Amyloid precursor protein (APP) RNAi agent composition and use method thereof

Abstract

The present application relates to double-stranded RNA (dsRNAi) agents and compositions targeting the APP gene, as well as methods of using the dsRNAi agents and compositions to inhibit the expression of the APP gene and methods of treating individuals suffering from APP-related diseases or disorders, such as cerebral amyloid angiopathy (CAA) and early-onset familial Alzheimer's disease (EOFAD or eFAD).

Description

Technical Field

[0001] This application is generally related to RNAi agents and methods for targeting APP.

[0002] sequence list

[0003] This application contains a sequence list, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on December 18, 2019, and is named 53433_500WO01_SequenceListing_ST25.txt, with a size of 632kB. Background Technology

[0004] The amyloid precursor protein (APP) gene encodes an intact membrane protein expressed in neurons and glial cells. While the primary function of APP remains unclear, secretase-cleaved forms of APP—particularly Aβ-cleaved forms, such as Aβ(1–42) (aka Aβ42) and Aβ(1–40) (aka Aβ40)—are commonly identified as major proteins in β-amyloid plaque mosaics and have long been described as being associated with the development and progression of Alzheimer's disease (AD) in affected individuals. Indeed, identifying β-amyloid plaque mosaics in individuals is essential for the pathological diagnosis of AD. The Aβ-cleaved forms of APP have been specifically described as playing a key, even causal, role in the development of two AD-related / associated diseases: cerebral amyloid angiopathy (CAA) and early-onset familial Alzheimer's disease (EOFAD or eFAD).

[0005] Using selective and effective inhibitors of APP to suppress its expression and / or activity, thereby blocking or inhibiting the production and / or level of the Aβ cleavage form of APP, can help prevent or treat a variety of APP-related diseases and conditions, including AD, CAA, and EOFAD.

[0006] Currently, treatment options for APP-related diseases and symptoms are both limited and largely ineffective. There are no therapies for hereditary CAA, and attempts to treat sporadic AD and Eofa's disease have so far proven unsuccessful—for example, all trials using BACE1 (β-secretase) inhibitors to treat sporadic AD have failed to date (Egan et al., The New England Journal of Medicine, 378:1691-1703; Hung and Fu. Journal of Biomedical Science, 24:47). Meanwhile, many Aβ-guided immunotherapies are at various stages of development, and many human γ-secretase inhibitor programs have been discontinued due to toxicity (Selkoe and Hardy. EMBO Molecular Medicine, 8:595-608). To date, approved drug treatments for APP-related diseases or symptoms are symptomatic rather than preventative or curative, and their effectiveness is limited, especially when APP-related diseases or symptoms develop in affected individuals. Therefore, treatment is needed for individuals with APP-related diseases and conditions, including, in particular, individuals with hereditary CAA and EOFAD. Summary of the Invention

[0007] This application provides an RNAi composition that affects the cleavage of the RNA transcript of the RNA precursor protein (APP) gene mediated by the RNA-induced silencing complex (RISC). The APP gene can be intracellular, for example, within the cells of an individual, such as a human. This application also provides methods for using the RNAi composition of this application to inhibit the expression of the APP gene and / or treat individuals who would benefit from inhibiting or reducing the expression of the APP gene, such as individuals who have or are susceptible to APP-related diseases, such as cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD), such as early-onset familial Alzheimer's disease (EOFAD).

[0008] Therefore, in one aspect, this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene, wherein the RNAi agent comprises a sense strand and an antisense strand, and wherein the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides differing by no more than 3 nucleotides from any antisense sequence listed in any of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B, 26, and 30. In some embodiments, differences in thymine to uracil and / or uracil to thymine between aligned (compared) sequences are not counted as differences in different nucleotides between aligned (compared) sequences.

[0009] Another aspect of this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from any sense strand sequence listed in any of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B, 26 and 30 by no more than 3 nucleotides; and wherein the antisense strand comprises at least 15 consecutive nucleotides that differ from any antisense strand nucleotide sequence listed in Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B, 26 and 30 by no more than 3 nucleotides.

[0010] In one embodiment, at least one of the sense strand and the antisense strand of the double-stranded RNAi agent includes one or more lipophilic moieties that bind to one or more internal nucleotide sites, optionally via a linker or vector.

[0011] Another aspect of this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from any nucleotide sequence of SEQ ID NOs:1 to 14 by no more than 3 nucleotides, wherein any substitution of uracil for thymine in SEQ ID NOs:1 to 14 (when comparing aligned sequences) is not considered to cause the difference from the SEQ ID NOs:1 to 14. NOs:1 to 14 differ by no more than 3 nucleotides; and wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any nucleotide sequence of SEQ ID NOs:15 to 28, wherein any substitution of uracil for thymine in SEQ ID NOs:15 to 28 (when comparing aligned sequences) is not considered to result in a difference of no more than 3 nucleotides from any nucleotide sequence of SEQ ID NOs:15 to 28, wherein at least one of the sense and antisense strands comprises one or more lipophilic moieties bound to one or more internal nucleotide positions, optionally via a linker or vector.

[0012] In one embodiment, the positive strand of the double-stranded RNAi agent comprises at least 15 consecutive nucleotides, which are associated with AD-392911, AD-392912, AD-392816, AD-392704, AD-392843, AD-392855, AD-392840, AD-392835, AD-392729, AD-392916, AD-392876, AD-392863, AD-392917, AD-392783, AD-392765, AD-392791, AD-392800, AD-392711, AD-392801, AD-392826, AD-392818, A D-392792, AD-392802, AD-392766, AD-392767, AD-392834, AD-392974, AD-392784, AD-392744, AD-392752, AD-392737, AD-392918, AD-392919, AD-392 A D-392703, AD-392715, AD-392836, AD-392966, AD-392832, AD-392972, AD-392961, AD-392967, AD-392894, AD-392864, AD-392865, AD-392922, AD-39 2833, AD-392968, AD-392962, AD-392963, AD-392969, AD-392973, AD-392923, AD-392866, AD-392877, AD-392707, AD-392926, AD-392927, AD-392717, AD-392700, AD-392878, AD-392718, AD-392929, AD-392819, AD-392745, AD-392770, AD-392806, AD-392771, AD-392820, AD-392821, AD-392786, AD-39 2772, AD-392699, AD-392868, AD-392719, AD-392880, AD-392930, AD-392932, AD-392869, AD-392870, AD-392896, AD-392720, AD-392746, AD-392773,AD-392807、AD-392730、AD-392721、AD-392933、AD-392881、AD-392897、AD-392898、AD-392899、AD-392935、AD-392882、AD-392738、AD-392739、AD-392936、AD-392900、AD-392901、AD-392937、AD-392883、AD-392975、AD-392938、AD-392902、AD-392941、AD-392942、AD-392943、AD-392944、AD-392903、AD-392775、AD-392758、AD-392945、AD-392884、AD-392947、AD-392748、AD-392759、AD-392837、AD-392970、AD-392976、AD-392965、AD-392831、AD-392904、AD-392885、AD-392886、AD-392776、AD-392887、AD-392722、AD-392760、AD-392731、AD-392709、AD-392723、AD-392948、AD-392724、AD-392949、AD-392725、AD-392950、AD-392732、AD-392726、AD-392862、AD-392951、AD-392871、AD-392872、AD-397183、AD-397175、AD-397177、AD-397176、AD-397260、AD-397266、AD-397267、AD-397178、AD-397180、AD-397184、AD-397179、AD-397224、AD-397225、AD-397203、AD-397185、AD-397195、AD-397204、AD-397191、AD-397251、AD-397240、AD-397205、AD-397254、AD-397259、AD-397247、AD-397233、AD-397181、AD-397196、AD-397197、AD-397226、AD-397212、AD-397182、AD-397227、AD-397217、AD-397213、AD-397229、AD-397264、AD-397265、AD-397209、AD-397192、AD-397210、AD-397219、AD-397214、AD-397220, AD-397230, AD-397231, AD-397193, AD-397190, AD-397200, AD-397248, AD-3972 07. AD-397211, AD-397243, AD-397246, AD-397223, AD-397202, AD-397256, AD-397257, AD-39 The nucleotide sequences of the positive strands of the duplexes 7258, AD-397250, AD-397244, AD-454972, AD-454973, AD-454842, AD-454843, AD-454844, AD-994379, AD-961583, AD-961584, AD-961585, or AD-961586 differ by no more than 3 nucleotides.

[0013] In another embodiment, the antisense strand of the double-stranded RNAi agent comprises at least 15 consecutive nucleotides, which are associated with AD-392911, AD-392912, AD-392816, AD-392704, AD-392843, AD-392855, AD-392840, AD-392835, AD-392729, AD-392916, AD-392876, AD-392863, AD-392917, AD-392783, AD-392765, AD-392791, AD-392800, AD-392711, AD-392801, AD-392826, AD-392818, AD-392792, AD-392802, AD-392766, AD-392767, AD-392834, AD-392974, AD-392784, AD-392744, AD-392752, AD-392737, AD-392918, AD-392919, AD-39 2803, AD-392804, AD-392827, AD-392828, AD-392785, AD-392829, AD-392920, AD-392921, AD-392768, AD-392805, AD-392769, AD-392753, AD-392714, AD-392703, AD-392715, AD-392836, AD-392966, AD-392832, AD-392972, AD-392961, AD-392967, AD-392894, AD-392864, AD-392865, AD-392922, AD-39 2833, AD-392968, AD-392962, AD-392963, AD-392969, AD-392973, AD-392923, AD-392866, AD-392877, AD-392707, AD-392926, AD-392927, AD-392717, AD-392700, AD-392878, AD-392718, AD-392929, AD-392819, AD-392745, AD-392770, AD-392806, AD-392771, AD-392820, AD-392821, AD-392786, AD-39 2772, AD-392699, AD-392868, AD-392719, AD-392880, AD-392930, AD-392932, AD-392869, AD-392870, AD-392896, AD-392720, AD-392746, AD-392773,AD-392807、AD-392730、AD-392721、AD-392933、AD-392881、AD-392897、AD-392898、AD-392899、AD-392935、AD-392882、AD-392738、AD-392739、AD-392936、AD-392900、AD-392901、AD-392937、AD-392883、AD-392975、AD-392938、AD-392902、AD-392941、AD-392942、AD-392943、AD-392944、AD-392903、AD-392775、AD-392758、AD-392945、AD-392884、AD-392947、AD-392748、AD-392759、AD-392837、AD-392970、AD-392976、AD-392965、AD-392831、AD-392904、AD-392885、AD-392886、AD-392776、AD-392887、AD-392722、AD-392760、AD-392731、AD-392709、AD-392723、AD-392948、AD-392724、AD-392949、AD-392725、AD-392950、AD-392732、AD-392726、AD-392862、AD-392951、AD-392871、AD-392872、AD-397183、AD-397175、AD-397177、AD-397176、AD-397260、AD-397266、AD-397267、AD-397178、AD-397180、AD-397184、AD-397179、AD-397224、AD-397225、AD-397203、AD-397185、AD-397195、AD-397204、AD-397191、AD-397251、AD-397240、AD-397205、AD-397254、AD-397259、AD-397247、AD-397233、AD-397181、AD-397196、AD-397197、AD-397226、AD-397212、AD-397182、AD-397227、AD-397217、AD-397213、AD-397229、AD-397264、AD-397265、AD-397209、AD-397192、AD-397210、AD-397219、AD-397214、AD-397220, AD-397230, AD-397231, AD-397193, AD-397190, AD-397200, AD-397248, AD-39 7207, AD-397211, AD-397243, AD-397246, AD-397223, AD-397202, AD-397256, AD-397257, The antisense nucleotide sequences of the double strands AD-397258, AD-397250, AD-397244, AD-454972, AD-454973, AD-454842, AD-454843, AD-454844, AD-994379, AD-961583, AD-961584, AD-961585, or AD-961586 differ by no more than 3 nucleotides.

[0014] Optionally, the double-stranded RNAi agent comprises at least one modified nucleotide.

[0015] In some embodiments, via logK ow The lipophilicity of the measured lipophilic portion exceeds 0.

[0016] In some embodiments, the hydrophobicity of the double-stranded RNAi agent, measured by the unbound portion in a plasma protein binding assay, exceeds 0.2. In related embodiments, the plasma protein binding assay uses electrophoretic mobility shift analysis of human serum albumin proteins.

[0017] In some embodiments, all nucleotides of the positive strand are modified nucleotides.

[0018] In some embodiments, substantially all nucleotides of the antisense strand are modified nucleotides. Optionally, all nucleotides of the sense strand are modified nucleotides.

[0019] In some embodiments, all nucleotides of the antisense strand are modified nucleotides. Optionally, all nucleotides of both the sense and antisense strands are modified nucleonucleotides.

[0020] In one embodiment, the modified nucleotide is a deoxynucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluorine modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a configuration-restricted nucleotide, a restricted ethyl nucleotide, a baseless nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, an aminophosphate ester, a non-natural base containing a nucleotide, a tetrahydropyran modified nucleotide, or a 1,5'-methyl-2 ... - Nucleotides modified with dehydrated hexitol, nucleotides modified with cyclohexenyl, nucleotides containing a 5'-thiophosphate group, nucleotides containing a 5'-methyl phosphate group, nucleotides containing a 5'-phosphate or a 5'-nucleotide phosphate analog, nucleotides containing a vinyl phosphate, nucleotides containing adenosine glycol nucleic acid (GNA), nucleotides containing the S-isomer of thymidine glycol nucleic acid (GNA), nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3' phosphate, or terminal nucleotides linked to cholesterol derivatives and / or dodecanoic acid bisdecylamino groups.

[0021] In related embodiments, the modified nucleotide is a 2'-deoxy-2'-fluorinated nucleotide, a 2'-deoxy-modified nucleotide, a 3'-terminal deoxythymidine nucleotide (dT), a locked nucleotide, a baseless nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, an aminophosphate ester, and / or a non-natural base containing a nucleotide.

[0022] In one embodiment, the modified nucleotide comprises a short sequence of 3'-terminal deoxythymidine nucleotide (dT).

[0023] In another embodiment, the modification on the nucleotide is a modification of 2'-O-methyl, 2'-fluorine, and GNA.

[0024] In another embodiment, the double-stranded RNAi agent comprises at least one intermolecular bond between phosphate-thionucleotides. Optionally, the double-stranded RNAi agent comprises 6 to 8 intermolecular bonds between phosphate-thionucleotides.

[0025] In some embodiments, the complementary region is at least 17 nucleotides long. Optionally, the complementary region is 19 to 23 nucleotides long. Optionally, the complementary region is 19 nucleotides long.

[0026] In one embodiment, each chain is no more than 30 nucleotides in length.

[0027] In another embodiment, at least one strand includes a 3' overhang of at least one nucleotide. Optionally, at least one strand includes a 3' overhang of at least two nucleotides.

[0028] In some embodiments, the double-stranded RNAi agent further comprises a C16 ligand that binds to the 3' end, 5' end, or 3' end and 5' end of the positive strand via a monovalent or branched divalent or trivalent linker.

[0029] In one embodiment, the ligand is

[0030]

[0031] Wherein, B is a nucleotide base or a nucleotide base analogue, optionally, wherein B is adenine, guanine, cytosine, thymine, or uracil.

[0032] In another embodiment, the complementary regions include any antonymous sequence of any of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B, 26 and 30.

[0033] In another embodiment, the complementary region is any antonymous sequence of any of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B, 26 and 30.

[0034] In some embodiments, the internal nucleotide positions include all positions except the two terminal positions at each end of the chain.

[0035] In relevant embodiments, the internal locations include all locations except the three end locations at each end of the chain. Optionally, the internal locations exclude the cutting site region of the justice chain.

[0036] In one embodiment, the internal positions exclude positions 9 to 12, which are counted starting from the 5' end of the justice chain.

[0037] In another embodiment, the internal positions exclude positions 11 to 13, counted from the 3' end of the sense chain. Optionally, the internal positions exclude the cleavage site region of the antisense chain.

[0038] In one embodiment, the internal positions exclude positions 12 to 14, which are counted starting from the 5' end of the antisense chain.

[0039] In another embodiment, the internal positions exclude positions 11 to 13, counted from the 3' end of the justice chain, and positions 12 to 14, counted from the 5' end of the antisense chain.

[0040] In another embodiment, one or more lipophilic portions are attached to one or more of the following internal locations: positions 4 to 8 and 13 to 18 of the positive strand, counting from the 5' end of each strand, and positions 6 to 10 and 15 to 18 of the negative strand. Optionally, one or more lipophilic portions are attached to one or more of the following internal locations: positions 5, 6, 7, 15, and 17 of the positive strand, counting from the 5' end of each strand, and positions 15 and 17 of the negative strand.

[0041] In some embodiments, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound. Optionally, the lipophilic moiety is a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytriphenylmethyl, or phenoxazine.

[0042] In some embodiments, the lipophilic portion comprises saturated or unsaturated C4 to C5 groups. 30 A hydrocarbon chain, and optional functional groups selected from hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide and / or alkyne.

[0043] In some embodiments, the lipophilic portion comprises saturated or unsaturated C6 to C6 moieties. 18 Hydrocarbon chain. Optionally, the lipophilic moiety comprises saturated or unsaturated C atoms. 16 Hydrocarbon chain. In one relevant embodiment, the lipophilic moiety is bound by a carrier that replaces one or more nucleotides in the internal position. In some embodiments, the carrier is a cyclic group that is pyrrolidyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolyl, isothiazolinyl, quinoxolinyl, pyridazinyl, tetrahydrofuranyl, or decahydronaphthalene; or an acyclic moiety based on a serinel or diethanolamine backbone.

[0044] In some embodiments, the lipophilic moiety is bound to the double-stranded RNAi agent via a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, click reaction product, or carbamate linker.

[0045] In one embodiment, the lipophilic portion binds to a nucleobase, a sugar moiety, or an internucleotide bond.

[0046] In another embodiment, the double-stranded RNAi agent further comprises a phosphate ester or phosphate ester analog at the 5'-terminus of the antisense strand. Optionally, the phosphate ester analog is 5'-vinyl phosphate (VP).

[0047] In some embodiments, the double-stranded RNAi agent further includes a targeting ligand that targets and mediates delivery to receptors in the CNS tissue. In one embodiment, the targeting ligand is a C16 ligand.

[0048] In some embodiments, the double-stranded RNAi agent further includes a targeting ligand that targets brain tissue.

[0049] In one embodiment, the lipophilic moiety or targeting ligand is bound via a biolytic linker, which is a functionalized monosaccharide or oligosaccharide of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose, or combinations thereof.

[0050] In related embodiments, the 3' end of the positive chain is protected by an end cap having a cyclic group having an amine, such cyclic group being pyrrolidyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolanecycloyl, oxazolinyl, isoxazolinyl, morpholinyl, thiazolyl, isothiazolyl, tetrahydrofuranyl, or decahydronaphthyl.

[0051] In one embodiment, the RNAi agent comprises at least one modified nucleotide, which is a 2'-O-methyl modified nucleotide, a 2'-fluorine modified nucleotide, a nucleotide comprising ethylene glycol nucleic acid (GNA), and / or a nucleotide comprising a vinyl phosphate ester. Optionally, the RNAi agent comprises at least one of the following modifications: a 2'-O-methyl modified nucleotide, a 2'-fluorine modified nucleotide, a nucleotide comprising ethylene glycol nucleic acid (GNA), and a nucleotide comprising a vinyl phosphate ester.

[0052] In another embodiment, the RNAi agent comprises, as Figure 1A , Figure 1B The patterns of modified nucleotides shown in Tables 2A, 5A, or 9 (where the positions of 2'-C16, 2'-O-methyl, GNA, thiophosphate, and 2'-fluorine modifications are as follows) Figure 1A , Figure 1B (As shown in Tables 2A, 5A, or 9, the individual nucleotide sequences of the RNAi agents presented are not relevant).

[0053] Another aspect of this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, wherein the antisense strand comprises a region partially complementary to the mRNA encoding APP, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (III):

[0054] Justice: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'

[0055] Antonym: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)

[0056] in:

[0057] i, j, k, and l are each independently 0 or 1;

[0058] p, p', q, and q' are each independently between 0 and 6;

[0059] N a and N a Each of the following independently represents an oligonucleotide sequence comprising 0 to 25 modified or unmodified nucleotides or combinations thereof, each sequence comprising at least two different modified nucleotides;

[0060] N b and N b Each represents an oligonucleotide sequence consisting of 0 to 10 modified or unmodified nucleotides or combinations thereof;

[0061] each n p n p '、n q and n q Each may or may not exist independently, representing a nucleotide at the overhang; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif of three consecutive nucleotides with the same modification.

[0062] N b The modifiers on Nb' are different from those on Y, and the modifiers on Nb' are different from those on Y'; and

[0063] The justice chain binds to at least one ligand.

[0064] In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1.

[0065] In another embodiment, k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1.

[0066] In some embodiments, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

[0067] In another embodiment, the YYY motif appears at or near the cleavage site of the justice chain.

[0068] In another embodiment, the Y'Y'Y' motif appears at positions 11, 12, and 13 at the 5'-end of the antisense chain. Optionally, Y' is 2'-O-methyl.

[0069] In some embodiments, equation (III) is represented by equation (IIIa):

[0070] Justice: 5'n p -N a -YYY-N a -n q 3'

[0071] Antonym: 3'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 5' (IIIa)

[0072] In another embodiment, equation (III) is represented by equation (IIIb):

[0073] Justice: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'

[0074] Antonym: 3'n p’ -N a’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’5' (IIIb)

[0075] Where N b and N b Each of these represents an oligonucleotide sequence consisting of 1 to 5 modified nucleotides.

[0076] In another embodiment, equation (III) is represented by equation (IIIc):

[0077] Justice: 5'n p -N a –XXX-N b -YYY-N a -n q 3'

[0078] Antonym: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5' (IIIc)

[0079] Where N b and N b Each of these represents an oligonucleotide sequence consisting of 1 to 5 modified nucleotides.

[0080] In some embodiments, equation (III) is represented by equation (IIId):

[0081] Justice: 5'n p -N a –XXX-N b -YYY-N b -ZZZ-N a -n q 3'

[0082] Antonym: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5' (IIId)

[0083] Where, N b and N b Each 'represents an oligonucleotide sequence consisting of 1 to 5 modified nucleotides, N' a and N a Each of these represents an oligonucleotide sequence consisting of 2 to 10 modified nucleotides.

[0084] In another embodiment, the length of the double-stranded region is 15 to 30 nucleotide pairs. Optionally, the length of the double-stranded region is 17 to 23 nucleotide pairs.

[0085] In some embodiments, the length of the double-stranded region is 17 to 25 nucleotide pairs. Optionally, the length of the double-stranded region is 23 to 27 nucleotide pairs.

[0086] In some embodiments, the length of the double-stranded region is 19 to 21 nucleotide pairs. Optionally, the length of the double-stranded region is 21 to 23 nucleotide pairs.

[0087] In some embodiments, each chain has 15 to 30 nucleotides. Optionally, each chain has 19 to 30 nucleotides.

[0088] In another embodiment, the nucleotide modification of the RNAi agent is LNA, ethylene glycol nucleic acid (GNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluorine, 2'-deoxy, and / or 2'-hydroxy, and combinations thereof. Optionally, the nucleotide modification includes 2'-O-methyl, 2'-fluorine, and / or GNA, and combinations thereof. In related embodiments, the modification on the nucleotide is 2'-O-methyl or 2'-fluorine modification.

[0089] In one embodiment, the RNAi agent includes a ligand that is or includes one or more C16 portions attached via a divalent or trivalent branched linker.

[0090] In some embodiments, the ligand is attached to the 3' end of the justice chain.

[0091] In some embodiments, the RNAi agent further comprises at least one phosphate-thioester or methylphosphate nucleotide internucleotide bond. In a relevant embodiment, the phosphate-thioester or methylphosphate nucleotide internucleotide bond is located at the 3' end of one strand. Optionally, the strand is an antisense strand. In another embodiment, the strand is a sense strand. In a relevant embodiment, the phosphate-thioester or methylphosphate nucleotide internucleotide bond is located at the 5' end of one strand. Optionally, the strand is an antisense strand. In another embodiment, the strand is a sense strand.

[0092] In another embodiment, the internucleotide bonds of the thiophosphate or methylphosphate are located at the 5' and 3' ends of one strand. Optionally, the strand is an antisense strand. In another embodiment, the strand is a sense strand.

[0093] In another embodiment, the base pair at position 1 of the 5'-end of the antisense strand of the RNAi agent duplex is an A:U base pair.

[0094] In some embodiments, the Y nucleotide contains a 2'-fluorine modification.

[0095] In some embodiments, the Y nucleotide contains a 2'-O-methyl modification.

[0096] In some embodiments, p' > 0. Alternatively, p' = 2.

[0097] In some embodiments, q' = 0, p = 0, q = 0, and the p' overhang nucleotide is complementary to the target mRNA.

[0098] In some embodiments, q' = 0, p = 0, q = 0, and the p' overhang nucleotide is not complementary to the target mRNA.

[0099] In one embodiment, the RNAi agent has a total of 21 nucleotides in the sense strand and a total of 23 nucleotides in the antisense strand.

[0100] In another embodiment, at least n p 'Linked to adjacent nucleotides via phosphate thioester bonding. Optionally, all n p They are all linked to adjacent nucleotides via thiophosphate bonds.

[0101] In some embodiments, the RNAi agent of this application is any one of those listed in Tables 2A, 2B, 3, 5A, 5B, 6 and / or 9.

[0102] In some embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand include modifications.

[0103] Another aspect of this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene in cells, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, wherein the antisense strand comprises a region partially complementary to the mRNA encoding APP, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (III):

[0104] Justice: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'

[0105] Antonym: 3'

[0106] n p '-N a'-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)

[0107] in:

[0108] i, j, k, and l are each independently 0 or 1;

[0109] p, p', q, and q' are each independently between 0 and 6;

[0110] N a and N a Each of the two oligonucleotide sequences independently represents an oligonucleotide sequence comprising 0 to 25 modified or unmodified nucleotides or combinations thereof, each sequence comprising at least two distinct modified nucleotides; Nb and Nb' each independently represent an oligonucleotide sequence comprising 0 to 10 modified or unmodified nucleotides or combinations thereof;

[0111] each n p n p '、n q and n q Each may or may not be present, independently representing the overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides, wherein the modification is a 2'-O-methyl or 2'-fluorine modification;

[0112] N b The modifiers on N are different from those on Y, N b The modification on ' is different from the modification on Y'; and wherein the justice chain binds to at least one ligand.

[0113] Another aspect of this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene in cells, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, wherein the antisense strand comprises a region partially complementary to the mRNA encoding APP, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (III):

[0114] Justice: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -Na -n q 3'

[0115] Antonym: 3'

[0116] n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)

[0117] in:

[0118] i, j, k, and l are each independently 0 or 1;

[0119] each n p n q and n q Each can exist or not, independently representing the overhanging nucleotide;

[0120] p, q, and q' are each independently between 0 and 6;

[0121] n p '>0 and at least one np' is linked to an adjacent nucleotide via a phosphate thioester bond;

[0122] N a and N a Each oligonucleotide sequence independently represents a sequence comprising 0 to 25 modified or unmodified nucleotides or combinations thereof, each sequence comprising at least two distinct modified nucleotides; N b and N b Each 'independently represents an oligonucleotide sequence, consisting of 0 to 10 modified or unmodified nucleotides or combinations thereof;

[0123] XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides, wherein the modification is a 2'-O-methyl, ethylene glycol nucleic acid (GNA), or 2'-fluorine modification;

[0124] N b The modifiers on N are different from those on Y, N b The modification on ' is different from the modification on Y'; and wherein the justice chain binds to at least one ligand.

[0125] Another aspect of this disclosure provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene in cells, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region partially complementary to the mRNA encoding APP, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (III):

[0126] Justice: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'

[0127] Antonym: 3'

[0128] n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)

[0129] in:

[0130] i, j, k, and l are each independently 0 or 1;

[0131] each n p n q and n q Each may or may not be present, independently representing the overhanging nucleotide; p, q, and q' are each independently 0 to 6;

[0132] n p '>0 and at least one n p 'Linked to adjacent nucleotides via thiophosphate bonds;'

[0133] N a and N a Each oligonucleotide sequence independently represents a sequence comprising 0 to 25 modified or unmodified nucleotides or combinations thereof, each sequence comprising at least two distinct modified nucleotides;

[0134] N b and N b Each 'independently represents an oligonucleotide sequence, consisting of 0 to 10 modified or unmodified nucleotides or combinations thereof;

[0135] XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides, wherein the modification is a 2'-O-methyl or 2'-fluorine modification;

[0136] N b The modifiers on N are different from those on Y, N b The modification on ' is different from the modification on Y'; and wherein the justice chain binds to at least one ligand, optionally wherein the ligand is one or more C16 ligands.

