Manifolds for MAPT adjustment

Abstract

We provide novel MAPT-targeting sequences. We also provide novel MAPT-targeting oligonucleotides for the treatment of neurodegenerative diseases. [Solution] A double-stranded (dsRNA) molecule comprising a sense strand and an antisense strand is provided, wherein the antisense strand comprises a sequence substantially complementary to one MAPT nucleic acid sequence from a specific sequence group.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 991,405, filed on March 18, 2020, and U.S. Provisional Application No. 63 / 071,106, filed on August 27, 2020, the entire disclosures of which are incorporated herein by reference.

[0002] Technical Field The present disclosure relates to novel MAPT - targeting sequences, novel branched oligonucleotides, and novel methods for treating and preventing MAPT - related neurodegeneration.

Background Art

[0003] Microtubule - associated protein tau (tau) is encoded by the MAPT gene located on chromosome 17q21 and is expressed throughout the central nervous system. The tau protein functions in the assembly and stabilization of microtubules in brain cells. Microtubules are essential for maintaining cell integrity, intracellular and intercellular transport, and promoting cell division. Therefore, microtubules are important for maintaining axonal transport and the structural integrity of cells. The tau protein is located within neurons, mainly within axons. The tau protein is also found in other nerve cells such as astrocytes and oligodendrocytes and performs similar functions.

[0004] Mutations within MAPT cause frontotemporal dementia with parkinsonism and progressive supranuclear palsy. Mutations within MAPT and hyperphosphorylated tau protein are further associated with Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and traumatic brain injury, affecting millions of people worldwide. Under pathological conditions, tau protein undergoes various intramolecular modifications, forming toxic oligomeric tau proteins and paired helical filaments, which further aggregate into neurofibrillary changes and form deposits in the brain (tauopathy). Since the regulation of tau is important for memory, tauopathy is associated with cognitive impairment. Effective treatments (both related to tau protein) are still lacking when stopping or reversing the progression of the highly prevalent Alzheimer's disease and Parkinson's disease. Therefore, it is necessary to efficiently and potently silence MAPT mRNA expression addressed in this application.

Summary of the Invention

[0005] In a first embodiment, the disclosure provides an RNA molecule having a nucleic acid sequence substantially complementary to any one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 292. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 295.

[0006] In another embodiment, the disclosure provides an RNA molecule having a nucleic acid sequence substantially complementary to any one of the MAPT nucleic acid sequences of SEQ ID NOs: 14-33, 299, and 302. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 24. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 26. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 32. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 33.In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 292. In some embodiments, the nucleic acid sequence is substantially complementary to the MAPT nucleic acid sequence of SEQ ID NO: 302.

[0007] In another embodiment, the Disclosure provides an RNA molecule having a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to any one of the nucleic acid sequences of SEQ ID NOs.34-46. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NOs.34. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 36. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 37. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 40.In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 41. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 42. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 43. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 45. In some embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to the nucleic acid sequence of SEQ ID NO: 46.

[0008] In one embodiment, the disclosure provides RNA molecules having a length of about 8 to about 80 nucleotides, and nucleic acid sequences substantially complementary to any one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295. In a particular embodiment, the RNA molecule has a length of about 8 to about 80 nucleotides (e.g., 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides). Otide (43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62 nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66 nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74 nucleotides, 75 nucleotides, 76 nucleotides, 77 nucleotides, 78 nucleotides, 79 nucleotides, or 80 nucleotides).

[0009] In certain embodiments, the RNA molecule is 10 to 50 nucleotides long (for example, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long).

[0010] In certain embodiments, the RNA molecule contains approximately 15 to approximately 25 nucleotides in length. In certain embodiments, the RNA molecule is 15 to 25 nucleotides in length (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).

[0011] In certain embodiments, the RNA molecule has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 14-33, 299, and 302.

[0012] In certain embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46. In certain embodiments, the RNA molecule has a nucleic acid sequence that is at least 90% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46 (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46). In certain embodiments, the RNA molecule has a nucleic acid sequence that is at least 95% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46 (for example, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46).

