg, about 0.1 to about 20m It is administered in doses of g / kg, approximately 0.25–20 mg / kg, approximately 0.5–20 mg / kg, approximately 0.75–20 mg / kg, approximately 1–20 mg / kg, approximately 1.5–20 mg / kg, approximately 2–20 mg / kg, approximately 2.5–20 mg / kg, approximately 3–20 mg / kg, approximately 3.5–20 mg / kg, approximately 4–20 mg / kg, approximately 4.5–20 mg / kg, approximately 5–20 mg / kg, approximately 7.5–20 mg / kg, approximately 10–20 mg / kg, or approximately 15–20 mg / kg. Intermediate values ​​and ranges between the listed values ​​are also intended to be part of this disclosure.

[0211] For example, dsRNA is approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5 It may be administered in doses of 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or approximately 10 mg / kg. Intermediate values ​​and ranges between the listed values ​​are also intended to be part of this disclosure.

[0212] In another embodiment, dsRNA is present in concentrations of approximately 0.5 to 50 mg / kg, 0.75 to 50 mg / kg, 1 to 50 mg / kg, 1.5 to 50 mg / kb, 2 to 50 mg / kg, 2.5 to 50 mg / kg, 3 to 50 mg / kg, 3.5 to 50 mg / kg, 4 to 50 mg / kg, 4.5 to 50 mg / kg, 5 to 50 mg / kg, 7.5 to 50 mg / kg, 10 to 50 mg / kg, 15 to 50 mg / kg, 20 to 50 mg / kg, 20 to 50 mg / kg, 25 to 50 mg / kg, and 25 to 50 mg / kg. g / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / mg, about 1.5 to about 45 mg / kb, about 2 to about 45 mg / kg, About 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 4 5 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / m g, about 1.5 to about 40 mg / kb, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about Approximately 40 mg / kg, approximately 15 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 30 to approximately 40 mg / kg, approximately 35 to approximately 40 mg / kg, approximately 0.5 to approximately 30 mg / kg, approximately 0.75 to approximately 30 m g / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.It is administered in doses of approximately 5-30 mg / kg, approximately 10-30 mg / kg, approximately 15-30 mg / kg, approximately 20-30 mg / kg, approximately 20-30 mg / kg, approximately 25-30 mg / kg, approximately 0.5-20 mg / kg, approximately 0.75-20 mg / kg, approximately 1-20 mg / kg, approximately 1.5-20 mg / kg, approximately 2-20 mg / kg, approximately 2.5-20 mg / kg, approximately 3-20 mg / kg, approximately 3.5-20 mg / kg, approximately 4-20 mg / kg, approximately 4.5-20 mg / kg, approximately 5-20 mg / kg, approximately 7.5-20 mg / kg, approximately 10-20 mg / kg, or approximately 15-20 mg / kg. Numbers and ranges that fall between the enumerated numbers are also intended to be part of this disclosure.

[0213] For example, the target population includes therapeutic doses such as approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5 RNAi agents such as 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or approximately 50 mg / kg can be administered. Intermediate values ​​and ranges between the listed values ​​are also intended to be part of this disclosure.

[0214] The pharmaceutical composition may be administered once daily, or the RNAi agent may be administered as two, three, or more subdoses at appropriate intervals throughout the day, or by continuous infusion or delivery via a controlled-release formulation. In this case, the amount of RNAi agent in each subdose must be correspondingly smaller in order to achieve the total daily dose. The administration unit may also be formulated to be delivered over several days, for example, using a conventional sustained-release formulation in which iRNA is released over a period of several days. Sustained-release formulations are well known in the art and are particularly useful for delivering drugs to specific sites and may be used, for example, with the drugs of this disclosure. In this embodiment, the administration unit contains a number of corresponding daily doses.

[0215] The effect of a single dose on APP levels may be long-lasting, for example, subsequent doses may be administered at intervals of 3, 4, or 5 days or less, or at intervals of 1, 2, 3, or 4 weeks or less, or at intervals of 1, 2, 3, 4, 5, or 6 months or less.

[0216] Such pharmaceutical compositions are formulated based on a delivery method. The formulations / pharmaceutical compositions disclosed herein are primarily formulated for injection, and for certain uses, for direct delivery into the CNS, such as by intrathecal or intravitreous injection, by infusion into the brain (e.g., the striatum), or by continuous pump infusion, etc. However, certain compositions disclosed herein may also be formulated for systemic administration via parenteral delivery, such as intravenous (IV) delivery, intramuscular (IM) delivery, or subcutaneous (subQ) delivery.

[0217] In certain embodiments, the pharmaceutical compositions of the Disclosure are substantially free of inorganic phosphoric acid. In some embodiments, the molar ratio of the nucleic acid therapeutic of the Disclosure in a divalent ion source versus formulation is greater than about 2:1.

[0218] In certain embodiments, the pharmaceutical compositions of this disclosure are pyrogen-free or non-pyrogenic.

[0219] The pharmaceutical compositions of this disclosure may be administered in doses sufficient to inhibit the expression of a target gene. Generally, preferred doses of the nucleic acid therapeutics of this disclosure are in the range of about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient per day, and generally in the range of about 1 to 50 mg per kilogram of body weight per day.

[0220] Repeated-dose regimens may include periodic administration of therapeutic doses of RNAi agents, for example, once a month to once every six months. In certain embodiments, RNAi agents are administered approximately once every quarter (i.e., approximately once every three months) to approximately twice a year.

[0221] After the initial treatment regimen (e.g., dose loading), treatment may be administered at a lower frequency.

[0222] In other embodiments, a single dose of the pharmaceutical composition may be long-lasting, for example, subsequent doses may be administered at intervals of 1, 2, 3, 4, 5, or 6 months or less, or longer. In some embodiments of the Disclosure, a single dose of the pharmaceutical composition of the Disclosure is administered once a month. In other embodiments of the Disclosure, a single dose of the pharmaceutical composition of the Disclosure is administered once a quarter to twice a year.

[0223] Those skilled in the art will recognize that certain factors, including but not limited to the severity of the disease or disability, prior treatment, the subject's overall health status or age, and the presence or absence of other diseases, may influence the dosage and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective amount of the composition may consist of a single treatment or a series of treatments.

[0224] The pharmaceutical compositions of this disclosure are primarily formulated for CNS delivery, administered via intracranial routes, for example, by intrathecal, intraparenchymal, or intraventricular administration.

[0225] RNAi drug formulations can be delivered to target specific tissues in the CNS (e.g., neuronal, glial, or vascular tissue in the brain), or both non-CNS organs (e.g., the liver) and the CNS.

[0226] Other formulations conforming to this disclosure (when prepared substantially without a source of inorganic phosphoric acid) are described in U.S. Provisional Patent Applications No. 61 / 018,616, filed 2 January 2008; No. 61 / 018,611, filed 2 January 2008; No. 61 / 039,748, filed 26 March 2008; No. 61 / 047,087, filed 22 April 2008; and No. 61 / 051,528, filed 8 May 2008. PCT Application No. PCT / US 2007 / 080331, filed 3 October 2007, also describes formulations conforming to this disclosure (when prepared substantially without a source of inorganic phosphoric acid).

[0227] The formulations described herein are intended to function with a wide variety of additional components, provided that such additional components do not impair efficacy / functionality of the pharmaceutical product. Examples of such additional components include, but are not limited to, fats, oils, waxes, fatty acids, aliphatic alcohols, aliphatic esters, water-retaining substances, hydrophilic colloids, preservatives, and antioxidants [Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199], as well as glucose and other sugar / carbon sources.

[0228] Suitable compositions and formulations for parenteral administration include, but are not limited to, those suitable for intraparenchymal administration [in the brain, e.g., intracerebral rebrovascular], intrathecal administration [e.g., lumbar puncture (LP) or intracisterna magna (ICM) injection], intradiscal administration, periganglionic administration, and / or intraventricular administration, as well as sterile aqueous solutions that may also contain buffers, diluents, and other suitable additives, such as, but are not limited to, permeability enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients. In certain embodiments herein, the compositions and formulations suitable for parenteral administration do not contain buffering components (e.g., phosphoric acid).

[0229] Examples of pharmaceutical compositions of this disclosure include, but are not limited to, solutions and emulsions. These compositions can be produced from a variety of components, including, but not limited to, pre-formed liquids.

[0230] Pharmaceutical formulations of this disclosure, which can be conveniently provided as unit-dose dosage forms, can be prepared based on prior art well known in the pharmaceutical industry. Such art involves associating an active ingredient with a pharmaceutical carrier(s) or excipient(s). Generally, formulations are prepared by homogeneously and tightly associating an active ingredient with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0231] The compositions of this disclosure may be formulated as suspensions in aqueous, non-aqueous, or mixed media. The aqueous suspensions may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. The suspensions may also contain stabilizers.

[0232] Excipients In contrast to carrier compounds, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspension, or any other pharmaceutically inert medium for delivering one or more nucleic acids to an animal. Excipients may be liquid or solid and are selected with the planned administration method in mind, so as to yield the desired bulk, consistency, etc., when integrated with the nucleic acid and other components of a given pharmaceutical composition. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0233] Suitable pharmaceutically acceptable excipients include, but are not limited to, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0234] Other Rules The compositions of this disclosure may additionally contain other additional components conventionally found in pharmaceutical compositions at levels of use established in the art. For example, the compositions may contain additional, compatible, pharmaceutically active substances, such as antipruritics, astringents, topical anesthetics, or anti-inflammatory agents. However, such substances, when added, must not unduly interfere with the biological activity of the components of the compositions of this disclosure, nor, in the case of this disclosure, provide a recognizable source of inorganic phosphoric acid. The formulations are sterilizable and, if desired, miscible with adjuvants, such as preservatives, stabilizers, emulsifiers, salts to affect osmotic pressure, colorants, flavorings, or aromatics, which do not cause adverse interactions with the nucleic acids of the formulation.

[0235] The aqueous suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. The suspension may also contain stabilizers.

[0236] In some embodiments, the pharmaceutical compositions studied herein include (a) one or more RNAi agents, and (b) one or more agents that function by a non-RNAi mechanism and are useful in treating a disease or disorder, such as APP-related neurodegenerative diseases. Examples of such agents include, but are not limited to, dopamine agonists and promoters, including, carbidopa-levodopa, levodopa, entacopon, tolcapone, opicapone, pramipexole, ropinirole, apomorphine, rotigotine, selegiline, rasagiline, safinamide, amantadine, istradefylline, trihexyphenidyl, benztropine, rivastigmine, donepezil, galantamine, and memantine.

[0237] The toxicity and therapeutic effects of such compounds are, for example, LD 50 (A lethal dose for 50% of the population) and ED 50 This can be determined in cell cultures or experimental animals using standard pharmaceutical procedures to determine the therapeutically effective dose (the dose effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, or LD50. 50 / ED 50 It can be expressed as a ratio. Compounds exhibiting a high therapeutic index are preferred.

[0238] Data obtained from cell culture assays and animal studies can be used when formulating a range of dosages for human use. The dosages of the compositions discussed in this disclosure are generally ED 50 The circulating concentration range is within the range of the substance and has little to no toxicity. The dosage may vary within this range depending on the dosage form adopted and the route of administration used. For any compound used in the methods described herein, the therapeutically effective dose may be initially estimated from a cell culture assay. The dose is determined in cell culture such as IC for the compound, or, where appropriate, for polypeptide products of the target sequence (e.g., achieving a reduction in polypeptide concentration).50 The circulating plasma concentration range can be formulated in animal models to achieve a range including (i.e., the concentration of the test compound that achieves half of the maximum inhibitory effect on the symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0239] As discussed above, in addition to its administration, the RNAi drug compositions described herein can be administered in combination with other known drugs effective in treating diseases or disorders. In any case, the administering physician may adjust the amount and timing of RNAi drug administration based on observed results, using standard efficacy indicators that are publicly known in the art or described herein.

[0240] Nucleic Acid Therapeutics as disclosed herein Certain aspects of this disclosure provide formulations suitable for delivering nucleic acid therapeutics to the CNS via intrathecal injection or direct intracerebral injection in certain embodiments. While RNAi agents are particularly exemplified, the compositions disclosed herein are expressly assumed to be employable for delivering a wide range of nucleic acid therapeutics to the CNS, optimally via injection. Exemplary nucleic acid therapeutics include, but are not limited to, dsRNA (e.g., siRNA), antisense oligonucleotides, decoys, miRNA, shRNA, guide RNA (gRNA), and ribozymes.

[0241] RNAi drugs dsRNA contains an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during the expression of the target gene. The complementary region is approximately 30 nucleotides or less in length (e.g., approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). When in contact with cells expressing the target gene, and assayed using methods such as PCR or branched DNA (bDNA), or by protein-based methods such as immunofluorescence analysis, or by techniques such as Western blotting or flow cytometry, the RNAi agent inhibits the expression of the target gene (e.g., the APP gene in humans, primates, non-primates, or birds) by at least approximately 10%.

[0242] A dsRNA contains two complementary RNA strands that hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementarity region that is substantially complementary to the target sequence and is generally fully complementary. The target sequence may be derived from the sequence of mRNA formed during the expression period of a target gene (e.g., the APP gene). The other strand (the sense strand) contains a region complementary to the antisense strand, and the two strands hybridize to form a double-stranded structure when combined, for example, under favorable conditions. As otherwise described herein and as known in the art, the complementary sequence of the dsRNA may also be included as a self-complementarity region of a single nucleic acid molecule, rather than being located on different oligonucleotides.

[0243] Generally, double-stranded structures are 15-30 base pairs long, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 1 These are 9-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In certain preferred embodiments, the double-stranded structures are of lengths 18 and 25 base pairs, for example, 18–25, 18–24, 18–23, 18–22, 18–21, 18–20, 19–25, 19–24, 19–23, 19–22, 19–21, 19–20, 20–25, 20–24, 20–23, 20–22, 20–21, 21–25, 21–24, 21–23, 21–22, 22–25, 22–24, 22–23, 23–25, 23–24, or 24–25 base pairs. Ranges and lengths intermediate to those listed above are also considered part of this disclosure.

[0244] Similarly, complementary regions to the target sequence are 15-30 nucleotides in length, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19- These ranges are 30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Ranges and lengths that fall between the ranges and lengths listed above are also considered part of this disclosure.

[0245] In some embodiments, the dsRNA is approximately 15 to 23 nucleotides in length, or approximately 25 and 30 nucleotides in length. Generally, the dsRNA is long enough to function as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than approximately 21 to 23 nucleotides can function as a substrate for Dicer. Also, as those skilled in the art will also recognize, the RNA region targeted for cleavage is very often a larger RNA molecule, often a part of an RNA molecule. Where applicable, a “part” of an mRNA target is a sequence of sufficient length of mRNA target that can serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0246] Those skilled in the art will know that a double-stranded region is the main functional part of dsRNA, for example, a double-stranded region of about 9 to 36 base pairs, for example, about 10 to 36, 11 to 36, 12 to 36, 13 to 36, 14 to 36, 15 to 36, 9 to 35, 10 to 35, 11 to 35, 12 to 35, 13 to 35, 14 to 35, 15 to 35, 9 to 34, 10 to 34, 11 to 34, 12 to 34, 13 to 34, 14 to 3 4, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24 It is also recognized that the double-stranded region is 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Therefore, in one embodiment, a dsRNA is an RNA molecule or complex of RNA molecules having a double-stranded region of more than 30 base pairs, processed into a functional double helix of, for example, 15-30 base pairs, to the extent that it targets the RNA of interest for cleavage. Therefore, those skilled in the art recognize that in one embodiment, miRNA is dsRNA. In another embodiment, dsRNA is not a naturally occurring miRNA. In another embodiment, RNAi agents useful for targeting the expression of a target gene (e.g., APP) are not generated within the target cell due to the cleavage of larger dsRNAs.

[0247] The dsRNAs described herein may further comprise one or more single-stranded nucleotide overhangs, e.g., 1, 2, 3, or 4 nucleotides. A dsRNA having at least one nucleotide overhang may exhibit unexpectedly superior inhibitory properties compared to its blunt-terminated counterpart. The nucleotide overhang may comprise or consist of a nucleotide / nucleoside analog containing a deoxynucleotide / nucleoside. The overhang(s) may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of the overhang may be located on the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0248] dsRNA can be synthesized by standard methods known in the art, as will be further discussed below, for example, by using automated DNA synthesizers, such as those commercially available from Biosearch, Applied Biosystems, Inc.

[0249] The RNAi agents of this disclosure may be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared individually. Next, the component strands are annealed. The individual strands of the siRNA compound may be prepared using solution-phase synthesis, solid-phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide strands containing non-natural or modified nucleotides can be readily prepared. The single-stranded oligonucleotides of this disclosure may be prepared using solution-phase synthesis, solid-phase organic synthesis, or both.

[0250] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence may be selected from the group of sequences presented in any one of Tables 16-19, and the corresponding nucleotide sequence of the antisense strand to the sense strand may be selected from the group of sequences in any one of Tables 16-19. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the mRNA sequence produced during APP gene expression. Accordingly, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide described as the sense strand (passenger strand) in any one of Tables 16-19, and the second oligonucleotide described as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 16-19. Accordingly, as an example, the following pairwise selections of sense and antisense strand sequences in Table 16: SEQ ID NOs. 1 and 8; SEQ ID NOs. 2 and 9; SEQ ID NOs. 3 and 10; SEQ ID NOs. 4 and 11; SEQ ID NOs. 5 and 12; SEQ ID NOs. 6 and 13; and SEQ ID NOs. 7 and 14 are explicitly assumed to form the double strands of this disclosure. Similarly, pairwise combinations of sense and antisense strands in Table 17 of this disclosure are also explicitly assumed, including, for example, a sense strand selected from Table 16 together with an antisense strand selected from Table 17, or vice versa.

[0251] In one embodiment, substantially complementary sequences of dsRNA are contained on different oligonucleotides. In another embodiment, substantially complementary sequences of dsRNA are contained on a single oligonucleotide.

[0252] Although the sequences in Tables 16 and 18 are described as modified and / or conjugated sequences, it is understood that the RNA of the RNAi drug of this disclosure, for example, the dsRNA of this disclosure, may include any one of the sequences specified in Tables 16 or 18, which are unmodified, unconjugated, and / or modified and / or conjugated, different from those described in the tables.

[0253] Those skilled in the art are well aware that dsRNAs having a double-stranded structure consisting of approximately 20–23 base pairs, for example, 21 base pairs, have been recognized as particularly effective in inducing RNA interference [Elbashir et al., (2001) EMBO J., 20:6877-6888]. However, it has been found that shorter or longer RNA double-stranded structures may also be effective [Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226]. In the embodiments described herein, due to the benefits of the properties of the oligonucleotide sequences presented herein, the dsRNAs described herein may include at least one strand having a minimum length of 21 nucleotides. It can be reasonably expected that shorter double-stranded structures, with only a few nucleotides removed from one or both ends, may be equally effective compared to the dsRNAs described above. Accordingly, dsRNAs having a sequence consisting of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from one of the sequences presented herein, and whose ability to inhibit the expression of a target gene does not differ in inhibition rate from dsRNAs containing the complete sequence by approximately 5, 10, 15, 20, 25, or 30%, are considered to be within the scope of this disclosure.

[0254] In addition, the exemplary RNAs described herein identify sites(s) within the APP transcript that are susceptible to RISC-mediated cleavage. Therefore, this disclosure further features RNAi agents that target these sites(s). As used herein, an RNAi agent is said to target a specific site within the RNA transcript if it promotes cleavage of the transcript at any of these specific sites. Such RNAi agents typically comprise at least about 15 consecutive nucleotides derived from one of the sequences presented herein, coupled with an additional nucleotide sequence taken from a region adjacent to a selected sequence within the APP gene.

[0255] The RNAi agents described herein may contain one or more mismatches with respect to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches with respect to the target sequence, the mismatches may be limited, as appropriate, to the last five nucleotides derived from either the 5' or 3' end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the APP gene region typically contains no mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an RNAi agent containing mismatches with respect to the target sequence is effective in inhibiting APP gene expression. In particular, if it is known that a specific complementary region within the APP gene exhibits polymorphic sequence variation within the population, it is important to investigate the effectiveness of RNAi agents containing mismatches in inhibiting APP gene expression.

[0256] This disclosure also presents a method for preparing a formulation, comprising annealing a sense strand and an antisense strand, vacuum lyophilizing the double-stranded solution to provide a double-stranded composition, and dissolving the double-stranded composition in an injection solution, wherein one of the sense strand and the antisense strand contains a lipophilic modification to form a double-stranded solution containing double-stranded RNA (dsRNA), the injection solution contains a divalent cation source (e.g., calcium) and does not contain phosphate buffer, and the double-stranded composition contains an antisense strand in a molar excess of 0-5% relative to the sense strand (e.g., about 1-2% molar excess). “Double-stranded solution” means any solution containing double-stranded RNA (dsRNA). “Double-stranded composition” means any composition containing dsRNA. In some embodiments, the double-stranded composition is prepared by vacuum lyophilization. In one embodiment, the double-stranded composition is a vacuum lyophilized powder. “Injection solution” is any solution used to dissolve the double-stranded composition. In some embodiments, the injection solution includes a divalent cation source. The divalent cation source is calcium, magnesium, copper, nickel, zinc, or strontium, and the divalent ion source may be calcium. In one embodiment, the injection solution does not contain phosphate buffer. In some embodiments, the double-stranded composition includes antisense chains in amounts of about 1-2% molar excess, 1-3% molar excess, 1-4% molar excess, 1-5% molar excess, 0-5% molar excess, 0-1% molar excess, 2-3% molar excess, 3-4% molar excess, 3-5% molar excess, 2-4% molar excess, and 2-5% molar excess relative to the sense chain. In one embodiment, the double-stranded composition includes antisense chains in an amount of about 1-2% molar excess relative to the sense chain. In another embodiment, the amount of antisense chains in excess of the sense chain is less than 1%.

[0257] The modified RNAi agents and other modified nucleic acid therapeutics disclosed herein In one embodiment, the RNA, e.g., dsRNA, of the nucleic acid therapeutic agent of the Disclosure (e.g., RNAi agent) is unmodified and, for example, known in the Art, and does not include the chemical modifications and / or conjugations described herein. In another embodiment, the RNA, e.g., dsRNA, of the nucleic acid therapeutic agent of the Disclosure is chemically modified to enhance stability or other beneficial properties. In certain embodiments of the Disclosure, substantially all of the nucleotides of the nucleic acid therapeutic agent of the Disclosure are modified. In other embodiments of the Disclosure, all of the nucleotides of the nucleic acid therapeutic agent of the Disclosure are modified. A nucleic acid therapeutic agent of the Disclosure in which "substantially all of the nucleotides are modified" may generally, but not all, be modified and may contain 5, 4, 3, 2 or fewer, or 1 unmodified nucleotide. In yet another embodiment of the Disclosure, the nucleic acid therapeutic agent of the Disclosure may contain 5, 4, 3, 2 or fewer, or 1 modified nucleotide.

[0258] The nucleic acids discussed herein may be synthesized and / or modified by well-established methods in the art, such as those described herein in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as modifications of the 5' end (phosphorylation, conjugation, reverse bond) or 3' end (conjugation, DNA nucleotide, reverse bond, etc.); base modifications, such as substitution with a stabilizing base, a destabilizing base, or a base that forms a base pair with a partly extended partner, base removal (debasing nucleotide), or base conjugation; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and / or skeleton modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of nucleic acid therapeutics useful in the embodiments described herein include, but are not limited to, RNA containing a modified skeleton or lacking natural internucleoside bonds. RNA having a modified skeleton includes, in particular, RNA that does not have a phosphorus atom in its skeleton. For the purposes of this specification and as is sometimes cited in the art, modified RNA that does not have a phosphorus atom in its internucleoside skeleton can also be considered an oligonucleoside. In some embodiments, the modified nucleic acid therapeutic has a phosphorus atom in its internucleoside skeleton.