[0137] Another aspect of this disclosure provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene in cells, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region partially complementary to the mRNA encoding APP, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (III):

[0138] Justice: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'

[0139] Antonym: 3'

[0140] n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)

[0141] in:

[0142] i, j, k, and l are each independently 0 or 1;

[0143] each n p n q and n q Each can exist or not, independently representing the overhanging nucleotide;

[0144] p, q, and q' are each independently between 0 and 6;

[0145] n p '>0 and at least one np' is linked to an adjacent nucleotide via a phosphate thioester bond;

[0146] N a and N a Each oligonucleotide sequence independently represents a sequence comprising 0 to 25 modified or unmodified nucleotides or combinations thereof, each sequence comprising at least two distinct modified nucleotides;

[0147] N b and N b Each 'independently represents an oligonucleotide sequence, consisting of 0 to 10 modified or unmodified nucleotides or combinations thereof;

[0148] XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif with three identical modifications on three consecutive nucleotides, wherein the modification is a 2′-O-methyl or a 2′-fluoro modification;

[0149] N b The modifiers on N are different from those on Y, N b The modifiers on ' are different from those on ';

[0150] Wherein, the positive chain includes at least one thiophosphate bond; and

[0151] The justice chain binds to at least one ligand, optionally wherein the ligand is one or more C16 ligands.

[0152] Another aspect of this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene in cells, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding APP, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (III):

[0153] Justice: 5'n p -N a -YYY-N a -n q 3'

[0154] Antonym: 3'n p '-N a '-Y'Y'Y'-N a '-n q '5' (IIIa)

[0155] in:

[0156] each np n q and n q Each can exist or not, independently representing the overhanging nucleotide;

[0157] p, q, and q' are each independently between 0 and 6;

[0158] n p '>0 and at least one np' is linked to an adjacent nucleotide via a phosphate thioester bond;

[0159] N a and N a Each oligonucleotide sequence independently represents a sequence comprising 0 to 25 modified or unmodified nucleotides or combinations thereof, each sequence comprising at least two distinct modified nucleotides;

[0160] YYY and Y'Y'Y' each independently represent a motif of three identical modifications on three consecutive nucleotides, wherein the modification is a 2'-O-methyl or 2'-fluorine modification;

[0161] Wherein, the positive chain includes at least one thiophosphate bond; and

[0162] The justice chain binds to at least one ligand, optionally wherein the ligand is one or more C16 ligands.

[0163] Another aspect of this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides differing from any nucleotide sequence of SEQ ID NOs:1 to 14 by no more than 3 nucleotides, and the antisense strand comprises ... NOs: at least 15 consecutive nucleotides of any nucleotide sequence from 15 to 28, differing by no more than 3 nucleotides, wherein substantially all nucleotides of the sense strand include modifications of 2'-O-methyl, GNA, and / or 2'-fluorine, wherein the sense strand contains a two-thiophosphate nucleotide bond at the 5' end, wherein substantially all nucleotides of the antisense strand include modifications selected from the group consisting of 2'-O-methyl and 2'-fluorine, wherein the antisense strand includes a two-thiophosphate nucleotide bond at the 5' end and a two-thiophosphate nucleotide bond at the 3' end, and wherein the sense strand is bound to one or more C16 ligands.

[0164] Another aspect of this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides differing from any nucleotide sequence of SEQ ID NOs:1 to 14 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotide sequences differing from any nucleotide sequence of SEQ ID NOs:15 to 28 by no more than 3 nucleotides, wherein the sense strand comprises at least one 3'-terminal deoxythymidine nucleotide (dT), and wherein the antisense strand comprises at least one 3'-terminal deoxythymidine nucleotide (dT).

[0165] In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

[0166] In another embodiment, each chain has 19 to 30 nucleotides.

[0167] In some embodiments, the antisense strand of the RNAi agent includes at least one thermally unstable modification of the double strand within the 5' region or the first 9 nucleotide positions of its precursor. Optionally, the thermally unstable modification of the double strand is one or more of the following:

[0168] and

[0169] In this case, B is a nucleobase.

[0170] Another aspect of this application provides cells containing the double-stranded RNAi agent of this application.

[0171] Another aspect of this application provides a pharmaceutical composition for inhibiting the expression of the APP gene, which includes the double-stranded RNAi agent of this application.

[0172] In one embodiment, the double-stranded RNAi agent is administered to a non-buffered solution. Optionally, the non-buffered solution is physiological saline or water.

[0173] In another embodiment, the double-stranded RNAi agent is administered together with a buffer solution. Optionally, the buffer solution comprises acetate, citrate, prolyl, carbonate, or phosphate, or any combination thereof. In another embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0174] Another aspect of this application provides a pharmaceutical composition comprising the double-stranded RNAi agent and lipid formulation of this application.

[0175] In one embodiment, the lipid formulation comprises LNP.

[0176] Another aspect of this application provides a method for inhibiting the expression of the amyloid precursor protein (APP) gene in cells, the method comprising: (a) contacting the cells with the double-stranded RNAi agent of this application or the pharmaceutical composition; and (b) maintaining the cells generated in step (a) for a sufficient time to allow time for degradation of the mRNA transcript of the APP gene, thereby inhibiting the expression of the APP gene in the cells.

[0177] In one embodiment, the cell is located in an individual. Optionally, the individual is a human.

[0178] In some embodiments, the individuals are rhesus monkeys, cynomolgus monkeys, mice, or rats.

[0179] In one embodiment, the human individual suffers from an app-related condition. Optionally, the app-related condition is cerebral amyloid angiopathy (CAA).

[0180] In another embodiment, the app-related condition is early-onset familial Alzheimer's disease (EOFAD). In another embodiment, the app-related condition is Alzheimer's disease (AD).

[0181] In some embodiments, the expression of the APP is inhibited by at least about 30% by an RNAi agent.

[0182] Another aspect of this application provides a method for treating an individual suffering from a condition in which reduced expression of the APP would benefit, the method comprising administering a therapeutically effective amount of the double-stranded RNAi agent or the pharmaceutical composition of this application to the individual, thereby treating the individual.

[0183] In some embodiments, the method further includes administering additional therapeutic agents to the individual.

[0184] In some embodiments, the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0185] In some embodiments, the double-stranded RNAi agent is administered intrathecally to an individual.

[0186] In some embodiments, administration of the double-stranded RNAi to an individual results in a reduction in Aβ accumulation. Optionally, administration of the double-stranded RNAi to an individual results in a reduction in the accumulation of Aβ(1-40) and / or Aβ(1-42).

[0187] In related embodiments, administration of dsRNA to an individual resulted in a reduction in the formation and / or accumulation of amyloid plaques in the individual.

[0188] In one embodiment, the method reduces the expression of target genes in brain or spinal cord tissue. Optionally, the brain or spinal cord tissue is the cortex, cerebellum, striatum, cervical vertebrae, lumbar vertebrae, and / or thoracic vertebrae.

[0189] Another aspect of this application provides a method for inhibiting APP expression in an individual, the method comprising: administering a therapeutically effective amount of the double-stranded RNAi agent of this application or a pharmaceutical composition of this application to the individual, thereby inhibiting APP expression in the individual.

[0190] Another aspect of this application provides a method for treating or preventing APP-related diseases or conditions in an individual, the method comprising administering a therapeutically effective amount of the double-stranded RNAi agent or pharmaceutical composition of this application to the individual, thereby treating or preventing APP-related diseases or conditions in the individual.

[0191] In some embodiments, the APP-related disease or condition is cerebral amyloid angiopathy (CAA) and / or Alzheimer's disease (AD). Optionally, the AD is early-onset familial Alzheimer's disease (EOFAD).

[0192] Another aspect of this application provides a kit for performing the methods of this application, the kit comprising: a) the double-stranded RNAi agent of this application, b) instructions for use, and c) optionally, a device for administering the double-stranded RNAi agent to an individual.

[0193] Another aspect of this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene, wherein the RNAi agent has a sense strand and an antisense strand, and wherein the antisense strand includes a complementary region comprising at least 15 consecutive nucleotides, which are associated with AD-392911, AD-392912, AD-392816, AD-392704, AD-392843, AD-392855, AD-392840, AD-392835, AD-392729, AD-392916, AD-392876, AD-392863, AD-392917, AD-392783, AD-3 92765, AD-392791, AD-392800, AD-392711, AD-392801, AD-392826, AD-392818, AD-392792, AD-392802, AD-392766, AD-392767, AD-392834, AD-39297 4. AD-392784, AD-392744, AD-392752, AD-392737, AD-392918, AD-392919, AD-392803, AD-392804, AD-392827, AD-392828, AD-392785, AD-392829, AD- 392920, AD-392921, AD-392768, AD-392805, AD-392769, AD-392753, AD-392714, AD-392703, AD-392715, AD-392836, AD-392966, AD-392832, AD-3929 A D-392923, AD-392866, AD-392877, AD-392707, AD-392926, AD-392927, AD-392717, AD-392700, AD-392878, AD-392718, AD-392929, AD-392819, AD-39 2745, AD-392770, AD-392806, AD-392771, AD-392820, AD-392821, AD-392786, AD-392772, AD-392699, AD-392868, AD-392719, AD-392880, AD-392930,AD-392932、AD-392869、AD-392870、AD-392896、AD-392720、AD-392746、AD-392773、AD-392807、AD-392730、AD-392721、AD-392933、AD-392881、AD-392897、AD-392898、AD-392899、AD-392935、AD-392882、AD-392738、AD-392739、AD-392936、AD-392900、AD-392901、AD-392937、AD-392883、AD-392975、AD-392938、AD-392902、AD-392941、AD-392942、AD-392943、AD-392944、AD-392903、AD-392775、AD-392758、AD-392945、AD-392884、AD-392947、AD-392748、AD-392759、AD-392837、AD-392970、AD-392976、AD-392965、AD-392831、AD-392904、AD-392885、AD-392886、AD-392776、AD-392887、AD-392722、AD-392760、AD-392731、AD-392709、AD-392723、AD-392948、AD-392724、AD-392949、AD-392725、AD-392950、AD-392732、AD-392726、AD-392862、AD-392951、AD-392871、AD-392872、AD-397183、AD-397175、AD-397177、AD-397176、AD-397260、AD-397266、AD-397267、AD-397178、AD-397180、AD-397184、AD-397179、AD-397224、AD-397225、AD-397203、AD-397185、AD-397195、AD-397204、AD-397191、AD-397251、AD-397240、AD-397205、AD-397254、AD-397259、AD-397247、AD-397233、AD-397181、AD-397196、AD-397197、AD-397226、AD-397212、AD-397182、AD-397227、AD-397217、AD-397213、AD-397229、AD-397264, AD-397265, AD-397209, AD-397192, AD-397210, AD-397219, AD-397214, AD-397220, AD-397230, AD- 397231, AD-397193, AD-397190, AD-397200, AD-397248, AD-397207, AD-397211, AD-397243, AD-397246, AD-397 The antisense strand nucleobase sequences of 223, AD-397202, AD-397256, AD-397257, AD-397258, AD-397250, AD-397244, AD-454972, AD-454973, AD-454842, AD-454843, AD-454844, AD-994379, AD-961583, AD-961584, AD-961585, or AD-961586 differ by no more than 3 nucleotides.

[0194] In one embodiment, the RNAi agent comprises one or more of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-C-alkyl modified nucleotides, and nucleotides comprising glycol nucleic acid (GNA), phosphate thioester (PS), and vinyl phosphate (VP). Optionally, the RNAi agent comprises at least one of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-C-alkyl modified nucleotides, and nucleotides comprising glycol nucleic acid (GNA), phosphate thioester, and vinyl phosphate (VP).

[0195] In another embodiment, the RNAi agent comprises four or more PS modifications, optionally six to ten PS modifications, optionally eight PS modifications.

[0196] In another embodiment, each sense and antisense strand of the RNAi agent includes a 5'-end and a 3'-end, and the RNAi agent includes eight PS modifications located at the penultimate and last internucleotide bonds at the 3'- and 5'-ends of each sense and antisense strand of the RNAi agent.

[0197] In another embodiment, each sense and antisense strand of the RNAi agent includes a 5'-terminus and a 3'-terminus, and the RNAi agent comprises only one nucleotide comprising GNA. Optionally, the GNA-containing nucleotide is located on the antisense strand at the position of the seventh nucleobase residue at the 5'-terminus of the antisense strand.

[0198] In another embodiment, each sense and antisense strand of the RNAi agent includes a 5'-terminus and a 3'-terminus, and the RNAi agent includes one to four 2'-C-alkyl-modified nucleotides. Optionally, the 2'-C-alkyl-modified nucleotide is a 2'-C16-modified nucleotide. Optionally, the RNAi agent includes a single 2'-C16-modified nucleotide. Optionally, the single 2'-C16-modified nucleotide is located on the sense strand at the sixth nucleotide position at the 5' end or at the terminal nucleotide position of the 5' end.

[0199] In another embodiment, each sense and antisense strand of the RNAi agent includes a 5' end and a 3' end, and the RNAi agent includes two or more 2'-fluorinated nucleotides. Optionally, each sense and antisense strand of the RNAi agent contains two or more 2'-fluorinated nucleotides. Optionally, the 2'-fluorinated nucleotides are located on the sense strand at nucleotide positions 7, 9, 10, and 11 at the 5' end of the sense strand and on the antisense strand at nucleotide positions 2, 14, and 16 at the 5' end of the antisense strand.

[0200] In another embodiment, each sense and antisense strand of the RNAi agent includes a 5'-end and a 3'-end, and the RNAi agent includes one or more VP modifications. Optionally, the RNAi agent includes a single VP modification at the 5'-end of the antisense strand.

[0201] In another embodiment, each sense and antisense strand of the RNAi agent comprises a 5' end and a 3' end, and the RNAi agent comprises two or more 2'-O-methyl modified nucleotides. Optionally, the RNAi agent comprises 2'-O-methyl modified nucleotides at all nucleobase positions not modified with 2'-fluorine, 2'-C-alkyl, or ethylene glycol nucleic acid (GNA). Optionally, two or more 2'-O-methyl modified nucleotides are located on the sense strand at positions 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 at the 5' end of the sense strand and on the antisense strand at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 at the 5' end of the antisense strand.

[0202] Another aspect of this application provides a double-stranded RNAi agent for inhibiting the expression of the amyloid precursor protein (APP) gene, wherein the RNAi agent comprises a sense strand and an antisense strand, and wherein the antisense strand comprises a region of at least 15 consecutive nucleotides in length, which corresponds to the following positions of APP NM_00484: 1891 to 1919; 2282 to 2306; 2464 to 2494; 2475 to 2638; 2621 to 2689; 2682 to 2725; 2705 to 2746; 2726 to 2771; 2754 to 2788; APP NM_00484. NM_00484 Positions 2782 to 2813; APP NM_00484 Positions 2801 to 2826; APP NM_00484 Positions 2847 to 2890; APP NM_00484 Positions 2871 to 2896; APP NM_00484 Positions 2882 to 2960; APP NM_00484 Positions 2942 to 2971; APP NM_00484 Positions 2951 to 3057; APP NM_00484 Positions 3172 to 3223; APP NM_00484 Positions 3209 to 3235; NM_00484 Positions 3256 to 3289; NM_00484 Positions 3302 to 3338; ​​APP NM_00484 Positions 3318 to 3353; APP NM_00484, positions 3334 to 3361; APP NM_001198823.1, positions 251 to 284; APP NM_001198823.1, positions 362 to 404; APP NM_001198823.1, positions 471 to 510; APP NM_001198823.1, positions 532 to 587; APP NM_001198823.1, positions 601 to 649; APP NM_001198823.1, positions 633 to 662; APP NM_001198823.1, positions 1351 to 1388; APP NM_001198823.1, positions 1609 to 1649; APP NM_001198823.1, positions 1675 to 1698; APP NM_001198823.1, positions 1752 to 1787; APP NM_001198823.1, positions 2165 to 2217; APP NM_001198823.1, positions 2280 to 2344 or APP NM_001198823.The target APP sequence at positions 2403 to 2431 is sufficiently complementary to influence APP knockout, and differs by no more than 3 nucleotides within the at least 15 consecutive nucleotides that are sufficiently complementary to the target APP sequence to influence APP knockout.

[0203] Another aspect of this application provides a double-stranded RNAi agent comprising one or more modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-C-alkyl modified nucleotides, and nucleotides comprising ethylene glycol nucleic acid (GNA), phosphate thioester (PS), and vinyl phosphate ester (VP). Optionally, the RNAi agent comprises at least one of each modification selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-C-alkyl modified nucleotides, and nucleotides comprising ethylene glycol nucleic acid (GNA), phosphate thioester, and vinyl phosphate ester (VP).

[0204] Another aspect of this application provides that the RNAi agent comprises four or more PS modifications, optionally six to ten PS modifications, optionally eight PS modifications.

[0205] Another aspect of this application provides that each sense strand and antisense strand of the RNAi agent comprises a 5'-end and a 3'-end, and wherein the RNAi agent comprises eight penultimate and last internucleotide bonds located at the respective 3'- and 5'-ends of the sense and antisense strands of the RNAi agent.

[0206] Another aspect of this application provides that each sense strand and antisense strand of the RNAi agent comprises a 5'-end and a 3'-end, and wherein the RNAi agent comprises only one nucleotide containing GNA, optionally wherein the GNA-containing nucleotide is located on the antisense strand at the position of the seventh nucleobase residue at the 5'-end of the antisense strand.

[0207] Another aspect of this application provides that each sense strand and antisense strand of an RNAi agent comprises a 5'-terminus and a 3'-terminus, and wherein the RNAi agent comprises one to four 2'-C-alkyl-modified nucleotides, optionally wherein the 2'-C-alkyl-modified nucleotide is a 2'-C16-modified nucleotide, optionally wherein the RNAi agent comprises a single 2'-C16-modified nucleotide, optionally located at the sixth nucleobase position of the 5'-terminus of the sense strand or at the terminal nucleobase position of the 5'-terminus on the sense strand.

[0208] Another aspect of this application provides an RNAi agent in which each sense strand and antisense strand comprises a 5'-terminus and a 3'-terminus, and wherein the RNAi agent comprises two or more 2'-fluorinated nucleotides, optionally wherein each sense strand and antisense strand of the RNAi agent comprises two or more 2'-fluorinated nucleotides, optionally wherein the 2'-fluorinated nucleotides are located on the sense strand at nucleotide positions 7, 9, 10, and 11 at the 5'-terminus of the sense strand and on the antisense strand at nucleotide positions 2, 14, and 16 at the 5'-terminus of the antisense strand.

[0209] Another aspect of this application provides that each sense strand and antisense strand of the RNAi agent comprises a 5'-end and a 3'-end, and wherein the RNAi agent comprises one or more VP modifications, optionally wherein the RNAi agent comprises a single VP modification at the 5'-end of the antisense strand.

[0210] Another aspect of this application provides that each sense strand and antisense strand of an RNAi agent comprises a 5'-terminus and a 3'-terminus, and wherein the RNAi agent comprises two or more 2'-O-methyl modified nucleotides, optionally wherein the RNAi agent comprises 2'-O-methyl modified nucleotides at all nucleobase positions not modified by 2'-fluorine, 2'-C-alkyl, or ethylene glycol nucleic acid (GNA), optionally wherein two or more 2'-O-methyl modified nucleotides are located on the sense strand at positions 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 at the 5'-terminus of the sense strand, and on the antisense strand at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 at the 5'-terminus of the antisense strand. BRIEF DESCRIPTION OF THE DRAWINGS

[0211] The following detailed description is given by way of example, but is not intended to limit this application to the specific embodiments described. The best understanding can be obtained by referring to the accompanying drawings, in which:

[0212] Figure 1A and Figure 1B A schematic diagram of a modified RNAi agent for an in vivo hsAPP knockout activity assay is shown.

[0213] Figure 2A and Figure 2B Showing the Figure 1A and Figure 1B The results of in vivo hsAPP knockout activity observed with the modified RNAi agent are shown in the figure.

[0214] Figure 3AThis paper presents a scheme for identifying human APP (hAPP) siRNAs that are effective in targeting hereditary cerebral amyloid angiopathy (hCAA).

[0215] Figure 3B This is a graph showing the percentage of remaining mRNA in Be(2)C cells during in vitro endogenous screening of hAPP siRNA at a concentration of 10 nM.

[0216] Figure 4A This is a schematic diagram illustrating the timing of APP siRNA transfection in BE(2)C neuronal cells. APP siRNA was transfected at 10, 1, and 0.1 nM and evaluated at 24 and 48 hours post-transfection.

[0217] Figure 4B The graph shows the application concentration of APP double-stranded siRNA and the percentage of remaining mRNA in BE(2)C cells 48 hours after transfection.

[0218] Figure 4C These are two images showing soluble APPα (top) and β (bottom) substances in the supernatant of BE(2)C cells 48 hours after transfection.

[0219] Figure 5A This diagram illustrates the design of a non-human primate (NHP) screening study for APP siRNA. Five compounds were evaluated, with five animals used in each experiment. A single intrathecal (IT) injection of 72 mg of the target compound was administered at the start of the study.

[0220] Figure 5B Two graphs showing the soluble APPα (top) and β (bottom) substances in BE(2)C (bottom) after 72 mg of AD-454972 targeting APP was administered to cynomolgus monkeys via IT administration.

[0221] Figure 5C The results of tissue mRNA knockout on day 29 after administration of 72 mg of AD-454972 targeting APP in cynomolgus monkeys are shown.

[0222] Figure 5D A schematic diagram showing the structure of the APP-targeting compound AD-454972 (top) and a table showing the delivery levels of the APP-targeting compound AD-454972 in tissues on day 29 after an IT administration of 72 mg of AD-454972 to APP in cynomolgus monkeys (bottom).

[0223] Figure 6 These are two graphs showing the results of CSF-soluble APPα and β (top) and CSFβ amyloid protein (bottom) collected 2 to 3 months after IT administration of 72 mg AD-454972 targeting APP in cynomolgus monkeys.

[0224] Figure 7A Two graphs show the CSF results of soluble APPα and β (top) and amyloid β38, 40 and 42 (bottom) collected and analyzed on days 8, 15 and 29 after IT administration of 72 mg AD-454842 targeting APP in cynomolgus monkeys.

[0225] Figure 7B A table showing the delivery levels of the AD-454842 compound in tissues on day 29 following IT administration of 72 mg of AD-454842 targeting the APP in cynomolgus monkeys.

[0226] Figure 8A Two graphs show the CSF results collected on days 8, 15, and 29 after IT administration of 72 mg AD-454843 targeting APP in cynomolgus monkeys, and the analysis of soluble APPα and β (top) and amyloid β38, 40, and 42 (bottom).

[0227] Figure 8B The results of tissue mRNA knockout on day 29 after administration of 72 mg of AD-454843 targeting APP in cynomolgus monkeys are shown.

[0228] Figure 8C This is a table showing the delivery levels of the AD-454843 compound in tissues on day 29 following IT administration of 72 mg of AD-454843 targeting APP in cynomolgus monkeys.

[0229] Figure 9A These are two graphs showing the results of CSF-soluble APPα and β (top) and CSFβ amyloid protein (bottom) collected 2 to 3 months after IT administration of 72 mg AD-454843 targeting APP in cynomolgus monkeys.

[0230] Figure 9B The results of tissue mRNA knockout on day 85 after administration of 72 mg of AD-454843 targeting APP in cynomolgus monkeys are shown.

[0231] Figure 10A Two graphs show the results of collecting and analyzing soluble APPα and β (top) and amyloid β38, 40 and 42 (bottom) on days 8, 15 and 29 after IT administration of 72 mg AD-454844 targeting APP in cynomolgus monkeys.

[0232] Figure 10B The results of tissue mRNA knockout on day 29 after administration of 72 mg of AD-454844 targeting APP in cynomolgus monkeys are shown.

[0233] Figure 10CA schematic diagram showing the structure of the APP-targeting compound AD-454844 (top) and a table showing the delivery levels of the AD-454844 compound in tissues on day 29 after an IT administration of 72 mg of AD-454844 targeting the APP in cynomolgus monkeys (bottom).

[0234] Figure 11A This is a table showing high levels of the compound delivery in tissues on day 29 after IT administration of 72 mg of the siRNA targeting APP in cynomolgus monkeys.

[0235] Figure 11B It shows that at a high level ( Figure 11A A figure showing the results of tissue mRNA knockout in cynomolgus monkeys on day 29 after IT administration of compounds targeting APP.

[0236] Figure 11C This demonstrates high levels of compound delivery in cynomolgus monkeys after IT administration of 72 mg of the targeted APP. Figure 11A Two graphs showing the CSF results of soluble APPα and β (top) and amyloid β38, 40 and 42 (bottom) collected and analyzed on days 8, 15 and 29.

[0237] Figure 12A These are two graphs showing the average values ​​from studies of five miRNA double strands. The top graph is a box plot of the results of the five compounds in cynomolgus monkeys at day 29 after IT administration of 72 mg of siRNA. The bottom graph is a box plot of the amount of remaining mRNA in each tissue in cynomolgus monkeys at day 29 after IT administration, relative to the control group.

[0238] Figure 12B These are two figures illustrating a repetitive miRNA double-stranded study, in which 72 mg of an APP-targeting siRNA compound was administered to cynomolgus monkeys via IT, with CSF collected on days 8, 15, and 29 and soluble APPα and β (top) and amyloid β38, 40, and 42 (bottom) analyzed.

[0239] Figure 13A The percentage of residual APP mRNA in striatal tissue of AD-454972, which targets APP, is shown 29 days after IT administration in cynomolgus monkeys.

[0240] Figure 13B The percentage of residual APP mRNA in striatal tissue 29 days after IT administration of AD-454973 targeting APP in cynomolgus monkeys is shown.

[0241] Figure 13C The percentage of residual APP mRNA in striatal tissue 29 days after IT administration of AD-454842, which targets APP, is shown.

[0242] Figure 13D The percentage of residual APP mRNA in striatal tissue 29 days after IT administration of AD-454843 targeting APP in cynomolgus monkeys is shown.

[0243] Figure 13E The percentage of residual APP mRNA in striatal tissue 29 days after IT administration of AD-454844 targeting APP in cynomolgus monkeys is shown.

[0244] Figure 14A and Figure 14B This is a schematic diagram of a modified RNAi agent that screens for AU-rich seeds in mice to target hsAPP knockout activity.

[0245] Figure 15 This image shows %hsAPP knockout in the livers of AAV8.HsAPP-CDS3TRNC mice treated with AU-rich seeds. PBS is included in the figure. The control group was AD-392927 (RLD592).

[0246] Figures 16A to 16D This is a schematic diagram of a modified lead RNAi agent used for screening hsAPP knockout activity in AAV mice.

[0247] Figure 17A and Figure 17B This is a diagram depicting %hsAPP knockout in the livers of AAV8.HsAPP-CDS3TRNC mice treated with a lead oligonucleotide. The diagram includes PBS and... Control group.

[0248] Figures 18A to 18D Screening for in vivo hsAPP knockout activity in AAV mice, based on the AD-886864 parent ( Figure 18A ), AD-886899 parent ( Figure 18B ), AD-886919 parent ( Figure 18C ) and AD-886823 parent ( Figure 18D A schematic diagram of modified lead RNAi agents grouped into families.

[0249] Figure 19 This is a protocol demonstrating the design of an APP knockout non-human primate (NHP) screening study for AD-454844 over a 4-month period, in which 60 mg of the target compound was injected intrathecally (IT) into cynomolgus monkeys at the start.