[0013] In certain embodiments, the RNA molecule includes single-stranded (ss)RNA or double-stranded (ds)RNA.

[0014] In certain embodiments, the RNA molecule is a dsRNA comprising a sense strand and an antisense strand. The antisense strand may contain a nucleic acid sequence substantially complementary to any one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295. For example, in certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 2. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 6. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 8. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 10. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 11. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 12. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 13. In certain embodiments, the nucleic acid sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 292. In certain embodiments, the nucleic acid sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 295.

[0015] In certain embodiments, the dsRNA includes an antisense strand complementary to at least 10, 11, 12, or 13 consecutive nucleotides of any one MAPT nucleic acid sequence of SEQ ID NOs: 1-13, 292, and 295. For example, in certain embodiments, the dsRNA includes an antisense strand complementary to a segment of 10-25 consecutive nucleotides of any one nucleic acid sequence of SEQ ID NOs: 1-13, 292, and 295 (for example, the segment of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 1, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, Segments of 23, 24 or 25 consecutive nucleotides, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 3, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 5, Segments of 22, 23, 24 or 25 consecutive nucleotides, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 6, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 7, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , a segment of consecutive nucleotides 21, 22, 23, 24 or 25, a segment of consecutive nucleotides 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 of the nucleic acid sequence of SEQ ID NO: 9, a segment of consecutive nucleotides 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 of the nucleic acid sequence of SEQ ID NO: 10, a segment of consecutive nucleotides 10, 11, 12, 13, 14, 15, 16, 17, 18,Segments of 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 12, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or (A segment of 25 consecutive nucleotides; a segment of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides in the nucleic acid sequence of SEQ ID NO: 292; a segment of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides in the nucleic acid sequence of SEQ ID NO: 295).

[0016] In certain embodiments, the dsRNA includes an antisense strand complementary to a segment of 15 to 35 consecutive nucleotides in any one of the nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295. For example, the antisense strand may complement a segment of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides in the nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 2, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 3, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides.In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 4, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 6, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides.In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 7, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 8, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 9, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides.In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 10, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 11, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 12, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides.In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 13, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 292. In certain embodiments, the antisense strand is complementary to the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 295.

[0017] In certain embodiments, the dsRNA includes an antisense strand having three or fewer mismatches with one of the MAPT nucleic acid sequences, SEQ ID NOs. 1-13, 292, and 295. For example, the antisense strand has 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO. 1. In certain embodiments, the antisense strand has 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO. 2. In certain embodiments, the antisense strand has 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO. 3. In certain embodiments, the antisense strand has 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO. 4. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 6. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 8. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 9.In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 10. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 11. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 12. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 13. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 292. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 295.

[0018] In certain embodiments, the dsRNA includes an antisense strand that is perfectly complementary to one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295.

[0019] In certain embodiments, the dsRNA molecule has an antisense strand that is at least 85% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs. In certain embodiments, the dsRNA molecule has an antisense strand that is at least 90% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46. In certain embodiments, the dsRNA molecule has an antisense strand that is at least 95% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46 (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs. 34-46). In certain embodiments, the dsRNA comprises an antisense strand having any one of the nucleic acid sequences of SEQ ID NOs. 34-46.

[0020] In certain embodiments, the antisense and / or sense strands include a length of approximately 13 to 35 nucleotides. For example, in certain embodiments, the antisense and / or sense strands are 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides long.