[0259] Modified RNA skeletons that do not contain a phosphorus atom have skeletons formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatom and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatom nucleoside bonds or heterocyclic nucleoside bonds. These include skeletons with morpholino bonds (partially formed from the sugar portion of the nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and other skeletons having mixed N, O, S, and CH2 component portions.

[0260] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, and 5,470,967. This includes, but is not limited to, the following publications: Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439. The entire contents of each publication are incorporated herein by reference.

[0261] Modified RNAs can also contain one or more substituted sugar moieties. Nucleic acid therapeutics, such as the dsRNAs discussed herein, can contain at the 2'-position one of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 alkyl, or C2-C 10 alkenyl, and alkynyl. Suitable exemplary modifications include O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are 1 to about 10. In other embodiments, the dsRNA is C1-C 10The 2' position contains one of the following substituents: lower alkyl, substituted lower alkyl, alkali, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of RNAi drugs, or group for improving the pharmacodynamic properties of RNAi drugs, and other substituents having similar properties. In some embodiments, as a modification, 2'-methoxyethoxy[2'-O-(2-methoxyethyl) or 2'-MOE also known as 2'-O-CH2CH2OCH3](Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., containing an alkoxy-alkoxy group. Another exemplary modification includes 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the following examples herein, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-C H2-O-CH2-N(CH2)2 [original: 2'-O--CH2--O--CH2--N(CH2)2]. Further exemplary modifications include 5'-Me-2'-F nucleotide, 5'-Me-2'-OMe nucleotide, 5'-Me-2'-deoxynucleotide (both R and S isomers in these three families); 2'-alkoxyalkyl; and 2'-O-NMA [N-methylacetamide, -OCH2C(O)N(H)Me].

[0262] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can be made at other positions on the RNA of nucleic acid therapeutics, particularly at the 3' position of the sugar on the 3' terminal nucleotide, or within 2'-5' linked dsRNA, and at the 5' position of the 5' terminal nucleotide. Nucleic acid therapeutics may also have sugar mimetic molecules, such as a cyclobutyl moiety instead of pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include U.S. Patents 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, and 5,5 Examples include, but are not limited to, patents 67,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920. Certain of these patents are owned by the same person as the present application. The entire contents of each of the above items are incorporated herein by reference.

[0263] The nucleic acid therapeutics of this disclosure may also include modifications or substitutions of nuclear bases (often simply referred to in the art as “bases”). As used herein, “unmodified” or “natural” nuclear bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nuclear bases include other synthetic and natural nuclear bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudolacil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, and 8-hydroxyl analogs (8-hydroxyl This includes anal, other 8-substituted adenines and guanines, 5-halos, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine, and 3-deazaguanine and 3-deazaadenine, etc.Further nuclear bases include those disclosed in U.S. Patent No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain nuclear bases among these are particularly useful for increasing the binding affinity of the oligomeric compounds discussed in this disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, containing 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of nucleic acid double helix by 0.6–1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and this is an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0264] Representative U.S. patents teaching the preparation of certain modified nuclear bases from the above-mentioned modified nuclear bases, as well as other modified nuclear bases, include U.S. Patents No. 3,687,808, No. 4,845,205, No. 5,130,30, No. 5,134,066, No. 5,175,273, No. 5,367,066, No. 5,432,272, No. 5,457,187, No. 5,459,255, No. 5,484,908, No. 5,502,177, No. 5,525,711, No. 5,552,540, and No. 5,587,46. Examples include, but are not limited to, No. 9, No. 5,594,121,5,596,091, No. 5,614,617, No. 5,681,941, No. 5,750,692, No. 6,015,886, No. 6,147,200, No. 6,166,197, No. 6,222,025, No. 6,235,887, No. 6,380,368, No. 6,528,640, No. 6,639,062, No. 6,617,438, No. 7,045,610, No. 7,427,672, and No. 7,495,088. The entire contents of each issue are incorporated herein by reference.

[0265] The nucleic acid therapeutics of this disclosure may also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety that includes an added crosslink connecting the 2' and 4' carbon atoms of the ribose moiety. This structure effectively "locks" the ribose into a 3' end conformation. It has been shown that adding locked nucleic acids to siRNA improves siRNA stability in serum and suppresses off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193].

[0266] The nucleic acid therapeutics of this disclosure may be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by crosslinking two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a crosslink that links two carbon atoms of a sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the crosslink links the 4' and 2' carbon atoms of the sugar ring. Thus, in some embodiments, the agents of this disclosure may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety that includes an added crosslink that links the 2' and 4' carbon atoms of the ribose moiety. In other words, an LNA is a nucleotide having a bicyclic sugar moiety that includes a 4'-CH2-O-2' crosslink. This structure effectively “locks” the ribose into a 3'-end structure. It has been shown that adding locked nucleic acids to siRNA improves siRNA stability in serum and suppresses off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193]. Examples of bicyclic nucleosides for use in the polynucleotides of this disclosure include, non-limitingly, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of this disclosure comprise one or more bicyclic nucleosides containing a 4'-2' bridge.Examples of such 4'-2' crosslinked bicyclic nucleosides include 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2' (also known as "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogs; see, for example, U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and its analogs; see, for example, U.S. Patent No. 8,278,283). See also); 4'-CH2-N(OCH3)-2' (and its analogs; see, e.g., U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application No. 2004 / 0171570); 4'-CH2-N(R)-O-2', where R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(-CH2)-2' (and its analogs; see, e.g., U.S. Patent No. 8,278,426), but are not limited to these. The entire contents of each of the above documents are incorporated herein by reference.

[0267] Additional representative U.S. patents and publications teaching the preparation of locked nucleic acid nucleotides include U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, and 7,399. U.S. Patent Applications No. 845, No. 7,427,672, No. 7,569,686, No. 7,741,457, No. 8,022,193, No. 8,030,467, No. 8,278,425, No. 8,278,426, No. 8,278,283; U.S. Patent Application No. 2008 / 0039618; and U.S. Patent Application No. 2009 / 0012281, among others. The entire contents of each of these applications are incorporated herein by reference.

[0268] Any of the above bicyclic nucleosides can be prepared to have one or more stereochemical sugar configurations, for example, α-L-ribofuranose or β-D-ribofuranose (see International Publication No. 99 / 14226).

[0269] The nucleic acid therapeutics of this disclosure may also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety including a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is an S-conformation denoted herein as “S-cEt”.

[0270] The nucleic acid therapeutics of this disclosure may also include one or more “structurally restricted nucleotides” (“CRNs”). CRNs are nucleotide analogs having a linker that connects the C2' and C4' carbons of ribose, or the C3 and C5' carbons of ribose. CRNs lock the ribose ring into a stable conformation and increase hybridization affinity to mRNA. The linker is long enough to position oxygen in an optimal position for stability and affinity, and suppresses ribose ring puckering.

[0271] Representative publications teaching the preparation of certain CRNs among those described above include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383 and PCT International Publication No. 2013 / 036868. The entire contents of each publication are incorporated herein by reference.

[0272] In some embodiments, the nucleic acid therapeutics of this disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA are acyclic unlocked nucleic acids in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) has been removed [see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated herein by reference].

[0273] Representative U.S. publications providing instruction on the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227, and U.S. Patent Publications 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020. The entire contents of each publication are incorporated herein by reference.

[0274] Modifications to the ends of RNA molecules that may stabilize them include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3'' phosphate, and reverse base dT (idT). Disclosure of these modifications can be found in PCT publication number International Publication 2011 / 005861.

[0275] Modified RNAi agents containing motifs from this disclosure In certain embodiments of this disclosure, the double-stranded RNAi agents of this disclosure include agents having chemical modifications, for example, as disclosed in International Publication No. 2013 / 075035, filed November 16, 2012, the entire contents of which are incorporated herein by reference. As shown herein and in PCT publication number International Publication No. 2013 / 075035, excellent results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense and / or antisense strands of the RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent may also be otherwise completely modified. Introducing such motifs will cause a break in the modification pattern (if any) of the sense and / or antisense strands. The RNAi agent may optionally be conjugated with, for example, a C16 ligand on the sense strand. The RNAi agent may optionally be modified by (S)-glycol nucleic acid (GNA) modification at, for example, one or more residues of the antisense strand. The obtained RNAi drug exhibits excellent gene expression inhibition activity.

[0276] More specifically, it was surprisingly discovered that the gene expression arrest activity of an RNAi drug is advantageously enhanced when the sense and antisense strands of a double-stranded RNAi drug are completely modified to have one or more motifs of three identical modifications on three consecutive nucleotides located at or near the cleavage site of at least one strand of the RNAi drug.

[0277] Accordingly, this disclosure presents a double-stranded RNAi agent having the ability to inhibit the expression of a target gene (i.e., the APP gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be in the range of 12 to 30 nucleotides in length. For example, each strand may be 14 to 30 nucleotides, 17 to 30 nucleotides, 25 to 30 nucleotides, 27 to 30 nucleotides, 17 to 23 nucleotides, 17 to 21 nucleotides, 17 to 19 nucleotides, 19 to 25 nucleotides, 19 to 23 nucleotides, 19 to 21 nucleotides, 21 to 25 nucleotides, or 21 to 23 nucleotides.

[0278] The sense strand and antisense strand generally form a duplex double-stranded RNA ("dsRNA"), also referred to herein as "dsRNA drug" or "RNAi drug." The terms "dsRNA drug," "RNAi drug," "iRNA drug," and "siRNA drug" are interchangeable herein. The double-stranded region of an RNAi drug may be a pair of 12–30 nucleotides in length. For example, the double-stranded region may be a pair of 14–30 nucleotides, a pair of 17–30 nucleotides, a pair of 27–30 nucleotides, a pair of 17–23 nucleotides, a pair of 17–21 nucleotides, a pair of 17–19 nucleotides, a pair of 19–25 nucleotides, a pair of 19–23 nucleotides, a pair of 19–21 nucleotides, a pair of 21–25 nucleotides, or a pair of 21–23 nucleotides. In another example, the double-stranded region is selected from lengths of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides.

[0279] In one embodiment, the RNAi agent may contain one or more overhang regions and / or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhang may be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. The overhang may result from one strand being longer than the other, or from a misalignment between two strands of equal length. The overhang may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence, or it may be a different sequence. The first and second strands may also be linked, for example, by additional bases to form a hairpin, or by other non-base linkers.

[0280] In one embodiment, the nucleotides within the overhang region of the RNAi drug may each independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar variants such as 2'-F (i.e., 2'-deoxy-2'fluoro), 2'-O-methyl (original text: 2'-Omethyl), thymidine (T), and any combination thereof.

[0281] For example, TT could be an overhang sequence to either end of either strand. The overhang could form a mismatch with the target mRNA, or it could be complementary to the targeted gene sequence, or it could be a different sequence altogether.

[0282] The 5' or 3' overhangs located on the sense strand, antisense strand, or both strands of an RNAi drug can be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides with a phosphorothioate between them, and the two nucleotides may be identical or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3' overhang is located within the antisense strand. In another embodiment, this 3' overhang is located within the sense strand.

[0283] RNAi drugs may contain only a single overhang that can enhance RNAi interference activity without affecting their overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand or the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt end at the 5' end. Without getting bogged down in theory, an asymmetric blunt end located at the 5' end of the antisense strand and an overhang at the 3' end of the antisense strand would facilitate the loading of the guide strand into the RISC process.

[0284] In one embodiment, the RNAi drug is a 19-nucleotide double-ended bluntmer, where the sense strand contains at least one motif of three 2'-F alterations on three consecutive nucleotides at positions 7, 8, and 9, starting from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl alterations on three consecutive nucleotides at positions 11, 12, and 13, starting from the 5' end.

[0285] In another embodiment, the RNAi drug is a 20-nucleotide double-ended bluntomer, where the sense strand contains at least one motif of three 2'-F variants on three consecutive nucleotides at positions 8, 9, and 10, starting from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl variants on three consecutive nucleotides at positions 11, 12, and 13, starting from the 5' end.

[0286] In yet another embodiment, the RNAi drug is a 21-nucleotide double-ended bluntomer, and the sense strand contains at least one motif of three 2'-F alterations on three consecutive nucleotides at positions 9, 10, and 11, starting from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl alterations on three consecutive nucleotides at positions 11, 12, and 13, starting from the 5' end.

[0287] In one embodiment, the RNAi drug comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F variants on three consecutive nucleotides at positions 9, 10, and 11, starting from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl variants on three consecutive nucleotides at positions 11, 12, and 13, starting from the 5' end, but one end of the RNAi drug is blunt-ended while the other end contains a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is located at the 3' end of the antisense strand. When the 2-nucleotide overhang is located at the 3' end of the antisense strand, there may be two phosphorothioate nucleotide interbonds between the three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. In one embodiment, the RNAi drug additionally has two phosphorothioate internucleotide bonds between three terminal nucleotides located at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, each nucleotide in the sense and antisense strands of the RNAi drug is a modified nucleotide, including nucleotides that are part of a motif. In one embodiment, each residue is independently modified, for example, with 2'-O-methyl or 3'-fluoro in an alternatingly repeating motif. The RNAi drug may further contain a ligand (a C16 ligand, as appropriate).

[0288] In one embodiment, the RNAi drug comprises a sense strand and an antisense strand, where the sense strand is 25–30 nucleotides long, starting from the 5' terminal nucleotide (position 1), and positions 1–23 of the first strand contain at least 8 ribonucleotides. The antisense strand is 36–66 nucleotides long, starting from the 3' terminal nucleotide, and contains at least 8 ribonucleotides at positions paired with positions 1–23 of the sense strand, forming a double helix. Here, at least the 3' terminal nucleotides of the antisense strand do not pair with the sense strand, and up to 6 consecutive 3' terminal nucleotides do not pair with the sense strand, thereby forming a 3' single-stranded overhang of 1–6 nucleotides. Here, the 5' end of the antisense strand contains 10–30 consecutive nucleotides that do not pair with the sense strand, thereby forming a 10–30 nucleotide single-stranded 5' overhang. Here, at least the 5' and 3' terminal nucleotides of the sense strand form base pairs with the nucleotides of the antisense strand when the sense strand and antisense strand are aligned to obtain maximum complementarity, thereby forming a substantially double-stranded region between the sense strand and the antisense strand. The antisense strand is also sufficiently complementary to the target RNA, with a length of at least 19 ribonucleotides along the antisense strand, so that the expression of the target gene is reduced when the double-stranded nucleic acid is introduced into mammalian cells. Here, the sense strand contains at least one motif of three 2'-F alterations on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl alterations on three consecutive nucleotides at or near the cleavage site.

[0289] In one embodiment, the RNAi drug comprises a sense strand and an antisense strand, where the RNAi drug comprises a first strand having a length of at least 25 and at most 29 nucleotides, and a second strand having a length of up to 30 nucleotides, with at least one motif of three 2'-O-methyl alterations on three consecutive nucleotides at positions 11, 12, and 13, starting from the 5' end. Here, the 3' end of the first strand and the 5' end of the second strand form blunt ends, and the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end. Here, the duplex region is at least 25 nucleotides long, and the second strand is sufficiently complementary to the target RNA along an antisense strand of at least 19 ribonucleotides in length, such that the expression of the target gene is reduced when the RNAi drug is introduced into mammalian cells. Furthermore, the dicer cleavage of the RNAi drug preferentially yields an siRNA containing the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. The RNAi drug may further contain a ligand.

[0290] In one embodiment, the sense strand of the RNAi drug contains at least one motif for three identical modifications on three consecutive nucleotides, one of which occurs at a cleavage site within the sense strand.

[0291] In one embodiment, the antisense strand of the RNAi drug may also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near a cleavage site within the antisense strand.

[0292] For RNAi drugs with a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are generally located around positions 10, 11, and 12, starting from the 5' end. Therefore, three identical modification motifs can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand. However, the counting starts either from the first nucleotide at the 5' end of the antisense strand, or from the first paired nucleotide located within the double-stranded region at the 5' end of the antisense strand. The cleavage sites within the antisense strand may vary depending on the length from the 5' end of the double-stranded region of the RNAi.

[0293] The sense strand of an RNAi drug may contain at least one motif of three identical modifications on three consecutive nucleotides located at the cleavage site of the strand. Similarly, the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides located at or near the cleavage site of the strand. When the sense and antisense strands form a dsRNA double helix, the sense and antisense strands may be aligned such that one of the three nucleotides on the sense strand and one of the three nucleotides on the antisense strand have at least one nucleotide overlap; that is, at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0294] In one embodiment, the sense strand of an RNAi drug may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near a cleavage site on the strand, while the other motifs may be wing modifications. In this specification, the term “wing modification” refers to a motif occurring in a different part of the strand than a motif located at or near a cleavage site on the same strand. The wing modification may be located near the first motif or separated by at least one or more nucleotides. If the motifs are located near each other, their chemistry may be different from each other; if the motifs are separated by one or more nucleotides, their chemistry may be identical or different. Two or more wing modifications may exist. For example, if two wing modifications exist, each wing modification may occur at one end relative to the first motif located at or near a cleavage site, or on either side of the lead motif.

[0295] Similar to the sense strand, the antisense strand of an RNAi drug may contain two or more motifs of three identical modifications on three consecutive nucleotides, with at least one motif occurring at or near a cleavage site on the strand. This antisense strand may also contain one or more wing modifications, aligned similarly to those present on the sense strand.

[0296] In one embodiment, a wing modification on the sense or antisense strand of an RNAi drug typically does not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0297] In another embodiment, the wing modification on the sense or antisense strand of the RNAi drug generally does not include paired nucleotides for the first one or two nucleotides in the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0298] When each of the sense and antisense strands of an RNAi drug contains at least one wing variant, the wing variant may be located at the same end of the double-stranded region, but with an overlap of one, two, or three nucleotides.

[0299] If each of the sense and antisense strands of an RNAi drug contains at least two wing modifications, the sense and antisense strands can be aligned such that each of the two modifications from one strand is located at one end of a double-stranded region having an overlap of one, two, or three nucleotides; each of the two modifications from one strand is located at the other end of a double-stranded region having an overlap of one, two, or three nucleotides; and the two modifications from one strand (two modifications one strand) are located within the double-stranded region on each side of a read motif having an overlap of one, two, or three nucleotides.

[0300] In one embodiment, the RNAi agent contains mismatches with the target in the double-stranded or combined form. Mismatches may occur in overhang regions or double-stranded regions. Base pairs can be ranked based on their tendency to promote dissociation or dissolution [e.g., based on the free energy of association or dissociation of a particular pairing. The simplest approach is to examine each pair individually; however, next-neighbor analysis or similar analytical methods may also be used]. With regard to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical pairing or non-canonical pairing (as otherwise described herein) are preferred over canonical (A:T, A:U, G:C) pairing; and pairings containing universal bases are preferred over canonical pairing.

[0301] In one embodiment, the RNAi agent includes, within the double-stranded region, at least one of the first 1, 2, 3, 4, or 5 base pairs independently selected from the group A:U, G:U, I:C, from the 5' end of the antisense strand, and a mismatch pair to facilitate the dissociation of the antisense strand, such as non-canonical pairing, non-canonical pairing, or pairing containing a universal base at the 5' end of the double-stranded region.

[0302] In one embodiment, in the antisense strand, the nucleotide located at position 1, starting from the 5' end of the double-stranded region, is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first, second, or third base pairs from the 5' end of the antisense strand in the double-stranded region is an A:U base pair. For example, the first base pair from the 5' end of the antisense strand in the double-stranded region is an A:U base pair.

[0303] In another embodiment, the nucleotide located at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide located at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, a short sequence consisting of deoxythymine nucleotides, for example, two dT nucleotides, is present at the 3' end of the sense strand and / or antisense strand.

[0304] Various published documents describe multimeric RNAi drugs that can be used in the method of this disclosure. Such publications include International Publication No. 2007 / 091269, U.S. Publication No. 7858769, International Publication No. 2010 / 141511, 2007 / 117686, 2009 / 014887, 2011 / 031520, 2013 / 074974, 2013 / 165816, 2016 / 028649, 2018 / 098328, 2019 / 126651, 2019 / 222479, 2019 / 217459, 2020 / 097044, and 2022 / 159158, the full contents of each of these publications are incorporated herein by reference.

[0305] In certain embodiments, the RNAi agents of the Disclosure may include a GalNAc ligand, even if such a GalNAc ligand is currently predicted to have limited value for the preferred intrathecal / CNS delivery pathway(s) of the Disclosure.

[0306] Modified RNAi drugs containing a phosphate moiety Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with the usual 3'-5' linkage, their 2'-5' linked analogues, and those with reverse polarity where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0307] Representative U.S. patents teaching the preparation of the above phosphorus-containing bond include U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, and 5 ,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476, 925, 5,519,126, 5,536,821, 5,541,316, 5,550,111, 5,563,253, 5,571,799, No. 5,587,361, No. 5,625,050, No. 6,028,188, No. 6,124,445, No. 6,160,109, No. 6,169,170, No. 6,17 No. 2,209, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590 This includes, but is not limited to, U.S. Patent Nos. 6,534,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE 39464. The entire contents of each of these patents are incorporated herein by reference.

[0308] Some embodiments of this disclosure include RNA having a phosphorothioate backbone, and heteroatom backbones, in particular -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as the methylene(methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [the natural phosphodiester backbone is represented as -OPO-CH2-], as well as oligonucleosides having an amide backbone, as referenced above in U.S. Patent No. 5,602,240. In some embodiments, the RNAs of this specification have a morpholino backbone structure, as referenced above in U.S. Patent No. 5,034,506.

[0309] Other modifications of the nucleic acid therapeutics of this disclosure include 5'-phosphate or 5'-phosphate mimetic, such as a 5'-terminal phosphate on the antisense strand of an RNAi drug, or a 5'-phosphate mimetic. A suitable 5'-phosphate mimetic is disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entirety of which is incorporated herein by reference.

[0310] RNAi drugs conjugated to ligands Another modification of the RNA in the RNAi drug composition of this disclosure involves chemically linking the RNA to one or more ligands, moieties, or conjugates that enhance RNAi activity, cell distribution, or intracellular uptake. Such parts include lipid portions, such as cholesterol portions [Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556], cholic acid [Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060], thioethers, such as beryl-S-tritylthiol [Manoharan et al., (1992) Ann. NY Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770], and thiocholesterol [Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538], aliphatic chains, e.g., dodecanediol or undecyl residues [Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54], phospholipids, e.g., di-hexadecyl-rac-glycerol, or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonic acid [Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., [18:3777-3783], polyamine or polyethylene glycol chain [Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973], or adamantane acetate [Manoharan et al., (1995) Tetrahedron Lett.Examples include, but are not limited to, the palmityl moiety [36:3651-3654], the palmityl moiety [Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237], or the octadecylamine or hexylamino-carbonyloxycholesterol moiety [Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937]. A wide range of ligands are explicitly assumed to be ligable to the dsRNAs of this disclosure, in addition to lipophilic moieties, GalNAc moieties, or, for example, other dsRNA ligation moieties (which are added with the intention of facilitating the delivery of such dsRNAs to target cells).

[0311] In one embodiment, the ligand alters the distribution, targeting, or lifespan of the RNAi drug into which it is incorporated. In a preferred embodiment, the ligand results in enhanced affinity for selected targets, such as molecules, cells or cell types, compartments, such as compartments in cells or organs, tissues, organs, or regions of the body, compared to species in which such ligand is absent. The preferred ligand does not participate in double-strand pair formation within double-stranded nucleic acids.

[0312] The oligonucleotides used in the conjugates of this disclosure can be conveniently and routinely prepared through well-known solid-phase synthesis techniques. Equipment for such synthesis is available from several vendors, including, for example, Applied Biosystems (Foster City, Calif.) and Cytiva Life Sciences. Any other means known in the art for such synthesis may be used additionally or alternatively. The use of similar techniques for preparing other oligonucleotides, such as phosphorothioates and alkylated derivatives, is also known.