[0250] Figures 20A to 20GThis figure shows data from in vivo screening of C16 siRNA conjugates, including parental AD-454855 and five other siRNA conjugates derived from the structure-activity relationship study of AD-454855. The figure depicts the percentage of soluble APPα and β collected from CSF on days 8, 15, and 19 after intrathecal administration of 60 mg of each compound. Figure 20A This is a graph showing the soluble APPα and β levels in two non-human primate individuals four months after AD-454844 administration. Figure 20B The percentages of soluble APPα and β collected from CSF at days 8, 15, and 19 following AD-454844 administration are shown. Figure 20C The graph shows the percentages of soluble APPα and β collected from CSF on days 8, 15, and 19 following administration of the 5'-terminal C16 siRNA conjugate AD-994379. Figure 20D The percentages of soluble APPα and β collected from CSF at days 8, 15, and 19 following AD-961583 administration are shown. Figure 20E The percentages of soluble APPα and β collected from CSF at days 8, 15, and 19 following AD-961584 administration are shown. Figure 20F The percentages of soluble APPα and β collected from CSF at days 8, 15, and 19 following AD-961585 administration are shown. Figure 20G The percentages of soluble APPα and β collected from CSF at days 8, 15, and 19 following AD-961586 administration are shown.

[0251] Figure 21A and 21B This is a schematic diagram of a C16-modified lead RNAi agent used for screening APP knockout activity in non-human primates. Figure 21A This is a schematic diagram of the parental internal C16 RNAi agent AD-454844 and the 5' end C16 siRNA agent AD-994379. Figure 21B This is a schematic diagram of the RNAi agents AD-961583, AD-961584, AD-961585, and AD-961586. Detailed Implementation

[0252] This application provides an RNAi composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the amyloid precursor protein (APP) gene. The APP gene can be intracellular, for example, within cells of an individual, such as human cells. This application also provides methods for using the RNAi composition of this application to inhibit APP gene expression and / or treat individuals with diseases that would benefit from inhibiting or reducing APP gene expression, such as APP-related diseases like cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD), such as early-onset familial Alzheimer's disease (EOFAD).

[0253] The RNAi agent of this application comprises an RNA strand (antisense strand) having a region of about 30 nucleotides or less in length, such as 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29. The regions are 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides in length, and are substantially complementary to at least a portion of the mRNA transcript of the APP gene.

[0254] In some embodiments, the RNAi agents of this application comprise an RNA strand (antisense strand) that may include a longer length, such as up to 66 nucleotides, for example, 36 to 66, 26 to 36, 25 to 36, 31 to 60, 22 to 43, or 27 to 53 nucleotides in length, having a region of at least 19 consecutive nucleotides, said region being substantially complementary to at least a portion of the mRNA transcript of the APP gene. These RNAi agents with longer antisense strands preferably include a second RNA strand (sense strand) of 20 to 60 nucleotides in length, wherein the sense and antisense strands form a doublet of 18 to 30 consecutive nucleotides.

[0255] The use of these RNAi reagents enables the targeted degradation of APP gene mRNA in mammals. In particular, very low doses of APP RNAi reagents can specifically and effectively mediate RNA interference (RNAi), thereby significantly suppressing APP gene expression. Using cell-based assays, the inventors have demonstrated that APP-targeting RNAi reagents can mediate RNAi, leading to significant suppression of APP gene expression. Therefore, methods and compositions comprising these RNAi reagents can treat individuals who would benefit from reduced APP protein levels and / or activity, such as individuals with APP-related diseases, such as CAA or AD, including, for example, EOFAD.

[0256] The following detailed description discloses compositions and methods for preparing and using compositions containing RNAi reagents to suppress the expression of the APP gene, as well as for treating individuals with diseases and conditions that would benefit from the suppression and / or reduction of said gene expression.

[0257] I. Definition

[0258] To facilitate a clearer understanding of this application, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of a parameter is referenced, the values ​​and ranges referenced are also intended to be part of this application.

[0259] The articles “a” and “an” are used in this text to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one element or more elements, such as a plural number of elements.

[0260] The term “including” is used herein to mean the phrase “including but not limited to” and may be used interchangeably with the phrase “including but not limited to”. The term “or” is used herein to mean the term “and / or” and may be used interchangeably with the term “and / or” unless the context clearly indicates otherwise.

[0261] The term “about” is used herein to indicate a typical tolerance range in the field. For example, “about” can be understood as approximately 2 standard deviations from the mean. In some embodiments, “about” means ±10%. In some embodiments, “about” means ±5%. When “about” appears before a series of numbers or ranges, it can be understood that “about” may modify each number in the series or range.

[0262] The term "at least" preceding a number or series of numbers is understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included, as is clear 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 nucleic acid molecule of 21 nucleotides" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" precedes a series of numbers or ranges, it should be understood that "at least" can modify each number in the series or range.

[0263] As used herein, “not greater than” or “less than” is understood to be the value adjacent to the phrase and the logically lower value or integer, logically from the context to zero. For example, a double strand with “not more than 2 nucleotides” overhangs has 2, 1, or 0 nucleotide overhangs. When “not more than” appears before a series of numbers or ranges, it can be understood that “not more than” can modify each number in the series or range.

[0264] The term "APP" refers to amyloid precursor protein (APP), also known as β-amyloid precursor protein, Alzheimer's amyloid protein, and cerebral angiomyolipoprotein, etc., having an amino acid sequence from any vertebrate or mammalian source, including but not limited to humans, cattle, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs, unless otherwise stated. The term also refers to fragments and variants of natural APP that maintain at least one in vivo or in vitro activity of natural APP (including, for example, fragments and variants of β-amyloid peptides (1-40), β-amyloid peptides (1-38)) and Aβ peptides in the form of β-amyloid peptides (1-42), including variants of APP fragments that maintain one or more activities of APP fragments with neurotoxic characteristics (e.g., variant forms of Aβ42 peptide explicitly covering the maintenance of neurotoxic characteristics). The term includes both the full-length unprocessed precursor form of APP and the mature form resulting from post-translational cleavage of a signal peptide. The term also includes peptides derived from APP through further cleavage, including, for example, Aβ peptides. The nucleotide and amino acid sequences of human APP can be found, for example, in GenBank accession number GI:228008405 (NM_201414; SEQ ID NO: 1). The nucleotide and amino acid sequences of human APP can also be found in, for example, GenBank accession number GI: 228008403 (NM_000484.3; SEQ ID NO: 2), GenBank accession number GI: 228008404 (NM_201413.2, SEQ ID NO: 3), GenBank accession number GI: 324021746 (NM_001136016.3, SEQ ID NO: 4), GenBank accession number GI: 228008402 (NM_001136129.2, SEQ ID NO: 5), GenBank accession number GI: 228008401 (NM_001136130.2, SEQ ID NO: 6), and GenBank accession number GI: 324021747 (NM_001136131.2, SEQ ID NO: 5). The following were found: NO: 7), GenBank login number GI: 324021737 (NM_001204301.1, SEQ ID NO: 8), GenBank login number GI: 324021735 (NM_001204302.1, SEQ ID NO: 9); and GenBank login number GI: 324021739 (NM_001204303.1, SEQ ID NO: 10), and GenBank login number GI: 1370481385 (XM_024452075.1, SEQ ID NO: 11).

[0265] The nucleotide and amino acid sequences of the cynomolgus monkey APP can be found, for example, in GenBank accession number GI: 982237868 (XM_005548883.2; SEQ ID NO: 12). The nucleotide and amino acid sequences of the mouse APP can be found, for example, in GenBank accession number GI: 311893400 (NM_001198823; SEQ ID NO: 13). The nucleotide and amino acid sequences of the rat APP can be found, for example, in GenBank accession number GI: 402692725 (NM_019288.2; SEQ ID NO: 14). Other instances of APP sequences can be easily obtained using publicly available databases such as GenBank, UniProt, and OMIM.

[0266] As used herein, the term "APP" also refers to naturally occurring DNA sequence variations in the APP gene, such as single nucleotide polymorphisms (SNPs) within the APP gene, that express specific polypeptides in cells. Many SNPs within the APP gene have been identified and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp). Non-restricted examples of SNPs within the APP gene can be found in NCBI dbSNP accessions rs193922916, rs145564988, rs193922916, rs214484, rs281865161, rs364048, rs466433, rs466448, rs532876832, rs63749810, rs63749964, rs63750064, rs63750066, rs63750151, and rs63750264. Found in rs63750363, rs63750399, rs63750445, rs63750579, rs63750643, rs63750671, rs63750734, rs63750847, rs63750851, rs63750868, rs63750921, rs63750973, rs63751039, rs63751122, and rs63751263. Certain instance-like rare APP variants previously described as playing a role in the development of EOFAD were identified in Hooli et al. (Neurology 78:1250-57). In addition, it has recently been found that various “non-canonical” APP variants containing exon in-linkage in sequenced cDNA are associated with somatic gene recombination in the brains of AD patients (PCT / US2018 / 030520, the entire contents of which are incorporated herein by reference).Examples of such "non-classic" app variants include cAPP-R3 / 16 (SEQ ID NO: 1865), cAPP-R3 / 16-2 (SEQ ID NO: 1866), cAPP-R2 / 18 (SEQ ID NO: 1867), cAPP-R6 / 18 (SEQ ID NO: 1868), cAPP-R3 / 14 (SEQ ID NO: 1869), cAPP-R3 / 17 (SEQ ID NO: 1870), cAPP-R1 / 11 (SEQ ID NO: 1871), cAPP-R1 / 13 (SEQ ID NO: 1872), cAPP-R1 / 11-2 (SEQ ID NO: 1873), cAPP-R1 / 14 (SEQ ID NO: 1874), cAPP-R2 / 17 (SEQ ID NO: 1875), cAPP-R2 / 16 (SEQ ID NO: 1876), and cAPP-R6 / 17 (SEQ ID NO: 1869). (SEQ ID NO: 1877), cAPP-R2 / 14 (SEQ ID NO: 1878), cAPP-R14 / 17-d8 (SEQ ID NO: 1879), and cAPP-D2 / 18-3 (SEQ ID NO: 1880). It is clearly anticipated that the RNAi agents of this application can be used to target “non-classical” APP variants and / or can be designed and used with RNAi agents optionally specific to such “non-classical” APP variants, optionally in combination with other RNAi agents of this application, including those targeting natural forms of APP. Such “non-classical” APP variants are described as being significantly absent in the tested HIV patient population, with a significantly reduced prevalence of AD in the HIV patient population compared to expected levels, suggesting that therapies commonly used to treat HIV patients with reverse transcriptase inhibitors and / or other antiretroviral drugs may also have a therapeutic / preventive effect on AD. Therefore, it is clearly anticipated that the RNAi agents of this application can be used optionally in combination with reverse transcriptase inhibitors and / or other antiretroviral therapies for therapeutic and / or preventive purposes.

[0267] As used herein, a “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during APP gene transcription, including mRNA that is a primary transcription product or an RNA processing product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for directed RNAi cleavage at or near a portion of the nucleotide sequence of the mRNA molecule formed during APP gene transcription.

[0268] The target sequence can be approximately 9 to 36 nucleotides in length, for example, approximately 15 to 30 nucleotides in length. For example, the target sequence length can be approximately 15 to 30 nucleotides, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides. Intermediate ranges and lengths within the ranges and lengths described above are also considered part of this application.

[0269] As used herein, the term "chain containing a sequence" refers to an oligonucleotide that contains a nucleotide chain described by a sequence referenced using standard nucleotide nomenclature.

[0270] “G,” “C,” “A,” “T,” and “U” typically represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, it should be understood that the term “ribonucleotide” or “nucleotide” can also refer to modified nucleotides, as further detailed below, or alternative substitutions (see, for example, Table 1). Those skilled in the art will readily understand that guanine, cytosine, adenine, thymidine, and uracil can be substituted with other parts without significantly altering the base-pairing properties of the oligonucleotide containing the nucleotide with such substitutions. For example, but not limited to, nucleotides containing inosine as a base can base-pair with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine can be substituted in the nucleotide sequence of the dsRNA characterized in this application by nucleotides containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively, to form a GU-wake base pair with the target mRNA. Sequences containing such substitutions are applicable to the compositions and methods described in this application.

[0271] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” are used interchangeably herein and refer to an RNA containing the terms defined herein and an agent that mediates the targeted cleavage of RNA transcripts via the RNA-inducible silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs the specific degradation of mRNA sequences. RNAi regulates, for example, the inhibition of APP expression in cells, such as within an individual, such as a mammalian individual.

[0272] In one embodiment, the RNAi agent of this application comprises single-stranded RNAi that interacts with a target RNA sequence, such as an APP target mRNA sequence, to guide the cleavage of the target RNA. Not wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is cleaved by a type III nuclease called Dicer into short double-stranded interfering RNA (siRNA) containing a sense strand and an antisense strand (Sharp et al., (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes these dsRNAs into short interfering RNAs of 19 to 23 base pairs, characterized by two 3' overhangs (Bernstein et al., (2001) Nature 409:363). These siRNAs are then integrated into an RNA-inducible mutagenesis complex (RISC), in which one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases in the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Therefore, in one respect, this application relates to single-stranded RNA (ssRNA) (the antisense strand of the siRNA duplex) produced within the cell and which promotes the formation of the RISC complex to achieve silencing of the target gene, namely the APP gene. Therefore, the term "siRNA" is also used herein to refer to RNAi as described above.

[0273] In another embodiment, the RNAi agent can be a single-stranded RNA introduced into a cell or organism to inhibit the target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs typically have 15 to 30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of which are incorporated herein by reference. Any antisense nucleotide sequence described herein may be used as the single-stranded siRNA described herein or chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0274] In another embodiment, the “RNAi agent” used in the compositions and methods of this application is double-stranded RNA and is referred to herein as a “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, described as having “sense” and “antisense” orientations relative to the target RNA, i.e., an APP gene. In some embodiments of this application, the double-stranded RNA (dsRNA) triggers the degradation of the target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0275] Generally, many nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, as used herein, an "RNAi agent" may include chemically modified ribonucleotides; an RNAi agent may include numerous modifications of multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, modified internucleotide bonds, and / or modified nucleobases. Thus, the term modified nucleotide includes substitution, addition, or removal of, for example, functional groups or atoms, of internucleotide bonds, sugar moieties, or nucleobases. Modifications applicable to agents of this application include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications used in siRNA-type molecules are included in the term "RNAi agent."

[0276] In some embodiments of this application, if a deoxynucleotide is present in the RNAi agent, which is considered to be a naturally occurring nucleotide form, it can be considered to constitute a modified nucleotide.

[0277] The double-stranded region can be of any length that allows for specific degradation of the target RNA via the RISC pathway, and the length can be approximately 9 to 36 base pairs, for example, approximately 15 to 30 base pairs, such as approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs, for example, approximately 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 To 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 1 The lengths are 9 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. Intermediate ranges and lengths within the ranges and lengths described above are also considered part of this application.

[0278] The two strands forming a double-stranded structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules. If the two strands are part of a larger molecule, and thus connected by a continuous nucleotide chain between the 3' end of one strand and the 5' end of the corresponding other strand to form a double-stranded structure, the connecting RNA strands are called a "hairpin loop." A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides. In some embodiments, a hairpin loop may contain 10 or fewer nucleotides. In some embodiments, a hairpin loop may contain 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 8 nucleotides.

[0279] When the two substantially complementary strands of dsRNA consist of separate RNA molecules, these molecules are not required, but can be covalently linked. In some embodiments where the two strands are covalently linked by a non-discontinuous nucleotide chain between the 3' end of one strand and the 5' end of the corresponding other strand, a manner different from forming a duplex structure, the linking structure is referred to as a "linker" (although note that certain other structures defined elsewhere herein may also be referred to as "linkers"). The 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 strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi may also contain one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, such as 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent contains a 5' overhang of at least one nucleotide. In some embodiments, at least one strand contains a 5' overhang of at least two nucleotides, such as 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, one strand of the RNAi agent contains an overhang of at least one nucleotide at both the 3' and 5' ends.

[0280] In one embodiment, the RNAi agent of this application is dsRNA, each strand of which contains 19 to 23 nucleotides, which interacts with a target RNA sequence, such as an APP target mRNA sequence, to guide the cleavage of the target RNA. Not wishing to be bound by theory, long double-stranded RNA introduced into the cell is cleaved into siRNA by a type III endonuclease called Dicer (Sharp et al., (2001) GenesDev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short interfering RNA of 19 to 23 base pairs, characterized by two 3' overhangs (Bernstein et al., (2001) Nature 409:363). The siRNA is then integrated into an RNA-inducible mutagenesis complex (RISC), in which one or more helicases unwind the siRNA double strand, allowing the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107:309). After binding to a suitable target mRNA, one or more endonucleases in RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0281] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of an RNAi agent such as dsRNA. For example, a nucleotide overhang exists when the 3' end of one strand of dsRNA extends beyond the 5' end of the other strand, or vice versa. dsRNA may contain at least one nucleotide overhang; or, the overhang may contain at least two, three, four, five, or more nucleotides. The nucleotide overhang may contain or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the nucleotide overhang may be present at the 5' end, 3' end, or both ends of the antisense or sense strand of dsRNA.

[0282] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' and / or 5' ends, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' and / or 5' ends, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In yet another embodiment, one or more nucleotides in the overhangs are replaced with nucleoside phosphate thioesters.

[0283] In some embodiments, the antisense strand of the dsRNA has 1 to 10 nucleotides protruding at the 3' and / or 5' ends, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides protruding at the 3' and / or 5' ends. In one embodiment, the sense strand of the dsRNA has 1 to 10 nucleotides protruding at the 3' and / or 5' ends, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides protruding at the 3' and / or 5' ends. In another embodiment, one or more nucleotides in the protruding ends are replaced with nucleoside phosphate thioesters.

[0284] In some embodiments, the protrusion on the sense strand or antisense strand, or both, may include an extension length longer than 10 nucleotides, such as 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In some embodiments, the extended protrusion is located on the sense strand of the bistrand. In some embodiments, the extended protrusion is located at the 3' end of the sense strand of the bistrand. In some embodiments, the extended protrusion is located at the 5' end of the sense strand of the bistrand. In some embodiments, the extended protrusion is located on the antisense strand of the bistrand. In some embodiments, the elongated protrusion is located at the 3' end of the antisense strand of the bistrand. In some embodiments, the elongated protrusion is located at the 5' end of the antisense strand of the bistrand. In some embodiments, one or more nucleotides in the protrusion are replaced by a nucleoside thiophosphate. In some embodiments, the protrusion includes a self-complementary portion, enabling the protrusion to form a hairpin structure stable under physiological conditions.

[0285] As used herein, the term "blunt" or "brief-end" for dsRNA refers to a dsRNA that has no unpaired nucleotide or nucleotide analogue at a given end, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. If both ends of a dsRNA are blunt, then the dsRNA is said to be blunt-ended. It is important to clarify that a "blunt" dsRNA is a dsRNA with blunt ends, meaning that neither end of the molecule has a nucleotide overhang. In most cases, such molecules are double-stranded along their entire length.

[0286] The term "antisense strand" or "guide strand" refers to the strand of an RNAi agent, such as dsRNA, which includes regions substantially complementary to the target sequence, such as APPmRNA.

[0287] As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein, such as a target sequence, such as an APP nucleotide sequence. When the complementary region is not perfectly complementary to the target sequence, the mismatch may be located in the internal or terminal regions of the molecule. Typically, the most tolerable mismatches are located in terminal regions, such as within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3' ends of an RNAi agent.

[0288] As used herein, the terms “sense chain” or “follower chain” refer to the chain of an RNAi agent that includes a region substantially complementary to the antisense chain region of the terms defined herein.

[0289] As used herein, the term "cleavage region" refers to the region immediately adjacent to the cleavage site. A cleavage site is the end of the target where cleavage occurs. In some embodiments, the cleavage region contains three bases immediately adjacent to the cleavage site at either end. In some embodiments, the cleavage region contains two bases immediately adjacent to the cleavage site at either end. In some embodiments, the cleavage site specifically occurs at the end bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region contains nucleotides 11, 12, and 13.

[0290] As used herein, unless otherwise stated, the term "complementarity," when used to describe a first nucleotide sequence associated with a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize and form a nucleotide sequence. As those skilled in the art will understand, a double-stranded structure containing an oligonucleotide or polynucleotide may exist under certain conditions. Such conditions may, for example, be stringent conditions, which may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12 to 16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989) Cold Spring Harbor Laboratory Press). Other conditions may be applied, such as physiologically relevant conditions that may be encountered in vivo. Those skilled in the art will be able to determine the set of conditions most suitable for testing the complementarity of the two sequences based on the final application of the hybridized nucleotides.

[0291] Complementary sequences within RNAi agents, such as those within dsRNAs as described herein, comprise base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence along the full length of one or both nucleotide sequences. Such sequences may be referred to herein as “perfectly complementary” to each other. However, when the first sequence is referred to herein as “fundamentally complementary” to the second sequence, the two sequences may be perfectly complementary, or they may form one or more, but for duplexes of up to 30 base pairs, hybridization typically does not exceed 5, 4, 3, or 2 mismatched base pairs, while retaining the ability to hybridize under conditions most relevant to its final application, such as repressing gene expression via a RISC pathway. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches in determining complementarity. For example, a dsRNA containing one 21-nucleotide oligonucleotide and another 23-nucleotide oligonucleotide, wherein the longer oligonucleotide contains a 21-nucleotide sequence perfectly complementary to the shorter oligonucleotide, for the purposes described herein.

[0292] As used herein, “complementary” sequences may also include base pairs formed entirely of non-Watson-Crick base pairs and / or non-natural and modified nucleotides, provided that the above-mentioned requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairs.

[0293] The terms “complementary,” “fully complementary,” and “fundamentally complementary” used in this article can be used for base matching between the sense and antisense strands of dsRNA, or between the antisense strand and the target sequence of an RNAi agent, as will be understood from the context of their use.

[0294] As used herein, a polynucleotide that is substantially complementary to at least a portion of messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding APP). For example, a polynucleotide is complementary to at least a portion of the APP mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding APP.

[0295] Therefore, in some embodiments, the antisense polynucleotide disclosed herein is completely complementary to the target APP sequence. In other embodiments, the antisense polynucleotide disclosed herein is substantially complementary to the target APP sequence and comprises a continuous nucleotide sequence that is at least about 80% complementary in its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 1 to 14, or complementary to the fragment of SEQ ID NO: 1 to 14, for example about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0296] In other embodiments, the antisense polynucleotide disclosed herein is substantially complementary to the target APP sequence and comprises a continuous nucleotide sequence that is at least about 80% complementary in its entire length to any of the positive strand nucleotide sequences in any of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B or 26, or to a fragment of any of the positive strand nucleotide sequences in any of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B or 26, for example about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0297] In one embodiment, the RNAi agent of this application comprises a positive strand substantially complementary to an antisense polynucleotide, which in turn is identical to the target APP sequence, and wherein the positive strand polynucleotide comprises a continuous nucleotide sequence, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98%, or about 99% complementary over its entire length to an equivalent region of the nucleotide sequence of any of the fragments in SEQ ID NOs: 15 to 28.

[0298] In one embodiment, at least partial inhibition of APP gene expression is assessed by a reduction in the amount of APP mRNA, which can be isolated or detected from a first cell or cell group in which the APP gene is transcribed, and which has been or has been treated to inhibit APP gene expression, compared to a second cell or cell group (control cells) that is substantially the same as the first cell or cell group but has been or has not been treated in this way. The degree of inhibition can be expressed as:

[0299]

[0300] As used herein, the phrase "contacting cells with an RNAi agent," such as dsRNA, includes contacting cells by any possible means. Contacting cells with an RNAi agent includes contacting cells with the RNAi agent in vitro or in vivo. Contact can be direct or indirect. Thus, for example, an individual performing the method may physically contact the RNAi agent with cells, or the RNAi agent may be placed in a situation that permits or results in its subsequent contact with cells.

[0301] In vitro cell contact can be performed, for example, by incubating the cells with an RNAi agent. In vivo cell contact can be performed, for example, by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another region, such as the central nervous system (CNS), optionally via intrathecal, intravitreal, or other injection, or by injection into the bloodstream or subcutaneous space, thereby allowing the agent to subsequently reach the tissue where the cells to be contacted are located. For example, the RNAi agent may contain and / or bind to a ligand, such as a lipophilic portion or a portion described below and further detailed, such as in U.S. Applications 62 / 668,072, 62 / 738,747, and / or 62 / 773,082, directing and / or otherwise stabilizing the RNAi agent at a terminal of interest, such as the CNS. Combinations of in vitro and in vivo contact methods are also possible. For example, cells can also be contacted with an RNAi agent in vitro and subsequently transplanted into an individual.

[0302] In one embodiment, contacting cells with an RNAi agent includes “introducing” or “delivering” the RNAi agent into cells by promoting or influencing uptake or absorption into the cells. Absorption or uptake of the RNAi agent can occur through a separate diffusion or active cell process, or via an adjuvant or device. Introducing the RNAi agent into cells can be done in vitro and / or in vivo. For example, for in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction of cells includes methods known in the art, such as electroporation and lipid transfection. Further methods are described below and / or are known in the art.

[0303] The terms "lipophile" or "lipophilic moiety" broadly refer to any compound or chemical fraction that has an affinity for lipids. One method for characterizing the lipophilicity of a lipophilic moiety is through the octanol-water partition coefficient logK. ow K ow The 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 under equilibrium conditions. It is a laboratory-measured property of a substance. However, it can also be predicted using coefficients attributed to the structural composition of the chemical substance, calculated using first-principles or empirical methods (e.g., see Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), the entire contents of which are incorporated herein by reference). It provides a thermodynamic measurement of a substance's tendency to prefer a non-aqueous or oily environment over water (i.e., its hydrophilic / lipophilic balance). In principle, when logK... ow When the value exceeds 0, the chemical substance is lipophilic. Typically, the lipophilic portion has a logK value exceeding 1, 1.5, 2, 3, 4, 5, or 10. ow For example, the logK of 6-aminohexanol. ow The expected value is approximately 0.7. Using the same method, the logK of cholesterol N-(hexyl-6-ol)carbamate was predicted. ow It is 10.7.

[0304] The lipophilicity of a molecule can be altered depending on the functional groups it carries. For example, adding a hydroxyl or amino group to the end of the lipophilic moiety can increase or decrease the partition coefficient of the lipophilic moiety (e.g., logK). ow )value.

[0305] Alternatively, the hydrophobicity of a double-stranded RNAi agent bound to one or more lipophilic moieties can be measured by its protein-binding properties. For example, in some embodiments, the unbound portion in a plasma protein binding assay of a double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of the double-stranded RNAi agent, and then positively correlated with the silencing activity of the double-stranded RNAi agent.

[0306] In one embodiment, the determined plasma protein binding assay is an electrophoretic mobility shift assay (EMSA) of human serum albumin proteins. Exemplary protocols for such binding assays are detailed in, for example, U.S. Patent Applications 62 / 668,072, 62 / 738,747, and / or 62 / 773,082. The hydrophobicity of the double-stranded RNAi agent is measured by the fraction of unbound siRNA in the binding assay, exceeding 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5, to enhance in vivo delivery of siRNA.

[0307] Therefore, binding the lipophilic portion to the internal location of the double-stranded RNAi agent provides optimal hydrophobicity for enhanced in vivo siRNA delivery.

[0308] The term "lipid nanoparticle" or "LNP" refers to a vesicle containing a lipid layer that encapsulates a drug-active molecule, such as a nucleic acid molecule, like an rNAi agent or RNAi agent transcribed from it, in a plasmid. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0309] As used herein, “individual” is an animal, such as a mammal, including primates (e.g., humans, non-human primates such as monkeys and chimpanzees), non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, mice, rats, horses, and whales), or birds (e.g., ducks or geese). In one embodiment, an individual is a human, such as a human being treated or evaluated for a disease, condition, or illness that would benefit from reduced APP expression; a human potentially benefiting from reduced APP expression; a human suffering from a disease, condition, or illness that could benefit from reduced APP expression; and / or a human being treated for a disease, condition, or illness that would benefit from reduced APP expression as described herein.

[0310] As used herein, the term “treatment” or “therapy” refers to a beneficial or desired outcome, including but not limited to the reduction or improvement of one or more symptoms associated with APP gene expression and / or APP protein production, such as APP-related diseases or conditions such as AD, CAA (e.g., hereditary CAA), and EOFAD. “Treatment” can also refer to an extension of survival compared to expected survival without treatment.

[0311] In the context of an individual's APP levels or disease markers or symptoms, the term "lower" means that the level is statistically significantly reduced. A reduction can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more. In some embodiments, the reduction is at least 20%. "Lower" in the context of an individual's APP levels preferably refers to a decrease to a level acceptable within the normal range for individuals without the condition.