[0021] In some embodiments of any one of the aforementioned embodiments, the antisense chain is 15 nucleotides long. In some embodiments, the antisense chain is 16 nucleotides long. In some embodiments, the antisense chain is 17 nucleotides long. In some embodiments, the antisense chain is 18 nucleotides long. In some embodiments, the antisense chain is 19 nucleotides long. In certain embodiments, the antisense chain is 20 nucleotides long. In certain embodiments, the antisense chain is 21 nucleotides long. In certain embodiments, the antisense chain is 22 nucleotides long. In some embodiments, the antisense chain is 23 nucleotides long. In some embodiments, the antisense chain is 24 nucleotides long. In some embodiments, the antisense chain is 25 nucleotides long. In some embodiments, the antisense chain is 26 nucleotides long. In some embodiments, the antisense chain is 27 nucleotides long. In some embodiments, the antisense chain is 28 nucleotides long. In some embodiments, the antisense chain is 29 nucleotides long. In some embodiments, the antisense chain is 30 nucleotides long. In some embodiments, the antisense chain is 31 nucleotides long. In some embodiments, the antisense chain is 32 nucleotides long. In some embodiments, the antisense chain is 33 nucleotides long. In some embodiments, the antisense chain is 34 nucleotides long. In some embodiments, the antisense chain is 35 nucleotides long. In some embodiments, the sense chain is 13 nucleotides long. In some embodiments, the sense chain is 14 nucleotides long. In certain embodiments, the sense chain is 15 nucleotides long. In certain embodiments, the sense chain is 16 nucleotides long. In certain embodiments, the sense chain is 18 nucleotides long. In certain embodiments, the sense chain is 20 nucleotides long. In some embodiments, the sense chain is 21 nucleotides long. In some embodiments, the sense chain is 22 nucleotides long. In some embodiments, the sense chain is 23 nucleotides long.In some embodiments, the sense strand is 24 nucleotides long. In some embodiments, the sense strand is 25 nucleotides long. In some embodiments, the sense strand is 26 nucleotides long. In some embodiments, the sense strand is 27 nucleotides long. In some embodiments, the sense strand is 29 nucleotides long. In some embodiments, the sense strand is 30 nucleotides long. In some embodiments, the sense strand is 31 nucleotides long. In some embodiments, the sense strand is 32 nucleotides long. In some embodiments, the sense strand is 33 nucleotides long. In some embodiments, the sense strand is 34 nucleotides long. In some embodiments, the sense strand is 35 nucleotides long.

[0022] In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 14 nucleotides long.

[0023] In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 15 nucleotides long.

[0024] In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 16 nucleotides long.

[0025] In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 17 nucleotides long.

[0026] In some embodiments, the antisense strand is 18 nucleotides long, and the sense strand is 18 nucleotides long.

[0027] In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 14 nucleotides long.

[0028] In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 15 nucleotides long.

[0029] In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 16 nucleotides long.

[0030] In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 17 nucleotides long.

[0031] In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 18 nucleotides long.

[0032] In some embodiments, the antisense strand is 19 nucleotides long, and the sense strand is 19 nucleotides long.

[0033] In certain embodiments, the antisense strand is 20 nucleotides long, and the sense strand is 15 or 16 nucleotides long.

[0034] In certain embodiments, the antisense strand is 21 nucleotides long, and the sense strand is 15 or 16 nucleotides long.

[0035] In certain embodiments, the antisense strand is 20 or 21 nucleotides long, and the sense strand is 15 nucleotides long.

[0036] In certain embodiments, the antisense strand is 20 or 21 nucleotides long, and the sense strand is 16 nucleotides long.

[0037] In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 14 nucleotides long.

[0038] In certain embodiments, the antisense strand is 20 nucleotides long and the sense strand is 15 nucleotides long.

[0039] In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 16 nucleotides long.

[0040] In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 17 nucleotides long.

[0041] In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 18 nucleotides long.

[0042] In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 19 nucleotides long.

[0043] In some embodiments, the antisense strand is 20 nucleotides long, and the sense strand is 20 nucleotides long.

[0044] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 14 nucleotides long.

[0045] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 15 nucleotides long.

[0046] In certain embodiments, the antisense strand is 21 nucleotides long and the sense strand is 16 nucleotides long.

[0047] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 17 nucleotides long.

[0048] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 18 nucleotides long.

[0049] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 19 nucleotides long.

[0050] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 20 nucleotides long.

[0051] In some embodiments, the antisense strand is 21 nucleotides long, and the sense strand is 21 nucleotides long.

[0052] In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 14 nucleotides long.

[0053] In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 15 nucleotides long.

[0054] In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 16 nucleotides long.

[0055] In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 17 nucleotides long.