[0313] In the ligand-conjugate oligonucleotides and ligand molecule-supported sequence-specific binding nucleosides of this disclosure, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer that utilizes a standard nucleotide or nucleoside precursor, or a nucleotide or nucleoside conjugate precursor already supporting a linking moiety, or a ligand nucleotide or nucleoside conjugate precursor already supporting a ligand molecule, or a non-nucleoside ligand-supported structural block.

[0314] When using nucleotide conjugate precursors that already support a linking moiety, the synthesis of sequence-specifically linked nucleosides is generally complete, and then the ligand molecule reacts with the linking moiety to form ligand-conjugate oligonucleotides. In some embodiments, the oligonucleotides or linked nucleosides of this disclosure are commercially available and are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, by adding them to standard and non-standard phosphoramidites routinely used in oligonucleotide synthesis.

[0315] A. Lipophilic part The term "lipophilic" or "lipophilic moiety" broadly refers to any compound or chemical part that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by its octanol-water partition coefficient logK. ow However, here, K owThe octanol-water partition coefficient is the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase in a two-phase system at equilibrium. The octanol-water partition coefficient is a property of a substance measured in the laboratory. However, this coefficient can also be predicted by using a coefficient attributed to the structural components of the chemical substance, calculated using first-principles or experimental methods [see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41: 1407-21 (2001), which is incorporated verbatim as reference herein]. This coefficient provides a thermodynamic indicator of a substance's tendency to prefer non-aqueous or oily environments over water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical substance has a logK ow When logK is greater than 0, it is lipophilic. Generally, the lipophilic portion is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow It has, for example, the logK of 6-aminohexanol. ow For example, it is predicted to be approximately 0.7. Using the same method, the logK of cholesteryl N-(hexane-6-ol) carbamate ow It is predicted to be 10.7.

[0316] The lipophilicity of a molecule can change with respect to the functional group it supports. For example, adding a hydroxyl group or an amine group to the end of the lipophilic portion can change the partition coefficient (e.g., logK) of the lipophilic portion. ow The value may increase or decrease.

[0317] Alternatively, the hydrophobicity of a double-stranded RNAi drug conjugated to one or more lipophilic moieties can be measured by its protein-binding properties. For example, in certain embodiments, the unbound fraction in a plasma protein-binding assay of a double-stranded RNAi drug may be determined to have a positive correlation with the relative hydrophobicity of the double-stranded RNAi drug, which in turn has a positive correlation with the drug's inhibitory activity.

[0318] Exemplary lipophilic portions and / or lipophilic modifications known in the art include, but are not limited to, saturated or unsaturated fatty acids, steroids, fat-soluble vitamins, phospholipids, sphingolipids, hydrocarbons, mono-, di-, and triglycerides, and their synthetic derivatives, cholesterol, C 10 ~C 26 Saturated fatty acids, C 10 ~C 26 unsaturated fatty acids, C 10 ~C 26 Examples include lipids selected from alkyl groups, triglycerides, tocopherols, or cholic acid. Particularly exemplary among the lipophilic moieties are fatty acids and adamantyl groups further comprising a “lipid conjugate” (LC) moiety, including the exemplary configuration for modification of the lipophilic / hydrophobic moiety of oligonucleotides as defined in PCT / US 2021 / 042469, where each ligand is independently a hydrogen atom or a hydrophobic moiety selected from an adamantyl group and a lipid moiety; and / or each LC is independently a saturated or unsaturated linear or branched C 1~50 A lipid conjugate moiety containing a hydrocarbon chain [wherein the 0-10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -NR-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-]; and / or LC is saturated or partially unsaturated linear or branched C 1~50A lipid conjugate moiety comprising a hydrocarbon chain [wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -NR-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-]; and / or the lipid conjugate moiety is formed from the coupling of a nucleic acid or analog thereof with a lipophilic compound. In some embodiments, LC is a lipid conjugate moiety comprising an esterified or amidated saturated linear fatty acid. In some embodiments, LC is -OC(O)CH3 or -NHC(O)CH3. In some embodiments, LC is -OC(O)C2H5 or -NHC(O)C2H5. In some embodiments, LC is -OC(O)C3H7 or -NHC(O)C3H7. In some embodiments, LC is -OC(O)C4H9 or -NHC(O)C4H9. In some embodiments, LC is -OC(O)C5H 11 or -NHC(O)C5H 11 In some embodiments, LC is -OC(O)C6H 13 Or -NHC(O)C6H 13 In some embodiments, LC is -OC(O)C7H 15 or -NHC(O)C7H 15 In some embodiments, LC is -OC(O)C8H 17 or -NHC(O)C8H 17 In some embodiments, LC is -OC(O)C9H 19 or -NHC(O)C9H 19 In some embodiments, LC is -OC(O)C 10 H 21 or -NHC(O)C 10 H 21 In some embodiments, LC is -OC(O)C 11 H 23 or -NHC(O)C 11 H 23 In some embodiments, LC is -OC(O)C 12 H 25 or -NHC(O)C 12 H 25In some embodiments, LC is -OC(O)C 13 H 27 or -NHC(O)C 13 H 27 In some embodiments, LC is -OC(O)C 14 H 29 or -NHC(O)C 14 H 29 In some embodiments, LC is -OC(O)C 15 H 31 or -NHC(O)C 15 H 31 In some embodiments, LC is -OC(O)C 16 H 33 or -NHC(O)C 16 H 33 In some embodiments, LC is -OC(O)C 17 H 35 or -NHC(O)C 17 H 35 In some embodiments, LC is -OC(O)C 18 H 37 or -NHC(O)C 18 H 37 In some embodiments, LC is -OC(O)C 19 H 39 or -NHC(O)C 19 H 39 In some embodiments, LC is -OC(O)C 20 H 41 or -NHC(O)C 20 H 41 In some embodiments, LC is -OC(O)C 21 H 43 or -NHC(O)C 21 H 43 In some embodiments, LC is -OC(O)C 22 H 45 or -NHC(O)C 22 H 45 In some embodiments, LC is -OC(O)C 23 H 47 or -NHC(O)C 23 H 47In some embodiments, LC is -OC(O)C 24 H 29 or -NHC(O)C 24 H 29 In some embodiments, LC is -OC(O)C 25 H 51 or -NHC(O)C 25 H 51 In some embodiments, LC is -OC(O)C 26 H 53 or -NHC(O)C 26 H 53 In some embodiments, LC is -OC(O)C 27 H 55 or -NHC(O)C 27 H 55 In some embodiments, LC is -OC(O)C 28 H 57 or -NHC(O)C 28 H 57 In some embodiments, LC is -OC(O)C 29 H 59 or -NHC(O)C 29 H 59 In some embodiments, LC is -OC(O)C 30 H 61 or -NHC(O)C 30 H 61 In some embodiments, LC is a lipid conjugate moiety containing esterified or amidated partially unsaturated linear fatty acids. In some embodiments, LC is esterified or amidated myristoleic acid. In some embodiments, LC is esterified or amidated palmitoleic acid. In some embodiments, LC is esterified or amidated sapienic acid. In some embodiments, LC is esterified or amidated oleic acid, i.e.

[0319] [ka] In some embodiments, LC is esterified or amidated elaidic acid. In some embodiments, LC is esterified or amidated vaccenic acid. In some embodiments, LC is esterified or amidated linoleic acid. In some embodiments, LC is esterified or amidated limoelaidic acid. In some embodiments, LC is esterified or amidated α-linolenic acid, i.e.

[0320] [ka] In some embodiments, LC is esterified or amidated arachidonic acid. In some embodiments, LC is esterified or amidated eicosapentaenoic acid, i.e.

[0321] [ka] In some embodiments, LC is esterified or amidated erucic acid. In some embodiments, LC is esterified or amidated docosahexaenoic acid, i.e.

[0322] [ka] In some embodiments, LC is an esterified or amidated adamantanecarboxylic acid. In some embodiments, LC is an esterified or amidated adamantaneacetic acid. In some embodiments, R 5 This is -C(O)(CH2)i-iodoadamantane.

[0323] Other forms of lipophilic portions that are known in the art and explicitly intended for use in the compositions and methods of this disclosure include, for example, the compound of formula (I) in PCT / US 2021 / 049880:

[0324] [ka] Or a pharmaceutically acceptable salt thereof. In the formula, R is -L A -R Z And; L A is a bond or R Z The divalent part that connects to Z; R Z L1 comprises an oligonucleotide-based agent; Z is CH, phenyl, or N; L1 and L2 are each independently linkers containing at least about 5 polyethylene glycol (PEG) units; and X and Y are each independently lipids containing about 10 to about 50 carbon atoms. In some embodiments, L1 and L2 each independently contain about 15 to about 100 PEG units. In some embodiments, L1 and L2 each independently contain about 20 to about 60 PEG units. In some embodiments, L1 and L2 each independently contain about 20 to about 30 PEG units. In other embodiments, L1 and L2 each independently contain about 40 to about 60 PEG units. Also, in some embodiments, one of L1 and L2 contains about 20 to about 30 PEG units and the other contains about 40 to about 60 PEG units.

[0325] Examples of the X and Y parts relating to formula (I) above in PCT / US No. 2021 / 049880 are, for example,

[0326] [Table 2] TIFF2026514044000023.tif211142 Includes TIFF2026514044000024.tif94150.

[0327] Other exemplary lipophilic / hydrophobic moieties intended for use in the compositions and methods disclosed herein include sterols (e.g., cholesterol), GM1, lipids, vitamins, small molecules, peptides, or combinations thereof. In some embodiments, the moiety is lipids. For example, in certain embodiments, the moiety is palmitoyl. In some embodiments, the moiety is sterols, e.g., cholesterol. Additional hydrophobic moieties include, for example, phospholipids, vitamin D, vitamin E, squalene, and fatty acids. In another exemplary embodiment, the oligonucleotide cargo is conjugated with myristic acid or a derivative thereof (e.g., myristoylated oligonucleotide cargo - see U.S. Patent No. 10,513,710).

[0328] As an additional exemplary structure of the lipophilic portion of PCT / US No. 2016 / 053836, Chol-TEG-:

[0329] [ka] TOCO-TEG-:

[0330] [ka] C10-TEG- and C16-TEG-:

[0331] [ka] (In the formula, n is 1 in "C10-TEG-" and n is 7 in "C16-TEG-")

[0332] For example, nucleotide modifications as defined in PCT / US No. 2020 / 046561 are expressly intended to be used in the compositions and methods of this disclosure.

[0333] In one embodiment, the determined plasma protein binding assay is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol for this binding assay is illustrated in detail, for example, in PCT / US 2019 / 031170. When measured by fraction of unbound siRNA in the binding assay, the hydrophobicity of double-stranded RNAi agents is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 in the case of in vivo delivery of enhanced siRNA.

[0334] Therefore, by conjugating the lipophilic portion to one or more internal positions of a double-stranded RNAi drug, optimal hydrophobicity for enhanced siRNA in vivo delivery can be obtained.

[0335] In certain embodiments, the lipophilic portion is an aliphatic compound, a cyclic compound, such as an alicyclic compound, or a polycyclic compound, such as a polycyclic alicyclic compound, such as a steroid (e.g., a sterol), or a linear or branched aliphatic hydrocarbon. The lipophilic portion generally includes a hydrocarbon chain that may be cyclic or acyclic. The hydrocarbon chain may include various substituents and / or one or more heteroatoms, such as an oxygen atom or a nitrogen atom. Such a lipophilic aliphatic portion may, non-limitingly, be saturated or unsaturated C4-C4. 30 Hydrocarbons (for example, C6~C 18 Hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes, and C 40 Examples include tetraterpenes and other polycyclic alicyclic hydrocarbons. For example, the lipophilic portion is C4-C 30 Hydrocarbon chains (for example, C4~C 30It may contain alkyl or alkenyl molecules. In some embodiments, the lipophilic portion is saturated or unsaturated C6-C6. 18 Hydrocarbon chains (e.g., linear C6-C6) 18 It contains alkyl or alkenyl compounds. In one embodiment, the lipophilic portion is saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C) 16 Contains alkyl or alkenyl compounds.

[0336] In some embodiments, the lipophilic portion is saturated or unsaturated C 10 ~C 30 Hydrocarbon chains, or saturated or unsaturated carbon 12 ~C 30 Hydrocarbon chains, or saturated or unsaturated carbon 14 ~C 30 Hydrocarbon chains, or saturated or unsaturated carbon 16 ~C 30 Hydrocarbon chains, or saturated or unsaturated carbon 18 ~C 30 Hydrocarbon chains, or saturated or unsaturated carbon 20 ~C 30 It contains hydrocarbon chains.

[0337] In other embodiments, the lipophilic portion is saturated or unsaturated C 10 ~C 24 Hydrocarbon chains, or saturated or unsaturated carbon 12 ~C 24 Hydrocarbon chains, or saturated or unsaturated carbon 14 ~C 24 Hydrocarbon chains, or saturated or unsaturated carbon 16 ~C 24 Hydrocarbon chains, or saturated or unsaturated carbon 18 ~C 24 Hydrocarbon chains, or saturated or unsaturated carbon 20 ~C 24 It contains hydrocarbon chains.

[0338] In other embodiments, the lipophilic portion is saturated or unsaturated C 10 Hydrocarbon chains, or saturated or unsaturated carbon 11 Hydrocarbon chains, or saturated or unsaturated carbon12 Hydrocarbon chains, or saturated or unsaturated carbon 13 Hydrocarbon chains, or saturated or unsaturated carbon 14 Hydrocarbon chains, or saturated or unsaturated carbon 15 Hydrocarbon chains, or saturated or unsaturated carbon 16 Hydrocarbon chains, or saturated or unsaturated carbon 17 Hydrocarbon chains, or saturated or unsaturated carbon 18 Hydrocarbon chains, or saturated or unsaturated carbon 19 Hydrocarbon chains, or saturated or unsaturated carbon 20 Hydrocarbon chains, or saturated or unsaturated carbon 21 Hydrocarbon chains, or saturated or unsaturated carbon 22 Hydrocarbon chains, or saturated or unsaturated carbon 23 Hydrocarbon chains, or saturated or unsaturated carbon 24 It contains hydrocarbon chains.

[0339] In other embodiments, the lipophilic portion is C 10 Alkyl chain, or C 11 Alkyl chain, or C 12 Alkyl chain, or C 13 Alkyl chain, or C 14 Alkyl chain, or C 15 Alkyl chain, or C 16 Alkyl chain, or C 17 Alkyl chain, or C 18 Alkyl chain, or C 19 Alkyl chain, or C 20 Alkyl chain, or C 21 Alkyl chain, or C 22 Alkyl chain, or C 23 Alkyl chain, or C 24 Each of the prior examples may, in other embodiments, be a linear alkyl chain (e.g., n-tetradecyl, or n-pentadecyl, or n-hexadecyl, or n-heptadecyl, or n-octadecyl, or n-nonadecyl, or n-eicosyl, or n-henicosanyl, n-docosanyl, or n-tricosanyl, or n-tetracosanyl).

[0340] In exemplary embodiments, modified nucleotides including lipophilic modifications have the following structure:

[0341] [ka] It may have the following: In the formula, B may be a modified nuclear base, and R L This is any of the lipophilic moieties of this specification that are appropriately linked to 2'-O via a carrier or linking group (for example, in certain embodiments, the lipophilic moiety is directly linked to 2'-O).

[0342] In a particular embodiment, R L C is appropriately substituted with one group selected from the group consisting of halogen, -C(O)OR, -OR, -NR2, -C(O)R, and -C(O)N(R)2. 10~24 It is an alkyl chain, where each R is independently hydrogen or carbon. 1~6 It is alkyl.

[0343] In a particular embodiment, R L C is appropriately substituted with a carboxyl group (e.g., an ω-carboxyl group). 10~24 It is an alkyl chain.

[0344] In a particular embodiment, R L C is appropriately substituted with a hydroxyl group (e.g., an ω-hydroxyl group). 10~24 It is an alkyl chain.

[0345] In a particular embodiment, R L ha-(CH2) n -OH, where n is 14-24 (e.g., 16 or 22). In a particular embodiment, R L ha-(CH2) n The compound is -COOH, where n is between 14 and 24 (for example, 16 or 22).

[0346] In a particular embodiment, R L teeth,

[0347] [Table 3] That is the case.

[0348] In certain embodiments, the compositions and methods of this disclosure include a C16 ligand. In exemplary embodiments, the C16 ligand of this disclosure has the following structure:

[0349] [ka] It has (here, uracil bases are given as an example below, but other linkages of the C16 ligand are conceivable for nucleotides corresponding to any base (C, G, A, etc.), and / or any other modifications presented herein, provided that the 2' ribolinking is maintained), and is linked at the 2' position of the ribo within the modified residue.

[0350] As shown above, the C16 ligand-modified residue provides a linear alkyl group at the 2'-ribo position of the exemplary residue that is modified in this way (in this case, uracil).

[0351] In further exemplary embodiments, a modified nucleotide including lipophilic modification has the following structure:

[0352] [Table 4] It may have any one of the following: In the formula, m is 0 to 8; n is 1 to 21; W is an alkyl group, e.g., C1 to C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl), etc. R, R', and R'' are each independently H, or an alkyl group, e.g., C1 to C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl), etc.; G is a lipophilic portion based on any embodiment of the Specified (e.g., G is saturated or unsaturated C 10 ~C 24 Hydrocarbon chains, or saturated or unsaturated carbon 12 ~C 24Hydrocarbon chains, or saturated or unsaturated carbon 14 ~C 24 Hydrocarbon chains, or saturated or unsaturated carbon 16 ~C 24 Hydrocarbon chains, or saturated or unsaturated carbon 18 ~C 24 Hydrocarbon chains, or saturated or unsaturated carbon 20 ~C 24 (It is a hydrocarbon chain.)

[0353] The lipophilic portion can be linked to the RNAi drug by any method known in the art, including via a functional group already present in the lipophilic portion or introduced into the RNAi drug, such as a hydroxyl group (e.g., -CO-CH2-OH). Examples of functional groups already present in the lipophilic portion or introduced into the RNAi drug include, but are not limited to, hydroxyls, amines, carboxylic acids, sulfonic acids, phosphoric acids, thiols, azides, and alkynes.

[0354] Conjugation between an RNAi drug and its lipophilic moiety can occur, for example, by forming an ether linkage, a carboxyl linkage, or a carbamoyl ester linkage between a hydroxyl group and an alkyl group R-, an alkanoyl group RCO-, or a substituted carbamoyl group RNHCO-. The alkyl group R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., linear or branched; and saturated or unsaturated). The alkyl group R may be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, henicosanil, docosanil, tricosanil, or tetracosanil groups.

[0355] In some embodiments, the lipophilic portion is conjugated to a double-stranded RNAi drug via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimidothioether, disulfide, phosphodiester, sulfonamide bond, click reaction product (e.g., triazole obtained from azido-alkyne cyclization), or carbamate.

[0356] In some embodiments, the lipophilic portion is conjugated to a double-stranded RNAi drug via a modified phosphodiester linkage. Examples of modified phosphodiester links include, but are not limited to,

[0357] [ka] Examples include: In the formula, each cleaved bond is attached to the preceding and following nucleotides (e.g., 3'-5'), and each X is independently O or S, and R N This refers to the lipophilic moieties described herein, such as butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, henicosanyl, docosanyl, tricosanyl, or tetracosanyl groups. For example, a modified nucleotide, as a modified phosphodiester, which includes a lipophilic modification, has the following structure:

[0358] [ka] The formula may have the following: In the formula, B may be a modified nuclear base, n is 1 to 21 (e.g., 5 to 19, or 9 to 19, or 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19), and R2 may be any functional group that is 2'-modified to the ribose sugar, e.g., hydrogen, halo, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl) modification, 2'-O-allyl modification, 2'-C-allyl modification, 2'-fluoro modification, 2'-ON-methylacetamide (2'-O-NMA) modification, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modification, 2'-O-aminopropyl (2'-O-AP) modification, or 2'-ara-F modification, etc. For example, R2' may be H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamide, O-dimethylaminoethoxyethyl, or O-aminopropyl. In some embodiments, the lipophilic moiety is conjugated to a double-stranded RNAi drug via a modified nuclear base, such as cytosine or uracil, which are substituted at position 5 with the lipophilic moiety described herein.

[0359] For example,

[0360] [ka] Examples include the following: In the formula, R is a lipophilic moiety as defined herein. R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamide, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nuclear base.

[0361] In some embodiments, the lipophilic portion is conjugated to one of the 3' ends of the sense and antisense chains via direct bonding or through a carrier or linker. For example, not limited to,

[0362] [ka] The following is an example of the formula. In the formula, the cleaved bond follows the 3' end (3'-OH) of the oligonucleotide via a phosphodiester or phosphothioate internucleotide bond; G is a linking group; Z is a bond, O, S, or N(H), and R L The following are:

[0363] [Table 5] This includes lipophilic portions, such as those described herein. In the formula, m is 0 to 8 and n is 1 to 21.

[0364] In some embodiments, the lipophilic portion is conjugated to one of the 5' ends of the sense and antisense chains via direct bonding or through a carrier or linker. For example, not limited to,

[0365] [ka] The following is an example of the formula: In the formula, the cleaved bond follows the 5' end (5'-OH) of the oligonucleotide via a phosphodiester or phosphothioate internucleotide bond; G is a linking group; Z is a bond, O, S, or N(H), and R L The following are:

[0366] [Table 6] This includes lipophilic portions, such as those described herein.

[0367] In some embodiments, the lipophilic portion is conjugated to one of the 5' ends of the sense and antisense chains via a direct bond to the 5' carbon at the 5' end. For example,

[0368] [Table 7]

[0369] In some embodiments, the lipophilic portion is conjugated to one of the 3' or 5' ends of the sense and antisense chains via direct bonding or through a carrier or linker. In some embodiments, the lipophilic portion is conjugated to one of the 3' ends of the sense and antisense chains via direct bonding or through a carrier or linker. In some embodiments, the lipophilic portion is conjugated to one of the 5' ends of the sense and antisense chains via direct bonding or through a carrier or linker. In some embodiments, the lipophilic portion is formula

[0370] [ka] or that of its salt. In the formula, X is O or S (e.g., S); L is a divalent linking group, e.g., C 1~20 Alkyl, C 1~10 Alkyl-SSC 1~10 It is alkyl. In one special case, the lipophilic group is formula

[0371] [ka] It is that of the formula. In the formula, X is O or S (for example, S), and R リガンド The selection is made from Table 1.

[0372] [Table 8] TIFF2026514044000043.tif253154 TIFF2026514044000044.tif253154 TIFF2026514044000045.tif225154

[0373] In another example, the lipophilic portion is attached to the 5' oxygen of the 5' terminal nucleotide, or the 3' oxygen of the 3' terminal nucleotide, as shown below:

[0374] [ka] It is one of them.

[0375] In some embodiments, the support or linker is a reverse-debased nucleotide, such as a reverse-debased deoxyribonucleotide or reverse-debased ribonucleotide, each linked to the remaining oligonucleotide via a phosphodiester (PO) or phosphorothioate (PS) bond. For example,

[0376] [ka] These are some examples, but are not limited to these.

[0377] In some embodiments, the lipophilic group is linked to the 5' oxygen of the 5' terminal nucleotide of the oligonucleotide, or to the 3' oxygen of the 3' terminal nucleotide, and formula

[0378] [ka] or that of its salt. In the formula, each X is independently O or S (for example, each is S), and R リガンド The group is selected from Table 1, and L is a divalent linking group, for example, C 1~20 Alkyl, C 1~10 Alkyl-SSC 1~10 It is alkyl. The preceding structure, as used herein, may be, for example, 5'-(L1)(inv)-.