[0312] As used herein, when “prevention” or “avoidance” is used to refer to a disease, condition, or illness, benefiting from reduced expression of the APP gene and / or production of the APP protein means reducing an individual’s likelihood of developing symptoms associated with such disease, condition, or illness, such as symptoms associated with APP gene expression, such as the presence of various forms of Aβ (e.g., Aβ38, Aβ40, and / or Aβ42), amyloid mosaicism and / or cerebral amyloid angiopathy (CAA), or Alzheimer’s disease (AD), including, for example, early-onset familial Alzheimer’s disease (EOFAD). Failure to develop a disease, condition, or illness, or a reduction in the development of symptoms associated with such disease, condition, or illness (e.g., a reduction of at least about 10% on a clinically accepted scale for the disease or illness), or a delay in the expression of symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0313] As used herein, the term "APP-related disease" refers to a disease or condition caused by or associated with the expression of the APP gene or the production of the APP protein. The term "APP-related disease" includes diseases, conditions, or ailments that would benefit from reduced expression, replication, or protein activity of the APP gene. Non-limiting examples of APP-related diseases include, for example, cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD), including, for example, early-onset familial Alzheimer's disease (EOFAD).

[0314] As used herein, “therapeutic effective dose” is intended to include an amount of RNAi agent that, when administered to an individual with APP-related conditions, is sufficient to treat the disease (e.g., by reducing, improving, or maintaining the existing disease or one or more symptoms of the disease). “Therapeutic effective dose” can vary depending on the RNAi agent, the method of administration, the disease and its severity and history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the individual being treated.

[0315] As used herein, “preventive effective dose” is intended to include the amount of RNAi agent sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to an individual with APP-related conditions. Improvement of the disease includes slowing the progression of the disease or reducing the severity of later-developed disease. “Preventive effective dose” may vary depending on the RNAi agent, the method of administration, the degree of disease risk, and medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, and any other individual characteristics of the patient to be treated.

[0316] "Therapeutic effective amount" or "prophylactic effective amount" also includes the amount of RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio suitable for any treatment. The RNAi agent used in the methods of this application may be administered in an amount sufficient to produce a reasonable benefit / risk ratio suitable for such treatment.

[0317] The phrase "pharmaceutically acceptable" as used in this article refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with tissues in human and animal individuals, within the bounds of reasonable medical judgment. This includes excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0318] As used herein, the phrase “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or excipient, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate, or zinc, or stearic acid), or solvent encapsulating material, which participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with other components of the formulation and harmless to the individual being treated. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, etc.; (4) astragalus gum powder; (5) malt; (6) gelatin; (7) lubricants, such as magnesium cellulose. (8) Excipients, such as cocoa butter, suppository wax, etc.; (9) Oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.; (10) Diols, such as propylene glycol; (11) Polyols, such as glycerol, sorbitol, mannitol, polyethylene glycol, etc.; (12) Esters, such as ethyl oleate, ethyl laurate, etc.; (13) Agar; (14) Buffers, such as magnesium hydroxide, aluminum hydroxide, etc.; (15) Alginate; (16) Atherless raw water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) pH buffer solution; (21) Polyester, polycarbonate and / or polyanhydride; (22) Fillers, such as peptides and amino acids. (23) Serum components, such as serum albumin, high-density lipoprotein (HDL) and low-density lipoprotein (LDL); (22) Other non-toxic compatible substances used in pharmaceutical preparations.

[0319] As used herein, the term "sample" includes a collection of similar fluids, cells, or tissues isolated from an individual, as well as fluids, cells, or tissues present within an individual. Examples of biological fluids include blood, serum and serous fluids, plasma, cerebrospinal fluid, eye discharge, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or localized regions. For example, a sample may originate from a specific organ, a portion of an organ, or fluids or cells within those organs. In some embodiments, the sample may be derived from the brain (e.g., the whole brain or portions of the brain or certain types of cells in the brain, such as neurons and glial cells (astrocytes, oligodendrocytes, microglia). In some embodiments, "sample from an individual" refers to blood or plasma drawn from an individual. In further embodiments, "sample from an individual" refers to brain tissue (or a subcomponent thereof) or retinal tissue (or a subcomponent thereof) derived from an individual.

[0320] II. Revealed RNAi Agents

[0321] This document describes an RNAi agent that inhibits the expression of the APP gene. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits the expression of the APP gene in cells, such as cells in an individual, such as mammals, such as human diseases associated with APP, such as cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD), including, for example, early-onset familial Alzheimer's disease (EOFAD). The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during the expression of the APP gene. The complementary region is about 30 nucleotides in length or less (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides in length or less). When in contact with cells expressing the APP gene, the RNAi agent inhibits the expression of the APP gene (e.g., human, primate, non-primate, or avian APP gene) by at least about 10%, for example by, for example, PCR- or branched-strand DNA (bDNA)-based methods, or protein-based methods, for example by immunofluorescence analysis, using techniques such as Western blotting or flow cytometry.

[0322] dsRNA comprises two complementary RNA strands that hybridize under dsRNA-dependent conditions to form a double-stranded structure. One strand of the dsRNA (antisense strand) includes a complementary region that is substantially complementary to, and usually perfectly complementary to, the target sequence. The target sequence can be derived from the mRNA sequence formed during the expression process of the APP gene. The other strand (sense strand) includes a region complementary to the antisense strand, such that when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure. As described elsewhere herein and known in the art, the complementary sequence of the dsRNA may also be included as a self-complementary region of a single nucleic acid molecule, rather than on a separate oligonucleotide.

[0323] The length of the double-stranded structure is typically between 15 and 30 base pairs, for example, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to The length between 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. In some preferred embodiments, the length of the double-stranded structure is between 18 and 25 base pairs, for example, lengths of 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 25, 22 to 24, 22 to 23, 23 to 25, 23 to 24, or 24 to 25 base pairs. Intermediate ranges and lengths within the ranges and lengths described above are also considered part of this application.

[0324] Similarly, the length of the region complementary to the target sequence is between 15 and 30 nucleotides, for example, in the ranges of 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, and 19 to 30. The lengths of nucleotides are 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22. Intermediate ranges and lengths within the ranges and lengths described above are also considered part of this application.

[0325] In some embodiments, the length of the dsRNA is between about 15 and about 23 nucleotides, or between about 25 and about 30 nucleotides. Generally, the length of the dsRNA is sufficient to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21 to 23 nucleotides can serve as substrates for Dicer. As will also be appreciated by those skilled in the art, the RNA region targeted for cleavage is typically a portion of a larger RNA molecule, usually an mRNA molecule. In relevant cases, the “portion” of the mRNA target is a contiguous sequence of the mRNA target that is long enough to make it a substrate for RNAi directed cleavage (i.e., cleavage via the RISC pathway).

[0326] Those skilled in the art will also recognize that double-stranded regions are the major functional parts of dsRNA, such as double-stranded regions 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, 13 to 34, 13 to 34, 14 to 35, 15 to 35, 9 to 34, 10 to 34, 11 to 34, 12 to 34, 13 to 34, 13 to 34, 15 to 35, 13 ...5 to 35, 13 to 34, 15 to 35, 4, 14 to 34, 15 to 34, 9 to 33, 10 to 33, 11 to 33, 12 to 33, 13 to 33, 14 to 33, 15 to 33, 9 to 32, 10 to 32, 11 to 32, 12 to 32, 13 to 32, 14 to 32, 15 to 32, 9 to 31, 10 to 31, 11 to 31, 12 to 31, 13 to 32, 14 to 31, 15 to 31, 15 to 30, 15 to 29, 15 to 28, 15 To 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. Therefore, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs is considered to be, for example, 15 to 30 base pairs, which are targeted at the desired RNA for cleavage. The base pairs are dsRNA. Therefore, those skilled in the art will recognize that, in one embodiment, miRNA is dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In yet another embodiment, the RNAi agent for targeting APP expression is not generated in target cells by cleaving larger dsRNAs.

[0327] The dsRNA described herein may also include one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can exhibit unexpectedly superior repressive properties relative to their blunt-ended counterparts. The nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present at the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0328] dsRNA can be synthesized using standard methods known in the art, as discussed further below, for example, by using an automated DNA synthesizer, which may be commercially available from companies such as Biosearch Applied Biosystems.

[0329] The RNAi agent of this application can be prepared using a two-step process. First, single strands of a double-stranded RNA molecule are prepared separately. Then, the constituent strands are annealed. The individual strands of the siRNA compound can be prepared using liquid-phase or solid-phase organic synthesis, or both. The advantage of organic synthesis is that it allows for the easy preparation of oligonucleotide chains containing non-natural or modified nucleotides. The single-stranded oligonucleotides of this application can be prepared using liquid-phase or solid-phase organic synthesis, or both.

[0330] In one respect, the dsRNA of this application comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence may be selected from any of the sequence groups provided in Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B and 26, and the corresponding nucleotide sequence of the antisense strand may be selected from any of the sequence groups in Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B and 26. In this respect, 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 generated in the expression of the APP gene. Therefore, in this respect, dsRNA will comprise two oligonucleotides, one of which is described as the sense strand (passenger strand) in any of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B and 26, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in any of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B and 26. Therefore, for example, the following pairwise selections of the justice and antisense chain sequences in Table 3 are explicitly covered to form the double strand of this application: SEQ ID NOs: 855 and 856; SEQ ID NOs: 857 and 858; SEQ ID NOs: 859 and 860; SEQ ID NOs: 861 and 862; SEQ ID NOs: 863 and 864; SEQ ID NOs: 865 and 866; SEQ ID NOs: 867 and 868; SEQ ID NOs: 869 and 870; SEQ ID NOs: 871 and 872; SEQ ID NOs: 873 and 874; SEQ ID NOs: 875 and 876; SEQ ID NOs: 877 and 878; SEQ ID NOs: 879 and 880; SEQ ID NOs: 881 and 882; SEQ ID NOs: 883 and 884; SEQ ID NOs: 885 and 886; SEQ ID SEQ ID NOs: 887 and 888; SEQ ID NOs: 889 and 890; SEQ ID NOs: 891 and 892; SEQ ID NOs: 893 and 894; SEQ ID NOs: 895 and 896; SEQ ID NOs: 897 and 898; SEQ ID NOs: 899 and 900; SEQ ID NOs: 901 and 902; SEQ ID NOs: 903 and 904; SEQ ID NOs: 905 and 906; SEQ ID NOs: 907 and 908; SEQ ID NOs: 909 and 910; SEQ ID NOs: 911 and 912; SEQ ID NOs: 913 and 914;SEQ ID NOs: 915 and 916; SEQ ID NOs: 917 and 918; SEQ ID NOs: 919 and 920; SEQ ID NOs: 921 and 922; SEQ ID NOs: 923 and 924; SEQ ID NOs: 925 and 926; SEQ ID NOs: 927 and 928; SEQ ID NOs: 929 and 930; SEQ ID NOs: 931 and 932; SEQ ID NOs: 933 and 934; SEQ ID NOs: 935 and 936; SEQ ID NOs: 937 and 938; SEQ ID NOs: 939 and 940; SEQ ID NOs: 941 and 942; SEQ ID NOs: 943 and 944; SEQ ID NOs: 945 and 946; SEQ ID NOs: 947 and 948; SEQ ID NOs: 949 and 950; SEQ ID SEQ ID NOs: 951 and 952; SEQ ID NOs: 953 and 954; SEQ ID NOs: 955 and 956; SEQ ID NOs: 957 and 958; SEQ ID NOs: 959 and 960; SEQ ID NOs: 961 and 962; SEQ ID NOs: 963 and 964; SEQ ID NOs: 965 and 966; SEQ ID NOs: 967 and 968; SEQ ID NOs: 969 and 970; SEQ ID NOs: 971 and 972; SEQ ID NOs: 973 and 974; SEQ ID NOs: 975 and 976; SEQ ID NOs: 977 and 978; SEQ ID NOs: 979 and 980; SEQ ID NOs: 981 and 982; SEQ ID NOs: 983 and 984; SEQ ID NOs: 985 and 986; SEQ ID NOs: 987 and 988; SEQ SEQ ID NOs: 989 and 990; SEQ ID NOs: 991 and 992; SEQ ID NOs: 993 and 994; SEQ ID NOs: 995 and 996; SEQ ID NOs: 997 and 998; SEQ ID NOs: 999 and 1000; SEQ ID NOs: 1001 and 1002; SEQ ID NOs: 1003 and 1004; SEQ ID NOs: 1005 and 1006; SEQ ID NOs: 1007 and 1008; SEQ ID NOs: 1009 and 1010; SEQ ID NOs: 1011 and 1012; SEQ ID NOs: 1013 and 1014; SEQ ID NOs: 1015 and 1016;SEQ ID NOs: 1017 and 1018; SEQ ID NOs: 1019 and 1020; SEQ ID NOs: 1021 and 1022; SEQ ID NOs: 1023 and 1024; SEQ ID NOs: 1025 and 1026; SEQ ID NOs: 1027 and 1028; SEQ ID NOs: 1029 and 1030; SEQ ID NOs: 1031 and 1032; SEQ ID NOs: 1033 and 1034; SEQ ID NOs: 1035 and 1036; SEQ ID NOs: 1037 and 1038; SEQ ID NOs: 1039 and 1040; SEQ ID NOs: 1041 and 1042; SEQ ID NOs: 1043 and 1044; SEQ ID NOs: 1045 and 1046; SEQ ID NOs: 1047 and 1048; SEQ ID SEQ ID NOs: 1049 and 1050; SEQ ID NOs: 1051 and 1052; SEQ ID NOs: 1053 and 1054; SEQ ID NOs: 1055 and 1056; SEQ ID NOs: 1057 and 1058; SEQ ID NOs: 1059 and 1060; SEQ ID NOs: 1061 and 1062; SEQ ID NOs: 1063 and 1064; SEQ ID NOs: 1065 and 1066; SEQ ID NOs: 1067 and 1068; SEQ ID NOs: 1069 and 1070; SEQ ID NOs: 1071 and 1072; SEQ ID NOs: 1073 and 1074; SEQ ID NOs: 1075 and 1076; SEQ ID NOs: 1077 and 1078; SEQ ID NOs: 1079 and 1080; SEQ ID SEQ ID NOs: 1081 and 1082; SEQ ID NOs: 1083 and 1084; SEQ ID NOs: 1085 and 1086; SEQ ID NOs: 1087 and 1088; SEQ ID NOs: 1089 and 1090; SEQ ID NOs: 1091 and 1092; SEQ ID NOs: 1093 and 1094; SEQ ID NOs: 1095 and 1096; SEQ ID NOs: 1097 and 1098; SEQ ID NOs: 1099 and 1100; SEQ ID NOs: 1101 and 1102; SEQ ID NOs: 1103 and 1104; SEQ ID NOs: 1105 and 1106; SEQ ID NOs: 1107 and 1108; SEQ ID NOs: 1109 and 1110;SEQ ID NOs: 1111 and 1112; SEQ ID NOs: 1113 and 1114; SEQ ID NOs: 1115 and 1116; SEQ ID NOs: 1117 and 1118; SEQ ID NOs: 1119 and 1120; SEQ ID NOs: 1121 and 1122; SEQ ID NOs: 1123 and 1124; SEQ ID NOs: 1125 and 1126; SEQ ID NOs: 1127 and 1128; SEQ ID NOs: 1129 and 1130; SEQ ID NOs: 1131 and 1132; SEQ ID NOs: 1133 and 1134; SEQ ID NOs: 1135 and 1136; SEQ ID NOs: 1137 and 1138; SEQ ID NOs: 1139 and 1140; SEQ ID NOs: 1141 and 1142; SEQ ID SEQ ID NOs: 1143 and 1144; SEQ ID NOs: 1145 and 1146; SEQ ID NOs: 1147 and 1148; SEQ ID NOs: 1149 and 1150; SEQ ID NOs: 1151 and 1152; SEQ ID NOs: 1153 and 1154; SEQ ID NOs: 1155 and 1156; SEQ ID NOs: 1157 and 1158; SEQ ID NOs: 1159 and 1160; SEQ ID NOs: 1161 and 1162; SEQ ID NOs: 1163 and 1164; SEQ ID NOs: 1165 and 1166; SEQ ID NOs: 1167 and 1168; SEQ ID NOs: 1169 and 1170; SEQ ID NOs: 1171 and 1172; SEQ ID NOs: 1173 and 1174; SEQ ID SEQ ID NOs: 1175 and 1176; SEQ ID NOs: 1177 and 1178; SEQ ID NOs: 1179 and 1180; SEQ ID NOs: 1181 and 1182; SEQ ID NOs: 1183 and 1184; SEQ ID NOs: 1185 and 1186; SEQ ID NOs: 1187 and 1188; SEQ ID NOs: 1189 and 1190; SEQ ID NOs: 1191 and 1192; SEQ ID NOs: 1193 and 1194; SEQ ID NOs: 1195 and 1196; SEQ ID NOs: 1197 and 1198; SEQ ID NOs: 1199 and 1200; SEQ ID NOs: 1201 and 1202; SEQ ID NOs: 1203 and 1204;SEQ ID NOs: 1205 and 1206; SEQ ID NOs: 1207 and 1208; SEQ ID NOs: 1209 and 1210; SEQ ID NOs: 1211 and 1212; SEQ ID NOs: 1213 and 1214; SEQ ID NOs: 1215 and 1216; SEQ ID NOs: 1217 and 1218; SEQ ID NOs: 1219 and 1220; SEQ ID NOs: 1221 and 1222; SEQ ID NOs: 1223 and 1224; SEQ ID NOs: 1225 and 1226; SEQ ID NOs: 1227 and 1228; SEQ ID NOs: 1229 and 1230; SEQ ID NOs: 1231 and 1232; SEQ ID NOs: 1233 and 1234; SEQ ID NOs: 1235 and 1236; SEQ ID SEQ ID NOs: 1237 and 1238; SEQ ID NOs: 1239 and 1240; SEQ ID NOs: 1241 and 1242; SEQ ID NOs: 1243 and 1244; SEQ ID NOs: 1245 and 1246; SEQ ID NOs: 1247 and 1248; SEQ ID NOs: 1249 and 1250; SEQ ID NOs: 1251 and 1252; SEQ ID NOs: 1253 and 1254; SEQ ID NOs: 1255 and 1256; SEQ ID NOs: 1257 and 1258; SEQ ID NOs: 1259 and 1260; SEQ ID NOs: 1261 and 1262; SEQ ID NOs: 1263 and 1264; SEQ ID NOs: 1265 and 1266; SEQ ID NOs: 1267 and 1268; SEQ ID SEQ ID NOs: 1269 and 1270; SEQ ID NOs: 1271 and 1272; SEQ ID NOs: 1273 and 1274; SEQ ID NOs: 1275 and 1276; SEQ ID NOs: 1277 and 1278; SEQ ID NOs: 1279 and 1280; SEQ ID NOs: 1281 and 1282; SEQ ID NOs: 1283 and 1284; SEQ ID NOs: 1285 and 1286; SEQ ID NOs: 1287 and 1288; SEQ ID NOs: 1289 and 1290; SEQ ID NOs: 1291 and 1292; SEQ ID NOs: 1293 and 1294; SEQ ID NOs: 1295 and 1296; SEQ ID NOs: 1297 and 1298;SEQ ID NOs: 1299 and 1300; SEQ ID NOs: 1301 and 1302; SEQ ID NOs: 1303 and 1304; SEQ ID NOs: 1305 and 1306; SEQ ID NOs: 1307 and 1308; SEQ ID NOs: 1309 and 1310; SEQ ID NOs: 1311 and 1312; SEQ ID NOs: 1313 and 1314; SEQ ID NOs: 1315 and 1316; SEQ ID NOs: 1317 and 1318; SEQ ID NOs: 1319 and 1320; SEQ ID NOs: 1321 and 1322; SEQ ID NOs: 1323 and 1324; SEQ ID NOs: 1325 and 1326; SEQ ID NOs: 1327 and 1328; SEQ ID NOs: 1329 and 1330; SEQ ID SEQ ID NOs: 1331 and 1332; SEQ ID NOs: 1333 and 1334; SEQ ID NOs: 1335 and 1336; SEQ ID NOs: 1337 and 1338; SEQ ID NOs: 1339 and 1340; SEQ ID NOs: 1341 and 1342; SEQ ID NOs: 1343 and 1344; SEQ ID NOs: 1345 and 1346; SEQ ID NOs: 1347 and 1348; SEQ ID NOs: 1349 and 1350; SEQ ID NOs: 1351 and 1352; SEQ ID NOs: 1353 and 1354; SEQ ID NOs: 1355 and 1356; SEQ ID NOs: 1357 and 1358; SEQ ID NOs: 1359 and 1360; SEQ ID NOs: 1361 and 1362; SEQ ID SEQ ID NOs: 1363 and 1364; SEQ ID NOs: 1365 and 1366; SEQ ID NOs: 1367 and 1368; SEQ ID NOs: 1369 and 1370; SEQ ID NOs: 1371 and 1372; SEQ ID NOs: 1373 and 1374; SEQ ID NOs: 1375 and 1376; SEQ ID NOs: 1377 and 1378; SEQ ID NOs: 1379 and 1380; SEQ ID NOs: 1381 and 1382; SEQ ID NOs: 1383 and 1384; SEQ ID NOs: 1385 and 1386; SEQ ID NOs: 1387 and 1388; SEQ ID NOs: 1389 and 1390; SEQ ID NOs: 1391 and 1392;SEQ ID NOs: 1393 and 1394; SEQ ID NOs: 1395 and 1396; SEQ ID NOs: 1397 and 1398; SEQ ID NOs: 1399 and 1400; and SEQ ID NOs: 1401 and 1402. Similarly, paired combinations of justice and antisense chains in Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B, and 26 of this application are also explicitly covered, including, for example, a justice chain selected from Table 2A and an antisense chain selected from Table 2B, and vice versa, etc.

[0331] In one embodiment, the basic complementary sequence of the dsRNA is contained on a single oligonucleotide. In another embodiment, the basic complementary sequence of the dsRNA is contained on a single oligonucleotide.

[0332] It should be understood that although the sequences in Tables 2A, 2B, 5A, 5B, 9, 10, 12, 14, 16A, 16B and 26 are described as modified and / or bound sequences, the present application, such as the dsRNA of the present application, may contain any of the sequences listed in any of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B and 26 that are unmodified, unbound and / or modified and / or bound differently from those described herein.

[0333] Those skilled in the art are well aware that dsRNAs with a double-stranded structure of about 20 to 23 base pairs (e.g., 21 base pairs) are considered particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have 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 above embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein may comprise at least one chain of at least 21 nucleotides in length. It is reasonable to expect that shorter double-stranded structures, with only a few nucleotides subtracted from one or both ends, may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from one of the sequences provided herein, whose ability to suppress APP gene expression varies, with suppression of more than about 5, 10, 15, 20, 25 or 30% from dsRNAs containing the full sequence being considered within the scope of this application.

[0334] Furthermore, the RNA identification described herein identifies RISC-mediated cleavage-sensitive ends in the APP transcript. Therefore, this application further describes RNAi agents targeting said ends. As used herein, if an RNAi agent promotes cleavage of the transcript at any location within said specific end, then the RNAi agent is said to target a specific end of the RNA transcript. Such RNAi agents typically comprise at least about 15 consecutive nucleotides from one of the sequences provided herein, which bind to an additional nucleotide sequence derived from a region adjacent to a selected sequence in the APP gene.

[0335] RNAi agents as described herein may contain one or more mismatches with the target sequence. In one embodiment, an RNAi agent as described herein contains no more than three mismatches. In some embodiments, if the antisense strand of the RNAi agent contains a mismatch with the target sequence, the mismatch may optionally be limited to the last five nucleotides from 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 region of the APP gene typically does not contain any mismatches within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch with the target sequence effectively inhibits the expression of the APP gene. It is important to consider the efficacy of RNAi agents with mismatches in inhibiting the expression of the APP gene, especially if the specific complementary region in the APP gene is known to have polymorphic sequence variations within the population.

[0336] III. The modified RNAi agent of this application

[0337] In one embodiment, the RNA of the RNAi agent of this application, such as dsRNA, is unmodified and does not contain, for example, chemical modifications known in the art and described herein, and / or bindings. In another embodiment, the RNA of the RNAi agent (e.g., dsRNA) of this application is chemically modified to enhance stability or other beneficial properties. In some embodiments of this application, substantially all nucleotides of the RNAi agent of this application are modified. In other embodiments of this application, all modified nucleotides of the RNAi agent of this application. Wherein “substantially all modified nucleotides” in the RNAi agent of this application is mostly, but not entirely, modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotide. In other embodiments of this application, the RNAi agent of this application may include no more than 5, 4, 3, 2, or 1 modified nucleotide.

[0338] Nucleic acids described in this application can be synthesized and / or modified by methods well established in the art, such as those described in "Current Schemes in Nucleic Acid Chemistry," which are incorporated herein by reference: Beaucage, SL et al., (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Modifications include, for example, terminal modifications, such as 5' terminal modifications (phosphorylation, binding, reverse linkage) or 3' terminal modifications (binding, DNA nucleotide, reverse linkage, etc.); base modifications, such as base substitutions with stabilizing bases, destabilizing bases, or base pairing with an expanded chaperone library, base removal (base-free nucleotides), or base binding; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and / or backbone modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of RNAi agents that can be used in the embodiments described herein include, but are not limited to, RNAs containing a modified backbone or without native nucleoside internucleotide bonds. RNAs with a modified backbone include those without phosphorus atoms in their backbone. For the purposes of this specification, and as sometimes cited in the art, modified RNAs that do not have phosphorus atoms in their internucleotide backbone may also be considered oligonucleotides. In some embodiments, the modified RNAi agent will have phosphorus atoms in its internucleotide backbone.

[0339] Modified RNA backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkyl phosphates, including 3'-alkylene phosphates and chiral phosphates, hypophosphite, aminophosphates, including 3'-aminoamino phosphates and aminoalkylamino phosphates, thiophosphoramides, thioalkyl phosphates, thioylalkyl phosphate triesters, and borate phosphates with normal 3'-5' linkages, their 2'-5' linkage analogs, and those with inverted polarity, wherein adjacent nucleoside unit pairs are linked at 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0340] Representative U.S. patents teaching the preparation of the aforementioned phosphorus-containing bonds include, but are not limited to, 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. U.S. Patent Nos. 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, and 5,571,799; U.S. Patent Nos. U.S. Patent No. 5,587,361; U.S. Patent No. 5,625,050; U.S. Patent No. 6,028,188; U.S. Patent No. 6,124,445; U.S. Patent No. 6,160,109; U.S. Patent No. 6,169,170; U.S. Patent No. 6,172,209; U.S. Patent No. 6,239,265; U.S. Patent No. 6,277,603; U.S. Patent No. 6,326,199; U.S. Patent No. 6,346,614; U.S. Patent No. 6,444,423; U.S. Patent No. 6,53 U.S. Patent Nos. 1,590, 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 RE39464, the entire contents of which are incorporated herein by reference.

[0341] The modified RNA backbone, excluding phosphorus atoms, has a backbone formed by bonds between short-chain alkyl or cycloalkyl nucleosides, mixed heteroatoms and alkyl or cycloalkyl nucleosides, or one or more short-chain heteroatoms or heterocyclic nucleosides. These include backbones with morpholine bonds (partially formed from the sugar moieties of nucleosides); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; methylacetyl and thiomethylacetyl backbones; methylene methylacetyl and thiomethylacetyl backbones; olefins containing the backbone; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other moieties mixed with N, O, S, and CH2 components.

[0342] Representative U.S. patents teaching the preparation of the aforementioned oligonucleotides include, but are not limited to, 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, 5,470,967, and others. U.S. Patent 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 of which are incorporated herein by reference.

[0343] In other embodiments, suitable RNA analogs are considered for use in RNAi agents, wherein the sugar and nucleoside bonds of the nucleotide units are inter-bonded, i.e., the backbone is replaced by novel groups. Base units are retained to hybridize with suitable nucleic acid target compounds. One such oligomeric compound, an RNA analog that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleobases are retained and directly or indirectly bound to the aza-nitrogen atoms of the backbone amide moiety. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Other PNA compounds suitable for RNAi agents of the present application are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0344] Some embodiments in this application include RNAs having a thiophosphate backbone and oligonucleotides having a heteroatom backbone, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as a methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [wherein the native phosphodiester backbone is represented as --O--P--O--CH2--], the amide backbones of U.S. Patent No. 5,489,677 and U.S. Patent No. 5,602,240. In some embodiments, the RNA characterized herein has the morpholine backbone structure of U.S. Patent No. 5,034,506.