[0056] In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 18 nucleotides long.

[0057] In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 19 nucleotides long.

[0058] In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 20 nucleotides long.

[0059] In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 21 nucleotides long.

[0060] In some embodiments, the antisense strand is 22 nucleotides long, and the sense strand is 22 nucleotides long.

[0061] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 14 nucleotides long.

[0062] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 15 nucleotides long.

[0063] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 16 nucleotides long.

[0064] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 17 nucleotides long.

[0065] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 18 nucleotides long.

[0066] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 19 nucleotides long.

[0067] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 20 nucleotides long.

[0068] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long.

[0069] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 22 nucleotides long.

[0070] In some embodiments, the antisense strand is 23 nucleotides long, and the sense strand is 23 nucleotides long.

[0071] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 14 nucleotides long.

[0072] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 15 nucleotides long.

[0073] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 16 nucleotides long.

[0074] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 17 nucleotides long.

[0075] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 18 nucleotides long.

[0076] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 19 nucleotides long.

[0077] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 20 nucleotides long.

[0078] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 21 nucleotides long.

[0079] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 22 nucleotides long.

[0080] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 23 nucleotides long.

[0081] In some embodiments, the antisense strand is 24 nucleotides long, and the sense strand is 24 nucleotides long.

[0082] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 14 nucleotides long.

[0083] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 15 nucleotides long.

[0084] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 16 nucleotides long.

[0085] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 17 nucleotides long.

[0086] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 18 nucleotides long.

[0087] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 19 nucleotides long.

[0088] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 20 nucleotides long.

[0089] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 21 nucleotides long.

[0090] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 22 nucleotides long.

[0091] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 23 nucleotides long.

[0092] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 24 nucleotides long.

[0093] In some embodiments, the antisense strand is 25 nucleotides long, and the sense strand is 25 nucleotides long.

[0094] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 14 nucleotides long.

[0095] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 15 nucleotides long.

[0096] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 16 nucleotides long.

[0097] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 17 nucleotides long.

[0098] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 18 nucleotides long.

[0099] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 19 nucleotides long.

[0100] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 20 nucleotides long.

[0101] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 21 nucleotides long.

[0102] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 22 nucleotides long.

[0103] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 23 nucleotides long.

[0104] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 24 nucleotides long.

[0105] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 25 nucleotides long.

[0106] In some embodiments, the antisense strand is 26 nucleotides long, and the sense strand is 26 nucleotides long.

[0107] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 14 nucleotides long.

[0108] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 15 nucleotides long.

[0109] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 16 nucleotides long.

[0110] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 17 nucleotides long.

[0111] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 18 nucleotides long.

[0112] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 19 nucleotides long.

[0113] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 20 nucleotides long.

[0114] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 21 nucleotides long.

[0115] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 22 nucleotides long.

[0116] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 23 nucleotides long.

[0117] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 24 nucleotides long.

[0118] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 25 nucleotides long.

[0119] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 26 nucleotides long.

[0120] In some embodiments, the antisense strand is 27 nucleotides long, and the sense strand is 27 nucleotides long.

[0121] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 14 nucleotides long.

[0122] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 15 nucleotides long.

[0123] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 16 nucleotides long.

[0124] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 17 nucleotides long.

[0125] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 18 nucleotides long.

[0126] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 19 nucleotides long.

[0127] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 20 nucleotides long.

[0128] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 21 nucleotides long.

[0129] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 22 nucleotides long.

[0130] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 23 nucleotides long.

[0131] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 24 nucleotides long.

[0132] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 25 nucleotides long.

[0133] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 26 nucleotides long.

[0134] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 27 nucleotides long.

[0135] In some embodiments, the antisense strand is 28 nucleotides long, and the sense strand is 28 nucleotides long.

[0136] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 14 nucleotides long.

[0137] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 15 nucleotides long.

[0138] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 16 nucleotides long.

[0139] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 17 nucleotides long.

[0140] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 18 nucleotides long.

[0141] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 19 nucleotides long.

[0142] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 20 nucleotides long.

[0143] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 21 nucleotides long.