[0379] For example, the lipophilic group is linked to the 5' oxygen of the 5' terminal nucleotide of the oligonucleotide, or to the 3' oxygen of the 3' terminal nucleotide, and the formula

[0380] [ka] It is that of the formula. In the formula, each X is either O or S (for example, each is S), and R リガンド The selection is made from Table 1. For example, R リガンド Table 2:

[0381] [Table 9] Examples include selection from TIFF2026514044000051.tif114154. The preceding structure, when used herein, may be, for example, 5'-(L1)(inv)-.

[0382] In another example, the lipophilic portion is one of the following, bonded to the 3' oxygen of the 3' terminal nucleotide or the 5' oxygen of the 5' terminal nucleotide:

[0383] [ka] or that of its salt. In the formula, each X is independently O or S (for example, each is S), and R リガンド The elements are selected from Table 1 (above), and L is a divalent linking group, for example, C 1~20 Alkyl, C 1~10 Alkyl-SSC 1~10 It is alkyl. The preceding structure, as used herein, may be, for example, -(inv)(L2)-3'.

[0384] In one particular example, the lipophilic portion is bonded to the 3' oxygen of the 3' terminal nucleotide, or to the 5' oxygen of the 5' terminal nucleotide, and the formula

[0385] [ka] or that of its salt. In the formula, each X is either O or S (for example, each is S), and R リガンド The selection is made from Table 1 (above). For example, Rリガンド Examples include the selection from Table 2 (above). The preceding structure, as used herein, may be -(inv)(L2)-3'.

[0386] In another embodiment, the lipophilic portion is a steroid, such as a sterol. The steroid is a polycyclic compound containing a perhydro-1,2-cyclopentanophenanthrene ring system. Examples of steroids, not limited to, include bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone. "Cholesterol derivative" refers to a cholesterol-derived compound obtained, for example, by substitution, addition, or removal of substituents.

[0387] In another embodiment, the lipophilic portion is the aromatic portion. In this context, the term "aromatic" broadly refers to mono- and polyaromatic hydrocarbons. The aromatic group is not limited to C6-C6 containing 1-3 aromatic rings which may be substituted as appropriate. 14 Examples include aryl moieties; "aralkyl" or "arylalkyl" groups containing an aryl group covalently linked to an alkyl group (each of which may be independently substituted or unsubstituted as appropriate); and "heteroaryl" groups. As used herein, the term "heteroaryl" refers to a group having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms, having 6, 10, or 14 π electrons shared within the cyclic array, and having 1 to about 3 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S) added to a carbon atom.

[0388] In this specification, "appropriately substituted" or "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic groups have 1 to about 4, preferably 1 to about 3, more preferably 1 to 2 nonhydrogen substituents. Suitable substituents include, but are not limited to, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano, and ureido groups. The term "acyl" refers to an alkyl group connected to another chemical moiety via a carbonyl group (C=O).

[0389] In some embodiments, the lipophilic moiety is an aralkyl group, for example, a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety binds in vivo to at least one protein. In certain embodiments, the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, vascular proteins, or cellular proteins. In certain embodiments, the structural features of the aralkyl group facilitate binding to albumin, immunoglobulins, lipoproteins, α-2-macroglobulins, or α-1-glycoproteins.

[0390] In certain embodiments, the ligand is naproxene or a structural derivative of naproxene. The synthesis procedure for naproxene can be found in U.S. Patents 3,904,682 and 4,009,197, which are incorporated herein by reference. Naproxene has the chemical name (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid, and its structure is

[0391] [ka] That is the case.

[0392] In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. The synthesis procedure for ibuprofen can be found in U.S. Patent No. 3,228,831, which is incorporated herein by reference. The structure of ibuprofen is:

[0393] [ka] That is the case.

[0394] Additional exemplary aralkyl groups are illustrated in U.S. Patent No. 7,626,014, which is incorporated herein by reference as is.

[0395] In another embodiment, suitable lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0396] In certain embodiments, two or more lipophilic moieties can be incorporated into a double-stranded RNAi drug, particularly when the lipophilicity or hydrophobicity of the lipophilic moieties is low. In one embodiment, two or more lipophilic moieties are incorporated into the same strand of the double-stranded RNAi drug. In one embodiment, one or more lipophilic moieties are incorporated into each strand of the double-stranded RNAi drug. In one embodiment, two or more lipophilic moieties are incorporated into the same position of the double-stranded RNAi drug (i.e., the same nuclear base, the same sugar moiety, or the same internucleosidic linkage). This can be achieved, for example, by conjugating two or more lipophilic moieties via a carrier, and / or by conjugating two or more lipophilic moieties via a branched linker, and / or by conjugating two or more lipophilic moieties via one or more linkers using one or more linkers that sequentially link the lipophilic moieties.

[0397] The lipophilic portion can be conjugated to the RNAi drug via direct linkage to the ribosugar of the RNAi drug. Alternatively, the lipophilic portion can be conjugated to the double-stranded RNAi drug via a linker or carrier.

[0398] In certain embodiments, the lipophilic portion may be conjugated to an RNAi drug via one or more linkers [tethers].

[0399] In one embodiment, the lipophilic portion is conjugated to a double-stranded RNAi drug via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimidothioether, disulfide, phosphodiester, sulfonamide bond, click reaction product (e.g., triazole obtained from azido-alkyne cycloaddition), or carbamate.

[0400] Exemplary linkers, tethers, carriers, nucleic acid modifiers, conjugates, ligands, and other components useful for achieving central nervous system-directed delivery of APP-targeted RNAi drugs of this disclosure are described in additional details, for example, in International Publication No. 2019 / 217459, the entire contents of which are incorporated herein by reference.

[0401] Additional lipophilic modified nucleotides include those described in International Publication No. 2021 / 092371 and U.S. Provisional Patent Application No. 63 / 357,379, filed June 30, 2022, entitled "MONOMERS AND METHODS FOR SYNTHESIS OF MODIFIED OLIGONUCLEOTIDES," the full contents of which are incorporated herein by reference.

[0402] Further exemplary lipid-modified double-chain motifs considered for use in the compositions and methods disclosed herein can be found in International Publication Nos. 2023 / 245060 and International Publication Nos. 2023 / 245061, which are incorporated herein by reference as they are.

[0403] Lipid conjugate In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds to a serum protein, such as human serum albumin (HSA). The HSA-binding ligand enables the vascular distribution of the conjugate to a target tissue, such as a non-renal target tissue of the body. In a particular embodiment, the target tissue may be the CNS, including glial cells of the brain. Other molecules that can bind to HSA can also be used as ligands. For example, neproxine or aspirin can be used. The lipid or lipid-based ligand can (a) increase the conjugate's resistance to degradation, (b) increase the targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, such as HSA.

[0404] Lipid-based ligands can be used to inhibit or control the binding of conjugates to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to target the kidneys and therefore less likely to be excreted from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used for kidney targeting by conjugates.

[0405] Lipid-based ligands may bind to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity so that the conjugate is preferentially distributed to non-renal tissue. However, it is preferable that the affinity is not so strong that HSA-ligand binding becomes irreversible.

[0406] In another preferred embodiment, the lipid-based ligand binds weakly to or does not bind at all to the HSA so that the conjugate is preferentially distributed to the kidney. Other parts that target renal cells can also be used instead of or in addition to the lipid-based ligand.

[0407] In another embodiment, the ligand is a portion taken up by target cells, such as proliferating cells, e.g., a vitamin. This is particularly useful for treating disorders characterized by unintended cell proliferation, such as malignant or non-malignant types of proliferation, e.g., cancer cell proliferation. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by target cells, e.g., brain cells. HSA and low-density lipoprotein (LDL) are also included.

[0408] In some embodiments, the carbohydrate conjugate of the RNAi agent of this disclosure further comprises one or more additional ligands, such as, but not limited to, PK modulators and / or cell-penetrating peptides.

[0409] Examples of additional carbohydrate conjugates (and linkers) suitable for use in this disclosure include those described in PCT publication numbers International Publication No. 2014 / 179620 and International Publication No. 2014 / 179627, the entire contents of which are incorporated herein by reference.

[0410] Exemplary lipid-modified double-stranded motifs For example, in a particular embodiment, the sense strand of the dsRNA drug follows the following modification pattern:

[0411] [Table 10] It has one of the following. In the formula, n is a 2'-O-methylnucleotide, Nf is a 2'-fluoromodified nucleotide, and The sense chain includes at least one of the following modifications (a) to (c): (a) A lipophilic modified nucleotide "(Lipo)" that is substituted at either position 4-8 or position 13-18, counting from the 5' end of the chain. For example, (Nhd)-2'-O-hexadecyl modified nucleotide, (Nda)-α2'-O-docosanyl modified nucleotide, (NhdOH)-2'-O-(ω-hydroxyhexadecyl) modified nucleotide), or (NdaOH)-a2'-O-(ω-hydroxydocosanyl) modified nucleotide These are some examples. (b) 5'-(L1)(inv)-(5'-5') linked to the 5' terminal nucleotide via a phosphodiester or phosphorothioate bond, as appropriate. (c) -(inv)(L2)-3'(3'-3') nucleotides linked to the 3' terminal nucleotide via a phosphodiester or phosphorothioate bond, as appropriate. During the ceremony, Each (inv) is a nucleotide in the reverse direction (e.g., a debased nucleotide in the reverse direction, a debased ribonucleotide in the reverse direction, a debased deoxyribonucleotide in the reverse direction, etc.), (L1) and (L2) are, independently, absent, hydrogen, or lipophilic groups (e.g., C 10 ~C 30 Alkyl or C 10 ~C 30 Alkenyl group, for example, C 16 Alkyl, C 16 Alkenil, C 18 Alkyl, C 18 Alkenil, C 20 Alkyl, C 20 Alkenil, C 22 Alkyl, C 22 Alkenil, C 24 Alkyl, C 24 Alkenil, C 15 Alkyl, C 15 Alkenil, C 17 Alkyl, C 17 Alkenil, C 19 Alkyl, C 19 Alkenil, C 21 Alkyl, C 21 Alkenil, C 23 Alkyl, or C 23 It is a ligand that contains alkenils.

[0412] In each of the preceding sense strands, each nucleotide is linked in series (i.e., in a 3'->5' configuration) via appropriately modified internucleotide bonds. For example, each nucleotide is linked by phosphodiester or phosphorothioate internucleotide bonds.

[0413] In a particular embodiment, counting from the 5' end of the oligonucleotide, the nucleotides at positions 1 and 2 are connected by phosphorothioate nucleotide bonds; the nucleotides at positions 2 and 3 are connected by phosphorothioate nucleotide bonds; and the remaining nucleotides are connected via phosphodiester bonds.

[0414] In a particular embodiment, counting from the 5' end of the oligonucleotide, the nucleotides at positions 1 and 2 are connected by phosphorothioate nucleotide bonds; the nucleotides at positions 2 and 3 are connected by phosphorothioate nucleotide bonds; the nucleotides at positions 3 and 4 are connected by phosphorothioate nucleotide bonds; and the remaining nucleotides are connected via phosphodiester bonds.

[0415] In certain embodiments, for nucleotides within a nucleotide of length m, the nucleotides at positions m-1 and m from the 5' end of the oligonucleotide are linked by phosphorothioate nucleotide bonds. That is, for nucleotides within a nucleotide of length 23, the nucleotides at positions 22 and 23 from the 5' end of the oligonucleotide are linked by phosphorothioate nucleotide bonds; and for nucleotides within a nucleotide of length 21, the nucleotides at positions 20 and 21 from the 5' end of the oligonucleotide are linked by phosphorothioate nucleotide bonds.

[0416] In a particular embodiment, for a nucleotide within a nucleotide of length m, the nucleotides at positions m-2 and m-1, counting from the 5' end of the oligonucleotide, are connected by phosphorothioate nucleotide bonds, and the nucleotides at positions m-1 and m are connected by phosphorothioate nucleotide bonds. That is, for a nucleotide within a nucleotide of length 23, the nucleotides at positions 21 and 22, counting from the 5' end of the oligonucleotide, are connected by phosphorothioate nucleotide bonds; and positions 22 and 23 are connected by phosphorothioate nucleotide bonds; and for a nucleotide within a nucleotide of length 21, the nucleotides at positions 20 and 21, counting from the 5' end of the oligonucleotide, are connected by phosphorothioate nucleotide bonds.

[0417] In other embodiments, each (inv) ligated to the 5' terminal nucleotide is linked via a phosphorothioate bond (5'-5').

[0418] In other embodiments, each (inv) linked to the 3' terminal nucleotide is connected via a phosphorothioate bond (3'-3').

[0419] In certain other embodiments, each (inv) linked to the 5' terminal nucleotide is connected via a phosphorothioate bond (5'-5'), and each (inv) linked to the 3' terminal nucleotide is connected via a phosphorothioate bond (3'-3').

[0420] For example, in a particular embodiment, the sense strand of the dsRNA drug has one of the following modification patterns (variables as defined above):

[0421] [Table 11] In formula TIFF2026514044000058.tif204143, s represents a phosphorothioate nucleotide bond.

[0422] In some embodiments, the antisense strand of the dsRNA drug has one of the following modification patterns:

[0423] [Table 12] n is a 2'-O-methyl-modified nucleotide; s is a phosphorothioate internucleotide bond (3'-5'); (dN) is a 2'-deoxynucleotide; Nf is a 2'-fluoro-modified nucleotide; (G) is a modification that is thermally destabilized, for example (Ngn)-glycolic acid, S-isomer; (N2p)-2' phosphate nucleotide (i.e., 3'-RNA linked in the 5' and 3' directions, respectively, by 3'-5' and 2'-5' nucleotide bonds); (Tna)-threose nucleotide (linked in the 5' and 3' directions, respectively, by 3'-3' and 2'-5' nucleotide bonds); (MM) Nuclear-base mismatch with the sense strand; (Nul) Unlocked nucleic acids, etc.; and Z is 5'-phosphate or a 5'-phosphate mimetic, for example VP is vinylphosphonate [e.g., 5'-(E)-vinylphosphonate]

[0424] [ka] or a salt thereof, wherein the 4'-CH2OH group in the ribose ring of the 5'-terminal nucleotide is substituted due to the preceding structure, or 5'-cyclopropylphosphonate' (5'-CP), or 4'-O-methylphosphonate (4'-OMP), or 4'-O-methylphosphonate methyl ester (4'-OMPMe), etc.

[0425] The structure of 5'-CP, when used herein,

[0426] [ka] or a salt thereof, where the cleaved bond is connected to the 4'-C of ribose (i.e., the preceding structure substitutes the 4'-CH2OH group in the ribose ring of the 5'-terminal nucleotide). The structure of 4'-OMP, as used herein,

[0427] [ka] or a salt thereof, where the cleaved bond is connected to the 4'-C of ribose (i.e., the preceding structure substitutes the 4'-CH2OH group in the ribose ring of the 5'-terminal nucleotide). The structure of 4'-OMPMe, as used herein,

[0428] [ka] or a salt thereof, in which the cleaved bond is connected to the 4'-C of ribose (i.e., the preceding structure substitutes the 4'-CH2OH group in the ribose ring of the 5' terminal nucleotide).

[0429] In each of the preceding antisense strands, each nucleotide is linked in series (i.e., in a 3'->5' manner) via phosphodiester or phosphorothioate nucleotide-nucleotide bonds.

[0430] In certain embodiments, counting from the 5' end of the oligonucleotide, the nucleotides at positions 1 and 2 are connected by phosphorothioate nucleotide bonds; the nucleotides at positions 2 and 3 are connected by phosphorothioate nucleotide bonds; and the remaining nucleotides are connected via phosphodiester bonds.

[0431] In certain embodiments, for nucleotides within a nucleotide of length m, the nucleotides at positions m-1 and m from the 5' end of the oligonucleotide are linked by phosphorothioate nucleotide-nucleotide bonds. That is, for nucleotides within a nucleotide of length 23 (e.g., an antisense strand), the nucleotides at positions 22 and 23 from the 5' end of the oligonucleotide are linked by phosphorothioate nucleotide-nucleotide bonds; and for nucleotides within a nucleotide of length 21 (e.g., a sense strand), the nucleotides at positions 20 and 21 from the 5' end of the oligonucleotide are linked by phosphorothioate nucleotide-nucleotide bonds.

[0432] In certain embodiments, for a nucleotide within a nucleotide of length m, the nucleotides at positions m-2 and m-1, counting from the 5' end of the oligonucleotide, are linked by phosphorothioate nucleotide-nucleotide bonds, and the nucleotides at positions m-1 and m are linked by phosphorothioate nucleotide-nucleotide bonds. That is, for a nucleotide within a nucleotide of length 23 (e.g., an antisense chain), the nucleotides at positions 21 and 22, counting from the 5' end of the oligonucleotide, are linked by phosphorothioate nucleotide-nucleotide bonds; and positions 22 and 23 are linked by phosphorothioate nucleotide-nucleotide bonds; and for a nucleotide within a nucleotide of length 21 (e.g., a sense chain), the nucleotides at positions 20 and 21, counting from the 5' end of the oligonucleotide, are linked by phosphorothioate nucleotide-nucleotide bonds.

[0433] In a particular embodiment, for a nucleotide within a nucleotide of length m, counting from the 5' end of the oligonucleotide: (a) The nucleotides at positions 1 and 2 are linked by a phosphorothioate nucleotide bond; (b) The nucleotides at positions 2 and 3 are linked by phosphorothioate nucleotide bonds; (c) The nucleotides at positions m-2 and m-1 are linked by phosphorothioate nucleotide bonds; and (d) The nucleotides at the m-1 and m positions are linked by phosphorothioate nucleotide bonds, The remaining nucleotides are linked via phosphodiester bonds.

[0434] In other words, for nucleotides within a nucleotide of length 23 (e.g., an antisense strand), the nucleotides at positions 1 and 2; 2 and 3; 21 and 22; and 22 and 23, counting from the 5' end of the oligonucleotide, are connected by phosphorothioate nucleotide bonds, while the remaining nucleotides are connected by phosphodiester bonds. Similarly, for nucleotides within a nucleotide of length 21 (e.g., a sense strand), the nucleotides at positions 1 and 2; 2 and 3; 19 and 20; and 20 and 21, counting from the 5' end of the oligonucleotide, are connected by phosphorothioate nucleotide bonds, while the remaining nucleotides are connected by phosphodiester bonds.

[0435] In a particular embodiment, for a nucleotide within a nucleotide of length m, counting from the 5' end of the oligonucleotide: (a) The nucleotides at positions 1 and 2 are linked by a phosphorothioate nucleotide bond; (b) The nucleotides at positions 2 and 3 are linked by phosphorothioate nucleotide bonds; (c) The nucleotides at positions 3 and 4 are linked by phosphorothioate nucleotide bonds; and (d) The nucleotides at the m-1 and m positions are linked by phosphorothioate nucleotide bonds, The remaining nucleotides are linked via phosphodiester bonds.

[0436] In other words, for a nucleotide with a length of 23 (e.g., an antisense strand), the nucleotides at positions 1 and 2; 2 and 3; 3 and 4; and 22 and 23, counting from the 5' end of the oligonucleotide, are connected by phosphorothioate nucleotide bonds, while the remaining nucleotides are connected by phosphodiester bonds. Similarly, for a nucleotide with a length of 21 (e.g., a sense strand), the nucleotides at positions 1 and 2; 2 and 3; 3 and 4; and 20 and 21, counting from the 5' end of the oligonucleotide, are connected by phosphorothioate nucleotide bonds, while the remaining nucleotides are connected by phosphodiester bonds.

[0437] For example, in some embodiments, the antisense strand of the dsRNA drug has one of the following modification patterns (variables as defined above):

[0438] [Table 13]

[0439] In further embodiments of the preceding exemplary sense strands and antisense strands, each of the sense strands S1 to S54 may be doubled with one of the antisense strands AS1 to AS44.

[0440] delivery The delivery of the RNAi drug (or other nucleic acid therapeutic agent) composition of this disclosure to cells, for example, a subject, such as a human subject (for example, a subject that needs it, such as a subject with a target gene-related disorder, such as AD, CAA, EOFAD, etc.), can be achieved in several different ways. For example, delivery may be carried out by contacting cells with the RNAi drug composition of this disclosure either in vitro or in vivo. In vivo delivery may be carried out by directly administering the RNAi drug, for example, a composition containing dsRNA, to the subject. Alternatively, in vivo delivery may be carried out indirectly by administering one or more vectors that encode and direct the expression of the RNAi drug.

[0441] Generally, methods for delivering nucleic acid molecules (in vitro or in vivo) can be modified for use with the RNAi drug compositions of this disclosure [see Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5): 139-144 and International Publication No. 94 / 02595, which are incorporated herein by reference]. For in vivo delivery, factors to be considered for delivering the RNAi drug composition include, for example, the biological stability of the delivered drug, prevention of nonspecific effects, and accumulation of the delivered drug in the target tissue. Nonspecific effects of RNAi drugs can be minimized by local administration, for example, direct injection or transplantation into tissue, or topical administration of the preparation. Local administration to the treatment site maximizes the local concentration of the drug, limiting exposure to systemic tissues that might otherwise be adversely affected or degraded by the drug, thus enabling the administration of RNAi drugs at a low total dose. Several studies have demonstrated successful knockdown of gene products when RNAi drugs are administered locally. For example, intravitreal injection of VEGF dsRNA into cynomolgus monkeys [Tolentino, MJ. et al., (2004) Retina 24: 132-138] and subretinal injection into mice [Reich, SJ. et al. (2003) Mol. Vis. 9: 210-216] both demonstrated inhibition of angiogenesis in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice reduces tumor volume [Pille, J. et al. (2005) Mol. Ther. 11: 267-274] and extends the survival period of tumor-bearing mice [Kim, WJ. et al., (2006) Mol. Ther. 14: 343-350; Li, S. et al., (2007) Mol. Ther. 15: 515-523].Regarding RNA interference, local delivery to the CNS by direct injection [Dorn, G. et al., (2004) Nucleic Acids 32: e49; Tan, PH. et al. (2005) Gene Ther. 12: 59-66; Makimura, H. et al. (2002) BMC Neurosci. 3: 18; Shishkina, GT., et al. (2004) Neuroscience 129: 521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101: 17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93: 594-602] and local delivery to the lungs by intranasal administration [Howard, KA. et al., The following studies have shown success: (2006) Mol. Ther. 14: 476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279: 10677-10684; Bitko, V. et al., (2005) Nat. Med. 11: 50-55.

[0442] Certain aspects of the present disclosure relate to a method for reducing the expression of a target gene within a cell or object, comprising contacting the cell or object with a double-stranded RNAi drug composition of the present disclosure. In one embodiment, the cell is an extrahepatic cell and may be a CNS cell.

[0443] Another aspect of the present disclosure relates to a method for reducing the expression of a target gene within a subject, comprising administering the double-stranded RNAi drug composition of the present disclosure to the subject.

[0444] Another aspect of the present disclosure relates to a method for treating a subject having a target gene-related disorder, comprising administering a therapeutically effective amount of a double-stranded RNAi drug-containing composition of the present disclosure to the subject, thereby treating the subject.

[0445] In one embodiment, the double-stranded RNAi agent is administered intrathecally. Intrathecal administration of the double-stranded RNAi agent allows the method to reduce the expression of target genes in the brain (e.g., striatum) or in spinal tissue, such as the cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

[0446] For the sake of clarity, the formulations, compositions, and methods described in this section are primarily considered in relation to modified siRNA compounds. However, it can be understood that such formulations, compositions, and methods are also applicable with other siRNA compounds, such as unmodified siRNA compounds, and such practices are also included in this disclosure.

[0447] The RNAi drug compositions of this disclosure can be further incorporated into pharmaceutical compositions suitable for parenteral administration. Such compositions generally comprise one or more species of RNAi drugs and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any solvent, antimicrobial and antifungal agent, isotonic agent, etc., that is compatible with the administration of the drug, except in certain embodiments, drugs containing inorganic phosphate are excluded [for example, in certain embodiments, phosphate-buffered saline (PBS) as an isotonic solution is excluded]. The use of such media and drugs for pharmaceutically active substances is well known in the art. Any conventional media or drug is intended for use in a composition unless it is incompatible with the active compound. Auxiliary active compounds may also be incorporated into the composition.