[0345] Modified RNA may also contain one or more substituted sugar moieties. RNAi agents characterized herein, such as dsRNA, may include one of the following at the 2'-position: OH, F, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. Typical suitable modifications include O[(CH2)] n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) nONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are 1 to approximately 10. In other embodiments, the dsRNA at the 2' position includes one of the following: C1 to C 10 Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl alkyl groups, O-alkylaryl or O-aryl alkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, information groups, intercalating agents, groups used to improve the pharmacokinetic properties of RNAi agents or groups used to improve the pharmacodynamic properties of RNAi agents, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy groups. Another exemplary modification is 2'-dimethylaminoethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the examples below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2. Further exemplary modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (R and S isomers in these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0346] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-O-hexadecyl, and 2'-fluorine (2'-F). Similar modifications can also be made at other positions on the RNA of the RNAi agent, particularly at the 3' end nucleotide or the 5' end of the sugar in the 2'-5' linked dsRNA. RNAi agents can also have sugar analogs, such as replacing the cyclobutyl moiety of pentofuranose. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 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 5,567. U.S. Patent Nos. 5,811; 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, some of which are common to this application. The entire contents of each of the foregoing are incorporated herein by reference.

[0347] The RNAi agents of this application may also include nucleobase (generally referred to in the art simply as "bases") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other adenine and guanine alkyl derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halogenated uracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, and cytosine. And thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogen, especially 5-bromine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazoguanine and 7-azaadenine, and 3-deazoguanine and 3-deazoadenine. Further nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in *Biochemistry, Biotechnology and Medicine*, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in *The Concise Encyclopedia Of Polymer Science and Engineering*, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613; and those disclosed by Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST. and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds characterized in this application. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitution has been shown to improve the stability of nucleic acid duplexes by 0.6 to 1.2 °C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and is an exemplary base substitution, even more particularly when combined with 2'-O-methoxyethyl sugar modification.

[0348] Representative U.S. patents teaching the preparation of certain of the aforementioned modified nucleosides and other modified nucleosides include, but are not limited to, those listed above: 3,687,808, 4,845,205; U.S. Patent No. 5,130,30; U.S. Patent No. 5,134,066; U.S. Patent No. 5,175,273; U.S. Patent No. 5,367,066; U.S. Patent No. 5,432,272; U.S. Patent No. 5,457,187; U.S. Patent No. 5,459,255; U.S. Patent No. 5,484,908; U.S. Patent No. 5,502,177; U.S. Patent No. 5,525,711; U.S. Patent No. 5,552,540; U.S. Patent No. 5,587,469; and U.S. Patent No. 5,594,120. U.S. Patent Nos. 1, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0349] The modified RNAi agents of this application may also include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides having a modified ribose moiety in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo configuration. It has been shown that adding locked nucleic acids to siRNA can increase the stability of siRNA in serum and reduce 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).

[0350] The modified RNAi agents of this application may also include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanyl ring modified by a two-atom bridging. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety containing a bridge connecting the two carbon atoms of the sugar ring, thus forming a bicyclic system. In some embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Therefore, in some embodiments, the agents of this application may include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides having a modified ribose moiety containing an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively “locks” the ribose in a 3'-endo configuration. It has been demonstrated that adding locked nucleic acids to siRNA can increase the stability of siRNA in serum and reduce 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 used in this application include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the antisense polynucleotide agent of this application comprises one or more bicyclic nucleosides containing a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleotides include, but are not limited to, 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 their analogues; see, for example, U.S. Patent No. 7,399,845); 4′-C (CH3)(CH3)—O-2′ (and its analogues; see, for example, U.S. Patent No. 8,278,283); 4′-CH2—N(OCH3)-2′ (and its analogues; see, for example, U.S. Patent No. 8,278,425); 4′-CH2—O—N(CH3)-2′ (see, for example, U.S. Patent Publication No. 2004 / 0171570); 4′-CH2—N(R)—O-2′, wherein R is H, Cl-C 12Alkyl or protecting groups (see, for example, U.S. Patent No. 7,427,672); 4′-CH2—C(H)(CH3)-2′ (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2—C(═CH2)-2′ (and the like; see, for example, U.S. Patent No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0351] Other representative U.S. patents and U.S. patent disclosures teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent No. 6,268,490; U.S. Patent No. 6,525,191; U.S. Patent No. 6,670,461; U.S. Patent No. 6,770,748; U.S. Patent No. 6,794,499; U.S. Patent No. 6,998,484; U.S. Patent No. 7,053,207; and U.S. Patent No. 7,034,133. U.S. Patent Nos. 7,084,125, 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, 2008 / 0039618, and 2009 / 0012281, the entire contents of each of these patents are incorporated herein by reference.

[0352] Any of the aforementioned bicyclic nucleotides having one or more stereochemical sugar configurations can be prepared, including, for example, α-L-ribofuranosyl and β-D-ribofuranosyl (see WO 99 / 14226).

[0353] The modified RNAi agent described in this application may also include one or more restricted ethyl nucleotides. As used herein, "restricted ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in the S configuration, referred to herein as "S-cEt".

[0354] The RNAi agent of this application may also include one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analog with a linker connecting the C2' and C4' carbons of the ribose or the C3 and -C5' carbons of the ribose. The CRN locks the ribose ring into a stable conformation and increases hybridization affinity with mRNA. The linker is long enough to place oxygen in an optimal position for stability and affinity, thereby reducing ribose ring wrinkling.

[0355] Representative publications teaching the preparation of certain of the aforementioned CRNs include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383 and PCT Publication No. WO 2013 / 036868, the entire contents of which are hereby incorporated herein by reference.

[0356] In some embodiments, the RNAi agent of this application comprises one or more monomers of UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any bonds of the sugar have been removed, forming unlocked “sugar” residues. In one instance, the UNA also includes monomers in which the bond between C1' and C4' has been removed (i.e., the covalent carbon-oxygen-carbon bond between C1' and C4' carbons). In another instance, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between 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, which are incorporated herein by reference).

[0357] Representative U.S. publications teaching UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publication No. 2013 / 0096289; U.S. Patent Publication No. 2013 / 0011922; and U.S. Patent Publication No. 2011 / 0313020, the entire contents of which are incorporated herein by reference.

[0358] Potential stabilizing modifications to the ends of RNA molecules may include N-(acetamidohexanoyl)-4-hydroxyproline (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyproline (Hyp-C6), N-(acetyl-4-hydroxyproline)(Hyp-NHAc), thymidine-2'-O-thymidine (ether), N-(aminohexanoyl)-4-hydroxyproline (Hyp-C6-amino), 2-docosyl-uridine-3"-phosphate, inverted dT (idT), etc. Disclosure of such modifications can be found in PCT Publication No. WO2011 / 005861.

[0359] Other modified RNAi agents of the present invention include 5' phosphate or 5' phosphate analogs, for example, an antisense strand on a 5' terminal phosphate or phosphate that mimics an RNAi agent. Suitable phosphate analogs are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0360] A. Modified RNAi agents containing the contents of this application

[0361] In certain aspects of this application, the double-stranded RNAi agent of this application comprises an agent having been chemically modified, such as the agent disclosed in WO 2013 / 075035, filed November 16, 2012, the entire contents of which are incorporated herein by reference. As shown herein and in PCT Publication WO 2013 / 075035, this is achieved by introducing one or more motifs with 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 be additionally fully modified. The introduction of these motifs disrupts the modification pattern of the sense and / or antisense strands (if present). The RNAi agent may optionally bind to a C16 ligand, for example, on the sense strand. The RNAi agent may optionally be modified with (S)-glycol nucleic acid (GNA), for example, on one or more residues of the antisense strand. The resulting RNAi agent expresses excellent gene silencing activity.

[0362] More specifically, it was surprisingly found that the gene silencing activity of the RNAi agent was significantly enhanced when the sense and antisense strands of the double-stranded RNAi agent were fully modified to have one or more motifs with three identical modifications on three consecutive nucleotides at or near at least one cleavage site.

[0363] Therefore, this application provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the APP gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. The length of each strand of the RNAi agent can be in the range of 12 to 30 nucleotides. For example, the length of each strand can be between 14 and 30 nucleotides, between 17 and 30 nucleotides, between 25 and 30 nucleotides, between 27 and 30 nucleotides, between 17 and 23 nucleotides, between 17 and 21 nucleotides, between 17 and 19 nucleotides, between 19 and 25 nucleotides, between 19 and 23 nucleotides, between 19 and 21 nucleotides, between 21 and 25 nucleotides, or between 21 and 23 nucleotides.

[0364] The sense and antisense strands typically form a double-stranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” The length of the double-stranded region in an RNAi agent can range from 12 to 30 nucleotide pairs. For example, the length of the double-stranded region can be 14 to 30 nucleotide pairs, 17 to 30 nucleotide pairs, 27 to 30 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 19 nucleotide pairs, 19 to 25 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 21 nucleotide pairs, 21 to 25 nucleotide pairs, or 21 to 23 nucleotide pairs. In another example, the length of the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides.

[0365] In one embodiment, the RNAi agent may include one or more overhanging regions and / or capping groups at the 3' end, 5' end, or both ends of one or both strands. The length of the overhangs can be 1 to 6 nucleotides, for example, 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 overhangs may result from one strand being longer than the other, or from two strands of the same length being interleaved. The overhangs may form a mismatch with the target mRNA, or they may be complementary to the targeted gene sequence, or they may be another sequence. The first and second strands may also be joined, for example, by forming a hairpin with an additional base, or by other non-base linkers.

[0366] In one embodiment, the nucleotides in the overhanging region of the RNAi agent can each be independently modified or unmodified nucleotides, including but not limited to 2'-sugar modified nucleotides, such as 2-F, 2'-O methyl, thymidine (T), and any combination thereof.

[0367] For example, TT can be a protruding sequence at any end of any strand. The protruding end can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence, or it can be another sequence.

[0368] The 5' or 3'-protrusion of the sense strand, antisense strand, or both strands of the RNAi agent may be phosphorylated. In some embodiments, the protruding region comprises two nucleotides with a phosphate thioester between them, wherein the two nucleotides may be the same or different. In one embodiment, the protrusion is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, the 3'-protrusion is located in the antisense strand. In one embodiment, the 3'-protrusion is located in the sense strand.

[0369] RNAi agents may contain only a single overhang, which can enhance the interfering activity of RNAi without affecting its overall stability. For example, the single-stranded overhang can be located at the 3' end of the sense strand, or at 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. Typically, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt end at the 5' end. While not wanting to be bound by theory, the asymmetric blunt end at the 5' end of the antisense strand and the overhang at the 3' end of the antisense strand are beneficial for loading the guide strand into the RISC process.

[0370] In one embodiment, the RNAi agent is a 19-nucleotide-long, blunt-ended molecule, wherein the sense strand contains at least one 2'-F modification motif at positions 7, 8, and 9 of the 5' end. The antisense strand contains at least one 2'-O-methyl modification at positions 11, 12, and 13 of the 5' end.

[0371] In another embodiment, the RNAi agent is a 20-nucleotide-long, dual-terminal blunt polymer, wherein the sense strand contains at least one 2'-F modified motif at positions 8, 9, and 10 at the 5' end. The antisense strand contains at least one 2'-O-methyl modified motif at positions 11, 12, and 13 at the 5' end.

[0372] In another embodiment, the RNAi agent is a blunt-ended, 21-nucleotide strand, wherein the sense strand contains at least one 2'-F modification motif at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one 2'-O-methyl modification at positions 11, 12, and 13 from the 5' end.

[0373] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains three consecutive 2'-F modifications of at least one motif at positions 9, 10, and 11 at the 5' end; and the antisense strand contains three consecutive 2'-O-methyl modifications of at least one motif at positions 11, 12, and 13 at the 5' end. One end of the RNAi agent is blunt, while the other end contains two nucleotide overhangs. Preferably, the two nucleotide overhangs are located at the 3' end of the antisense strand. When the two nucleotide overhangs are at the 3' end of the antisense strand, there may be two phosphate thioester nucleotide inter-bonds between the three terminal nucleotides, where two of the three nucleotides are overhang nucleotides, and the third nucleotide is the next pairing nucleotide to the overhang nucleotide. In one embodiment, the RNAi agent further comprises two phosphate thioester nucleotide inter-bonds between the three terminal nucleotides at 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 agent, including nucleotides that are part of a motif, is a modified nucleotide. In one embodiment, each residue is independently modified with a 2'-O-methyl or 3'-fluorine, for example, in an alternating motif. Optionally, the RNAi agent also comprises a ligand (optionally, a C16 ligand).

[0374] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues long, starting at the 5' terminal nucleotide (position 1) of the first strand, with positions 1 to 23 comprising at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues long, starting at the 3' terminal nucleotide, and comprising at least 8 ribonucleotides at positions 1 to 23 of the sense strand to form a double strand; wherein at least the 3' terminal nucleotide of the antisense strand is unpaired from the sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired from the sense strand, thereby forming a 1 to 6 nucleotide 3' single-stranded overhang; wherein the 5' end of the antisense strand comprises 10 Up to 30 consecutive nucleotides unpaired with the sense strand, forming a single-stranded 5' overhang of 10 to 30 nucleotides; wherein, when the sense and antisense strands are aligned for maximum complementarity, at least the 5' and 3' terminal nucleotides of the sense strand pair with the nucleotide bases of the antisense strand, thereby forming a substantially double-stranded region between the sense and antisense strands; when the double-stranded nucleic acid is introduced into mammalian cells, the antisense strand is sufficiently complementary to the target RNA along at least 19 antisense strand lengths of ribonucleotides to reduce the expression of the target gene; and wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, wherein at least one motif is present at or near the cleavage site. The antisense strand contains three 2'-O-methyl modifications of at least one motif on three consecutive nucleotides at or near the cleavage site.

[0375] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the RNAi agent comprises a first strand of at least 25 and at most 29 nucleotides in length and a second strand of at most 30 nucleotides in length, and at least one 2'-O-methyl modified motif on three consecutive nucleotides at positions 11, 12, and 13 at the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, the 3' end of the second strand is 1 to 4 nucleotides longer than the first strand, wherein at least 19 nucleotides of at least 25 nucleotides in length along the second strand are sufficiently complementary to the target mRNA to reduce the expression of the target gene when the RNAi agent is introduced into mammalian cells, and wherein the RNAi cleavage agent preferentially produces siRNA containing the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. Optionally, the RNAi agent further comprises a ligand.

[0376] In one embodiment, the positive strand of the RNAi agent contains at least one motif with three identical modifications on three consecutive nucleotides, wherein one of the motifs appears at the cleavage site of the positive strand.

[0377] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif with three identical modifications on three consecutive nucleotides, wherein one of the motifs appears at or near the cleavage site of the antisense strand.

[0378] For RNAi agents with a double-stranded region of 17 to 23 nucleotides in length, the cleavage site of the antisense strand is typically located near positions 10, 11, and 12 at the 5' end. Therefore, three identically modified motifs may appear at positions 9, 10, and 11; positions 10, 11, and 12; positions 11, 12, and 13; positions 12, 13, and 14; or positions 13, 14, and 15 of the antisense strand, counted from the first nucleotide at the 5' end of the antisense strand, or counted from the first paired nucleotide within the 5'-end double-stranded region of the antisense strand. The cleavage site in the antisense strand may also vary depending on the length of the 5' end of the RNAi double-stranded region.

[0379] The sense strand of an RNAi agent may contain at least one motif with three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may contain at least one motif with three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense and antisense strands form a duplex, they may be arranged such that at least one nucleotide overlaps between one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand, i.e., 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.

[0380] In one embodiment, the positive strand of the RNAi agent may contain more than one of three identical modified motifs on three consecutive nucleotides. The first motif may appear at or near the cleavage site of the strand, and the other motifs may be wing modifications. The term "wing modification" as used herein refers to a motif appearing in another part of the strand, separated from the first motif at or near the cleavage site of the same strand. The wing modification is adjacent to or separated from the first motif by at least one or more nucleotides. When the motifs are directly adjacent to each other, their chemical properties differ from each other, and when the motifs are separated by one or more nucleotides, their chemical properties may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may occur at one end relative to the first motif, which is located at or near the cleavage site or on either side of the leading motif.

[0381] Similar to the sense strand, the antisense strand of an RNAi agent may contain one or more motifs of the same modification on three consecutive nucleotides, wherein at least one motif appears at or near the cleavage site of the strand. The antisense strand may also contain one or more wing modifications aligned in a manner similar to wing modifications that may be present on the sense strand.

[0382] In one embodiment, wing modifications on the sense or antisense strand of the RNAi agent are typically not included at the 3' end, 5' end, or the first or two terminal nucleotides at both ends of the strand.

[0383] In another embodiment, the wing modifications on the sense or antisense strand of the RNAi agent are typically not included in the first or two paired nucleotides within the double-stranded regions at the 3' end, 5' end, or both ends of the strand.

[0384] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modification may fall at the same end of the double-stranded region and have an overlap of 1, 2 or 3 nucleotides.

[0385] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be arranged such that each pair of modifications from one strand falls at one end of the double-stranded region with an overlap of one, two, or three nucleotides; the two modifications from one strand fall at the other end of the double-stranded region with an overlap of one, two, or three nucleotides; and the two modifications from one strand fall on each side of the leader motif with an overlap of one, two, or three nucleotides in the double-stranded region.

[0386] In one embodiment, the RNAi agent comprises a mismatch with the target, in the duplex, or a combination thereof. Mismatches may occur at the overhanging ends or in the duplex. Base pairs can be ordered according to their tendency to promote dissociation or unwinding (e.g., based on the association or dissociation free energy of a particular pair; the simplest method is to examine these pairs on a single-pair basis, although next-neighbor or similar analyses can also be used). Regarding 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, such as atypical or non-typical pairings (as described elsewhere herein), are preferred over typical (A:T, A:U, G:C) pairings; and pairings containing universal bases are preferred over typical pairings.

[0387] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs from the first 5 of the double-stranded region at the 5' end of the antisense strand, independently selected from: A:U, G:U, I:C, and mispairing, such as atypical or non-typical pairings or pairings containing universal bases, to promote the dissociation of the antisense strand at the 5' end of the double-stranded structure.

[0388] In one embodiment, the first nucleotide in the double-stranded region starting from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the double-stranded region starting from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region starting from the 5' end of the antisense strand is an AU base pair.

[0389] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). In one embodiment, there is a short sequence of deoxythymidine nucleotides, such as two dT nucleotides at the 3' end of the sense and / or antisense strands.

[0390] In one embodiment, the justice chain sequence can be represented by equation (I):

[0391] 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' (I)

[0392] in:

[0393] i and j are each independently 0 or 1;

[0394] p and q are each independently between 0 and 6;

[0395] N a Each oligonucleotide sequence independently represents a sequence containing 0 to 25 modified nucleotides, and each sequence contains at least two different modified nucleotides;

[0396] N b Each independently represents an oligonucleotide sequence containing 0-10 modified nucleotides;

[0397] n p and n q Each nucleotide independently represents a single overhanging nucleotide;

[0398] Where N b Y has no identical modifiers; and

[0399] XXX, YYY, and ZZZ each independently represent a motif consisting of three identical modifications on three consecutive nucleotides. Preferably, YYY represents all nucleotides modified with 2'-F.

[0400] In one embodiment, N a and / or N b This includes modifications for alternating patterns.

[0401] In one embodiment, the YYY motif appears at or near the cleavage site of the positive strand. For example, when the RNAi agent has a double-stranded region of 17 to 23 nucleotides in length, the YYY motif may appear at or near the cleavage site of the positive strand, counting from the first nucleotide from the 5' end (e.g., it may appear at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13); or alternatively, it may be counted from the first paired nucleotide from the 5' end within the double-stranded region.

[0402] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Therefore, the justice chain can be represented by the following formula:

[0403] 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib);

[0404] 5'n p -N a -XXX-N b -YYY-N a -n q 3' (Ic); or

[0405] 5'np -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id).

[0406] When the justice chain is represented by equation (Ib), N b This indicates an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides.

[0407] Each N a It can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0408] When the justice chain is represented as (Ic), N b This represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a It can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0409] When the justice chain is represented as equation (Id), each N b Independently representing an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Preferably, N b It can be 0, 1, 2, 3, 4, 5, or 6. Each N a It can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0410] Each of X, Y, and Z can be the same as or different from each other.

[0411] In other embodiments, i is 0 and j is 0, and the justice chain can be represented by the following formula:

[0412] 5'n p -N a -YYY-N a -n q 3' (Ia).

[0413] When the justice chain is represented by equation (Ia), each N a It can independently represent oligonucleotide sequences containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0414] In one embodiment, the antisense strand sequence of RNAi can be represented by formula (II):

[0415] 5'

[0416] n q’ -N a ′-(Z'Z′Z′) k -N b ′-Y′Y′Y′-N b ′-(X′X′X′) l -N′ a -n p ′3' (II)

[0417] in:

[0418] k and l are each independently 0 or 1;

[0419] p' and q' are each independently between 0 and 6;

[0420] Each N a 'Independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides;

[0421] Each N b 'Independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides;

[0422] Each n p 'and n q 'Independently represents a single overhanging nucleotide;'

[0423] Where N b 'and Y' have no identical modifiers;

[0424] as well as

[0425] X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif consisting of three identical modifications on three consecutive nucleotides.

[0426] In one embodiment, N a 'and / or N b 'Including modifications for alternating patterns.'

[0427] The Y'Y'Y' motif appears at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a double-stranded region of 17 to 23 nucleotides in length, the Y'Y'Y' motif may occur at positions 9, 10, 11, 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, adjacent to the 5'-end 1 st Nucleotide counting begins; or optionally, starting from the 5' end double-stranded region. stCounting the start of paired nucleotides. Preferably, the Y'Y'Y' motif appears at positions 11, 12, and 13.

[0428] In one embodiment, the Y'Y'Y' motif is a 2'-OMe modified nucleotide.

[0429] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0430] Therefore, an antisense chain can be represented by the following formula:

[0431] 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3' (IIb);

[0432] 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3' (IIc); or

[0433] 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3' (IId).

[0434] When the antisense chain is represented by the following equation (IIb), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a 'Independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.'

[0435] When the antisense chain is expressed as in equation (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a 'Independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.'

[0436] When the antisense chain is represented as in equation (IId), each N b'Independently represents 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a Independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Preferably, N b It is 0, 1, 2, 3, 4, 5, or 6.

[0437] In other embodiments, k is 0 and l is 0, and the antisense chain can be represented by the following formula:

[0438] 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3' (Ia).

[0439] When the antisense chain is represented as equation (IIa), each N a 'Independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.'

[0440] Each of X', Y', and Z' can be the same as or different from each other.

[0441] Each nucleotide in the sense and antisense strands can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxy, or 2'-fluorine. For example, each nucleotide in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluorine. In particular, each X, Y, Z, X', Y', and Z' can represent either a 2'-O-methyl modification or a 2'-fluorine modification.

[0442] In one embodiment, when the double-stranded region is 21 nt, the positive strand of the RNAi agent may contain a YYY motif appearing at positions 9, 10, and 11 of the strand, counting from the first nucleotide at the 5' end, or optionally, counting from the first paired nucleotide within the double-stranded region; Y represents a 2'-F modification. The positive strand may additionally contain an XXX motif or a ZZZ motif as a wing modification at the other end of the double-stranded region; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0443] In one embodiment, the antisense strand may include a Y'Y'Y' motif appearing at chain positions 11, 12, 13, counted starting from the first nucleotide at the 5' end, or optionally, counted starting from the first paired nucleotide within the double-stranded region, with Y' representing a 2'-O-methyl modification. The antisense strand may additionally include an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the other end of the double-stranded region; X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0444] The justice chain represented by any one of the above equations (Ia), (Ib), (Ic), and (Id) forms a double chain with the antisense chain represented by any one of the above equations (IIa), (IIb), (IIc), and (IId).

[0445] Therefore, the RNAi agent used in the method of this application may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex is represented by formula (III):

[0446] Justice: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'

[0447] Antonym: 3'

[0448] n p '-N a '-(X'X′X′) k -N b '-Y′Y′Y′-N b '-(Z′Z′Z′) l -N a '-n q '5'

[0449] (III)

[0450] in:

[0451] i, j, k, and l are each independently 0 or 1;

[0452] p, p', q, and q' are each independently between 0 and 6;

[0453] Each N a and N a 'Independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides;

[0454] Each N b and N b 'Independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides;

[0455] in

[0456] each n p '、n p n q 'and n q Each may or may not be present, independently representing the overhanging nucleotide; and

[0457] XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif consisting of three identical modifications on three consecutive nucleotides.

[0458] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0459] Instance combinations of the sense and antisense strands that form an RNAi duplex include the following:

[0460] 5'n p -N a -YYY-N a -n q 3'

[0461] 3'n p '-N a '-Y'Y'Y'-N a 'n q '5'

[0462] (IIIa)

[0463] 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'

[0464] 3'n p '-N a '-Y'Y'Y'-N b '-Z'Z'Z'-N a 'n q '5'

[0465] (IIIb)

[0466] 5'np -N a -XXX-N b -YYY-N a -n q 3'

[0467] 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N a '-n q '5'

[0468] (IIIc)

[0469] 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'

[0470] 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N b '-Z'Z'Z'-N a -n q '5'

[0471] (IIId)

[0472] When the RNAi agent is represented by formula (IIIa), each N a Independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0473] When the RNAi agent is represented by formula (IIIb), each N b Independently represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. Each N a Independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0474] When the RNAi agent is represented as shown in formula (IIIc), each N b 、N b 'Independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a Independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0475] When the RNAi agent is represented as shown in formula (IIId), each N b N b 'Independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a N a 'Independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. N a N a '、N b and N b Each of the characters in 'independently includes the modification of the alternation pattern.

[0476] In one embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluorine modification. In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is 2'-O-methyl or 2'-fluoro and n p '>0 and at least one n p 'Linked to adjacent nucleotides via phosphate thioester bonding. In yet another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification, n p '>0 and at least one n p 'It is linked to an adjacent nucleotide via a phosphate thioester bond, and the positive strand is attached to one or more C16 (or related) moieties via a divalent or trivalent side linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification, n p '>0 and at least one n p 'Linked to adjacent nucleotides via thiophosphate bonds, the sense strand includes at least one thiophosphate bond, and the sense strand is bound to one or more C16 (or related) moieties, optionally attached via a linker through a divalent or trivalent branch.'

[0477] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a The modification is a 2'-O-methyl or 2'-fluoro modification, n p '>0 and at least one n p It is linked to an adjacent nucleotide via a thiophosphate bond, the positive strand contains at least one thiophosphate bond, and the positive strand is linked to one or more C16 (or related) portions connected by divalent or trivalent branched linkers.

[0478] In one embodiment, the RNAi agent is a multimer comprising at least two duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer also comprises a ligand. Each duplex may target the same gene or two different genes; or each duplex may target the same gene at two different target ends.

[0479] In one embodiment, the RNAi agent is a multimer comprising three, four, five, six, or more duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are linked by linkers. The linkers may be cleavable or non-cleavable. Optionally, the multimer also comprises a ligand. Each duplex may target the same gene or two different genes; or each duplex may target the same gene at two different target ends.

[0480] In one embodiment, two RNAi agents represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are interconnected at one or both of their 5' and 3' ends and optionally bind to a ligand. Each agent may target the same gene or two different genes; or each agent may target the same gene at two different target ends.

[0481] Various public documents describe multimeric RNAi agents that can be used in the methods described in this application. Such publications include WO2007 / 091269, U.S. Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, the entire contents of which are incorporated herein by reference. In some embodiments, the RNAi agent of this application may include a GalNAc ligand, even though such GalNAc ligands are currently expected to have limited value for the preferred intrathecal / CNS delivery pathway of this application.

[0482] As described in more detail below, RNAi agents comprising a conjugate of one or more carbohydrate moieties with an RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moieties will be linked to a modified subunit of the RNAi agent. For example, the ribose of one or more ribonucleotide subunits of a dsRNA agent can be replaced by another moiety, such as a non-carbohydrate (preferably cyclic) carrier linked to a carbohydrate ligand. The ribonucleotide subunits in which the ribose of the subunits has been so replaced are referred to herein as ribose-substituted modified subunits (RRMS). The cyclic carrier can be a carbocyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic system, or it can contain two or more rings, such as a fused ring. The cyclic carrier can be a fully saturated cyclic system, or it can contain one or more double bonds.