[0448] The primary route of administration for the pharmaceutical compositions of this disclosure is via parenteral administration, such as intrathecal injection; however, the pharmaceutical compositions of this disclosure may be administered in several ways depending on whether local or systemic treatment is desired and the area being treated. Parenteral administration includes intravenous infusion, subcutaneous injection, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

[0449] Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions (except in certain embodiments, which exclude drugs containing inorganic phosphoric acid), as well as diluents and other suitable additives.

[0450] Parenteral formulations may contain sterile aqueous solutions, as well as diluents and other suitable additives. Intracerebroventricular injection is facilitated, for example, by an intracerebroventricular catheter connected to a reservoir. For intravenous use, the total concentration of the solute may be controlled to make the preparation isotonic.

[0451] In one embodiment, the administration of a dsRNA compound, such as a double-stranded siRNA compound or ssiRNA compound, is parenteral, and is, for example, intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, or intraocular injection. The administration may be provided by the subject or by another person, such as a healthcare provider. The selected mode of delivery is discussed in more detail below.

[0452] Intrathecal administration In certain embodiments, nucleic acid drug formulations (e.g., double-stranded RNAi drug compositions) are delivered by intrathecal injection (i.e., injection into cerebrospinal fluid impregnating brain and spinal cord tissue). Intrathecal injection of nucleic acid (e.g., RNAi) drugs into cerebrospinal fluid can be performed as a bolus injection or via a minipump implanted under the skin, which can enable regular and consistent delivery of nucleic acids into the cerebrospinal fluid. Cerebrospinal fluid circulation originates from the choroid plexus where cerebrospinal fluid is produced, flows down to the spinal cord and surrounding spinal dorsal root ganglia, then passes through the cerebellum and over the cerebral cortex to the arachnoid granules, where cerebrospinal fluid can be drained from the CNS. Thus, depending on the size, stability, and solubility of the injected compound, the intrathecally delivered molecules can act on their targets throughout the CNS.

[0453] In some embodiments, intrathecal administration is via a pump. The pump may be a surgically implanted osmotic pump. In one embodiment, the osmotic pump is implanted in the subarachnoid space of the spinal canal to facilitate intrathecal administration.

[0454] In some embodiments, intrathecal administration is via an intrathecal delivery system for pharmaceuticals, which includes a reservoir containing a certain volume of pharmaceutical agent and a pump configured to deliver a portion of the pharmaceutical agent contained in the reservoir. More detailed information regarding this intrathecal delivery system can be found in International Publication No. 2015 / 116658, which is incorporated as is for reference.

[0455] The amount of nucleic acid drug (e.g., RNAi drug) administered intrathecally may vary depending on the target gene, and the appropriate amount to be applied may also be determined individually for each target gene. In several embodiments, this amount is in the range of 10 μg to 100 mg / mL of injection, may be 50 μg to 150 mg / mL, or even 20 mg to 100 mg / mL of injection.

[0456] mRNA knockdown to treat related diseases As used herein, “Subject” means an animal, such as a mammal, and includes primates (e.g., humans, non-human primates, such as monkeys and chimpanzees) or non-primates (e.g., rats or mice). In preferred embodiments, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced target gene expression; a human being at risk of a disease, disorder, or condition that would benefit from reduced target gene expression; a human being having a disease, disorder, or condition that would benefit from reduced target gene expression, or a human being treated for a disease, disorder, or condition that would benefit from reduced target gene expression as described herein.

[0457] As used herein, the terms “treating” or “treatment” mean a beneficial or desirable outcome, including, but not limited to, the reduction or improvement of one or more symptoms associated with a disease or disorder. In certain embodiments, “treating” or “treatment” means a beneficial or desirable outcome, including, but not limited to, the reduction or improvement of one or more symptoms associated with, for example, APP-related diseases or disorders associated with the regulation of APP gene expression and / or APP protein production, such as, for example, Alzheimer's disease (AD), cerebral amyloid vascular disease (CAA, e.g., hereditary CAA), early-onset familial Alzheimer's disease (EOFAD or eFAD), early-onset Alzheimer's disease (EOAD), familial Alzheimer's disease, or late-onset Alzheimer's disease. “Treatment” may also mean life extension compared to the expected lifespan if no treatment is performed.

[0458] In the context of target genes within a subject, or disease markers, or symptom levels, the term “reduction” refers to a statistically significant decrease in such levels. A reduction may be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the reduction is at least 20%. In certain embodiments, the reduction in disease markers, such as protein levels or gene expression levels, is at least 50%. In the context of target gene levels within a subject, “reduction” may be a reduction to a level acceptable as within the normal range in individuals without such disorder. In certain embodiments, “reduction” is a reduction in the difference between the level of a marker or symptom in a person suffering from a disease and an acceptable level within the normal range for that person, for example, between the level of memory or cognition decline in an individual with Alzheimer’s disease and the level of memory or cognition decline in an individual without Alzheimer’s disease or an individual with symptoms that are within the normal range.

[0459] Where “prevention” or “prevention of” is used herein, when used in connection with a disease or disorder, the disease or disorder is one in which a subject is susceptible to, for example, a target gene-related disorder due to genetic factors or age, and who does not yet meet the diagnostic criteria for the target gene-related disorder, and who benefits from reduced expression of the target gene or production of the target protein in such a subject. Where used herein, prevention may be understood as the administration of a drug to a subject who does not yet meet the diagnostic criteria for a target gene-related disorder in order to delay the onset of the subject or reduce the likelihood thereof. Since the drug is a pharmaceutical substance, its administration will generally be understood as being under the direction of a healthcare professional capable of identifying a subject who does not yet meet the diagnostic criteria for the target gene-related disorder as susceptible to developing the target gene-related disorder.

[0460] [Table 14] TIFF2026514044000066.tif253154 TIFF2026514044000067.tif243154 TIFF2026514044000068.tif246154 TIFF2026514044000069.tif141160

[0461] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of CAA and AD, including hereditary CAA and EOFAD, as well as APP-related disorders or disorders, including sporadic and / or late-onset AD. Hereditary CAA (hCAA) is a vascular proteinopathy in which the amyloid treatment hypothesis is relatively simple and clinically testable. hCAA is a devastating and rare disease, and no existing therapies exist. Both biochemical and imaging biomarkers exist to clinically validate anti-APP siRNA-mediated treatment of hCAA. Various types of hCAA are described, including sporadic CAA, HCHWA Dutch and Italian EOFAD, LOAD, and trisomy 21 as an Aβ-related form of hAPP. hCAA may be associated with AD, EOFAD, and / or Down syndrome, among other conditions.

[0462] In particular, soluble forms of APP, including APPα and APPβ, may be useful as cerebrospinal fluid (CSF) biomarkers for evaluating the effectiveness of APP knockdown. Further discussion of hCAA and APP can be found in the references, especially in PCT / US 19 / 67449.

[0463] The available assay methods can also be used to detect soluble APP levels in human CSF samples. In particular, sAPP and sAPPβ are soluble forms of APP and have been identified as useful pharmacodynamic (PD) biomarkers. Analytes have also been detected in non-human primate (NHP) CSF samples, and such assay methods enable efficacy testing in NHP. Detection of Aβ40 / 42 / 38 peptides and total tau / P181 tau is also described.

[0464] Animal models of CAA have been identified, making it possible to determine the effect of APP knockdown on CAA pathology and identify translationable biomarkers. In particular, several rodent models expressing mutant human APP, including Tg-SwDI / NOS2- / -, and exhibiting CAA pathology have been developed [Hall and Roberson. Brain Res Bull. 2012; 88(1): 3-12; Attems et al., Nephrology and Applied Neurobiology, 2011, 37, 75-93].

[0465] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of SOD1-related diseases or disorders. The protein encoded by the SOD1 gene is superoxide dismutase 1, which binds to both copper and zinc ions. Rare transcript variants have been reported for this gene. The SOD1 protein is one of two isozymes that facilitate the removal of free superoxide radicals in the body by converting naturally occurring superoxide radicals into molecular oxygen and hydrogen peroxide. Mutations in SOD1 have been suggested as a cause of familial amyotrophic lateral sclerosis (ALS).

[0466] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of SCN9a-related diseases or disorders. The SCN9a gene encodes a voltage-opening sodium channel that plays a crucial role in nociceptive signaling. Mutations within this gene have been associated with primary erythromelalgia, channelopathy-associated insensitivity to pain, and paroxysmal severe pain.

[0467] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of HTT-related diseases or disorders. HTT (huntingtin) is a disease gene associated with Huntington's disease, a neurodegenerative disorder characterized by the loss of neurons in the striatum and thought to be caused by an extended, unstable trinucleotide repeat within the HTT gene that is translated as a polyglutamine repeat in the protein product. The HTT locus is a large locus, approximately 180 kb in length and containing 67 exons. While not bound by theory, the genetic defect that causes Huntington's disease does not necessarily result in the loss of transcription, but it may confer new properties to the mRNA or alter the function of the protein.

[0468] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of APOE-related disease or disorder. The protein encoded by the APOE gene is the major apolipoprotein of chylomicrons. This protein binds to specific liver and peripheral cell receptors and is essential for the normal catabolism of triglyceride-rich lipoprotein components. Mutations in APOE lead to familial abnormal β-lipoproteinemia or type III hyperlipoproteinemia (HLP III), resulting in increased plasma cholesterol and triglycerides as a result of impaired cleavage of chylomicrons and VLDL remnants.

[0469] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of LRRK2-related disease or disorder. The LRRK2 gene is a member of the leucine-rich repeat kinase family and encodes a protein comprising an ankyrin repeat region, a leucine-rich repeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, an MLK-like domain, and a WD40 domain. The LRRK2 protein is primarily located in the cytoplasm but also associates with the outer mitochondrial membrane. Mutations within this gene have been associated with Parkinson's disease.

[0470] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of PRNP-related diseases or disorders. The PRNP protein encodes a membrane glycosylphosphatidylinositol-anchored glycoprotein that tends to aggregate into a rod-like structure. The PRNP protein contains an extremely unstable region consisting of five serial octapeptide repeats. This gene is located on chromosome 20, approximately 20 kbp upstream of a gene encoding a biochemically and structurally similar protein to the one encoded by this gene. Mutations in the repeat region of this gene, as well as in other regions, have been associated with Creutzfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Streussler disease, Huntington's disease-like disorder type 1, and Kuru disease.

[0471] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of SCD5-related diseases or disorders. Stearoyl-CoA desaturase is a membrane-endogenous protein of the intracytoplasmic reticular structure that catalyzes the formation of monounsaturated fatty acids from saturated fatty acids. Four SCD isoforms (SCD1-SCD4) have been identified in mice, while only two SCD isoforms (SCD1 and SCD5) have been identified in humans. SCD1 shares approximately 85% amino acid identity with all four mouse SCD isoforms, as well as rat SCD1 and SCD2. However, SCD5 shares only limited homology with rodent SCD and appears to be unique to primates. Diseases associated with SCD5 include deafness, autosomal dominant inheritance, and chronic maxillary sinusitis.

[0472] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of GPR75-related diseases or disorders. GPR75 encodes a member of the G protein-coupled receptor family. GPR proteins are cell surface receptors that activate guanine nucleotide-binding proteins upon ligand binding. GPR75 is activated by the chemokine CCL5 / RANTES. Though not theoretically bound, GPR75 is thought to couple with the heterotrimeric Gq protein, where activation stimulates inositol triphosphate production and calcium recruitment. Together with CCL5 / RANTES, GPR75 may play a role in neuronal survival through activation of downstream signaling pathways involving PI3, Akt, and MAP kinases. CCL5 / RANTES may also regulate insulin secretion by pancreatic islet cells through activation of this receptor. GPR75 is thought to play a role in obesity.

[0473] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of MAPT-related diseases or disorders. The MAPT gene encodes microtubule-associated protein tau (MAPT), whose transcript undergoes controlled, complex alternative splicing, giving rise to several mRNA species. MAPT transcripts are differentially expressed in the nervous system depending on the neuronal maturation stage and neuron type. MAPT gene mutations have been associated with several neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, frontotemporal dementia, corticobasal degeneration, and progressive supranuclear palsy.

[0474] Certain aspects of this disclosure relate to RNAi drug-mediated knockdown of SNCA-related diseases or disorders. SNCA (synuclein alpha) is a member of the synuclein family, which also includes beta and gamma-synucleins. Synucleins are abundantly expressed in the brain, and alpha and beta-synucleins selectively inhibit phospholipase D2. SNCA may play a role in integrating presynaptic signaling and membrane transport. Defects in SNCA have been suggested to be related to the pathogenesis of Parkinson's disease. In addition, SNCA peptides are a major component of amyloid plaques present in the brains of patients with Alzheimer's disease.

[0475] Therefore, APP has been identified as a target for hereditary cerebral amyloid vascular disease (CAA). Mutations within APP that have been reported to cause severe forms of CAA include A692G (Flemish type), E693Q (Dutch type), E693K (Italian type), and D694N (Iowa type). On the other hand, mutations within APP that have been described to cause early-onset AD include E665D, K670N, M671L (Swedish type), T714A (Iranian type), T714I (Austrian type), V715M (French type), V715A (German type), I716V (Florida type), I716T, V717I (London type), V717F, V717G, and V717L. In particular, the APP E693Q (Dutch type) mutation causes severe CAA with some parenchymal neurofibrillary tangles; E693Q increases amyloid-beta aggregation and toxicity; E693K (Italian type) is similar, but E693G (Arctic type), E693A, and E693 delta mutations cause EOFAD with little or no CAA; and APP D694N (Iowa type) causes severe CAA with typical AD pathology. In addition to the above point mutations, APP duplication causing APP overexpression has also been identified as causing Aβ deposition. On the other hand, there are no known APP mutations that inhibit or delay APP-hCAA. In addition to APP mutants, AβCAA has also been observed with PSEN1 (L282V) and PSEN2 (N141I) mutations. On the other hand, ApoEε2 (unrelated to AD) and ApoEε4 (AD-dependent) have also been reported as risk factors for CAA [Rensink A et al., Brain Research Reviews, 43 (2) 2003].

[0476] Certain aspects of this disclosure relate to targeting APP for the purpose of knockdown in individuals with APP-hCAA. Given the current lack of disease-modifying therapies for CAA, there is a need for such agents. In certain embodiments, the RNAi agents of this disclosure should achieve approximately 60–80% knockdown of both mutant and wild-type APP levels throughout the CNS.

[0477] The term "APP" refers to amyloid precursor protein (APP), also known by various names, particularly amyloid-beta precursor protein, Alzheimer's disease amyloid protein, and cerebral vascular amyloid peptide, and unless otherwise specified, refers to a polypeptide having an amino acid sequence derived from any vertebrate or mammal, including but not limited to humans, cattle, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs. The term refers to fragments and variants of native APP that maintain at least one in vivo or in vitro activity of native APP (in particular, including, for example, the forms of beta-amyloid peptides (1-40), beta-amyloid peptides (1-38), and beta-amyloid peptides (1-42) of Aβ peptide), and includes variants of APP fragments that retain the activity of one or more APP fragments that are neurotoxic as a characteristic (for example, a variant form of Aβ42 peptide that retains neurotoxic characteristics is explicitly assumed). The term encompasses the full-length, untreated precursor form of APP, as well as the mature form resulting from post-translational cleavage of the signal peptide. The term also includes peptides derived from APP via further cleavage, such as the Aβ peptide. The nucleotide and amino acid sequences of human APP can be found, for example, in GenBank accession number GI:228008405(NM_201414).The nucleotide and amino acid sequences of human APP are, for example, those of GenBank accession numbers GI:228008403(NM_000484.4);GenBank accession number GI:228008404(NM_201413.3);GenBank accession number GI:324021746(NM_001136016.3);GenBank accession number GI:228008402(NM_001136129.3);GenBank accession number GI:228008401(NM_001136130. 3);GenBank accession number GI:324021747(NM_001136131.3);GenBank accession number GI:324021737(NM_001204301.2);GenBank accession number GI:324021735(NM_001204302.2);and GenBank accession number GI:324021739(NM_001204303.2);and GenBank accession number GI:1370481385(XM_024452075.1). Examples of additional APP sequences are readily available using publicly available databases, such as GenBank, UniProt, and OMIM.

[0478] As used herein, the term "APP" also refers to specific polypeptides expressed in cells due to naturally occurring DNA sequence variations in the APP gene, such as single nucleotide polymorphisms within the APP gene. A large number of SNPs have been identified within the APP gene, which can be found, for example, in NCBIdbSNP (see, for example, www.ncbi.nlm.nih.gov / snp). Non-exclusive examples of SNPs within the APP gene include, for example, NCBIdbSNP acceptance numbers rs193922916, rs145564988, rs193922916, rs214484, rs281865161, rs364048, rs466433, rs466448, rs532876832, rs63749810, rs63749964, rs63750064, rs63750066, and rs63750151. These variants may be found in rs63750264, rs63750363, rs63750399, rs63750445, rs63750579, rs63750643, rs63750671, rs63750734, rs63750847, rs63750851, rs63750868, rs63750921, rs63750973, rs63751039, rs63751122, and rs63751263. Certain rare, exemplary APP variants that have been previously described as playing a role in the development of EOFAD were identified by Hooli et al. (Neurology 78: 1250-57). In addition, various “non-classical” APP variants with intraexonal junctions in sequenced cDNA have recently been identified as being associated with the occurrence of somatic recombination in the brains of AD patients (PCT / US No. 2018 / 030520, incorporated verbatim for reference herein). Examples of such “non-classical” APP variants include cAPP-R3 / 16, cAPP-R3 / 16-2, cAPP-R2 / 18, cAPP-R6 / 18, cAPP-R3 / 14, cAPP-R3 / 17, cAPP-R1 / 11, cAPP-R1 / 13, cAPP-R1 / 11-2, cAPP-R1 / 14, cAPP-R2 / 17, cAPP-R2 / 16, cAPP-R6 / 17, cAPP-R2 / 14, cAPP-R14 / 17-d8, and cAPP-D2 / 18-3.It is expressly assumed that the RNAi agents of this disclosure can be used to target “non-classical” APP variants, and / or that RNAi agents that are appropriately specific to such “non-classical” APP variants can be appropriately designed and used in combination with other RNAi agents of this disclosure, including those that target the native forms of APP. It is stated that such “non-classical” APP variants are not particularly present in the HIV patient population assayed, and the prevalence of AD in this HIV patient population was significantly lower than expected, suggesting that reverse transcriptase inhibitors and / or other antiretroviral therapies commonly used to treat HIV patients likely play a therapeutic / preventive role in AD as well. Therefore, it is expressly assumed that the RNAi agents of this disclosure can be appropriately employed in combination with reverse transcriptase inhibitors and / or other antiretroviral therapies for therapeutic and / or preventive purposes.

[0479] Humans with heterozygous APP mutations exist in the general population with a pLI score of 0.3; however, human APP knockouts have not yet been identified.

[0480] The following are some pharmacological attempts to treat human CAA: Ponetsumab. This amyloid-beta 40 antibody was tested by Pfizer in 36 individuals with late-onset CAA. Three infusions of either ponezumab or placebo over a 60-day course were evaluated for changes in cerebrovascular reactivity as measured by bold fMRI, as well as changes in cerebral edema, infarction, Aβ, cognitive changes, and other secondary outcomes. Ponetsumab showed a difference between drug and placebo, but did not meet the primary endpoint. BAN2401. Although systemically delivered amyloid-beta therapeutic antibodies have been identified as safe, they may cause focal cerebral edema. In a recent Phase II 18-mo trial of BAN2401 in LOADs, the incidence of SAEs was 17.6% with placebo and 15.5% with the highest dose (10 mg / kg every two weeks). Amyloid-related imaging abnormalities-edema (ARIA-E) occurred in 14.6% of APOE4 carriers at the highest dose.

[0481] In animal models of CAA, ponezumab was found to be effective in reducing amyloid-beta loading and vascular reactivity in mouse models of CAA (Bales, 2018). Furthermore, it has been noted that overall APP knockout mice are survivable.

[0482] The following exemplary biomarkers and pathological data also provide further insight into the key role of amyloid-beta protein in the pathogenesis of CAA: In contrast to Aβ42 in parenchymal AD, a genetic form of "pure" CAA (i.e., lacking parenchymal plaque amyloid) has been observed, characterized by a dominance of Aβ40 deposition within amyloid; CAA is observed as "non-tauopathy," and in contrast to the elevated levels observed in AD, T-tau and P-tau levels in CSF are normal; A negative correlation between increased brain amyloid load measured by PiB PET and decreased CSF Aβ40 levels has been identified as specific to CAA; and In vitro and in vivo experimental data provided further support for the prion hypothesis in CAA, which posits that Aβ40 containing hereditary CAA mutations tends to misfold, inducing misfolding within the WT protein, and consequently, both are present within amyloid fibrils (similar to transthyretin (TTR)).

[0483] RNAi-mediated drug-mediated treatment for EOFAD is also explicitly envisioned. Similar to hCAA, EOFAD is a devastating and rare disease, and as with hCAA, the causal role of APP is well established. Furthermore, phenotyping of the disease can be performed with greater accuracy and in a shorter timeframe than, for example, sporadic and / or late-onset AD (and, where appropriate, late-onset AD with severe CAA as a subclass of late-onset AD). EOFAD is a progressive dementia-like neurodegenerative disease in young adults, with an age of onset between 60 and under 65 years, sometimes under 55 years.

[0484] The prevalence of EOFAD is estimated at 41.2 per 100,000 people in the risk group (i.e., those aged 40-59 years), and 61% of those with EOFAD had a family history of EOFAD (of which 13% had a family history of the disease spanning three generations). EOFAD accounts for less than 3% of all AD cases (Bird, Genetics in Medicine, 10: 231-239; Brien and Wang. Annu Rev Neu Sci, 2011, 34: 185-204; NCBI Gene Reviews).

[0485] As described above, specific APP mutations causing EOFAD have been identified, including E665D, K670N, M671L (Swedish type), T714A (Iranian type), T714I (Austrian type), V715M (French type), V715A (German type), I716V (Florida type), I716T, V717I (London type), V717F, V717G, and V717L, which provide human genetic validation for the APP target (OMIM 104300). In addition, dominant amyloid-beta precursor protein mutations causing EOFAD and CAA have also been identified.

[0486] While not strictly adhering to theory, the etiology of Alzheimer's disease (AD) is thought to begin in the hippocampus, a bulge of gray matter located directly above the bilateral ventricles. Degeneration of this tissue is thought to cause the memory loss characteristic of early-stage AD. The mechanisms of neurodegeneration at the protein level have been a subject of much debate, but the association of APP duplication with EOFAD suggests that APP overexpression may be sufficient to cause AD (Haass and Selkoe. Nature Reviews Molecular Cell Biology, 8: 101-112).

[0487] In contrast to EOFAD and CAA, the pathogenesis of sporadic AD is still not understood, and the clinically defined population of sporadic AD is likely mechanistically heterogeneous.

[0488] Certain aspects of this disclosure are directed toward targeting APP for knockdown in individuals with EOFAD. More generally, there is a need for such drugs because only symptomatic treatments (with limited efficacy) exist for AD, particularly EOFAD. In certain embodiments, the RNAi drugs of this disclosure should achieve approximately 60–80% knockdown of both mutant and WT APP levels throughout the CNS. Further observational findings from human genetics that mention the potential therapeutic efficacy of APP-targeted therapies capable of knocking down APP levels in CNS cells include the identification of the A673T mutation in the general population that protects carriers from AD and dementia (Jonsson et al. Nature Letter, 488. doi:doi:10.1038 / nature11283). The A673T substitution is adjacent to a β-secretase cleavage site and has been described as resulting in a 40% reduction in amyloid-beta in cell assays. Therefore, dominant inhibitory APP point mutations appear to protect families from AD, further supporting the idea that RNAi-mediated APP knockdown may exert similar protective and / or therapeutic effects in at least certain forms of AD, including EOFAD.

[0489] APP knockout mice have been shown to be viable as a support for the early stages of APP-targeted RNAi drug development (OMIM 104300), and are expected to be effectively used as a model system during lead compound development. In contrast to mice, humans with heterozygous APP mutations exist within the general population with an EXAC score of 0.3, but human APP knockouts have not yet been identified. APP and MAPT peptides in CSF are listed as biomarkers that can be used in the development of APP-targeted RNAi drugs, and these should enable rapid evaluation even in genetically homogeneous populations, yielding useful efficacy (Mo et al. (2017) Alzheimers & Dementia: Diagnosis, Assessment & Disease Monitoring, 6: 201-209).

[0490] As noted above, attempts to treat sporadic forms of AD and EOFAD have proven unsuccessful to date. For example, all trials of BACE1 (β-secretase) inhibitors (BACE1i) aimed at treating sporadic AD have so far failed (Egan et al. The New England Journal of Medicine, 378: 1691-1703; Hung and Fu. Journal of Biomedical Science, 24: 47). No completed trials of such BACEi tests target genetically defined populations exist (only trials have been initiated). Notably, the most recent BACE1i trial demonstrated that verubecestat reduced amyloid-beta levels by 60% in a population selected based on age and clinical criteria suggesting a possible diagnosis of AD (Egan et al. The New England Journal of Medicine, 378: 1691-1703; Hung and Fu. Journal of Biomedical Science, 24: 47). Meanwhile, among Aβ-targeted immunotherapies, one such therapy demonstrated proof-of-concept in a recent trial targeting sporadic AD, justifying the commencement of an ongoing Phase III trial (Selkoe and Hardy. EMBO Molecular Medicine, 8: 595-608). Given its role in APP cleavage, γ-secretase has also been targeted in certain AD-targeted trials. However, to date, there are no completed trials of gamma-secretase inhibitors targeting genetically defined populations, and several programs have been discontinued due to toxicity (Selkoe and Hardy).

[0491] Therefore, there is a need for pharmaceutically acceptable formulated drugs that can treat or prevent APP-related diseases or disorders in affected individuals.

[0492] It is expressly assumed that all APP-related diseases or disorders can ultimately be targeted using the RNAi drug compositions of this disclosure. Specifically, given the diagnostic / phenotyping challenges currently faced for these particular APP-related diseases, it is also assumed that the RNAi drug compositions of this disclosure can target sporadic CAA, as well as sporadic and / or late-onset AD (and further assume that diagnostics for these diseases will continue to improve).

[0493] kit In certain embodiments, this disclosure presents a kit comprising a suitable container for housing a nucleic acid drug pharmaceutical formulation [e.g., an siRNA compound, e.g., a double-stranded siRNA compound, or a precursor to an siRNA compound (e.g., a precursor, e.g., a larger siRNA compound that can be processed into an siRNA compound, or an siRNA compound, e.g., a double-stranded siRNA compound, or DNA encoding an siRNA compound or its precursor)] and instructions for use thereof. In certain embodiments, individual components of the pharmaceutical formulation may be provided in a single container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation individually in two or more containers, e.g., one container for the nucleic acid drug (e.g., an siRNA compound) preparation and at least another container for the carrier compound. The kit may be packaged in several different configurations, e.g., one or more containers housed in a single box. Different components can be combined, for example, according to instructions provided with the kit. Components can be combined, for example, for preparing and administering a pharmaceutical composition, according to methods described herein. The kit may also include a delivery device.

[0494] In some embodiments, this disclosure further presents methods for using RNAi agents or pharmaceutical compositions thereof in combination with other pharmaceuticals or other therapeutic methods, such as known pharmaceuticals or therapeutic methods, such as those currently employed to treat these diseases, to treat subjects who would benefit from reduced or inhibited APP expression, such as subjects with APP-related neurodegenerative diseases. For example, in certain embodiments, an RNAi agent targeting APP is administered in combination with agents useful for treating APP-related neurodegenerative diseases, such as those separately described herein or known in the art. For example, suitable additional agents and therapeutic methods for treating subjects who would benefit from reduced APP expression, such as subjects with APP-related neurodegenerative diseases, may include agents currently used to treat symptoms of APP. The RNAi agent and the additional therapeutic agent may be administered simultaneously or in the same combination, for example, intrathecally, or the additional therapeutic agent may be administered as part of a separate composition, or at a separate time, or by other methods known in the art or described herein.

[0495] Exemplary additional therapeutic and therapeutic agents include dopamine modulators, in particular, such as carbidopa-levodopa, levodopa, entacopon, tolcapone, opicapon, pramipexole, ropinirole, apomorphine, rotigotine, selegiline, rasagiline, safinamide, amantadine, istradefylline, trihexyphenidyl, benztropine, rivastigmine, donepezil, galantamine, and memantine, as well as physical, occupational, and speech therapies, exercise programs (including cardiopulmonary, resistance, flexibility, and walking balance exercises), and deep brain stimulation (DBS) with the implantation of electrodes in targeted areas of the brain.

[0496] In one embodiment, the method involves administering the composition described herein such that the expression of a target gene is reduced for at least one month. In a particular embodiment, the expression is reduced for at least two, three, or six months.

[0497] RNAi agents useful for the methods and compositions described herein may specifically target the RNA (primary or post-processed) of a target gene (e.g., APP). Compositions and methods for inhibiting the expression of such genes using RNAi agents may be prepared and carried out as described herein.

[0498] Administration of dsRNA according to the method of this disclosure may result in a reduction in the severity, signs, symptoms, or markers of such disease or disorder in patients with target gene-associated disorders. "Reduction" in this context means a statistically significant or clinically significant decrease of such level. The reduction may be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

[0499] The effectiveness of treatment or prevention of a disease can be evaluated, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dosage of medicine required to maintain the treatment effect, levels of disease markers, or any other measurable parameters appropriate for a given disease to which treatment or prevention is targeted. Monitoring the effectiveness of treatment or prevention by measuring any one of such parameters, or any combination of such parameters, is well within the scope of the skills of those skilled in the art. For example, the effectiveness of treatment for APP-related neurodegenerative disease can be evaluated, for example, by regularly monitoring the subject's cognition, learning, or memory. By comparing subsequent readings with initial readings, the physician is given an indicator of whether the treatment is effective. Monitoring the effectiveness of treatment or prevention by measuring any one of such parameters, or any combination of such parameters, is well within the scope of the skills of those skilled in the art. With regard to the administration of RNAi agents or pharmaceutical compositions targeting APP, "effective against" APP-related neurodegenerative disease means that, when administered in a clinically appropriate manner, it will produce beneficial effects for at least a statistically significant proportion of patients, such as improvement of symptoms, cure, disease reduction, life extension, improved quality of life, or other effects that are generally positively perceived by physicians familiar with the treatment of APP-related neurodegenerative disease and related causes.

[0500] The effectiveness or preventive effect of a treatment becomes apparent when there is a statistically significant improvement in one or more parameters of the disease state, or when there is no worsening or onset of symptoms that would normally be expected. For example, a favorable change of at least 10% in a measurable parameter of the disease, and, where appropriate, a favorable change of at least 20%, 30%, 40%, 50%, or more, may be an indicator of an effective treatment. The effectiveness of a given RNAi drug or a formulation thereof can also be determined using an experimental animal model for the given disease, as is well known in the art. When using an experimental animal model, the effectiveness of the treatment is demonstrated when a statistically significant decrease is observed in a marker or symptom.

[0501] Alternatively, effectiveness may be measured by a reduction in disease severity, as determined by those skilled in the art based on clinically accepted disease severity assessment scales. For example, if any positive change, such as a reduction in disease severity, is observed when measured using an appropriate scale, it indicates that treatment using the RNAi agents or RNAi drug formulations described herein is appropriate.

[0502] The target population may be administered a therapeutic dose of dsRNA, such as approximately 0.01 mg / kg to approximately 200 mg / kg.

[0503] RNAi agents may be administered intrathecally at regular intervals over a period of time, via intravitreous injection, or by intravenous infusion. In certain embodiments, after an initial treatment regimen, treatment may be administered less frequently. Administration of RNAi agents can reduce, for example, the levels of target genes in the patient's cells, tissues, blood, CSF samples, or other compartments by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99% or more. In preferred embodiments, administration of RNAi agents can reduce, for example, the levels of target transcripts and / or proteins in the patient's cells, tissues, blood, CSF samples, or other compartments by at least 50%.

[0504] Before administering the full dose of an RNAi drug, patients may be given a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects such as allergic reactions. In another example, patients may be monitored for undesirable immunostimulatory effects, such as increased cytokine levels (e.g., TNF-alpha or INF-alpha).

[0505] Alternatively, RNAi agents may be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver a desired dose, e.g., a monthly dose of RNAi agent. Injections may be repeated over a period of time. Administration may be repeated periodically. In certain embodiments, after an initial treatment regimen, treatment may be administered less frequently. Repeated-dose regimens may include administration of therapeutic doses of RNAi agent performed periodically, e.g., once a month, or extended to once a quarter, twice a year, or once a year. In certain embodiments, RNAi agents are administered approximately once a month to approximately once a quarter (i.e., approximately once every three months).

[0506] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. When practicing or testing the RNAi agents and methods discussed herein, similar or equivalent methods and materials may be used, but preferred methods and materials are described below. All published documents, patent applications, patents, and other reference materials mentioned herein are incorporated as informative documents. In case of any discrepancy, including definitions, this specification shall prevail. In addition, materials, methods, and examples are illustrative and not intended to limit.

[0507] definition To make this disclosure easier to understand, certain terms are defined first. In addition, it should be noted that whenever numerical values ​​or ranges of numerical values ​​for parameters are enumerated, intermediate numerical values ​​and ranges of those enumerated are also intended to be part of this disclosure.

[0508] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements, such as multiple elements.

[0509] The term "including" is used herein to mean the idiomatic expression "including but not limited to," and is also interchangeable with it.

[0510] The term "or" is used herein to mean and is interchangeable with the term "and / or" unless the context otherwise clearly indicates.

[0511] The term “about” is used herein to mean within a general acceptable range in the art. For example, “about” may be understood as within about two standard deviations from the mean. In certain embodiments, “about” means ±10%. In certain embodiments, “about” means ±5%. When “about” precedes a set of numbers or ranges, it is understood that “about” may modify each of those numbers or ranges.

[0512] The term "at least," placed before a number or a range of numbers, is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included as is evident from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, "at least 18 nucleotides in a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" is placed before a range of numbers, it is understood to modify each number in the range.

[0513] As used herein, “no more than” is understood to be a number adjacent to the phrase and a number or integer value less than or equal to zero, which can be logically inferred from the context. For example, a double strand with an overhang of “no more than 2 nucleotides” has an overhang of 2, 1, or 0 nucleotides. If “no more than” precedes a series of numbers or a range, it is understood to be able to modify each number in the series or range. As used herein, a range includes both an upper and lower limit.

[0514] If a discrepancy occurs between the displayed target site and the nucleotide sequence of the sense or antisense strand, the displayed sequence takes precedence.

[0515] In the event of a discrepancy between the chemical structure and the chemical name, the chemical structure takes precedence.

[0516] As used herein, “excess” is any amount by which one component is in excess of another component. “Excess” can be measured / estimated by non-denaturing ion-pair reverse phase HPLC (nd-IPRP), which approximates, if not equal to, molar excess. Therefore, the terms “molar excess” and “excess” are used interchangeably herein. For example, an excess of antisense strands relative to sense strands, or an excess of sense strands relative to antisense strands. The amount of molar excess can be any amount. An excess can be approximately 1-2% molar excess, 1-3% molar excess, 1-4% molar excess, 1-5% molar excess, 0-5% molar excess, 0-1% molar excess, 2-3% molar excess, 3-4% molar excess, 3-5% molar excess, 2-4% molar excess, 2-5% molar excess, 0-10% molar excess, 5-10% molar excess, 2-6% molar excess, 3-7% molar excess, or 4-8% molar excess with respect to one component relative to the second component (e.g., antisense chain relative to sense chain, or sense chain relative to antisense chain).

[0517] The term "inorganic phosphoric acid," as used herein, means free phosphoric acid (PO4) in the solutions and / or compositions of this disclosure, as determined by means known in the art, for example, taking a measured amount of aqueous sample and adding ammonium heptamolybdate reagent into a mixing tube. 3- This refers to the total amount of ). Next, the tube is stoppered and shaken vigorously. The concentrated tin chloride reagent and the freshly prepared diluted tin chloride reagent from distilled water are added to the mixture in the tube. This produces a blue color (due to the formation of molybdenum blue), and the intensity of this blue color indicates the amount of phosphoric acid in the boiler water. The absorbance of the blue solution can be measured with a colorimeter, and the phosphoric acid concentration in the original solution can be calculated. Alternatively, a direct (but approximate) reading of the phosphoric acid concentration can be obtained by using a Lovibond comparameter. In certain embodiments, the total amount of inorganic phosphoric acid is less than 100 parts per million (ppm) or less than 0.64 μg / L.

[0518] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, and includes mRNA, which is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence is at least long enough to function as a substrate for RNAi directive cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the target gene. In one embodiment, the target sequence is located within the protein-coding region of the target gene. In another embodiment, the target sequence is located within the 3'UTR of the target gene.

[0519] The target sequence can be approximately 9-36 nucleotides in length, for example, approximately 15-30 nucleotides. For example, the target sequence can be approximately 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, The target sequence may be 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. In some embodiments, the target sequence is about 19-30 nucleotides in length. In other embodiments, the target sequence is about 19-25 nucleotides in length. In yet another embodiment, the target sequence is about 19-23 nucleotides in length. In some embodiments, the target sequence is a nucleotide of approximately 21 to 23 in length. Ranges and lengths intermediate to those listed above are also considered part of this disclosure.

[0520] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a chain of nucleotides described by a sequence named using standard nucleotide nomenclature.

[0521] "G," "C," "A," "T," and "U" generally represent ribonucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively, in the context of modified or unmodified nucleotides. However, the terms "ribonucleotide" or "nucleotide" are understood to also refer to modified nucleotides or substitutional regions, as further detailed below (see, for example, Table 15).

[0522] As used herein, the term “modified nucleotide” independently refers to a nucleotide having a modified sugar moiety, a modified nucleotide-nucleotide bond, or a modified nuclear base, or a combination thereof. Therefore, the term “modified nucleotide” encompasses, for example, the substitution, addition, or removal of a functional group or atom to the nucleoside bond, sugar moiety, or nuclear base. Suitable modifications for use in the drugs of this disclosure include all types of modifications disclosed herein or known in the art. Any such modification, when used in an siRNA-type molecule, is encompassed in “RNAi drug” in light of the purposes of this specification and the claims.

[0523] As used herein, the term “2'-deoxynucleotide” refers to a 2'-deoxyribonucleotide or a 2'-deoxynucleotide containing a ribose analog. In certain embodiments, “2'-deoxynucleotide” refers to a 2'-deoxyribonucleotide. When present in an RNAi drug, a 2'-deoxy variant is understood to be a modified nucleotide.

[0524] The term “blunt-ended” or “blunt-ended,” as used herein in relation to dsRNA, means that at a given end of dsRNA, there are no unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhangs. One or both ends of dsRNA can be blunt-ended. If both ends of dsRNA are blunt-ended, dsRNA is said to be blunt-ended. For clarity, a “blunt-ended” dsRNA is one in which both ends are blunt, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded along their entire length.

[0525] The terms "antisense strand" or "guide strand" refer to the strand of an RNAi drug, such as dsRNA, that contains a region substantially complementary to the target sequence, such as APP mRNA.

[0526] As used herein, the term “complementary region” refers to a region on an antisense strand that is substantially complementary to a sequence, such as a target sequence, such as an APP nucleotide sequence, as defined herein. If the complementary region is not perfectly complementary to the target sequence, the mismatch may be located in an internal or terminal region of the molecule. Generally, most acceptable mismatches are located in terminal regions, such as the 5' or 3' ends of an RNAi drug, specifically within the 5, 4, 3, or 2 nucleotides.

[0527] The terms “sense strand” or “passenger strand,” as used herein, refer to a strand of an RNAi drug that contains a region substantially complementary to the antisense strand (as defined herein).

[0528] As used herein, “substantially all nucleotides are modified” means that the nucleotides are not completely but are largely modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0529] As used herein, the term “cleavage region” refers to the region immediately adjacent to a cleavage site. A cleavage site is the site on the target where the cleavage occurs. In some embodiments, the cleavage region includes three bases immediately adjacent to either end of the cleavage site. In some embodiments, the cleavage region includes two bases immediately adjacent to either end of the cleavage site. In some embodiments, the cleavage site occurs specifically at the binding site of nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0530] As used herein, and unless otherwise indicated, the term “complementary” means, as will be obvious to those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions to form a double-stranded structure, when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Such conditions may be, for example, strict conditions, which may include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing (see, for example, “Molecular Cloning,” A Laboratory Manual, Sambrook, et al. (1989), Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically significant conditions that may be encountered within living organisms, are also applicable. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences, based on the final intended use of the hybridized nucleotides.

[0531] In RNAi agents, for example, in dsRNA as described herein, complementary sequences include base pairing of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence, over the full length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary.” However, where herein the first sequence is referred to as “substantially complementary” with respect to the second sequence, the two sequences may be fully complementary, or they may form one or more mismatched base pairs, but generally not more than 5, 4, 3, or 2, when hybridizing to form a double helix of up to 30 base pairs, while retaining their ability to hybridize under conditions most relevant to their ultimate use, e.g., gene expression inhibition via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs when hybridized, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing one oligonucleotide of length 21 nucleotides and another oligonucleotide of length 23 nucleotides can still be called “fully complementary” in light of the purposes described herein, if the longer oligonucleotide contains a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide.

[0532] When used herein, “complementary” sequences may include, or be formed entirely from, non-Watson-Crick base pairs, or base pairs formed from non-natural and modified nucleotides, provided that the above requirements regarding their hybridizing ability are met. Examples of such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen-type base pairings.

[0533] The terms “complementary,” “fully complementary,” and “substantially complementary” as used herein may be used in reference to base matching between the sense strand and antisense strand of a dsRNA, or between the antisense strand and target sequence of an RNAi drug, as is evident from the context of their use.

[0534] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding the target protein). For example, a polynucleotide is complementary to at least a portion of the target gene mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the target protein.

[0535] The idiomatic expression "contacting cells with an RNAi agent (e.g., dsRNA)" as used herein includes contacting cells by any possible means. Contacting cells with an RNAi agent includes contacting cells with an RNAi agent in vitro or in vivo. Contact may be carried out directly or indirectly. For example, an RNAi agent may be brought into physical contact with cells by an individual performing the method, or an RNAi agent may be placed in a situation that allows or causes it to subsequently come into contact with cells.

[0536] In vitro cell contact may be performed, for example, by incubating cells with an RNAi drug. In vivo cell contact may be performed, for example, by injecting the RNAi drug into or near the tissue in which the cells are located, or by injecting the RNAi drug into another region, such as the central nervous system (CNS), via intrathecal, intravitreous, or other injection, or into the bloodstream or subcutaneous space, as appropriate, so that the drug subsequently reaches the tissue in which the cells to be contacted are located. For example, the RNAi drug may contain or be coupled with a ligand, such as a lipophilic moiety(single or multiple) described below and incorporated herein by reference, for example, PCT / US 2019 / 031170, which induces the RNAi drug to a target site, such as the CNS, or otherwise stabilizes it there. In some embodiments, the RNAi agent may contain or be coupled with a ligand, such as one or more GalNAc derivatives described below, that induces the RNAi agent to a target site, such as the liver, or otherwise stabilizes it there. In other embodiments, the RNAi agent may contain or be coupled with one or more lipophilic moieties and one or more GalNAc derivatives. Combinations of in vitro and in vivo contact methods are also possible. For example, cells may be contacted with the RNAi agent in vitro and then transplanted into a target.

[0537] In one embodiment, contacting an RNAi agent with a cell includes "introducing or delivering an RNAi agent into the cell" by promoting or facilitating its uptake or absorption into the cell. The absorption or uptake of the RNAi agent may occur through unassisted diffusion or active cellular processes, or by adjuvants or devices. The introduction of an RNAi agent into cells may be in vitro or in vivo. For example, in in vivo introduction, the RNAi agent may be injectable into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further approaches are described below herein or are known in the art.

[0538] The term "artificial cerebrospinal fluid (aCSF)" refers to a solution prepared using a representative hCSF composition that closely approximates the electrolyte concentration of human cerebrospinal fluid (hCSF) and may contain glucose. Exemplary ion concentrations prepared in high-purity water are given in mM units: Na + as 150;K + As 3.0; Ca 2+ as 1.4; Mg 2+ As 0.8; P as 1.0; Cl - This includes 155 (for example, available from Tocris Bioscience® ACSF, catalog number 3525, Thermo Fisher Scientific, Hampton, NH, USA). An exemplary aCSF formulation contains 127 mM NaCl; 1.0 mM KCl; 1.2 mM KH2PO4; 26 mM NaHCO3; 10 mM D-glucose; 2.4 mM CaCl2; and 1.3 mM MgCl2, with pH and oxygen levels stabilized by bubbling with carbogen (95% O2 and 5% CO2).

[0539] A solution is considered "isotonic with respect to aCSF" when its effective osmolality is equal to that of aCSF. For example, if the concentration of a solute outside the cell is equal to the concentration of a solute inside the cell, then the solution on either side of the cell membrane is isotonic.

[0540] If two substantially complementary strands of dsRNA are contained within separate RNA molecules, these molecules do not need to be covalently linked, but it is possible. If the two strands are covalently linked by means other than an uninterrupted nucleotide chain between the 3' end of one strand and the 5' end of the other, the connecting structure is called a "linker." RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest dsRNA strand, excluding all overhangs present in the double helix. In addition to the double helix structure, dsRNA can contain one or more nucleotide overhangs.

[0541] The term "linker" or "bonding group" refers to an organic part that connects two parts of a compound, for example, by covalently linking two parts of a compound. Linkers are formed by direct bonds, or atoms, such as oxygen or sulfur, units, such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or chains of atoms, such as, without limitation, substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, substituted or unsubstituted alkynyls, arylalkyls, arylalkenyls, arylalkynyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, aryl, heteroaryl, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroarylalkenyls Teloarylalkynyl, alkenyl heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkenyl heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkylhererocyclylalkynyl, alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkenyl heterocyclylalkynyl These generally include quinyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, etc., where one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycles. Here, R8 is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker is approximately 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

[0542] As used herein, the expression “as appropriate” means that at least one hydrogen present on a group (e.g., the carbon of an alkyl, alkenyl, or alkynyl group) is substituted with an acceptable substituent, such as a substituent that, when substituted, results in a stable compound, such as a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, or other reactions. Heteroatoms, such as nitrogen, may have substituents, such as any preferred substituents described herein that satisfy the bonding value of the heteroatom and cause the formation of a stable moiety. For example, each example of an alkyl group may be independently substituted as appropriate, i.e., it may be unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents ("substituted alkyl"). Suitable substituents include hydroxyl, nitro, amino (e.g., -NH2 or dialkylamino), imino, cyano, halo (e.g., F, Cl, Br, I, etc.), haloalkyl (e.g., -CCl3, -CF3, etc.), thio, sulfonyl, thioamide, amidino, imidino, oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamide, carboxyl, formyl, alkyl, alkoxy, alkoxyalkyl, alkylcarbonyl, alkylcarbonyloxy (e.g., -OCOR), aminocarbonyl, arylcarbonyl, aralkylcarbonyl, carbonylamino, heteroarylcarbonyl, and he Examples of such compounds include, but are not limited to, teloaralkylcarbonyl, alkylthio, aminoalkyl, cyanoalkyl, carbamoyl (e.g., -NHCOOR- or -OCONHR-), urea (e.g., -NHCONHR-), cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, (-O), thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, nitro, amino, heterocyclic, -CN, etc.When used herein, "alkyl" can be combined with other groups, such as the groups presented above that form functionalized alkyl groups.