[0483] Ligands can be linked to polynucleotides via a carrier. The carrier includes (i) at least one “backbone linker,” preferably two “backbone linkers,” and (ii) at least one “shared linker.” As used herein, a “backbone linker” refers to a functional group, such as a hydroxyl group, or generally, a bond suitable for incorporating the carrier into the backbone, such as a phosphate ester or a modified phosphate ester, for example, the sulfur-containing backbone of a ribonucleic acid. In some embodiments, a “shared linker” (TAP) refers to a constituent ring atom of the cyclic carrier, such as a carbon atom or a heteroatom (different from the atom providing the backbone linker), which connects to a selected portion. The portion can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected portion is linked to the cyclic carrier via an inserted ligand. Thus, cyclic carriers typically include functional groups, such as amino groups, or generally provide bonds suitable for binding or binding another chemical entity, such as a ligand, to the constituent ring.

[0484] RNAi agents can bind to ligands via a carrier, wherein the carrier can be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolyl, isothiazolinyl, quinoxolinyl, pyridazinyl, tetrahydrofuran, and decahydronaphthalene; preferably, the acyclic group is selected from the serine backbone or the diethanolamine backbone.

[0485] In some specific embodiments, the RNAi agent used in the methods of this application is an agent from the group listed in any one of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10 to 15, 16A, 16B, and 26. These agents may also contain ligands.

[0486] IV. App knockout therapy for app-related diseases

[0487] Certain aspects of this application relate to the knockout of APP-related diseases or conditions mediated by RNAi agents, including CAA and AD, including hereditary CAA and EOFAD, as well as sporadic and / or late-onset AD.

[0488] Hereditary CAA (hCAA) is an angioprotein disorder, and the amyloid-based treatment hypothesis is relatively simple and clinically testable. It is a devastating rare disease with no current cure. Biochemical and imaging biomarkers exist for clinical validation of anti-APP siRNA-mediated hCAA therapy.

[0489] One specific type of hCAA under consideration for treatment with the RNAi agents described in this application is "Dutch-type" AβhCAA, with an estimated patient population of several hundred, primarily located in the Netherlands and Western Australia. What makes hCAA unique among APP-related diseases is its purely vascular nature: in CAA, amyloid fibrils are deposited in the small arteries and capillaries of the central nervous system parenchyma and pia mater, leading to cognitive decline in CAA patients due to cerebral ischemia and microbleeds. CAA is present in over 80% of all AD individuals (25% of AD individuals have moderate to severe CAA), and the incidence of CAA increases with age, with an incidence of approximately 50% in individuals over 70 years of age.

[0490] The following are typical expressions of hereditary CAA:

[0491] Amyloid-β-sporadic CAA, HCHWA-Dutch and Italian EOFAD, LOAD, Trisomy 21

[0492] ABr-familial British dementia

[0493] ADan - Familial Danish Dementia

[0494] Cystatin C-HCHWA-Icelandic type (HCHWA-hereditary cerebral hemorrhage with amyloidosis)

[0495] Gel protein - Familial amyloidosis - Finnish type

[0496] Prion protein - prion disease

[0497] Thyroxine transporter protein-hereditary systemic amyloidosis

[0498] As mentioned above, Aβ-hCAA (also known as APP-hCAA) is a rapidly progressive dementia associated with intracranial hemorrhage. Known indications for CAA include APP-hCAA and sporadic CAA. Other possible indications for CAA include: CAA associated with EOFAD (PSEN1; APP; PSEN2); CAA associated with Down syndrome; and CAA associated with late-onset Alzheimer's disease (which is common, as mentioned above).

[0499] Using APP-hCAA as an indicator, the prevalence of APP-hCAA is unclear; however, pure APP-hCAA is less common than EOFAD (Dutch hCAA (related to the APP E693Q mutation) has been reported in hundreds of individuals). Typically, the onset of APP-hCAA symptoms occurs between the ages of 35 and 45; APP-hCAA usually develops into severe CVA within 2 to 5 years, with the peak age of death from CVA being 55 years.

[0500] Sporadic CAA has a relatively high prevalence as an indication: it is a common cause of lobar hemorrhage (ICH) in older adults. It is also a rapidly progressing disease, with 86 out of 316 patients (36%) experiencing recurrent ICH during a 5-year mean follow-up period (Van Etten et al., 2016 Neurology). In one study, the cumulative dementia incidence of sporadic CAA was observed to be 14% at 1 year and 73% at 5 years (Xiong et al., 2017 J Cerebr Blood Flow Metab). Sporadic CAA also largely overlaps with AD, as late-stage CAA has been identified in approximately 25% of AD brain cases; however, less than 50% of CAA cases actually meet the pathological criteria for AD.

[0501] In order to evaluate the efficacy of APP knockout in individuals treated with the RNAi agent described in this application, soluble forms of APP, particularly including APPα and APPβ, are clearly considered as cerebrospinal fluid (CSF) biomarkers for assessing APP knockout efficiency.

[0502] The generation, elimination, and deposition of β-amyloid in the CAA: Growing evidence suggests that the primary source of Aβ is neurons. It is produced by the cleavage of amyloid precursor protein (APP) by β- and γ-secretases in a sequence proportional to neuronal activity. Aβ is eliminated from the brain via four main pathways: (a) proteolytic degradation by endopeptidases (e.g., enkephalinase and insulin-degrading enzyme (IDE)); (b) receptor-mediated clearance of brain parenchymal cells (microglia, astrocytes, and to a lesser extent, neurons); (c) active transport across the blood-brain barrier (BBB) ​​into the bloodstream; and (d) elimination along perivascular pathways, with interstitial fluid draining from the brain. Specialized carriers (e.g., ApoE) and / or receptor transport mechanisms (e.g., low-density lipoprotein receptor (LDLR) and LDLR-associated protein (LRP1)) are involved in all major cellular clearance pathways. Angiogenesis is promoted by factors that increase the Aβ40:Aβ42 ratio (while increased Aβ42 leads to oligomerization and amyloid plaque mosaicism) and obstructs perivascular channels. As clearance mechanisms fail with age, Aβ increasingly enters the basement membrane of brain capillaries and arterioles via perivascular drainage pathways, leading to cardiac aneurysmia (CAA). The ApoE allele has different effects on various molecular and cellular processes of Aβ production, elimination, and deposition, increasing or decreasing the risk of CAA (Charidimou A et al., J Neurol Neurosurg Psychiatry 2012; 83:124-137).

[0503] The sequential cleavage of APP occurs via two pathways. The APP protein family is believed to possess a large, biologically active N-terminal extracellular domain and a short C-terminus, containing a key tyrosine-glutamate-asparagine-proline-threonine-tyrosine (YENPTY; SEQ ID NO: 1863) adaptor X11 and Fe65-binding protein sorting domain. The resulting Aβ peptide cleavage product begins within the extracellular domain and continues into the transmembrane region. In one pathway, APP is cleaved by α-secretase, followed by γ-secretase in the non-amyloid processing of APP. In the second pathway, the amyloid formation process of APP involves BACE1 cleavage, followed by γ-secretase. Both methods yield soluble extracellular domains (sAPPα and sAPPβ) and identical intracellular C-terminal fragments (AICD; SEQ ID NO: 1864; Thinakaran and Koo. J. Biol. Chem. 283: 29615–19; Reinhard et al., The EMBO Journal, 24: 3996-4006; Walsh et al., Biochemical Society Transactions, 35: 416-420; O'Brien and Wong. Annu Rev Neurosci. 34: 185-204).

[0504] Histopathology of CAA includes morphological changes in the vessel wall (as shown by hematoxylin-eosin staining) and Aβ deposition. Significant structural alterations and double barreling have been observed in leptomeningeal arterioles (Charidimou et al., J Neurol Neurosurg Psychiatry 83:124-137). In mild and moderate CAA, only minimal structural changes were detected; however, in advanced CAA, significant structural alterations have been detected, the most extreme being double barreling (separation and stratification of the outer media). Similar pathological extent of CAA-related changes in leptomeningeal arterioles has also been observed using immunohistochemical detection of Aβ. In mild CAA, patchy deposits of amyloid protein are observed in the examined vessel wall. Moderate CAA expresses denser amyloid protein deposition across the entire vessel wall, while severe CAA expresses double-globular vessels and endothelial involvement. Pathological findings of CAA in cortical arterioles suggest that Aβ deposition is proportional to disease severity. Moderate CAA shows panmural deposition of Aβ as well as Aβ deposition in the surrounding brain parenchyma, while in severe CAA, double barrel vessels are observed, although this is less common compared to leptomeningeal vessels (Charidimou et al.).

[0505] The pathogenesis of CAA has also been investigated. Beta-amyloid produced by the brain parenchyma is normally cleared via the perivascular pathway. Overproduction of Aβ and delayed drainage of Aβ in certain CAA variants have been observed, leading to amyloid deposition in the media of small arteries in the central nervous system. Soluble and insoluble amyloid fibrils have been identified as toxic to vascular smooth muscle; these fibrils replace these cells, resulting in loss of vascular reactivity. Further damage to the endothelium has been observed leading to microbleeds, microinfarctions, and tissue destruction leading to dementia. Further progression results in cerebral hemorrhage, which is often observed to be fatal. CAA has been observed most frequently in the occipital lobe, less frequently in the hippocampus, cerebellum, basal ganglia, and is generally not found in the deep central gray matter, subcortical white matter, and brainstem (Charidimou et al.).

[0506] Numerous potential outcome markers have been identified for the performance of CAA in human studies. In addition to symptomatic intracerebral hemorrhage, microbleeds, white matter leukoderma (WMH), and amyloid imaging are associated with disease severity and progression (Greenburg et al., Lancet Neurol 13:419–28).

[0507] Available assays can also be used to detect soluble APP levels in human cerebrospinal fluid samples. In particular, sAPP and sAPPβ, soluble forms of APP, have been identified as PD (pharmacodynamic) biomarkers. Assays have also been detected in non-human primate (NHP) CSF samples, and such assays can be used in efficacy studies in NHP. Detection of Aβ40 / 42 / 38 peptides and total tau / P181Tau has also been described and is being performed in current studies.

[0508] Imaging biomarkers can also be used in CAA research because cerebrovascular function has been identified as a reflection of the pathology of CAA. Imaging has been specifically used to measure oxygen level-dependent (BOLD) signals following visual stimulation (Van Opstal et al., Thelancet Neurology; 16(2); 2017; Peca S et al., Neurology. 2013; 81(19); Switcher A et al., NeuroImage Clinical; 2016). Reduced functional MRI activation has been observed in CAA patients when BOLD fMRI is performed on individuals with CAA (assessing oxygen level-dependent functional MRI responses in groups that assess motor and visual tasks). In particular, activity in visual cortex BOLD fMRI has been observed to be associated with higher volume WMH and higher microbleed counts (Peca et al., Neurology 2013; 81(19); Switcher et al., NeuroImage Clinical 2016).

[0509] Animal models of CAA have also been described, allowing for the determination of the impact of APP knockout on CAA pathology and the identification of translatable biomarkers. In particular, several rodent animal models expressing mutant human APP and exhibiting CAA pathology have been developed, including Tg-SwDI / NOS2- / -. In Tg-SwDI / NOS2- / - mice, Aβ levels increase with age. Perivascular hyperphosphorylated tau protein is also associated with capillary amyloid, not only in Tg-SwDI / NOS2- / - mice but also in human CAA type 1 samples (Hall and Roberson. Brain ResBull. 2012; 88(1):3–12; Attems et al., Nephrology and Applied Neurobiology, 2011, 37, 75-93). The CVN mouse model of AD (APPSDI / NOS2KO) also expressed the phenotype, including amyloid plaques in the hippocampus, thalamus, and cortex, increased tissue inflammation, and behavioral deficits. A transgenic rat model (carrying the hAPP mutation) was also developed.

[0510] Therefore, APP has been identified as a target for hereditary cerebral amyloid angiopathy (CAA). APP mutations leading to severe forms of CAA, including A692G (Flemish), E693Q (Netherlands), E693K (Italy), and D694N (Iowa), have been reported. Meanwhile, APP mutations leading to early-onset AD, including E665D, K670N, M671L (Sweden), T714A (Iran), T714I (Austria), V715M (France), V715A (Germany), I716V (Florida), I716T, V717I (London), V717F, V717G, and V717L, have been described. In particular, the APP E693Q (Netherlands) mutation leads to severe CAA with almost no substantial neurofibrillary tangles; E693Q increases β-amyloid aggregation and toxicity; E693K (Italy) is similar, but the E693G (Arctic), E693A, and E693delta mutations lead to EOFAD with no or very little CAA; and APP D694N (Iowa) leads to severe CAA with typical AD pathology. In addition to the point mutations mentioned above, APP duplication leading to APP overexpression has also been identified as contributing to Aβ deposition. Meanwhile, no known APP mutations have been described that can prevent or delay APP-hCAA. In addition to APP mutants, PSEN1 (L282V) and PSEN2 (N141I) mutations in AβCAA have also been observed. Meanwhile, ApoEε2 (independent of AD) and ApoEε4 (dependent of AD) have also been reported as risk factors for CAA (Rensink A et al., Brain Research Reviews, 43(2) 2003).

[0511] Certain aspects of this application relate to targeting APP for knockout in individuals with APP-hCAA. Such agents are needed because there are currently no disease-modifying therapies targeting CAA. In some embodiments, the RNAi agent of this application should provide approximately 60 to 80% knockout of mutant and WT APP levels throughout the CNS.

[0512] Humans with heterozygous APP mutations exist in the general population with a pLI score of 0.3; however, no human APP gene knockout has been found to date.

[0513] Pharmacological attempts to treat CAA in humans include:

[0514] Pfizer investigated an amyloid-40 antibody, Ponezumab, in 36 individuals with late-onset CAA. Three infusions of Ponezumab or placebo were evaluated over a 60-day period to measure cerebrovascular reactivity and changes in cerebrovascular reactivity using BOLD fMRI. Secondary outcomes included edema, infarction, Aβ, cognitive changes, and other secondary outcomes. Ponezumab showed a difference between the drug and placebo, but the primary endpoint was not met.

[0515] BAN2401—a systemically delivered therapeutic antibody against β-amyloid—has been shown to be safe, but may cause focal cerebral edema. In the most recent LOAD of BAN2401 in a phase II 18-month trial, the incidence of SAE (subjective encephalopathy) was 17.6% with placebo and 15.5% with the highest dose (10 mg / kg every two weeks). Amyloid-associated imaging abnormalities-edema (ARIA-E) was 14.6% in APOE4 carriers at the highest dose.

[0516] Compared to animal CAA models, ponezumab was effective in reducing β-amyloid load and vascular reactivity in CAA mouse models (Bales, 2018). Meanwhile, the global APP knockout mouse model has also been considered a viable option.

[0517] The following illustrative biomarkers and pathological data further validate the major role of β-amyloid in the pathogenesis of CAA:

[0518] The genetically observed “pure” CAA (i.e., lacking parenchymal mosaic amyloid) is characterized by predominantly Aβ40 deposition in amyloid, rather than Aβ42 in parenchymal AD;

[0519] It has been observed that CAA is not "tauopathy," and the levels of T-tau and P-tau in CSF are normal, contrary to the elevated levels observed in AD.

[0520] The negative correlation between increased brain amyloid burden and decreased CSF Aβ40 levels, as measured by PiB PET, has been identified as unique to CAA; and

[0521] In vitro and in vivo experimental data provide increasing support for the prion hypothesis in CAA, with Aβ40 containing genetic CAA mutations exhibiting a tendency to misfold and induce WT protein misfolding, both of which are present in amyloid fibrils (similar to transthyretin (TTR)).

[0522] As disclosed in the following examples, this application provides a number of mouse / rat cross-reactive APP-targeting duplexes (including, for example, AD-397177, AD397192, AD-397196, AD-397182, AD397190, AD-397265, and AD-397203), which are evaluated on day 21 after administration of a single dose of 2 mg / Kg, based on screening results obtained from APP liver mRNA. This application also provides numerous human / cynomolgus monkey cross-reactive duplexes (including, for example, AD-392911, AD-392912, AD-392703, AD-392866, AD-392927, AD-392913, AD-392843, AD-392916, AD-392714, AD-392844, AD-392926, AD-392824, AD-392704, and AD-392790), based on screening results obtained for the treatment of primary cynomolgus monkey hepatocytes and human BE(2)C cells.

[0523] RNAi-mediated EOFAD knockout was also explicitly considered. Like hCAA, EOFAD is a devastating and rare disease. For hCAA, the causal role of APP has been established, and EOFAD is associated with sporadic and / or late-onset AD (optionally late-onset AD with severe CAA as a subclass of late-onset AD). EOFAD is a progressive neurodegenerative dementia that occurs in young adults, with an age of onset between 60 and 65 years, typically before 55 years.

[0524] The prevalence of EOFAD is estimated at 41.2 per 100,000 people (i.e., those aged 40 to 59 years), with 61% of those affected having a positive family history of EOFAD (13% of whom have been affected across three generations). EOFAD accounts for less than 3% of all AD cases (Bird, Genetics in Medicine, 10:231–239; Brien and Wang, Annu RevNeu Sci, 2011, 34:185-204; NCBI Gene Reviews).

[0525] Human genome validation of the APP target (OMIM 104300) has been provided, and certain APP mutations leading to EOAAD have been identified, including E665D, K670N, M671L (Sweden), T714A (Iran), T714I (Austria), V715M (France), V715A (German), I716V (Florida), I716T, V717I (London), V717F, V717G, and V717L as described above. Furthermore, dominant β-amyloid precursor protein mutations leading to EOAAD and CAA have also been identified.

[0526] Unwilling to be bound by theory, they believe that the pathogenesis of AD begins in the hippocampus, the gray matter crest immediately above the lateral ventricles. Degeneration of this tissue is thought to contribute to the memory loss characteristic of early disease. Although the mechanisms of neurodegeneration at the protein level remain highly controversial, the APP repetition associated with EOFAD suggests that APP overexpression may be sufficient to cause AD. (Haass and Selkoe. Nature Reviews Molecular Cell Biology, 8:101-112).

[0527] Compared to EOFAD and CAA, the pathogenesis of sporadic AD is unclear, and the clinically defined sporadic AD population may be heterogeneous in terms of mechanism.

[0528] Certain aspects of this application relate to targeting APP for knockout in individuals with EOFAD. There is a need for such agents because for the more prevalent AD, especially EOFAD, only symptomatic treatments exist (with limited efficacy). In some embodiments, the RNAi agent of this application should provide approximately 60 to 80% knockout of mutant and WT APP levels throughout the CNS. Further observation from human genetics suggests that APP-targeted therapies capable of reducing APP levels in CNS cells may have therapeutic efficacy, namely, the identification of an A673T mutation that protects AD and dementia carriers in the general population (Jonsson et al., Nature Letters, 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 β-amyloid protein in cellular assays. Therefore, dominant-negative APP point mutants appear to protect families from AD, further reinforcing the idea that RNAi-mediated APP knockout can play a similar protective and / or therapeutic role in at least some forms of AD, including EOFAD.

[0529] In the initial stages of developing RNAi agents targeting APP, the feasibility of APP knockout mice (OMIM104300) has been noted, which promises to allow the feasible use of mice as a model system in lead compound development processes. In contrast to mice, while heterozygous APP mutations with an EXAC score of 0.3 exist in the general population, no human APP gene knockout has been identified to date. Biomarkers that could be used to develop RNAi agents targeting APP include APP and MAPT peptides in CSF, which should allow for rapid assessment and useful efficacy even in genetically homogeneous populations (Mo et al., (2017) Alzheimers & Dementia: Diagnosis, Assessment & Disease Monitoring, 6:201-209).

[0530] As mentioned above, attempts to treat sporadic AD and Eofa's disease have so far proven unsuccessful—for example, all trials of BACE1 (β-secretase) inhibitors (BACE1i) for the treatment of sporadic AD have failed to date (Egan et al., The New England Journal of Medicine, 378:1691-1703; Hung and Fu. Journal of Biomedical Science, 24:47). In such BACEi tests, studies have not yet been completed in genetically defined populations (clinical studies only). Notably, recent BACE1i studies have shown that verubecestat (a new drug) reduced amyloid levels by 60% in populations selected based on age and clinical criteria that suggest 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, a popular Aβ-guided immunotherapy has demonstrated proof of concept in recent sporadic AD trials, supporting ongoing Phase III clinical trials (Selkoe and Hardy. EMBO Molecular Medicine, 8:595-608). Given its role in APP cleavage, γ-secretase has also become a target for some AD-guided trials. However, to date, no γ-secretase inhibitor trials have been completed in genetically defined populations; and some programs have been discontinued due to toxicity (Selkoe and Hardy).

[0531] Therefore, there is a need for medications that can treat or prevent APP-related diseases or conditions in affected individuals.

[0532] It is clearly anticipated that the RNAi agents using the content of this application will eventually be able to target all APP-related diseases or conditions—specifically, the RNAi agents of this application are also expected to target sporadic CAA and sporadic and / or late-onset AD, even taking into account the current diagnostic / expressional problems faced by these specific APP-related diseases (further anticipating that the diagnosis of these diseases will continue to improve).

[0533] V. Ligand-binding RNAi agents

[0534] Another modification of the RNA of the RNAi agent described in this application relates to the chemical linking of one or more ligands, portions or conjugates that enhance RNAi activity, cellular distribution or cellular uptake to the RNA. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), bile acids (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4: 1053-1060), thioethers, such as beryl-S-triphenylmethylthiol (Manoharan et al., (1992) Ann. NY Acad. Sci., 660: 306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3: 2765-2770), sulfur cholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20: 533-538), and fatty acid chains, such as 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, such as di-hexadecyl-racemic-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-phosphate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmitic moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or octadecylamine or hexylamino-carbonyl-oxocholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0535] In one embodiment, the ligand alters the distribution, targeting, or lifetime of the RNAi agent it incorporates. In a preferred embodiment, the ligand provides enhanced affinity to selected targets such as molecules, cells or cell types, compartments such as cellular or organ compartments, tissues, organs, or body regions, for example, compared to species in which such ligands are absent. Preferred ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0536] Ligands may include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin, N-acetamidoglucose, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, including synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include: polyethyleneimine, polylysine (PLL), arginine, spermidine, polyamines, pseudopeptide-polyamines, pseudopeptide-polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0537] Ligands may also include targeting groups, such as cell or tissue targets, such as lectins, glycoproteins, lipids, or proteins, for example, antibodies that bind to specific cell types, such as kidney cells. Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide analogs.

[0538] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), and lipophilic molecules such as cholesterol, bile acids, adamantine, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O (hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, and O3-(oleoyl) Lithocholic acid (O3-(oleoyl)cholic acid, dimethoxytriphenylmethyl or phenoxazine) and peptide conjugates (e.g., antennal peptide, Tat peptide), alkylating agents, phosphates, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled substances, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraza macrocyclic Eu3+ complexes), dinitrophenyl, HRP or AP.

[0539] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with a specific affinity for a coligand, or antibodies that bind to specific cell types, such as CNS cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide substances, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose.

[0540] The ligand can be a substance, such as a drug, that increases the uptake of RNAi agents by cells by disrupting the cellular cytoskeleton, for example, by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. Drugs can be, for example, paclitaxel, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, phycobilin A, phalloidin, swinholide A, indanocine, or myosin.

[0541] In some embodiments, the ligand linked to the RNAi agent described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, bile acids, lithocholic acids, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides containing multiple thiophosphate bonds are also known to bind to serum proteins; therefore, short oligonucleotides, such as those of about 5, 10, 15, or 20 bases, containing multiple thiophosphate bonds in their backbone, are also suitable as ligands in this application (e.g., as PK-regulating ligands). Furthermore, aptamers that bind serum components (e.g., serum proteins) are also suitable as PK-regulating ligands in the embodiments described herein.

[0542] The ligand-bound oligonucleotides of this application can be synthesized using oligonucleotides with overhanging reactive functional groups, such as those derived from linker molecules attached to oligonucleotides (as described below). The reactive oligonucleotides can react directly with commercially available ligands, synthetic ligands with multiple protecting groups, or ligands with linker moieties.

[0543] The oligonucleotides used in the conjugates of this application can be conveniently and routinely prepared using well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, CA). Any other methods known in the art for such synthesis may be used additionally or alternatively. Similar techniques are also known for the preparation of other oligonucleotides, such as phosphate thioesters and alkylated derivatives.

[0544] In the ligand-bound oligonucleotides and sequence-specific ligand-molecule-linked nucleosides of this application, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotides or nucleoside precursors, or nucleotides or nucleoside conjugate precursors that already have sequence-specific ligands, linking parts with ligand molecules, ligand-nucleotides or nucleoside-conjugate precursors, or structural units with non-nucleoside ligands.

[0545] When using a nucleotide conjugate precursor that already contains a linker, the synthesis of a sequence-specific linked nucleoside is typically performed, and then the ligand molecule reacts with the linker to form a ligand-bound oligonucleotide. In some embodiments, in addition to standard and non-standard phosphorus amides that are commercially available and routinely used in oligonucleotide synthesis, the oligonucleotides or linked nucleosides of this application are synthesized using phosphorus amides derived from the ligand-nucleoside conjugate via an automated synthesizer.

[0546] A. Lipophilic portion

[0547] In some embodiments, the lipophilic moiety is an aliphatic, cyclic (e.g., alicyclic) or polycyclic (e.g., polyalicyclic) compound, such as a steroid (e.g., a sterol) or a straight-chain or branched aliphatic hydrocarbon. The lipophilic moiety may typically comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic moietyes include, but are not limited to, saturated or unsaturated C4-C... 30 Hydrocarbons (e.g., C6-C) 18 Hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C464 ... 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes and C 40 Tetraterpenes and other polycyclic hydrocarbons. For example, the lipophilic moiety can contain C4 to C5. 30 Hydrocarbon chains (e.g., C4 to C5) 30 Alkyl or alkenyl). In some embodiments, the lipophilic moiety comprises saturated or unsaturated C6 to C6 groups. 18 Hydrocarbon chains (e.g., linear C6 to C5) 18 (alkyl or alkenyl). In one embodiment, the lipophilic moiety comprises saturated or unsaturated C. 16 Hydrocarbon chains (e.g., linear C) 16 (alkyl or alkenyl).

[0548] The lipophilic moiety can be linked to the RNAi agent by any method known in the art, including via a functional group already present in the lipophilic moiety or introduced into the RNAi agent, such as a hydroxyl group (e.g., —CO—CH2—OH). Functional groups already present in the lipophilic moiety or introduced into the RNAi agent include, but are not limited to, hydroxyl groups, amines, carboxylic acids, sulfonates, phosphates, thiols, azides, and alkynes.

[0549] The binding of the RNAi agent to the lipophilic moiety can occur, for example, by forming an ether or carboxylic acid or carbamate bond between the hydroxyl group and an alkyl R-, alkylyl RCO-, or a substituted carbamoyl RNHCO-. The alkyl R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chain or branched; and saturated or unsaturated). The alkyl R can be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, or octadecyl, etc.

[0550] In some embodiments, the lipophilic portion is bound to a double-stranded RNAi agent via a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, click reaction (e.g., triazole from azide-alkyne cycloaddition), or carbamate.

[0551] In another embodiment, the lipophilic moiety is a steroid, such as a sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentylphenanthrene ring system. Steroids include, but are not limited to, bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxin, testosterone, cholesterol, and cationic steroids, such as cortisone. "Cholesterol derivative" refers to compounds derived from cholesterol, for example, through substitution, addition, or removal of substituents.

[0552] In another embodiment, the lipophilic portion is the aromatic portion. In this context, the term "aromatic" refers broadly to monoaromatic and polyaromatic hydrocarbons. Aromatic groups include, but are not limited to, C6-C groups comprising one to three aromatic rings. 14 The aryl moiety may optionally be substituted; the "aralkyl" or "aralkyl" group comprises an aryl group covalently linked to an alkyl group, either of which may be optionally substituted or unsubstituted independently; and the "heteroaryl" group. 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 in the cyclic array; and having, in addition to a carbon atom, 1 to about 3 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0553] As used herein, a “substituted” alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is a group having 1 to about 4, preferably 1 to about 3, more preferably 1 or 2 non-hydrogen substituents. Suitable substituents include, but are not limited to, halogens, hydroxyl groups, nitro groups, haloalkyl groups, alkyl groups, alkyl groups, aryl groups, aralkyl groups, alkoxy groups, arylcarbamoyl groups, aminoalkyl groups, alkoxycarbonyl groups, carboxyl groups, hydroxyalkyl groups, alkylsulfonyl groups, arylsulfonyl groups, alkylsulfonamide groups, arylalkylsulfonamide groups, alkylcarbonyl groups, acyloxy groups, cyano groups, and urea groups.

[0554] In some embodiments, the lipophilic moiety is an aralkyl group, such as a 2-arylpropionyl moiety. The structural features of the aralkyl group are chosen such that the lipophilic moiety binds to at least one protein in vivo. In some embodiments, the structural features of the aralkyl group are chosen such that the lipophilic moiety binds to serum, vascular, or cellular proteins. In some embodiments, the structural features of the aralkyl group facilitate binding to albumin, immunoglobulins, lipoproteins, α-2-macroglobulin, or α-1-glycoprotein.

[0555] In some embodiments, the ligand is naproxen or a structural derivative of naproxen. The synthetic process for naproxen can be found in U.S. Patent Nos. 3,904,682 and 4,009,197, the entire contents of which are incorporated herein by reference. The chemical name of naproxen is (S)-6-methoxy-α-methyl-2-naphthylacetate, and its structural formula is [insert structural formula here].

[0556] In some embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. The synthetic process for ibuprofen can be found in U.S. Patent No. 3,228,831, the entire contents of which are incorporated herein by reference. The structural formula of ibuprofen is...

[0557] Other exemplary aralkyl groups are described in U.S. Patent No. 7,626,014, which is incorporated herein by reference in its entirety.

[0558] In another embodiment, suitable lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, ibuprofen, naproxen, dimethoxytriphenylmethyl, or phenoxazine.

[0559] In some embodiments, more than one lipophilic moiety may be incorporated into the double-stranded RNAi agent, particularly when the lipophilic moiety is of low lipophilicity or hydrophobicity. In one embodiment, two or more lipophilic moieties are incorporated into the same strand of the double-stranded RNAi agent. In one embodiment, each strand of the double-stranded RNAi agent incorporates one or more lipophilic moieties. In one embodiment, two or more lipophilic moieties are incorporated into the same position of the double-stranded RNAi agent (i.e., the same nucleotide, the same sugar moiety, or the same internucleotide bond). This can be achieved, for example, by binding two or more lipophilic moieties via a vector and / or by binding two or more lipophilic moieties via a branched linker and / or by binding two or more lipophilic moieties via one or more linkers having one or more consecutively connected lipophilic moieties.

[0560] The lipophilic moiety can bind to RNAi agents by directly linking to the ribose of the RNAi agent. Alternatively, the lipophilic moiety can bind to double-stranded RNAi agents via a linker or vector.

[0561] In some embodiments, the lipophilic portion can bind to the RNAi agent via one or more linkers (linkage strands).

[0562] In one embodiment, the lipophilic moiety is bound to a double-stranded RNAi agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, click reaction product (e.g., triazole from azide-alkyne cycloaddition), or carbamate.

[0563] The exemplary linkers, ligands, vectors, nucleic acid modifications, conjugates, ligands, and other parts of RNAi agents for the targeted delivery of the APPs of this application to the central nervous system are, for example, U.S. Applications Nos. 62 / 668,072, 62 / 738,747, and / or 62 / 773,082, the entire contents of which are incorporated herein by reference.

[0564] B. Lipid conjugates

[0565] In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. This lipid or lipid-based molecule preferably binds to a serum protein, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to be distributed vascularly to target tissues, such as non-renal target tissues of the body. In some embodiments, the target tissue may be the CNS, including glial cells of the brain. Other molecules that can bind HSA may also be used as ligands. For example, neproxin or aspirin may be used. Lipid or lipid-based ligands may (a) increase resistance to conjugate degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to modulate binding to serum proteins, such as HSA.

[0566] Lipid-based ligands can inhibit, for example, control the binding of the conjugate to the target tissue. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to target the kidney and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the conjugate to the kidney.

[0567] Optionally, lipid-based ligands bind to HSA. Preferably, the ligands bind to HSA with sufficient affinity such that the conjugate is preferably distributed to non-renal tissues. However, the affinity is preferably not strong enough to prevent reversing HSA-ligand binding.

[0568] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to the HSA, such that the conjugate is preferably distributed to the kidney. Other portions targeting renal cells may also be used instead of the lipid-based ligand, or ligands other than the lipid-based ligand.

[0569] On the other hand, ligands are the portions absorbed by target cells, such as proliferating cells, like vitamins. These are particularly useful for treating diseases characterized by unwanted cell proliferation, such as malignant or non-malignant cell types, such as cancer cells. Examples of vitamins include vitamins A, E, and K. Other examples of vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients absorbed by target cells, such as brain cells. Also included are HSA and low-density lipoprotein (LDL).

[0570] C. Cell permeabilizer

[0571] On the other hand, the ligand is a cell permeabilizer, preferably a helical cell permeabilizer. Preferably, the agent is amphiphilic. Exemplary agents are peptides, such as tat or antennopedia. If the agent is a peptide, it can be modified, including peptide analogs, transformation isomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent, which preferably has both a lipophilic and a lipophobic phase.

[0572] The ligand can be a peptide or a peptide analog. A peptide analog (also referred to herein as an oligopeptide analog) is a molecule capable of folding into a defined three-dimensional structure similar to that of a natural peptide. The attachment of peptides and peptide analogs to RNAi agents can influence the pharmacokinetic distribution of RNAi agents, such as by enhancing cellular recognition and absorption. The peptide or peptide analog moiety can be approximately 5 to 50 amino acids in length, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0573] Peptides or peptide analogs can be, for example, cell-permeable peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., primarily composed of Tyr, Trp, or Phe). The peptide moiety can be a dendritic peptide, a restricted peptide, or a cross-linked peptide. In another alternative, the peptide moiety may include a hydrophobic membrane translocation sequence (MTS). An example peptide containing a hydrophobic MTS is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 29). RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 30)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide that can carry highly polar molecules, including peptides, oligonucleotides, and transcellular proteins across the membrane. For example, sequences from HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 31) and Drosophila antennal foot protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 32)) have been found to function as delivery peptides. Peptides or peptide analogs can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or monobead-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Examples of peptides or peptide analogs tethered to dsRNA agents via incorporated monomeric units for cell targeting purposes are arginine-glycine-aspartic (RGD) peptides or RGD analogs. The length of the peptide moiety can range from about 5 amino acids to about 40 amino acids. The peptide moiety can have structural modifications, for example, to increase stability or direct conformational properties. Any structural modifications described below can be utilized.

[0574] The RGD peptides used in the compositions and methods of this application may be linear or cyclic and may be modified, for example, by glycosylation or methylation, to facilitate targeting of specific tissues. RGD-containing peptides and peptide analogs may include D-amino acids, as well as synthetic RGD analogs. In addition to RGD, other portions of integrin ligands may be used. Preferred conjugates of said ligands target PECAM-1 or VEGF.

[0575] "Cell-permeable peptides" are capable of penetrating cells, such as microbial cells, like bacterial or fungal cells, or mammalian cells, like human cells. Microbial cell-permeable peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), peptides containing disulfide bonds (e.g., α-defensins, β-defensins, or bacteriocins), or peptides containing only one or two major amino acids (e.g., PR-39 or indomethacin). Cell-permeable peptides may also include nuclear localization signals (NLS). For example, cell-permeable peptides can be amphiphilic peptides, such as MPG, which derives from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0576] D. Carbohydrate complexes and ligands

[0577] In some embodiments of the compositions and methods of this application, the RNAi agent oligonucleotide further comprises a carbohydrate. Carbohydrate-bound RNAi agents facilitate in vivo delivery of nucleic acids and are compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" means a compound that is itself composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) bonded to each carbon atom; or a compound having a carbohydrate portion as part thereof, said carbohydrate portion being composed of one or more monosaccharide units, each monosaccharide unit having at least six carbon atoms (which may be linear, branched, or cyclic), each carbon atom bonded to an oxygen, nitrogen, or sulfur atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides comprising about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars with C5 or more (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars with two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0578] In one embodiment, the carbohydrate conjugate used in the composition and methods of this application is a monosaccharide.

[0579] In some embodiments, the compositions and methods of this application include a C16 ligand. In exemplary embodiments, the C16 ligand of this application has the following structure (described below for uracil bases, but applicable to any base (C, G, A, etc.) and / or any other modifications as described herein, provided the 2' ribose linkage is retained) and is linked at the 2' position of the ribose within such modified residues:

[0580]

[0581] Chemical formula: C 25 H 43 N2O8P

[0582] Precision quality: 530.2757

[0583] Molecular weight: 530.5913

[0584] As shown above, the residues modified with C16 ligands have a straight-chain alkyl group at the 2'-ribose position of such an example residue (in this case, uracil).

[0585] In some embodiments, a carbohydrate conjugate of the RNAi agent of this application may also include, as described above, for example, but not limited to, one or more additional ligands of a PK regulator and / or a cell permeation peptide.

[0586] Other carbohydrate conjugates (and linkers) applicable to this application include those described in PCT Publications WO 2014 / 179620 and WO 2014 / 179627, the entire contents of which are incorporated herein by reference.

[0587] In some embodiments, the compositions and methods of this application include vinyl phosphate (VP) modification of the RNAi agents as described herein. In exemplary embodiments, the vinyl phosphate of this application has the following structure:

[0588]

[0589] The vinyl phosphate of this application can be linked to the antisense or sense strand of the dsRNA of this application. In some preferred embodiments, the vinyl phosphate of this application is linked to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0590] The compositions and methods of this application also consider vinyl phosphate modification. An exemplary vinyl phosphate structure is:

[0591]

[0592] E. Thermally unstable modification

[0593] In some embodiments, dsRNA molecules can optimize RNA interference to reduce or suppress off-target gene silencing by incorporating a thermally unstable modification in the seed region of the antisense strand (i.e., positions 2 to 9 at the 5' end of the antisense strand). It has been found that dsRNAs having an antisense strand containing at least one double-stranded thermally unstable modification within the first 9 nucleotide positions (counting from the 5' end) of the antisense strand reduce off-target gene silencing activity. Therefore, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five, or more) double-stranded thermally unstable modification within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more thermally unstable modifications of the double-stranded modification are located at positions 2 to 9, or preferably positions 4 to 8, from the 5' end of the antisense strand. In some further embodiments, the thermally unstable modification of the double-stranded modification is located at positions 6, 7, or 8, from the 5' end of the antisense strand. In other embodiments, the thermally unstable modification of the double-stranded modification is located at position 7, from the 5' end of the antisense strand. The term "thermally unstable modification" includes modifications that result in a lower total melting temperature (Tm) for the dsRNA (preferably a Tm 1, 2, 3, or 4 degrees lower than the Tm of the dsRNA) and modifications that do not involve such modifications. In some embodiments, the thermally unstable modification of the duplex is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0594] Thermally unstable modifications may include, but are not limited to, baseless modifications; mismatches with opposite nucleotides in the opposite strand; and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNA) or glycol nucleic acids (GNA).

[0595] Examples of base-free modifications include, but are not limited to, the following:

[0596]

[0597] Where R = H, Me, Et, or OMe; R' = H, Me, Et, or OMe; R = H, Me, Et, or OMe

[0598]

[0599] B can be a modified or unmodified nucleobase.

[0600] Examples of sugar modifications include, but are not limited to, the following:

[0601]

[0602] B can be a modified or unmodified nucleobase.

[0603] In some embodiments, the thermally unstable modification of the duplex is selected from:

[0604]

[0605] B represents a modified or unmodified nucleobase, and the asterisk on each structure represents R, S, or a racemic mixture.

[0606] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose, for example, wherein any bonds between ribose carbons (e.g., C1' to C2', C2' to C3', C3' to C4', C4' to O4', or C1' to O4') are absent and / or at least one of the ribose carbons or oxygen (e.g., C1', C2', C3', C4', or O4') is independently or in combination absent from the nucleotide. In some embodiments, an acyclic nucleotide is... Where B is a modified or unmodified nucleobase, and R... 1 and R 2 Independently, it is H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term “UNA” refers to an unlocked acyclic nucleic acid in which any bond of the sugar has been removed, forming an unlocked “sugar” residue. In one instance, UNA also includes monomers in which the bond between C1’ and C4’ has been removed (i.e., the covalent carbon-oxygen-carbon bond between C1’ and C4’ carbons). In another instance, the C2’-C3’ bond of the sugar (i.e., the covalent carbon-carbon bond between C2’ and C3’ carbons) has been removed (see Mikhailov et al., Tetrahedron Letters, 26(17):2059(1985); and Fluiter et al., Mol. Biosyst., 10:1039(2009), which are incorporated herein by reference in their entirety). Acyclic derivatives offer greater skeletal flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked by 2'-5' or 3'-5' bonds.

[0607] The term "GNA" refers to ethylene glycol nucleic acid, a polymer similar to DNA or RNA, but with a different "backbone" composed of repeating glycerol units linked by phosphodiester bonds.

[0608]

[0609] The thermally unstable modification of the duplex can be a mismatch (i.e., a non-complementary base pair) between the opposite nucleotides in the thermally unstable nucleotide and the dsRNA duplex. Exemplary mismatched base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatched base pairings known in the art are also applicable to this invention. Mismatches can occur between nucleotides of naturally occurring nucleotides or modified nucleotides; that is, mismatched base pairings can occur between nucleobases from individual nucleotides, regardless of modifications to the ribose of the nucleotide. In some embodiments, the dsRNA molecule contains at least one nucleobase in the mismatched pair, namely a 2'-deoxynucleobase; for example, the 2'-deoxynucleobase is in the positive strand.

[0610] In some embodiments, thermally unstable modifications of the duplex in the antisense seed region include nucleotides with impaired WCH-bonding to complementary bases on the target mRNA, such as:

[0611]

[0612] Further examples of non-base nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0613] Thermally unstable modifications may also include general base and phosphate modifications that have the ability to reduce or eliminate hydrogen bonding with the relative base.

[0614] In some embodiments, thermally unstable modifications of the duplex include nucleotides with atypical bases, such as, but not limited to, nucleobase modifications that impair or completely eliminate the ability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex, as described in WO 2010 / 0011895, which is incorporated herein by reference in its entirety. Example nucleobase modifications are:

[0615]

[0616] In some embodiments, the thermally unstable modification of the duplex in the seed region of the antisense strand includes one or more nucleotides complementary to bases on the target mRNA, for example:

[0617]

[0618] Wherein, R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0619] Compared to natural phosphodiester bonds, the following are examples of phosphate ester modifications that are known to reduce the thermal stability of the dsRNA duplex:

[0620]

[0621] The alkyl group of R can be a C1-C6 alkyl group. Specific alkyl groups of R include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0622] As those skilled in the art will recognize, given that the functional role of nucleosides defines the specificity of the RNAi agents of this application, and that nucleobase modifications can be performed in various ways as described herein, such as introducing destabilizing modifications into the RNAi agent, for example, to enhance the on-target effect relative to off-target effects, the range of modifications available and commonly present on the RNAi agents of this application tends to be much larger than that of non-nucleobase modifications, such as modifications to the glycosyl and / or phosphate backbone of polynucleotides. Other parts of this application describe such modifications in more detail and explicitly contemplate RNAi agents of this application having native or modified nucleosides, as described above and / or elsewhere herein.

[0623] In addition to the antisense strand containing thermally unstable modifications, dsRNA may also contain one or more stable modifications. For example, dsRNA may contain at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stable modifications. Unrestricted, stable modifications can be present on a single strand. In some embodiments, both the sense and antisense strands contain at least two stable modifications. Stable modifications can occur on any nucleotide of the sense or antisense strand. For example, stable modifications can occur on every nucleotide of the sense and / or antisense strands; each stable modification can alternate on the sense or antisense strand; or the sense or antisense strand may contain alternating patterns of stable modifications. The alternating pattern of stable modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of stable modifications on the sense strand may vary relative to the alternating pattern of stable modifications on the antisense strand.

[0624] In some embodiments, the antisense chain includes at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Unrestricted, the stabilizing modifications in the antisense chain can be present at any position. In some embodiments, the antisense includes stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense includes stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In still other embodiments, the antisense includes stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0625] In some embodiments, the antisense strand includes at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification may be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., at a position -1 or +1 away from the destabilizing modification. In some embodiments, the antisense strand includes a stabilizing modification at each of the 5' and 3' ends of the destabilizing modification, i.e., at positions -1 and +1 away from the destabilizing modification.

[0626] In some embodiments, the antisense chain contains at least two stabilization modifiers at the 3' end of the destabilization modifier, i.e. at the +1 and +2 positions of the destabilization modifier position.

[0627] In some embodiments, the justice chain includes at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifiers. Unrestricted, stabilizing modifiers in the justice chain can be present at any position. In some embodiments, the justice chain includes stabilizing modifiers at positions 7, 10, and 11 from the 5' end. In some other embodiments, the justice chain includes stabilizing modifiers at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the justice chain includes stabilizing modifiers at positions opposite to or complementary to positions 11, 12, and 15 from the 5' end of the antisense chain. In some other embodiments, the justice chain includes stabilizing modifiers at positions opposite to or complementary to positions 11, 12, 13, and 15 from the 5' end of the antisense chain. In some embodiments, the justice chain includes segments with two, three, or four stabilizing modifiers.

[0628] In some embodiments, the justice chain does not contain stabilizing modifications at positions opposite to or complementary to the thermally unstable modifications of the bichain in the antisense chain.

[0629] Exemplary thermally stable modifications include, but are not limited to, 2'-fluorine modification. Other thermally stable modifications include, but are not limited to, LNA.

[0630] In some embodiments, the dsRNA of this application comprises at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2'-fluoronucleotides. There is no limitation; all 2'-fluoronucleotides can be present on a single strand. In some embodiments, both the sense and antisense strands comprise at least two 2'-fluoronucleotides. 2'-fluoro modifications can occur on any nucleotide of the sense or antisense strand. For example, 2'-fluoro modifications can occur on every nucleotide of the sense and / or antisense strands; each 2'-fluoro modification can alternate on the sense or antisense strand; or the sense or antisense strand may contain two 2'-fluoro modifications in alternating patterns. The alternating pattern of 2'-fluoro modifications on the sense strand can be the same as or different from that on the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand can be relative to the 2'-fluoro modifications on the antisense strand.

[0631] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoronucleotides. There is no limitation; the 2'-fluoro modification in the antisense strand can be present at any position. In some embodiments, positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand comprise 2'-fluoronucleotides. In some other embodiments, positions 2, 6, 14, and 16 at the 5' end of the antisense strand comprise 2'-fluoronucleotides. In still other embodiments, positions 2, 14, and 16 at the 5' end of the antisense strand comprise 2'-fluoronucleotides.

[0632] In some embodiments, the antisense strand comprises at least one 2'-fluoronucleotide adjacent to the destabilizing modification. For example, the 2'-fluoronucleotide may be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., at a position -1 or +1 from the destabilizing modification position. In some embodiments, the antisense strand comprises a 2'-fluoronucleotide at each of the 5' and 3' ends of the destabilizing modification, i.e., at positions -1 and +1 starting from the destabilizing modification position.

[0633] In some embodiments, the antisense strand contains at least two 2'-fluoronucleotides at the 3' end of the destabilization modification, i.e. at the +1 and +2 positions of the destabilization modification site.

[0634] In some embodiments, the positive strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoronucleotides. Without limitation, the 2'-fluoro modification in the positive strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoronucleotides at positions 7, 10, and 11 at the 5' end. In some other embodiments, the positive strand comprises 2'-fluoronucleotides at positions 7, 9, 10, and 11 at the 5' end. In some embodiments, the positive strand comprises 2'-fluoronucleotides at positions opposite to or complementary to positions 11, 12, and 15 at the 5' end of the antisense strand. In some other embodiments, the positive strand comprises 2'-fluoronucleotides at positions opposite to or complementary to positions 11, 12, 13, and 15 at the 5' end of the antisense strand. In some embodiments, the positive strand comprises blocks of two, three, or four 2'-fluoronucleotides.

[0635] In some embodiments, the sense strand does not contain a 2'-fluoronucleotide at a position in the antisense strand that is opposite to or complementary to the thermally unstable modification of the bistrand.

[0636] In some embodiments, the dsRNA molecule of this application comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand comprises at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present in the seed region of the antisense strand (i.e., at positions 2 to 9 of the 5' end of the antisense strand), wherein one end of the dsRNA is blunt and the other end comprises a 2-nt overhang, wherein the dsRNA optionally further has at least one of the following features (e.g., one, two, three, four). (i) The antisense contains 2, 3, 4, 5, or 6 2'-fluorine modifications; (ii) The antisense contains 1, 2, 3, 4, or 5 phosphate thioester nucleotides bonded together; (iii) The sense strand binds to the ligand; (iv) The sense strand contains 2, 3, 4, or 5 2'-fluorine modifications; (v) The sense strand contains 1, 2, 3, 4, or 5 phosphate thioester nucleotides bonded together; (vi) The dsRNA contains at least four 2'-fluorine modifications; (vii) The dsRNA has a blunt end at the 5' end of the antisense strand. Preferably, the 2nt overhang is located at the 3' end of the antisense.

[0637] In some embodiments, the dsRNA molecule of this application comprises a sense strand and an antisense strand, wherein: the sense strand is 25 to 30 nucleotide residues in length, wherein positions 1 to 23 of the sense strand, starting from the 5' terminal nucleotide (position 1), contain at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, starting from the 3' terminal nucleotide, and contains at least 8 ribonucleotides at positions 1 to 23 of the sense strand to form a double strand; wherein at least the 3' terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; wherein the 5' end of the antisense strand contains 10 Up to 30 consecutive nucleotides unpaired with the sense strand, forming a single-stranded 5' overhang of 10 to 30 nucleotides; wherein, when the sense and antisense strands are aligned for maximum complementarity, at least the 5' and 3' terminal nucleotides of the sense strand are paired with nucleotide bases of the antisense strand, thereby forming a substantially double-stranded region between the sense and antisense strands; when said double-stranded nucleic acid is introduced into mammalian cells, the antisense strand is sufficiently complementary to the target RNA along at least 19 antisense strand lengths of ribonucleotides to reduce the expression of the target gene; and wherein the antisense strand contains at least one thermally unstable nucleotide, wherein at least one thermally unstable nucleotide is in the seed region of the antisense strand (i.e., at positions 2 to 9 at the 5' end of the antisense strand). For example, the thermally unstable nucleotides occur between positions 14 to 17 opposite or complementary to the 5' end of the sense strand, and the dsRNA optionally also has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluorine modifications; (ii) the antisense contains 1, 2, 3, 4, or 5 phosphate thioester nucleotides bonded together; (iii) the sense strand is bound to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluorine modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphate thioester nucleotides bonded together; (vi) the dsRNA contains at least four 2'-fluorine modifications; and (vii) the dsRNA contains a double-stranded region of 12 to 30 nucleotide pairs in length.

[0638] In some embodiments, the dsRNA molecule of this application comprises a sense strand and an antisense strand, wherein the dsRNA molecule comprises a sense strand of at least 25 and at most 29 nucleotides in length and an antisense strand of at most 30 nucleotides in length, wherein the nucleotides having the sense strand comprise modified nucleotides that are susceptible to enzymatic degradation from the 5' end position 11, wherein the 3' end of the sense strand and the 5' end of the antisense strand form blunt ends and the antisense strand is 1 to 4 nucleotides longer than the sense strand at its 3' end, wherein when the dsRNA molecule is introduced into mammalian cells, it reduces the expression of the target gene, and wherein the cleavage of the dsRNA by a cleavage enzyme preferentially produces the siRNA antisense strand containing the 3' end, thereby reducing the expression of the target gene in mammals, wherein the length of the double-stranded region is at least 25 nucleotides, and the antisense strand is coupled to the target mRNA along at least 19 nt of the antisense strand length. The NA is fully complementary, wherein the antisense strand contains at least one thermally unstable nucleotide, wherein the at least one thermally unstable nucleotide is located in the seed region of the antisense strand (i.e., position 2 to 9 of the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one of the following features (e.g., one, two, three, four, five, six, or all seven): (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluorine modifications; (ii) the antisense contains 1, 2, 3, 4, or 5 phosphate thioester nucleotides bonded together; (iii) the sense strand is bound to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluorine modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphate thioester nucleotides bonded together; (vi) the dsRNA contains at least four 2'-fluorine modifications; and (vii) the dsRNA has a double-stranded region of 12 to 29 nucleotide pairs in length.

[0639] In some embodiments, each nucleotide in the sense and antisense strands of the dsRNA molecule can be modified. Each nucleotide can be modified with the same or different modifications, which may include one or more changes to one or two of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens; changes to the composition of the ribose, for example, changing the 2' hydroxyl group on the ribose; replacing the phosphate portion with a "dephosphorylated" linker batch; modifying or replacing naturally occurring bases; and replacing or modifying the ribose-phosphate backbone.

[0640] Nucleic acids are polymers of subunits, and many modifications occur at repetitive positions within the nucleic acid, such as modifications to the base or phosphate groups or non-linked O nucleotides of the phosphate group. In some cases, modifications occur at all subject positions in the nucleic acid, but in many cases not. For example, modifications may occur only at the 3' or 5' end positions, or only in terminal regions, such as positions on terminal nucleotides or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of RNA or only in single-stranded regions of RNA. For example, phosphate thioester modifications at non-linked O positions may occur only at one or both ends, or only in terminal regions, such as positions on terminal nucleotides or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in both double-stranded and single-stranded regions, particularly at the ends. The 5' end or end may be phosphorylated.

[0641] It is possible, for example, to include specific bases at the overhang, or to include modified nucleotides or nucleotide substitutes at single-stranded overhangs, such as at the 5' or 3' overhang, or both, to enhance stability. For example, it may be necessary to include purine nucleotides at the overhang. In some embodiments, all or some bases at the 3' or 5' overhang may be modified, for example, with the modifications described herein. Modifications may include, for example, modification at the 2' position of the ribose using modifications known in the art, such as using deoxyribonucleotides, ribose modified with 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl groups instead of nucleobases, and modifications in the phosphate group, such as thiophosphate modifications. The overhang does not need to be homologous to the target sequence.

[0642] In some embodiments, each residue of the sense and antisense strands is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluorine. The strands may contain multiple modifications. In some embodiments, each residue of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluorine. It should be understood that these modifications are complementary to at least one thermally unstable modification present in the bistrand in the antisense strand.

[0643] At least two different modifications are typically present on the sense and antisense strands. These two modifications can be 2'-deoxy, 2'-O-methyl, or 2'-fluorine modifications, acyclic nucleotides, or other modifications. In some embodiments, the sense and antisense strands each contain two different modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue of the sense and antisense strands is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluorine nucleotide, a 2'-ON-methylacetamide (2'-O-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl (2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. Similarly, it should be understood that these modifications complement at least one thermally unstable modification of the bistrand present in the antisense strand.

[0644] In some embodiments, the dsRNA molecule of this application contains alternating patterns of modification, particularly in the regions B1, B2, B3, B1', B2', B3', and B4'. As used herein, the term "alternating motif" or "alternative pattern" refers to having one or more modified motifs, each modification occurring on an alternating nucleotide on one strand. Alternating nucleotides can refer to every other nucleotide or every three nucleotides, or a similar pattern. For example, if A, B, and C represent modifications to a substance of a nucleotide, the alternating motif could be "ABABABABABAB…," "AABBAABBAABB…," "AABAABAABAAB…," "AAABAAABAAAB…," "AAABBBAAABBB…," or "ABCABCABCABC…," etc.

[0645] The types of modifications contained in an alternating motif can be the same or different. For example, if A, B, C, and D each represent a type of modification on a nucleotide, the alternating pattern, i.e., the modification every other nucleotide, can be the same, but each sense or antisense strand can be chosen from several modification possibilities within the alternating motif, such as “ABABAB…”, “ACACAC…”, “BDBDBD…”, or “CDCDCD…”, etc.