[0543] In one embodiment, the target gene-associated disease or disorder is one of Alzheimer's disease (AD), cerebral amyloid vascular disease (CAA, e.g., hereditary CAA), early-onset familial Alzheimer's disease (EOFAD or eFAD), early-onset Alzheimer's disease (EOAD), familial Alzheimer's disease, or late-onset Alzheimer's disease.

[0544] When used herein, “therapeutic dose” is intended to include the amount of RNAi agent that, when administered to a subject with a target gene-associated disease, is sufficient to treat the disease (e.g., by reducing, improving, or maintaining the symptoms of the existing disease, or one or more symptoms of the disease). “Therapeutic dose” may vary depending on the RNAi agent, the method of drug administration, the disease and its severity, as well as the history, age, weight, family history, genetic structure, type of prior or concomitant treatment (if any), and other individual characteristics of the subject being treated.

[0545] When used herein, “a prophylactically effective dose” is intended to include the amount of an RNAi agent that, when administered to a subject with a target gene-associated disorder, is sufficient to prevent or improve the disease, or one or more symptoms of the disease. Improvement of the disease includes slowing the course of the disease or reducing the severity of late-onset disease. “A prophylactically effective dose” may vary depending on the RNAi agent, the method of administration, the degree of the disease risk, and the patient’s history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the patient being treated.

[0546] The “therapeutic dose” or “prophylactic dose” also includes the amount of RNAi agent that produces some desirable local or systemic effect in a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent employed in the manner of this disclosure may be administered in an amount sufficient to be considered reasonable in terms of the benefit / risk ratio applicable to such treatment.

[0547] The idiomatic expression "pharmaceutically acceptable" is used herein to refer to a compound, substance, composition, or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, inflammation, allergic reactions, or other problems or complications, and that is balanced by a reasonable benefit / risk ratio, within the bounds of reasonable medical judgment.

[0548] As used herein, the idiomatic term "pharmaceutically acceptable salt" refers to both pharmaceutically acceptable acid addition salts and base addition salts and solvated compounds. Such pharmaceutically acceptable salts include acids, such as hydrogen chloride, phosphoric acid, hydrobromic acid, sulfuric acid, sulfinic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, nitric acid, benzoic acid, citric acid, tartaric acid, maleic acid, hydroiodic acid, etc., and alkanonic acids, such as acetic acid, HOOC-(CH2) n Examples of salts include -COOH (where n is 0 to 4). Examples of non-toxic medicinal base addition salts include salts of bases such as lithium, sodium, potassium, calcium, magnesium, and ammonium. Those skilled in the art will recognize a wide variety of non-toxic, pharmaceutically acceptable addition salts.

[0549] The term “sample,” as used herein, includes similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present within the subject. Examples of biological fluids include blood, serum, and serosal fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples derived from tissue, organs, or local areas. For example, a sample may originate from a specific organ, a part of an organ, or fluids or cells within such an organ. In certain embodiments, a sample may originate from the brain [e.g., the whole brain or a specific segment of the brain, e.g., the striatum, or specific types of cells within the brain, e.g., neurons and glial cells (astrocytes, oligodendrocytes, microglia)]. In other embodiments, “sample derived from subject” refers to liver tissue (or its subcomponents) derived from the subject. In some embodiments, “sample derived from subject” refers to blood taken from the subject or plasma or serum derived therefrom. In further embodiments, “sample derived from subject” means brain tissue (or its subcomponents) or retinal tissue (or its subcomponents) derived from subject.

[0550] Although the sequences in Tables 16 and 18 are described as modified or conjugated sequences, the RNA of the RNAi agent of this disclosure, e.g., the dsRNA of this disclosure, is understood to include any one of the sequences defined in Tables 16-19, which are unmodified, unconjugated, or modified or conjugated in a manner different from those described therein. That is, the modified sequences presented in Tables 16 and 18 do not require the indicated hexadecyl lipophilic moiety or any ligand. The lipophilic ligand may be included at any position presented in this application. [Examples]

[0551] [Example 1] Materials and methods This embodiment describes methods for designing, synthesizing, and selecting exemplified APP-targeted RNAi agents, as well as improved formulation processes disclosed herein for preparing pharmaceutical products containing RNAi agents.

[0552] Reagent suppliers Unless otherwise specified herein, such reagents are available from any supplier of reagents for molecular biology, provided they meet the quality / purity standards applicable in molecular biology.

[0553] siRNA drugs siRNA drugs targeting the human amyloid-beta precursor protein gene (APP) are described in International Publication No. 2020 / 132227 (International Application No. PCT / US 2019 / 067449); International Publication No. 2022 / 165172 (International Application No. PCT / US 2022 / 014309); and International Publication No. 2023 / 039503 (International Application No. PCT / US 2022 / 076159), each of which is incorporated herein by reference.

[0554] siRNA drugs targeting the human superoxide dismutase 1 gene (SOD1) are described in International Publication No. 2022 / 174000 (International Application No. PCT / US 2022 / 016046), which is incorporated herein by reference as is.

[0555] siRNA drugs targeting the human huntingtin gene (HTT) are described in International Publication No. 2021 / 087036 (International Application No. PCT / US 2020 / 057849), International Publication No. WO2022 / 212231 (International Application No. PCT / US 2022 / 022093), and International Publication No. 2023 / 076450 (International Application No. PCT / US 2022 / 047986, filed on 27 October 2022, entitled "HUNTINGTIN (HTT) iRNA AGENT COMPOSITIONS AND METHODS OF USE THEREOF"), each of which is incorporated herein by reference.

[0556] Synthesis of siRNA drugs All oligonucleotides were prepared on an automated solid-phase synthesizer using a general-purpose or specially designed support. [See, for example, F. Eckstein (ed.), Oligonucleotides and Analogues, a Practical Approach (Oxford University Press, New York 1991)]. Drugs containing 3'-RNA may be prepared based on the process disclosed in International Publication No. 2021 / 108291, which is incorporated herein by reference as is.

[0557] APP single-stranded annealing was performed on a Tecan liquid handling robot. Sense and antisense single-stranded APP were combined in equimolar ratios in a 96-well plate and buffered with 10×PBS to a final double-stranded concentration of 10 μM in 1×PBS. After combining complementary single-stranded APP, the 96-well plate was sealed and heated in a 100°C oven for 40 minutes, then allowed to slowly rise to room temperature for 2-3 hours before being used directly for in vitro screening assays at appropriate concentrations.

[0558] Detailed lists of modified APP sense strand and antisense strand sequences are shown in Figure 1A and Table 16, and detailed lists of unmodified APP sense strand and antisense strand sequences are shown in Table 17.

[0559] APP pharmaceutical products (DP) were prepared from vacuum lyophilized powder of APP active pharmaceutical ingredient (DS). Formulation of APP DP began by dissolving powdered APP DS (e.g., AD-961583, AD-454973, AD-454843, AD-961584, AD-961585, AD-961586, AD-454844, AD-1302922, AD-1302923, or AD-1999409, etc.) in buffer and thoroughly mixing it manually at ambient temperature to a concentration of approximately 80 mg / mL of ALN DS. If necessary, the pH was adjusted to 6.8 ± 0.2 using a 0.25 M solution of sodium hydroxide or hydrochloric acid. The concentration of APP DS in the solution was measured, and AD-961583 (free acid form) was adjusted to a final target concentration of approximately 60 mg / mL using buffer. The concentration was determined by an assay based on UV spectrophotometry. To reduce bioburden, the solution was passed through a 0.22 μm polyethersulfone (PES) membrane filter and filtered again to ensure sterility. The sterile filtered solution was aseptically filled into Type I glass vials and aseptically completed using a fluoropolymer-coated stopper and an aluminum overseal. After aseptic filling, vials were sampled and inspected for any damage to the container closure or any obvious granular material.

[0560] Calcium exchange experiment For each double-stranded molecule, dose adjustments were performed with the goal of achieving a 5% excess of each sense strand. This excess was confirmed by incorporating nd-IPRP (non-denaturing ion-pair reversed-phase liquid chromatography) before annealing. Once it was confirmed that the ratio was close to the target, annealing was performed.

[0561] After double-strand formation, approximately 5 g of each double-strand was quantitatively portioned for use in calcium exchange. The amount of substance was determined by calculation using the single-strand solution concentration, dose adjustment amount, and single-strand conversion coefficient to calculate the volume required for approximately 5 g of double-strand. Two calcium solutions (1 mM and 20 mM) were prepared by dissolving calcium chloride dihydrate in a RODI while mixing, and were used after filtration. For reference, the pH and conductivity of each calcium solution were measured.

[0562] The same calcium exchange procedure was performed on all four double-stranded cells. Calcium exchange was carried out using a standard UF apparatus equipped with a 5kD MWCO Sartorius Hydrosart membrane cassette, applying standard cross-flow filtration (CFF) and transmembrane pressure (TMP) parameters. To maintain the dialysate filtration volume (DV), the target concentration in the retaining solution tank was set to 30 mg / mL. Based on the determined DV, UF exchange was performed by performing a 15 DV exchange using 20 mM CaCl2, followed by a 10 DV exchange using 1 mM CaCl2. To evaluate the completion of the exchange, the conductivity of the permeate was monitored to ensure that the conductivity of the permeate was close to that of each feed solution at the end of each exchange volume. The retaining solution tank was also monitored for precipitation or turbidity, but no process adjustments were made regardless of the observations. After the 1 mM CaCl2 exchange was completed, the material was collected and set aside. An additional rinse with 1 mM CaCl2 was performed, but it did not merge with the initial sample.

[0563] For the evaluation of calcium exchange, a ThermoScientific Orion AQ4500 Turbidity Meter was used in conjunction with nd-IPRP analysis. For each calcium form double-stranded molecule, a series of samples were prepared by quantitatively dispensing 10 mL of the calcium form double-stranded solution into a Falcon tube four times. Next, the amount of sodium antisense strands was gradually increased in 100 μL increments, i.e., in the range of 100–400 μL, while adjusting the dose for each fixed amount. The samples were capped, vortexed, and heated on a preheated hot plate set to 85°C for 5 minutes (the caps were removed at this time to prevent pressure buildup). After 5 minutes, the samples were removed from the hot plate and allowed to cool to room temperature on a bench. Once at room temperature, nd-IPRP samples were prepared for each sample and analyzed to confirm the presence of an excess of single strands. The remaining samples were transferred to a washed vial for turbidity measurement (the vial was dedicated to the ThermoScientific Orion AQ4500 instrument) and left undisturbed for at least one night. One hour before turbidity measurement, the samples were gently mixed by inverting them 10 times to avoid introducing air bubbles, and then all samples were analyzed together. Initial data analysis included graphing turbidity (y-axis) against single-strand excess (x-axis: negative for sense strand excess, positive for antisense strand excess) by nd-IPRP.

[0564] Particulate matter reduction protocol Double-stranded antisense was annealed with an excess of sense strands. The degree of sense strand excess was confirmed by non-denaturing ion pair reversed-phase high-performance liquid chromatography (nd-IPRP) analysis. Next, calcium exchange was performed on the double-stranded antisense by ultrafiltration according to an established protocol. Then, the precipitate suspension / turbidity solution was collected and its dissolution was initiated. In the dissolution test, the precipitate suspension / turbidity solution was quantitatively divided into a series of vials, and various amounts of antisense were successively added to the vials. After incubation, turbidity was measured to determine the effectiveness of the particulate matter reduction strategy.

[0565] Formulations discussed in the examples As shown in Figure 1B, the “original” formulations (both placebo and pharmaceutical product) contained disodium hydrogen phosphate and sodium dihydrogen phosphate as their respective sources of inorganic phosphate. Formulation 1: The inorganic phosphate sources were removed from the placebo ("F1") and pharmaceutical product ("F1DP") formulations, but all other components were retained. Formulation 2: Not only were the inorganic phosphate sources removed from the placebo ("F2") and pharmaceutical product ("F2DP") formulations, but the potassium chloride and magnesium chloride components were also removed, so that only sodium chloride and calcium chloride remained in the placebo / diluted formulation (F2), and only sodium chloride, calcium chloride, and APP API (RNAi drug) remained in the pharmaceutical product formulation (F2DP). The compositions of F3DP to F8FP were identical to F1DP, except for variations in the relative amounts of the sense and antisense strands, as summarized below:

[0566] [Table 15] [Example 2] The phosphate-free solution improved the iRNA drug formulation properties.

[0567] Granular formation occurred in phosphate-buffered formulations of APP-targeted iRNA drugs prepared for central nervous system (CNS) delivery via intrathecal administration. These granular formations occurred in an isotonic aqueous buffer (containing sodium, potassium, magnesium, calcium, and phosphate) based on artificial cerebrospinal fluid (aCSF) containing 60 mg / mL of APP-targeted drug substance (AD-961583 double-stranded, as free acid, as shown in Table 16 below), prepared for intrathecal (IT) injection. The observed granular formations appeared to be a result of calcium phosphate accumulation as a precipitate, significantly reducing the efficacy of the APP-targeted formulation when applied clinically. Therefore, a search for improved iRNA drug product formulations, particularly for iRNA drug substances intended for intrathecal and / or central nervous system (CNS) delivery, was initiated.

[0568] While calcium phosphate particles were considered the primary source of granular material observed in aCSF-based formulations, it had also been identified that including calcium as a supplement was useful in mitigating certain clinical signs of intrathecal formulation-induced effects (e.g., tremors, convulsions, etc.) observed when such iRNA pharmaceutical formulations were injected into the CNS of animals.

[0569] To formulate the iRNA drug substance, we investigated phosphate-free solutions. Specifically, we removed the sodium phosphate excipient from the original granular aCSF-based iRNA drug test formulation solution. The original pharmaceutical product formulation (containing 60 mg / mL of the iRNA drug AD-961583) contained 0.5 mM disodium hydrogen phosphate, 0.2 mM sodium dihydrogen phosphate, 97.6 mM sodium chloride, 1.9 mM potassium chloride, 0.5 mM magnesium chloride, and 13.0 mM calcium chloride, while the "F1" pharmaceutical product ("F1DP") formulation (containing 60 mg / mL of the iRNA drug AD-961583) contained 97.6 mM sodium chloride, 1.9 mM potassium chloride, 0.5 mM magnesium chloride, and 13.0 mM calcium chloride (Figure 1B). On the other hand, the “F2” pharmaceutical product (“F2DP”) formulation (which also contained the iRNA drug AD-961583 at 60 mg / mL) contained only 97.6 mM sodium chloride and 13.0 mM calcium chloride. In such phosphate-free pharmaceutical product formulations, it was observed that the APP-targeted active pharmaceutical ingredient (60 mg / mL AD-961583) inherently possessed buffering capacity comparable to the disodium hydrogen phosphate and sodium dihydrogen phosphate buffers removed from the original aCSF-based formulations when forming the “F1DP” and “F2DP” formulations. This identified that the free phosphate-containing components of the original formulation(s) could be removed without significantly affecting the buffering capacity.

[0570] Removing phosphates (disodium hydrogen phosphate and sodium dihydrogen phosphate) from the original DP formulation, and further removing not only all phosphates but also potassium chloride and magnesium chloride from the F1DP formulation, had only minimal effect on the osmotic pressure of the F1DP and F2DP formulations compared to the initial aCSF-based DP formulation. The F1DP formulation was found to be the closest to the original aCSF-based formulation among the novel formulations tested, while the F2DP formulation was substantially a physiological saline formulation with the required calcium concentration.

[0571] Diluted formulations corresponding to the above-mentioned novel DP formulations (F1DP and F2DP) were also developed and tested in parallel. As described above, the sodium phosphate excipient found in the original aCSF-based formulations was removed to minimize the formation of calcium phosphate precipitate during the dilution process at use. The phosphate-free placebo formulations F1 and F2 maintained the same formulation components as the corresponding pharmaceutical products F1DP and F2DP, respectively (Figure 1B). In particular, the F1 placebo / diluted formulation contained 150.1 mM sodium chloride, 3.0 mM potassium chloride, 0.8 mM magnesium chloride, and 1.4 mM calcium chloride, while the F2 placebo / diluted formulation contained only 150.1 mM sodium chloride and 1.4 mM calcium chloride (Figure 1B). The pH range of each of these formulations was similar to that of physiological saline solution.

[0572] Stress and stability tests were conducted on each of the above formulations, revealing that the phosphate-free novel formulations of this disclosure possess improved properties (in particular, the property of not forming granules under the relevant test conditions) that enable enhanced use of each of them as pharmaceutical product formulations.

[0573] The stability tests of the modified formulations tested included the following: (1) a comprehensive visual inspection of the formulations in the vials before the start of the tests; (2) the commencement of long-term stability tests for up to 5 years at 5°C, 25°C, and 30°C (at 1, 3, 6, 9, 12, 18, 24, 36, 48, and 60 months); (3) the commencement of accelerated stability tests for 6 months at 40°C (at 0, 1, 3, and 6 months); (4) testing of samples using the same time intervals and methods as those used in tests to identify harmful particulate matter formation in aCSF-based formulations, with the addition of high-precision particle counting (HIAC) tests for all time points and storage conditions; and (5) additional visual inspections of samples stored at 40°C at 2, 4, 6, and 8 hours and 1, 3, and 7 days after the start of the tests.

[0574] The following stress tests were also performed on the formulations of this disclosure: (1) Stress testing was initiated after a comprehensive visual inspection of the formulations in the vials; (2) Stress was first applied to the formulations by incubation at -20°C. If no granular material was observed, stress testing was performed, including under -20°C conditions. If granular material was observed at -20°C, the stress test temperatures used in the assay were limited to temperatures above 0°C.

[0575] Therefore, Stage 1 of the stress test was a thermal stress test, which involved freezing the formulation at -...

Claims

1. (a) A double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, wherein one of the sense strand or antisense strand of the dsRNA contains at least one lipophilic modification, and the other strand of the dsRNA does not contain a lipophilic modification, (b) Divalent ion source and A composition comprising, (i) Substantially all of the sense or antisense strands of the dsRNA in the composition that contain at least one lipophilic modification are doubled with a strand that does not contain the lipophilic modification, or (ii) A sense strand or antisense strand of a dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to a strand without a lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand without a lipophilic modification is present in a molar excess compared to a strand of dsRNA containing at least one lipophilic modification. composition.

2. (a) A double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, wherein one of the sense strand or antisense strand of the dsRNA contains at least one lipophilic modification, and the other strand of the dsRNA does not contain a lipophilic modification, (b) Divalent ion source and A composition comprising, A sense or antisense strand of a dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to a dsRNA strand without a lipophilic modification, a dsRNA strand without a lipophilic modification is present in an equimolar amount relative to the sense or antisense strand of the dsRNA containing at least one lipophilic modification, or a dsRNA strand without a lipophilic modification is present in a molar excess compared to the sense or antisense strand of the dsRNA containing at least one lipophilic modification. composition.

3. The composition according to claim 1 or claim 2, which is substantially free of inorganic phosphoric acid.

4. The composition according to any one of claims 1 to 3, wherein the sense strand of the dsRNA contains at least one lipophilic modification, and the antisense strand of the dsRNA does not contain any lipophilic modifications.

5. The composition according to any one of claims 1 to 4, wherein there is a molar excess of antisense chains compared to sense chains.

6. The composition according to claim 5, wherein there is at least 0.1% molar excess antisense chains compared to the sense chain, there may be at least 0.2% molar excess antisense chains compared to the sense chain, there may be at least 0.3% molar excess antisense chains compared to the sense chain, there may be at least 0.4% molar excess antisense chains compared to the sense chain, there may be at least 0.5% molar excess antisense chains compared to the sense chain, there may be at least 1% molar excess antisense chains compared to the sense chain, there may be at least 2% molar excess antisense chains compared to the sense chain, there may be at least 3% molar excess antisense chains compared to the sense chain, there may be at least 4% molar excess antisense chains compared to the sense chain, and there may be about 5% or more molar excess antisense chains compared to the sense chain.

7. The composition according to any one of claims 1 to 3, wherein the antisense strand of the dsRNA contains at least one lipophilic modification, and the sense strand of the dsRNA does not contain a lipophilic modification, the antisense strand of the dsRNA may contain at least one lipophilic modification, and there may be a molar excess of the sense strand compared to the antisense strand.

8. The composition according to claim 7, wherein there is at least 0.1% molar excess of sense chains compared to the antisense chain, there may be at least 0.2% molar excess of sense chains compared to the antisense chain, there may be at least 0.3% molar excess of sense chains compared to the antisense chain, there may be at least 0.4% molar excess of sense chains compared to the antisense chain, there may be at least 0.5% molar excess of sense chains compared to the antisense chain, there may be at least 1% molar excess of sense chains compared to the antisense chain, there may be at least 2% molar excess of sense chains compared to the antisense chain, there may be at least 3% molar excess of sense chains compared to the antisense chain, there may be at least 4% molar excess of sense chains compared to the antisense chain, and there may be about 5% or more molar excess of sense chains compared to the antisense chain.

9. The composition according to any one of the claims, formulated for use in a route of administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, intra-articular, and intrathecal.

10. Lipophilic modification, saturated or unsaturated C ４ ~C ３０ Hydrocarbons, C as appropriate ４ ~C ３０ Alkyl or alkenyl, optionally linear C ６ ~C １８ Alkyl or alkenyl, as appropriate, C １６ The composition according to any one of the claims, wherein the alkyl group is optionally linked to the 2'-ribo position of a nucleic acid residue of dsRNA.

11. The composition according to any one of the claims, wherein the divalent ion source is selected from the group consisting of magnesium, calcium, copper, nickel, zinc, and strontium.

12. The composition according to any one of the claims, wherein the molar ratio of divalent ions to dsRNA is greater than approximately 2:

1.

13. The composition according to any one of the claims, which is substantially free of inorganic phosphoric acid and / or contains less than 100 ppm of inorganic phosphoric acid, optionally less than 50 ppm of inorganic phosphoric acid, optionally less than 10 ppm of inorganic phosphoric acid, optionally less than 5 ppm of inorganic phosphoric acid, and optionally free of inorganic phosphoric acid.

14. The composition according to any one of the claims, wherein the molar ratio of divalent ion source to dsRNA is greater than approximately 2.5:1, may be greater than approximately 3.0:1, may be greater than approximately 3.5:1, or may be greater than approximately 4.0:

1.

15. The composition according to any one of the above claims, wherein the molar ratio of divalent ion source to dsRNA is about 2:1 to about 10:1, may be about 3:1 to about 10:1, may be about 3:1 to about 9:1, may be about 3:1 to about 8:1, may be about 3:1 to about 7:1, may be about 3:1 to about 6:1, or may be about 3:1 to about 5:

1.

16. The composition according to any one of the claims, wherein the dsRNA comprises 5'-phosphate or a 5'-phosphate mimetic variant.

17. 5'-phosphate mimic variants, 【Chemistry 1】 Therefore, due to the preceding structure, the 4'-CH group within the ribose ring of the 5' terminal nucleotide ２ The composition according to claim 16, wherein the OH group is substituted.

18. The composition according to claim 16, wherein the phosphonic acid variant is a 5'-phosphonic acid variant.

19. The composition according to claim 16, wherein the phosphonic acid variant is a 5'-vinylphosphonate variant, or a 5'-(E)-vinylphosphonate variant.

20. The composition according to claim 16, wherein the phosphoric acid mimic modifier is a 5'-vinyl phosphate modifier.

21. The composition according to any one of the claims, further comprising a diluent.

22. The composition according to claim 21, which is isotonic with respect to cerebrospinal fluid (CSF).

23. The composition according to claim 22, further comprising a sodium source, a potassium source, a magnesium source, and a calcium source.