[0646] In some embodiments, the dsRNA molecule of this application comprises a modification pattern of alternating motifs on the sense strand, relative to the modification pattern of alternating motifs on the antisense strand. This transition allows the modification set of nucleotides on the sense strand to correspond to a different modification set of nucleotides on the antisense strand, and vice versa. For example, when the sense strand from a double-stranded region pairs with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may begin with “ABABAB” at the 5'-3' position of the strand, and the alternating motif in the antisense strand may begin with “BABABA” at the 3'-5' position of the strand. As another example, in the double-stranded region, the alternating motif in the sense strand may begin with “AABBAABB” at the 5'-3' position of the strand, and the alternating motif in the antisense strand may begin with “BBAABBAA” at the 3'-5' position of the strand, resulting in a complete or partial transfer of the modification pattern between the sense and antisense strands.

[0647] The dsRNA molecule of this application may further comprise at least one phosphate thioester or methyl phosphate nucleotide inter-bond. The phosphate thioester or methyl phosphate nucleotide inter-bond modification may occur at any position on any nucleotide of the sense or antisense strand, or both. For example, the inter-nucleotide inter-bond modification may occur on each nucleotide of the sense and / or antisense strand; each inter-nucleotide inter-bond modification may occur in an alternating pattern on the sense or antisense strand; or the sense or antisense strand may contain two inter-nucleotide inter-bond modifications in alternating patterns. The alternating pattern of the inter-nucleotide inter-bond modification on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of the inter-nucleotide inter-bond modification on the sense strand may vary relative to the alternating pattern of the inter-nucleotide inter-bond modification on the antisense strand.

[0648] In some embodiments, the dsRNA molecule includes phosphate-thiophosphate or methylphosphate nucleotide inter-nucleotide (PTMN) bonds in the overhang region. For example, the overhang region contains two nucleotides with a phosphate-thiophosphate or methylphosphate nucleotide inter-nucleotide bond between them. PTMN bonds can also be made to link the overhang nucleotide to a terminal pairing nucleotide within the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides can be linked by a phosphate-thiophosphate or methylphosphate nucleotide inter-nucleotide bond, and optionally, additional phosphate-thiophosphate or methylphosphate nucleotide inter-nucleotide bonds can link the overhang nucleotide to a pairing nucleotide immediately adjacent to the overhang nucleotide. For example, at least two phosphate-thiophosphate nucleotide inter-nucleotide bonds may exist between the three terminal nucleotides, wherein two of the three nucleotides are overhang nucleotides and the third is a pairing nucleotide adjacent to the overhang nucleotide. Preferably, these three terminal nucleotides may be located at the 3' end of the antisense strand.

[0649] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphate thioester or methyl phosphate nucleotides separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate ester nucleotides, wherein one of the phosphate thioester or methyl phosphate nucleotides is located at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphate thioester, methyl phosphate, and phosphate ester nucleotides or an antisense strand comprising phosphate thioester, methyl phosphate, or phosphate ester bonds.

[0650] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphate thioester or methyl phosphate nucleotides separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate ester nucleotides, wherein one of the phosphate thioester or methyl phosphate nucleotides is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphate thioester, methyl phosphate, and phosphate ester nucleotides, or an antisense strand comprising phosphate thioester, methyl phosphate, or phosphate ester bonds.

[0651] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphate thioester or methyl phosphate nucleotides separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate ester nucleotides, wherein one of the phosphate thioester or methyl phosphate nucleotides is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphate thioester, methyl phosphate, and phosphate ester nucleotides or an antisense strand comprising phosphate thioester, methyl phosphate, or phosphate ester nucleotides.

[0652] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphate thioester or methyl phosphate nucleotides separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate ester nucleotides, wherein one of the phosphate thioester or methyl phosphate nucleotides is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphate thioester, methyl phosphate, and phosphate ester nucleotides, or an antisense strand comprising any one of phosphate thioester, methyl phosphate, or phosphate ester nucleotides.

[0653] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphate thioester or methyl phosphate nucleotides separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate nucleotides, wherein one of the phosphate thioester or methyl phosphate nucleotides is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphate thioester, methyl phosphate, and phosphate nucleotides, or an antisense strand comprising phosphate thioester, methyl phosphate, or phosphate bonds.

[0654] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphate-thiophosphate or methylphosphate nucleotides separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate nucleotides, wherein one of the phosphate-thiophosphate or methylphosphate nucleotides is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphate-thiophosphate, methylphosphate, and phosphate nucleotides, or an antisense strand comprising phosphate-thiophosphate, methylphosphate, or phosphate nucleotides.

[0655] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphate-thiophosphate or methylphosphate nucleotides separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate ester nucleotides, wherein one of the phosphate-thiophosphate or methylphosphate nucleotides is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphate-thiophosphate, methylphosphate, and phosphate ester nucleotides, or an antisense strand comprising a phosphate-thiophosphate, methylphosphate, or phosphate ester bond.

[0656] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphate thioester or methyl phosphate nucleotides separated by 1, 2, 3, 4, 5, or 6 phosphate ester nucleotides, wherein one of the phosphate thioester or methyl phosphate nucleotides is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphate thioester, methyl phosphate, and phosphate ester nucleotides, or an antisense strand comprising phosphate thioester, methyl phosphate, or phosphate ester bonds.

[0657] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphate thioester or methyl phosphate nucleotides separated by one, two, three, or four phosphate ester nucleotides, wherein one of the phosphate thioester or methyl phosphate nucleotides is located in an oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphate thioester, methyl phosphate, and phosphate ester nucleotides, or an antisense strand comprising phosphate thioester, methyl phosphate, or phosphate ester nucleotides.

[0658] In some embodiments, the dsRNA molecule of this application further comprises one or more phosphate thioester or methyl phosphate nucleotide inter-linking modifications at 1 to 10 terminal positions of the sense and / or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked by phosphate thioester or methyl phosphate nucleotide inter-linking at one or both ends of the sense and / or antisense strands.

[0659] In some embodiments, the dsRNA molecule of this application further comprises one or more phosphate thioester or methyl phosphate nucleotide inter-linking modifications within 1 to 10 regions of the inner region of the duplex of each sense strand and / or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked by phosphate thioester or methyl phosphate nucleotide inter-linking at positions 8 to 16 from the 5' end of the sense strand in the duplex region; the dsRNA molecule may optionally further comprise one or more phosphate thioester or methyl phosphate nucleotide inter-linking modifications within 1 to 10 terminal positions.

[0660] In some embodiments, the dsRNA molecule of this application further includes 1 to 5 phosphate thioester or methyl phosphate nucleotide inter-linking modifications at positions 1 to 5 on the sense strand and 1 to 5 phosphate thioester or methyl phosphate nucleotide inter-linking modifications at positions 18 to 23 on the sense strand (counted from the 5' end), and 1 to 5 phosphate thioester or methyl phosphate nucleotide inter-linking modifications at positions 1 and 2 on the antisense strand, and 1 to 5 modifications at positions 18 to 23 on the antisense strand (counted from the 5' end).

[0661] In some embodiments, the dsRNA molecule of this application further comprises one phosphate thioester nucleotide inter-linking modification at positions 1 to 5 and one phosphate thioester or methyl phosphate nucleotide inter-linking modification at positions 18 to 23 of the sense strand (counting from the 5' end), and one phosphate thioester nucleotide inter-linking modification at positions 1 and 2 of the antisense strand, and two phosphate thioester or methyl phosphate nucleotide inter-linking modifications at positions 18 to 23 of the antisense strand (counting from the 5' end).

[0662] In some embodiments, the dsRNA molecule of this disclosure further includes two inter-thiophosphate nucleotide binding modifications at positions 1 to 5 on the sense strand and one inter-thiophosphate nucleotide binding modification (counted from the 5' end) at positions 18 to 23 on the sense strand, as well as one inter-thiophosphate nucleotide binding modification at positions 1 and 2 and two inter-thiophosphate nucleotide binding modifications at positions 18 to 23 (counted from the 5' end) on the antisense strand.

[0663] In some embodiments, the dsRNA molecule of this application further comprises two phosphate thioester nucleotides bonded to each other at positions 1 to 5 of the sense strand and two phosphate thioester nucleotides bonded to each other at positions 18 to 23 of the sense strand (counted from the 5' end), as well as one phosphate thioester nucleotide bonded to each other at positions 1 and 2 and two phosphate thioester nucleotides bonded to each other at positions 18 to 23 of the antisense strand (counted from the 5' end).

[0664] In some embodiments, the dsRNA molecule of this application further comprises two phosphate thioester nucleotide inter-linking modifications at positions 1 to 5 of the sense strand and two phosphate thioester nucleotide inter-linking modifications at positions 18 to 23 of the sense strand (counting from the 5' end), as well as one phosphate thioester nucleotide inter-linking modification at positions 1 and 2 and one phosphate thioester nucleotide inter-linking modification at positions 18 to 23 of the antisense strand (counting from the 5' end).

[0665] In some embodiments, the dsRNA molecule of this application further includes one phosphate nucleotide inter-linking modification at positions 1 to 5 on the sense strand and one phosphate nucleotide inter-linking modification at positions 18 to 23 on the sense strand (counting from the 5' end), as well as two phosphate nucleotide inter-linking modifications at positions 1 and 2 and two phosphate nucleotide inter-linking modifications at positions 18 to 23 on the antisense strand (counting from the 5' end).

[0666] In some embodiments, the dsRNA molecule of this application further has one phosphate nucleotide inter-linking modification (counted from 5' to the end) at positions 1 to 5 and 18 to 23 on the sense strand, two phosphate nucleotide inter-linking modifications at positions 1 and 2, and one phosphate nucleotide inter-linking modification at positions 18 to 23 on the antisense strand (counted from the 5' end).

[0667] In some embodiments, the dsRNA molecule of this application further includes a phosphate thionucleotide inter-linking modification at positions 1 to 5 (counting from the 5' end) on the sense strand, and two phosphate thionucleotide inter-linking modifications at positions 1 and 2, and one phosphate thionucleotide inter-linking modification at positions 18 to 23 (counting from the 5' end) on the antisense strand.

[0668] In some embodiments, the dsRNA molecule of this application further comprises two inter-thiophosphate nucleotide bonding modifications at positions 1 to 5 (counting from the 5' end) on the sense strand, one inter-thiophosphate nucleotide bonding modification at positions 1 and 2, and two inter-thiophosphate nucleotide bonding modifications at positions 18 to 23 (counting from the 5' end) on the antisense strand.

[0669] In some embodiments, the dsRNA molecule of this application further includes two inter-thiophosphate nucleotide binding modifications (counted from the 5' end) at positions 1 to 5 and 18 to 23 on the sense strand, and two inter-thiophosphate nucleotide binding modifications at positions 1 and 2, and one inter-thiophosphate nucleotide binding modification at positions 18 to 23 on the antisense strand (counted from the 5' end).

[0670] In some embodiments, the dsRNA molecule of this application further includes two inter-thiophosphate nucleotide binding modifications at positions 1 to 5 on the sense strand and one inter-thiophosphate nucleotide binding modification (counted from the 5' end) at positions 18 to 23 on the sense strand, as well as two inter-thiophosphate nucleotide binding modifications at positions 1 and 2 and two inter-thiophosphate nucleotide binding modifications at positions 18 to 23 on the antisense strand (counted from the 5' end).

[0671] In some embodiments, the dsRNA molecule of this application further includes two phosphate thionucleotide inter-linked modifications at positions 1 to 5 on the sense strand and one phosphate thionucleotide inter-linked modification (counted from the 5' end) at positions 18 to 23 on the sense strand, as well as one phosphate thionucleotide inter-linked modification at positions 1 and 2 and two phosphate thionucleotide inter-linked modifications at positions 18 to 23 (counted from the 5' end) on the antisense strand.

[0672] In some embodiments, the dsRNA molecule of this application further includes inter-thiophosphate nucleotide bonding modifications at positions 1 and 2, inter-thiophosphate nucleotide bonding modifications at positions 20 and 21 of the sense strand (counting from the 5' end), and inter-thiophosphate nucleotide bonding modifications at positions 1 and 21 of the antisense strand (counting from the 5' end).

[0673] In some embodiments, the dsRNA molecule of this application further includes an inter-nucleotide thiophosphate bonded modification at position 1, an inter-nucleotide thiophosphate bonded modification at position 21 of the sense strand (counting from the 5' end), two inter-nucleotide thiophosphate bonded modifications at positions 1 and 2, and two inter-nucleotide thiophosphate bonded modifications at positions 20 and 21 of the antisense strand (counting from the 5' end).

[0674] In some embodiments, the dsRNA molecule of this application further includes two phosphate thioester nucleotide inter-linked modifications at positions 1 and 2 on the sense strand, two phosphate thioester nucleotide inter-linked modifications at positions 21 and 22 on the sense strand (counted from the 5' end), and a phosphate thioester nucleotide inter-linked modification at position 1 and a phosphate thioester nucleotide inter-linked modification at position 21 on the antisense strand (counted from the 5' end).

[0675] In some embodiments, the dsRNA molecule of this application further includes two phosphate thioester nucleotide inter-linked modifications at positions 1 and 2 of the sense strand, two phosphate thioester nucleotide inter-linked modifications at positions 21 and 22 of the sense strand (counted from the 5' end), and a phosphate thioester nucleotide inter-linked modification at position 1 and a phosphate thioester nucleotide inter-linked modification at position 21 of the antisense strand (counted from the 5' end).

[0676] In some embodiments, the dsRNA molecule of this application further includes an inter-thiophosphate nucleotide bonded modification at position 1, and an inter-thiophosphate nucleotide bonded modification at position 21 on the sense strand (counted from the 5' end), and two inter-thiophosphate nucleotide bonded modifications at positions 1 and 2 and at positions 23 and 23 are antisense strands (counted from the 5' end).

[0677] In some embodiments, the dsRNA molecule of this application further includes an inter-thiophosphate nucleotide bonded modification at position 1 of the sense strand and an inter-thiophosphate nucleotide bonded modification at position 21 of the sense strand (counted from the 5' end), and two inter-thiophosphate nucleotide bonded modifications at positions 1 and 2 and at positions 23 and 23 are antisense strands (counted from the 5' end).

[0678] In some embodiments, the compounds of this application comprise a pattern of skeletal chiral centers. In some embodiments, a common pattern of the main-chain chiral center comprises at least 5 internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the main-chain chiral center comprises at least 6 internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the main-chain chiral center comprises at least 7 internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the main-chain chiral center comprises at least 8 internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the main-chain chiral center comprises at least 9 internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the main-chain chiral center comprises at least 10 internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the main-chain chiral center comprises at least 11 internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the main-chain chiral center comprises at least 12 internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the main-chain chiral center comprises at least 13 internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the main-chain chiral center comprises at least 14 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of the main-chain chiral center includes at least 15 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of the main-chain chiral center includes at least 16 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of the main-chain chiral center includes at least 17 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of the main-chain chiral center includes at least 18 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of the main-chain chiral center includes at least 19 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of the main-chain chiral center includes no more than 8 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of the main-chain chiral center includes no more than 7 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of the main-chain chiral center includes no more than 6 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of the main-chain chiral center includes no more than 5 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of the main-chain chiral center includes no more than 4 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of the main-chain chiral center in the Rp configuration includes no more than 3 internucleotide bonds. In some embodiments, the common pattern of the main-chain chiral center in the Rp configuration includes no more than 2 internucleotide bonds. In some embodiments, the common pattern of the main-chain chiral center in the Rp configuration includes no more than 1 internucleotide bond. In some embodiments, the common pattern of the main-chain chiral center includes no more than 8 achiral internucleotide bonds (as a non-limiting example, a phosphodiester).In some embodiments, a common pattern of a main-chain chiral center comprises no more than 7 achiral nucleotide bonds. In some embodiments, a common pattern of a main-chain chiral center comprises no more than 6 achiral nucleotide bonds. In some embodiments, a common pattern of a main-chain chiral center comprises no more than 5 achiral nucleotide bonds. In some embodiments, a common pattern of a main-chain chiral center comprises no more than 4 achiral nucleotide bonds. In some embodiments, a common pattern of a main-chain chiral center comprises no more than 3 achiral nucleotide bonds. In some embodiments, a common pattern of a main-chain chiral center comprises no more than 2 achiral nucleotide bonds. In some embodiments, a common pattern of a main-chain chiral center comprises no more than 1 achiral nucleotide bond. In some embodiments, a common pattern of a main-chain chiral center comprises at least 10 nucleotide bonds and no more than 8 achiral nucleotide bonds in the Sp configuration. In some embodiments, a common pattern of a main-chain chiral center comprises at least 11 nucleotide bonds and no more than 7 achiral nucleotide bonds in the Sp configuration. In some embodiments, a common pattern of main-chain chiral centers comprises at least 12 intermolecular bonds and no more than 6 achiral intermolecular bonds in the Sp configuration. In some embodiments, a common pattern of main-chain chiral centers comprises at least 13 intermolecular bonds and no more than 6 achiral intermolecular bonds in the Sp configuration. In some embodiments, a common pattern of main-chain chiral centers comprises at least 14 intermolecular bonds and no more than 5 achiral intermolecular bonds in the Sp configuration. In some embodiments, a common pattern of main-chain chiral centers comprises at least 15 intermolecular bonds and no more than 4 achiral intermolecular bonds in the Sp configuration. In some embodiments, the nucleotide linkages in the Sp configuration are optionally continuous or discontinuous. In some embodiments, the nucleotide linkages in the Rp configuration are optionally continuous or discontinuous. In some embodiments, the achiral intermolecular linkages are optionally continuous or discontinuous.

[0679] In some embodiments, the compounds of this application comprise stereochemical blocks. In some embodiments, the blocks are Rp blocks because the internucleotide linkage of each nucleotide in the block is Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the blocks are Sp blocks because the internucleotide linkage of each nucleotide in the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, the provided oligonucleotide comprises Rp and Sp blocks. In some embodiments, the provided oligonucleotide comprises one or more Rp blocks but does not comprise Sp blocks. In some embodiments, the provided oligonucleotide comprises one or more Sp blocks but does not comprise Rp blocking. In some embodiments, the provided oligonucleotide comprises one or more PO blocks, wherein the internucleotide linkage is a native phosphate ester linkage.

[0680] In some embodiments, the compounds of this application comprise a 5'-block that is an Sp block, wherein each sugar moiety contains a 2'-F modification. In some embodiments, the 5'-block is an Sp block, wherein each intermolecular bond is a modified intermolecular bond and each sugar moiety contains a 2'-F modification. In some embodiments, the 5'-block is an Sp block, wherein each intermolecular bond is a phosphate thioester bond and each sugar moiety contains a 2'-F modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block, wherein each sugar moiety contains a 2'-F modification. In some embodiments, the 3'-block is an Sp block, wherein each intermolecular bond is a modified intermolecular bond and each sugar moiety contains a 2'-F modification. In some embodiments, the 3'-block is an Sp block, wherein the internucleotide bond is a phosphate thioester bond and each sugar moiety contains a 2'-F modification. In some embodiments, the 3'-block comprises four or more nucleoside units. In some embodiments, the 3'-block comprises five or more nucleoside units. In some embodiments, the 3'-block comprises six or more nucleoside units. In some embodiments, the 3'-block comprises seven or more nucleoside units.

[0681] In some embodiments, the compounds of this application comprise a nucleoside or oligonucleotide followed by a specific type of internucleotide bond in a region, such as a native phosphate bond, a modified internucleotide bond, an Rp chiral internucleotide bond, a Sp chiral internucleotide bond, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a native phosphate bond (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a native phosphate bond (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a native phosphate bond (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a native phosphate bond (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a native phosphate bond (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0682] In some embodiments, the antisense strand comprises phosphate thionucleotide inter-linking between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, wherein the antisense strand comprises at least one thermally unstable modification of the bistrand located in the seed region of the antisense strand. That is, positions 2 to 9 at the 5' end of the antisense strand, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluorine modifications; (ii) the antisense contains 3, 4, or 5 phosphate thioester nucleotide bonds; (iii) the sense strand binds to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluorine modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphate thioester nucleotide bonds; (vi) the dsRNA contains at least four 2'-fluorine modifications; (vii) the dsRNA contains a double-stranded region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0683] In some embodiments, the antisense strand comprises an inter-nucleotide thiophosphate bond between nucleotide positions 1 ...

Claims

1. A double-stranded RNAi agent for inhibiting the expression of amyloid precursor protein (APP) gene, wherein: The double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein: (a) the nucleotide sequence of the sense strand is 5'-GGCUACGAAAAUCCAACCUAA-3' (SEQ ID NO: 2735), wherein the sense strand is bound to a saturated or unsaturated C16 hydrocarbon chain; and (b) the nucleotide sequence of the antisense strand consists of the nucleotide sequence of 5'-UUAGGUTGGAUTUUCGUAGCCGU-3' (SEQ ID NO: 2743), Wherein, all nucleotides of the sense strand and all nucleotides of the antisense strand are independently modified by a nucleotide modification selected from the group consisting of 2'-O-methyl modification, 2'-fluoro modification, 2'-deoxy modification, 2'-O-hexadecyl modification and glycol nucleic acid (GNA) modification.

2. The double-stranded RNAi agent according to claim 1, wherein The antisense strand comprises one or more lipophilic moieties bound to one or more non-terminal nucleotide positions, optionally via a linker or carrier.

3. The double-stranded RNAi agent according to claim 1 or 2, in, The nucleotide modification includes a short sequence of 3' terminal deoxythymidine nucleotide (dT); further comprising at least one phosphorothioate internucleotide linkage; wherein the double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages; wherein at least one strand comprises a 3' overhang of at least 2 nucleotides; Wherein, the C16 hydrocarbon chain is wherein B is a nucleotide base or a nucleotide base analog, Optionally, wherein B is selected from the group consisting of adenine, guanine, cytosine, thymine and uracil; wherein the C16 hydrocarbon chain is bound by a carrier that replaces one or more nucleotides in the internal nucleotide position, Optionally, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolyl, isothiazolyl, quinoxalinyl, pyridazinyl, tetrahydrofuranyl and decahydronaphthyl; or a cyclic group based on the acyclic part of the serinol skeleton or the diethanolamine skeleton; wherein the C16 hydrocarbon chain is bound to the double-stranded RNAi agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bonding, the product of a click reaction, or a carbamate; wherein the C16 hydrocarbon chain is bound to a nucleobase, a sugar moiety or an internucleoside linkage; further comprising a phosphate or phosphate analog at the 5'-end of said antisense strand, Optionally, wherein the phosphate analog is 5'-vinyl phosphate (VP); wherein the C16 hydrocarbon chain is bound via a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharide or oligosaccharide of galactosamine, glucosamine, glucose, galactose, mannose and combinations thereof; and / or The 3' end of the sense strand is protected by an end cap having an amine cyclic group, wherein the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolyl, isothiazolyl, tetrahydrofuranyl and decahydronaphthyl.

4. The double-stranded RNAi agent according to claim 1 or 2, in, The C16 hydrocarbon chain is attached to the 3' end or the 5' end of the sense strand; wherein the agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage, Optionally, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3'-end of one strand, wherein the strand is the antisense strand or the strand is the sense strand, Optionally, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5'-end of one strand, wherein the strand is the antisense strand or the strand is the sense strand; and / or Optionally, wherein the phosphorothioate or methylphosphonate internucleotide linkages are located at the 5'- and 3'-ends of one strand.

5. The double-stranded ribonucleic acid (RNAi) agent according to claim 1 or 2, in, All nucleotides of the sense strand contain a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-end, wherein all nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, and / or Wherein, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3'-end.

6. The double-stranded RNAi agent according to claim 1, in, The antisense strand of the RNAi agent comprises at least one thermolabile modification of the duplex in the first 9 nucleotide positions of the 5' region or its precursor; and / or Optionally, wherein the RNAi agent comprises a and The thermolabile modification of the duplex of the group consisting of Wherein, B is a nucleobase.

7. The double-stranded RNAi agent according to claim 1, wherein The sense strand is a modified nucleotide sequence of 5'-gsgscua(Chd)gadAadAuccaaccusasa-3' (SEQ ID NO: 2734), and the antisense strand is a modified nucleotide sequence of 5'-VPusUfsaggu(Tgn)ggaudTuUfcdGuagccsgsu-3' (SEQ ID NO: 2742), wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C and U, respectively; s is a thiophosphate bond; Uf is 2'-fluoro U; dA, dG and dT are 2'-deoxy A, G and T, respectively; (Chd) is 2'-O-hexadecylcytidine-3'-phosphate; (Tgn) is thymidine-glycol nucleic acid (GNA) S-isomer; VP is 5'-vinyl phosphate.

8. The RNAi agent according to claim 1, wherein The sense strand consists of a modified nucleotide sequence of 5'-gsgscua(Chd)gadAadAuccaaccusasa-3' (SEQ ID NO: 2734); and the antisense strand consists of a modified nucleotide sequence of 5'-VPusUfsaggu(Tgn)ggaudTuUfcdGuagccsgsu-3' (SEQ ID NO: 2742), wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C and U, respectively; s is a phosphorothioate bond; Uf is 2'-fluoro U; dA, dG and dT are 2'-deoxy A, G and T, respectively; (Chd) is 2'-O-hexadecylcytidine-3'-phosphate; (Tgn) is thymidine-glycol nucleic acid (GNA) S-isomer; and VP is 5'-vinyl phosphate.

9. A cell comprising the double-stranded RNAi agent according to any one of claims 1 to 8. 10 . A pharmaceutical composition for inhibiting the expression of APP gene, comprising the double-stranded RNAi agent according to any one of claims 1 to 8.

11. The pharmaceutical composition according to claim 10, wherein The double-stranded RNAi agent is administered into a non-buffered solution; Wherein, the non-buffered solution is physiological saline or water; wherein the double-stranded RNAi agent is administered together with a buffer solution, Optionally, wherein the buffer solution comprises acetate, citrate, prolamin, carbonate or phosphate or any combination thereof; and / or Optionally, wherein the buffer solution is phosphate buffered saline (PBS).

12. A pharmaceutical composition comprising a double-stranded RNAi agent according to any one of claims 1 to 8, and a lipid preparation, Optionally, wherein The lipid formulation comprises LNPs.

13. Use of the double-stranded RNAi agent according to any one of claims 1 to 8 or the pharmaceutical composition according to any one of claims 10 to 12 in the preparation of a medicament for inhibiting the expression of the amyloid precursor protein (APP) gene in cells in vitro; or Use of a double-stranded RNAi agent according to any one of claims 1 to 8 or a pharmaceutical composition according to any one of claims 10 to 12 for the preparation of a medicament for inhibiting the expression of APP in an individual suffering from cerebral amyloid angiopathy (CAA), early-onset familial Alzheimer's disease (EOFAD) or Alzheimer's disease (AD); or A use of a double-stranded RNAi agent according to any one of claims 1 to 8 or a pharmaceutical composition according to any one of claims 10 to 12 in the preparation of a medicament for treating or preventing cerebral amyloid angiopathy (CAA), early-onset familial Alzheimer's disease (EOFAD) or Alzheimer's disease (AD) in an individual.

14. The use according to claim 13, wherein The cells are from an individual suffering from cerebral amyloid angiopathy (CAA), early-onset familial Alzheimer's disease (EOFAD), or Alzheimer's disease (AD), Optionally, wherein the individual is a human, Optionally, wherein the individual is selected from the group consisting of rhesus monkeys, cynomolgus monkeys, mice and rats, wherein the expression of APP is inhibited by at least about 30%; wherein the dosage of the drug is about 0.01 mg / kg to about 50 mg / kg; wherein the drug is in a form suitable for intrathecal administration; wherein the drug results in a reduction in Aβ accumulation, Optionally, the medicament results in a reduction in the accumulation of Aβ(1-40) and / or Aβ(1-42); wherein the medicament results in a reduction in the formation and / or accumulation of amyloid plaques in the individual; wherein the drug reduces the expression of a target gene in brain or spinal tissue; and / or Wherein, the brain or spinal tissue is selected from the group consisting of cortex, cerebellum, striatum, cervical vertebrae, lumbar vertebrae and thoracic vertebrae.

15. A method for inhibiting the expression of amyloid precursor protein (APP) gene in cells in vitro, the method comprising: (a) contacting the cell with the pharmaceutical composition according to any one of claims 10 to 12; as well as (b) maintaining the cell produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the APP gene, thereby inhibiting the expression of the APP gene in the cell.

16. The method according to claim 15, wherein: The APP expression is inhibited by at least about 30%.

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