24. The composition according to claim 23, comprising sodium chloride, magnesium chloride, potassium chloride, and calcium chloride.

25. The composition according to any one of the claims, having a pH between approximately 4 and approximately 10, which may be approximately 6 to approximately 10, or approximately 6.5 to approximately 8.

0.

26. The composition according to any one of the above claims, having an osmotic pressure of approximately 200 to 400 mOsm / kg.

27. The composition according to any one of the above claims, which does not contain hydrogen phosphate or dihydrogen phosphate.

28. A composition according to any one of the claims, which does not contain a buffer.

29. The composition according to any one of the claims, further comprising a stabilizer selected from the group consisting of sucrose, glucose, mannitol, sorbitol, polyethylene glycol (PEG), histidine, arginine, lysine, phospholipids, trehalose, and combinations thereof.

30. The composition according to any one of the claims, comprising more than 1 mg of dsRNA per 1 ml of composition, which may also comprise more than 5 mg of dsRNA per 1 ml of composition, which may also comprise more than 10 mg of dsRNA per 1 ml of composition, which may also comprise more than 25 mg of dsRNA per 1 ml of composition, which may also comprise more than 50 mg of dsRNA per 1 ml of composition, which may also comprise about 60 mg of dsRNA per 1 ml of composition.

31. The composition according to any one of the claims, wherein the dsRNA is AD-961583, AD-1395762, or AD-1498524.

32. (a) A double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, wherein one of the sense strand and the antisense strand is a dsRNA comprising at least one lipophilic modification, (b) Sodium chloride in a concentration of approximately 80 mM to approximately 110 mM, (c) Approximately 8.0 mM to approximately 20.0 mM calcium chloride A composition comprising the above, wherein the composition contains approximately 50 mg to approximately 70 mg of dsRNA per 1 ml of the composition.

33. The composition according to claim 32, which is substantially free of inorganic phosphoric acid and / or contains less than 100 ppm of inorganic phosphoric acid, optionally less than 50 ppm of inorganic phosphoric acid, optionally less than 10 ppm of inorganic phosphoric acid, optionally less than 5 ppm of inorganic phosphoric acid, and optionally free of inorganic phosphoric acid.

34. The sense strand of the dsRNA contains at least one lipophilic modification, and the antisense strand of the dsRNA does not contain any lipophilic modifications. The composition according to claim 32 or claim 33.

35. The composition according to any one of claims 32 to 34, wherein there is a molar excess of antisense chains compared to sense chains.

36. The composition according to claim 35, wherein there is at least a 0.1% molar excess of antisense chains compared to the sense chain, there may be at least a 0.2% molar excess of antisense chains compared to the sense chain, there may be at least a 0.3% molar excess of antisense chains compared to the sense chain, there may be at least a 0.4% molar excess of antisense chains compared to the sense chain, there may be at least a 0.5% molar excess of antisense chains compared to the sense chain, there may be at least a 1% molar excess of antisense chains compared to the sense chain, there may be at least a 2% molar excess of antisense chains compared to the sense chain, there may be at least a 3% molar excess of antisense chains compared to the sense chain, there may be at least a 4% molar excess of antisense chains compared to the sense chain, and there may be about 5% or more excess antisense chains compared to the sense chain.

37. (d) Potassium chloride in a concentration of approximately 1.0 mM to approximately 2.5 mM, (e) Approximately 0.1 mM to 1.0 mM magnesium chloride The composition according to any one of claims 32 to 36, further comprising:

38. The composition according to any one of claims 32 to 37, comprising approximately 60 mg of dsRNA per 1 ml of the composition.

39. The composition according to any one of claims 32 to 38, comprising approximately 97.6 mM sodium chloride.

40. The composition according to any one of claims 32 to 39, comprising approximately 13.0 mM calcium chloride.

41. A composition according to any one of claims 32 to 40, comprising approximately 1.9 mM potassium chloride.

42. A composition according to any one of claims 32 to 41, comprising approximately 0.5 mM magnesium chloride.

43. A pharmaceutical composition for intrathecal administration targeting dsRNA, as described in any one of claims 32 to 42.

44. The composition according to claim 43, wherein the subject is a mammal, and the subject may be a human.

45. The composition according to any one of claims 32 to 44, further comprising a diluent.

46. The composition according to claim 45, which is isotonic with respect to CSF.

47. The composition according to any one of claims 32 to 46, having a pH of about 6 to about 10, and possibly having a pH of about 6.5 to about 8.

0.

48. The composition according to any one of claims 32 to 47, having an osmotic pressure of approximately 200 to 400 mOsm / kg.

49. A composition according to any one of claims 32 to 48, which does not contain hydrogen phosphate or dihydrogen phosphate.

50. A composition according to any one of claims 32 to 49, which does not contain a buffer.

51. The composition according to any one of claims 32 to 50, further comprising a stabilizer selected from the group consisting of sucrose, glucose, mannitol, sorbitol, polyethylene glycol (PEG), histidine, arginine, lysine, phospholipids, trehalose, and combinations thereof.

52. The composition according to any one of claims 32 to 51, wherein the dsRNA is AD-961583.

53. (a) dsRNA selected from the group consisting of AD-961583, AD-454973, AD-454843, AD-961584, AD-961585, and AD-961586, (b) A source of calcium ions, (c) Dilutant and A composition for intrathecal administration containing a calcium ion to dsRNA molar ratio greater than 3:

1.

54. (a) dsRNA selected from the group consisting of AD-1395718, AD-1395724, AD-1395731, AD-1395738, AD-1395743, AD-1395756, AD-1395760, AD-1395762, AD-1395764, and AD-1395771, (b) A source of calcium ions, (c) Dilutant and A composition for intrathecal administration containing a calcium ion to dsRNA molar ratio greater than 3:

1.

55. (a) dsRNA selected from the group consisting of AD-1019448, AD-1019465, AD-1271082, AD-1271083, AD-1271084, AD-1271085, AD-1498524, AD-1498526, and AD-1498528, (b) A source of calcium ions, (c) Dilutant and A composition for intrathecal administration containing a calcium ion to dsRNA molar ratio greater than 3:

1.

56. The composition according to any one of claims 53 to 55, which is substantially free of inorganic phosphoric acid and / or contains less than 100 ppm of inorganic phosphoric acid, optionally less than 50 ppm of inorganic phosphoric acid, optionally less than 10 ppm of inorganic phosphoric acid, optionally less than 5 ppm of inorganic phosphoric acid, and optionally free of inorganic phosphoric acid.

57. The composition according to any one of claims 53 to 56, which is isotonic with respect to CSF.

58. The composition according to any one of claims 53 to 57, further comprising a sodium source, a potassium source, a magnesium source, and a calcium source.

59. The composition according to claim 58, comprising sodium chloride, magnesium chloride, potassium chloride, and calcium chloride.

60. The composition according to any one of claims 53 to 59, having a pH of about 6 to about 10, and possibly having a pH of about 6.5 to about 8.

0.

61. The composition according to any one of claims 53 to 60, having an osmotic pressure of approximately 200 to 400 mOsm / kg.

62. A composition according to any one of claims 53 to 61, which does not contain hydrogen phosphate or dihydrogen phosphate.

63. A composition according to any one of claims 53 to 62, which does not contain a buffer.

64. The composition according to any one of claims 53 to 63, comprising more than 1 mg of dsRNA per 1 ml of composition, more than 5 mg of dsRNA per 1 ml of composition, more than 10 mg of dsRNA per 1 ml of composition, more than 25 mg of dsRNA per 1 ml of composition, more than 50 mg of dsRNA per 1 ml of composition, or approximately 60 mg of dsRNA per 1 ml of composition.

65. The composition according to any one of claims 53 to 64, wherein the dsRNA is AD-961583.

66. The composition according to any one of claims 53 to 65, wherein the antisense strand of dsRNA is present in equimolar or molar excess amounts compared to the sense strand of dsRNA.

67. A composition comprising double-stranded ribonucleic acid (dsRNA) having the ability to anneal to amyloid precursor protein (APP) mRNA and reduce its expression, wherein the dsRNA comprises a sense strand and an antisense strand, and one of the sense strand or antisense strand of the dsRNA contains at least one lipophilic modification, and the other strand of the dsRNA does not contain a lipophilic modification, and (i) Substantially all of the sense or antisense strands of the dsRNA in the composition that contain at least one lipophilic modification are doubled with a strand that does not contain the lipophilic modification, or (ii) The sense or antisense strand of the dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to the strand without the lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand without the lipophilic modification is present in a molar excess compared to the strand of the dsRNA containing at least one lipophilic modification. composition.

68. The composition according to claim 67, further comprising a divalent ion source.

69. The composition according to claim 67 or claim 68, wherein the dsRNA is selected from the group consisting of AD-961583, AD-454973, AD-454843, AD-961584, AD-961585, and AD-961586.

70. A composition comprising double-stranded ribonucleic acid (dsRNA) having the ability to anneal to superoxide dismutase 1 (SOD1) mRNA and reduce its expression, wherein the dsRNA comprises a sense strand and an antisense strand, one of the sense strand or antisense strand of the dsRNA contains at least one lipophilic modification, and the other strand of the dsRNA does not contain a lipophilic modification, and (i) Substantially all of the sense or antisense strands of the dsRNA in the composition that contain at least one lipophilic modification are doubled with a strand that does not contain the lipophilic modification, or (ii) The sense or antisense strand of the dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to the strand without the lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand without the lipophilic modification is present in a molar excess compared to the strand of the dsRNA containing at least one lipophilic modification. composition.

71. The composition according to claim 70, further comprising a divalent ion source.

72. The composition according to claim 70 or claim 71, wherein the dsRNA is selected from the group consisting of AD-1395718, AD-1395724, AD-1395731, AD-1395738, AD-1395743, AD-1395756, AD-1395760, AD-1395762, AD-1395764, and AD-1395771.

73. A composition comprising double-stranded ribonucleic acid (dsRNA) having the ability to anneal to huntingtin (HTT) mRNA and reduce its expression, wherein the dsRNA comprises a sense strand and an antisense strand, one of the sense strand or antisense strand of the dsRNA contains at least one lipophilic modification, and the other strand of the dsRNA does not contain a lipophilic modification, and (i) Substantially all of the sense or antisense strands of the dsRNA in the composition that contain at least one lipophilic modification are doubled with a strand that does not contain the lipophilic modification, or (ii) The sense or antisense strand of the dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to the strand without the lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand without the lipophilic modification is present in a molar excess compared to the strand of the dsRNA containing at least one lipophilic modification. composition.

74. The composition according to claim 73, further comprising a divalent ion source.

75. The composition according to claim 73 or claim 74, wherein the dsRNA targets a sequence within exon 1 of the huntingtin gene.

76. The composition according to any one of claims 73 to 75, wherein the dsRNA is selected from the group consisting of AD-1019448, AD-1019465, AD-1271082, AD-1271083, AD-1271084, AD-1271085, AD-1498524, AD-1498526, and AD-1498528.

77. A composition comprising a double-stranded ribonucleic acid (dsRNA) including a sense strand and an antisense strand, wherein one of the sense strand or antisense strand of the dsRNA contains at least one lipophilic modification in one or more internal residues of the sense strand or antisense strand, and the other strand of the dsRNA does not contain a lipophilic modification, (i) Substantially all of the sense or antisense strands of the dsRNA in the composition that contain at least one lipophilic modification are doubled with a strand that does not contain the lipophilic modification, or (ii) The sense or antisense strand of the dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to the strand without the lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand without the lipophilic modification is present in a molar excess compared to the strand of the dsRNA containing at least one lipophilic modification. composition.

78. The composition according to claim 77, further comprising a divalent ion source.

79. The composition according to claim 77 or claim 78, wherein the dsRNA comprises at least one lipophilic modification in one or more internal residues of the sense strand, the dsRNA may also comprise at least one lipophilic modification at any one of positions 4-8 or 13-18 counting from the 5' end of the strand, and the dsRNA may also comprise at least one lipophilic modification at position 6 counting from the 5' end of the strand.

80. The composition according to claim 77 or claim 78, wherein the dsRNA comprises at least one lipophilic modification in one or more internal residues of the antisense strand.

81. At least one lipophilic modification is a saturated or unsaturated C ４ -C ３０ hydrocarbon, optionally C ４ -C ３０ alkyl or alkenyl, optionally linear C ６ -C １８ alkyl or alkenyl, optionally containing C １６ alkyl, and optionally, at least one lipophilic modification is linked to the 2'-ribo position of the nucleic acid residue of the dsRNA. The composition according to any one of claims 77 to 80.

82. A composition comprising a double-stranded ribonucleic acid (dsRNA) including a sense strand and an antisense strand, wherein one of the sense strand or antisense strand of the dsRNA contains at least one lipophilic modification at one or more terminal residues of the sense strand or antisense strand, and the other strand of the dsRNA does not contain a lipophilic modification, (i) Substantially all of the sense or antisense strands of the dsRNA in the composition that contain at least one lipophilic modification are doubled with a strand that does not contain the lipophilic modification, or (ii) The sense or antisense strand of the dsRNA containing at least one lipophilic modification is present in a molar excess of less than 1% compared to the strand without the lipophilic modification, the sense strand and antisense strand are present in equimolar amounts, or the strand without the lipophilic modification is present in a molar excess compared to the strand of the dsRNA containing at least one lipophilic modification. composition.

83. The composition according to claim 82, further comprising a divalent ion source.

84. The composition according to claim 82 or claim 83, wherein the dsRNA comprises at least one lipophilic modification at the 5' terminal(s) and / or 3' terminal(s) of the sense strand, and the dsRNA may also comprise at least one lipophilic modification at the 3' terminal of the sense strand.

85. The composition according to any one of claims 82 to 84, wherein the dsRNA comprises at least one lipophilic modification at the 5' terminal residue of the sense strand.

86. The composition according to claim 82 or claim 83, wherein the dsRNA comprises at least one lipophilic modification at the 5' terminal residue(s) and / or 3' terminal residue(s) of the antisense strand, and the dsRNA may also comprise at least one lipophilic modification at the 3' terminal residue of the antisense strand.

87. The composition according to any one of claims 82, 83, or 86, wherein the dsRNA comprises at least one lipophilic modification at the 5' terminal residue of the antisense strand.

88. At least one lipophilic modification is saturated or unsaturated C ４ ~C ３０ Hydrocarbons, C as appropriate ４ ~C ３０ Alkyl or alkenyl, optionally linear C ６ ~C １８ Alkyl or alkenyl, as appropriate, C １６ The composition according to any one of claims 67 to 87, comprising an alkyl group, wherein at least one lipophilic modification is optionally linked to the 2'-ribo position of a nucleic acid residue of dsRNA.

89. A solid prepared by vacuum freeze-drying of the composition according to any one of the above claims.

90. (a) A diluent containing a divalent cation source, (b) Double-stranded ribonucleic acid (dsRNA) including sense strand and antisense strand A kit comprising a dsRNA containing at least one modified nucleotide that is not a 2'-deoxynucleotide, and a molar ratio of divalent cation source to dsRNA greater than 2:

1.

91. The kit according to claim 90, wherein the diluent is substantially free of inorganic phosphoric acid and / or contains less than 100 ppm of inorganic phosphoric acid, optionally less than 50 ppm of inorganic phosphoric acid, optionally less than 10 ppm of inorganic phosphoric acid, optionally less than 5 ppm of inorganic phosphoric acid, and optionally the diluent does not contain inorganic phosphoric acid.

92. A kit comprising (a) the solid matter described in claim 89 and (b) a diluent.

93. The kit according to claim 92, wherein the diluent is substantially free of inorganic phosphoric acid and / or contains less than 100 ppm of inorganic phosphoric acid, optionally less than 50 ppm of inorganic phosphoric acid, optionally less than 10 ppm of inorganic phosphoric acid, optionally less than 5 ppm of inorganic phosphoric acid, and optionally the diluent does not contain inorganic phosphoric acid.

94. A method for treating a subject having a disorder that would benefit from reduced expression of a target gene, comprising administering a therapeutically effective amount of the composition described in any one of claims 1 to 88 to the subject, thereby treating the subject.

95. The method according to claim 94, wherein the subject is a human.

96. The method according to claim 94 or claim 95, wherein the target gene is amyloid precursor protein (APP), superoxide dismutase 1 (SOD1), or the huntingtin gene, and optionally exon 1 of the huntingtin gene.

97. The method according to any one of claims 94 to 96, wherein the subject is suffering from an APP-related disease.

98. The method according to claim 97, wherein the APP-related disease is cerebral amyloid vascular disease (CAA).

99. The method according to claim 97, wherein the APP-related disease is early-onset familial Alzheimer's disease (EOFAD).

100. The method according to claim 97, wherein the APP-related disease is Alzheimer's disease (AD), early-onset Alzheimer's disease (EOAD), familial Alzheimer's disease, or late-onset Alzheimer's disease.

101. The method according to any one of claims 96 to 100, wherein APP expression is inhibited by at least about 30%.

102. The method according to any one of claims 94 to 101, further comprising administering an additional therapeutic agent to the target.

103. The method according to any one of claims 94 to 102, wherein the dsRNA of the composition is administered in a dose of about 0.1 mg / kg to about 50 mg / kg.

104. The method according to any one of claims 94 to 103, wherein the composition is administered intrathecally to a subject.

105. A method for inhibiting APP expression in a subject, comprising administering a therapeutically effective amount of the composition described in any one of claims 1 to 88 to the subject, thereby inhibiting APP expression within the subject.

106. A method for treating or preventing APP-related diseases or disorders in a subject, comprising administering a therapeutically effective amount of the composition described in any one of claims 1 to 88 to the subject, thereby treating or preventing APP-related diseases or disorders in the subject.

107. The method according to claim 106, wherein the APP-related disease or disorder is selected from the group consisting of cerebral amyloid vascular disease (CAA), Alzheimer's disease (AD), early-onset familial Alzheimer's disease (EOFAD), early-onset Alzheimer's disease (EOAD), familial Alzheimer's disease, and late-onset Alzheimer's disease.

108. The method according to claim 106 or claim 107, wherein the composition is administered by intrathecal injection, and the intrathecal injection may be performed in conjunction with intravenous administration.

109. The method according to claim 108, wherein intrathecal administration is performed without intravenous administration.

110. The method according to any one of claims 106 to 109, wherein administration of the composition causes a reduction in the intensity, severity, or frequency of, or a delay in the onset of, at least one symptom or feature of an APP-related disease or disorder.

111. The method according to any one of claims 106 to 110, wherein the composition does not cause serious side effects in the subject when administered, and the subject does not experience tremors or convulsions when the composition is administered to the subject.

112. The method according to any one of claims 106 to 111, wherein the composition is administered at intervals selected from once every two weeks, once every month, once every two months, once every three months, once every four months, once every five months, and once every six months.

113. A method for administering dsRNA to a subject in need thereof, comprising intrathecal administration of a composition according to any one of claims 1 to 88 to the subject, thereby administering dsRNA to the subject.

114. The method according to claim 113, wherein the subject is a human.

115. The method according to claim 113 or claim 114, wherein the dsRNA targets the amyloid precursor protein (APP) gene, the superoxide dismutase 1 (SOD1) gene, or the huntingtin gene, and optionally, exon 1 of the huntingtin gene is the target.

116. The method according to any one of claims 113 to 115, wherein the dsRNA of the composition is administered in a dose of about 0.1 mg / kg to about 50 mg / kg.

117. A method for inhibiting the expression of SOD1 in target cells or tissues, comprising administering to the target a composition according to any one of claims 1 to 88 in an amount sufficient to reduce the expression of SOD1 in the target cells or tissues, thereby inhibiting the expression of SOD1 in the target cells or tissues.

118. The method according to claim 117, wherein the subject is a human.

119. The method according to claim 117 or claim 118, wherein the composition is administered intrathecally to a subject.

120. The method according to any one of claims 117 to 119, wherein the administration reduces the level of SOD1 mRNA in the target cells or tissue by at least 50%, and optionally at least 80%, compared to a suitable control.

121. The method according to any one of claims 117 to 120, wherein the dsRNA of the composition is administered in a dose of about 0.1 mg / kg to about 50 mg / kg.

122. A method for inhibiting the expression of HTT in target cells or tissue, comprising administering to the target a composition according to any one of claims 1 to 66 in an amount sufficient to reduce the expression of HTT in the target cells or tissue, thereby inhibiting the expression of HTT in the target cells or tissue.

123. The method according to claim 122, wherein the subject is a human.

124. The method according to claim 122 or claim 123, wherein the composition is administered intrathecally to the subject.

125. The method according to any one of claims 122 to 124, wherein the composition comprises a dsRNA that targets exon 1 of HTT.

126. The method according to any one of claims 122 to 124, wherein the administration reduces the level of HTT mRNA in the target cells or tissue by at least 50%, and optionally at least 80%, compared to a suitable control.

127. The method according to any one of claims 94 to 126, wherein the dsRNA of the composition is administered in a dose of about 0.1 mg / kg to about 50 mg / kg.

128. A kit for carrying out the method described in any one of claims 94 to 127, a) A composition containing dsRNA, b) Instructions for use and c) Means for administering the composition to the target, as appropriate. A kit that includes this.

129. A method for reducing or preventing particle formation in a solution containing a divalent ion source and double-stranded ribonucleic acid (dsRNA) having a sense strand and an antisense strand, wherein the dsRNA contains at least one lipophilic modification of the sense strand or antisense strand, and either the sense strand or the antisense strand does not contain a lipophilic modification, and the method comprises maintaining the sense strand or antisense strand of the dsRNA containing the lipophilic modification in a molar excess of less than 1% compared to the strand without the lipophilic modification, thereby reducing or preventing particle formation in a solution containing a divalent ion source and double-stranded ribonucleic acid (dsRNA).

130. The method according to claim 129, wherein the sense chain and the antisense chain are present in equimolar amounts.

131. The method according to claim 129, wherein strands without lipophilic modifications are present in a molar excess compared to strands of dsRNA containing at least one lipophilic modification.

132. The method according to any one of claims 129 to 131, wherein the sense chain comprises at least one lipophilic modification, and the antisense chain does not comprise any lipophilic modifications.

133. The method according to any one of claims 129 to 131, wherein the antisense chain includes lipophilic modification and the sense chain does not include lipophilic modification.

134. The method according to any one of claims 129 to 133, wherein the divalent ion source is calcium, magnesium, copper, nickel, zinc, or strontium.

135. The method according to any one of claims 129 to 134, wherein at least one lipophilic modification is C16 or a longer lipophilic modification.

136. A method for preparing a pharmaceutical product, To form a double-stranded solution containing double-stranded RNA (dsRNA), the sense strand and antisense strand are annealed, To provide a double-stranded composition, the double-stranded solution is vacuum freeze-dried, Dissolving the double-stranded composition in the injection solution and It contains, and one of the sense chain and the antisense chain contains lipophilic modification, The injection solution contains a divalent cation source (e.g., calcium) and does not contain phosphate buffer. and a method wherein the double-stranded composition contains an antisense chain in an antisense chain in an amount of 0 to 5% molar excess relative to the sense chain (for example, about 1 to 2% molar excess).

137. The method according to claim 136, wherein the divalent ion source is calcium, magnesium, copper, nickel, zinc, or strontium.

138. The method according to claim 136 or 137, wherein the double-stranded composition comprises an antisense chain in an antisense chain in an excess of about 1-2% molars relative to the sense chain.

139. The method according to any one of claims 136 to 138, wherein the sense chain comprises at least one lipophilic modification, and the antisense chain does not comprise any lipophilic modifications.

140. The method according to any one of claims 136 to 139, wherein at least one lipophilic modification is C16 or a longer lipophilic modification.

141. The method according to any one of claims 136 to 140, wherein the dsRNA is selected from the group consisting of AD-961583, AD-454973, AD-454843, AD-961584, AD-961585, and AD-961586.

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