[0144] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 22 nucleotides long.

[0145] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 23 nucleotides long.

[0146] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 24 nucleotides long.

[0147] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 25 nucleotides long.

[0148] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 26 nucleotides long.

[0149] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 27 nucleotides long.

[0150] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 28 nucleotides long.

[0151] In some embodiments, the antisense strand is 29 nucleotides long, and the sense strand is 29 nucleotides long.

[0152] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 14 nucleotides long.

[0153] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 15 nucleotides long.

[0154] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 16 nucleotides long.

[0155] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 17 nucleotides long.

[0156] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 18 nucleotides long.

[0157] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 19 nucleotides long.

[0158] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 20 nucleotides long.

[0159] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 21 nucleotides long.

[0160] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 22 nucleotides long.

[0161] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 23 nucleotides long.

[0162] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 24 nucleotides long.

[0163] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 25 nucleotides long.

[0164] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 26 nucleotides long.

[0165] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 27 nucleotides long.

[0166] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 28 nucleotides long.

[0167] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 29 nucleotides long.

[0168] In some embodiments, the antisense strand is 30 nucleotides long, and the sense strand is 30 nucleotides long.

[0169] In certain embodiments, the dsRNA contains a double-stranded region of 14 to 30 base pairs (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs). In certain embodiments, the dsRNA contains a double-stranded region of 14 base pairs. In certain embodiments, the dsRNA contains a double-stranded region of 15 base pairs. In certain embodiments, the dsRNA contains a double-stranded region of 16 base pairs. In certain embodiments, the dsRNA contains a double-stranded region of 17 base pairs. In certain embodiments, the dsRNA contains a double-stranded region of 18 base pairs. In some embodiments, the dsRNA contains a double-stranded region of 19 base pairs. In some embodiments, the dsRNA contains a 20-base pair double-stranded region. In some embodiments, the dsRNA contains a 21-base pair double-stranded region. In some embodiments, the dsRNA contains a 22-base pair double-stranded region. In some embodiments, the dsRNA contains a 23-base pair double-stranded region. In some embodiments, the dsRNA contains a 24-base pair double-stranded region. In some embodiments, the dsRNA contains a 25-base pair double-stranded region. In some embodiments, the dsRNA contains a 26-base pair double-stranded region. In some embodiments, the dsRNA contains a 27-base pair double-stranded region. In some embodiments, the dsRNA contains a 28-base pair double-stranded region. In some embodiments, the dsRNA contains a 29-base pair double-stranded region. In some embodiments, the dsRNA contains a 30-base pair double-stranded region.

[0170] In certain embodiments, the dsRNA contains a blunt end. In certain embodiments, the dsRNA contains at least one single-stranded nucleotide overhang. In certain embodiments, the dsRNA contains a single-stranded nucleotide overhang of approximately 2 to 5 nucleotides.

[0171] In certain embodiments, the dsRNA contains naturally occurring nucleotides.

[0172] In certain embodiments, the dsRNA includes at least one modified nucleotide.

[0173] In certain embodiments, the modified nucleotides include 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debased nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramides, non-natural bases including nucleotides, or mixtures thereof.

[0174] In certain embodiments, the dsRNA includes at least one modified nucleotide linkage.

[0175] In certain embodiments, the modified nucleotide linkages include phosphorothioate nucleotide linkages. In certain embodiments, the dsRNA includes 4–16 phosphorothioate nucleotide linkages (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphorothioate linkages). In certain embodiments, the dsRNA includes 8–13 phosphorothioate nucleotide linkages (e.g., 9, 10, 11, 12, or 13 phosphorothioate linkages).

[0176] In certain embodiments, the dsRNA includes at least one modified nucleotide linkage of formula I: [ka] (In the formula, B is the base pair portion; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH; X is a halo, hydroxy, and C 1-6 Selected from the group consisting of alkoxys, Y is O - OH, OR, NH- NH2, S - Selected from the group consisting of , and SH, Z is selected from the group consisting of O and CH2; R is a protecting group, [ka] (where is any double bond).

[0177] In a particular embodiment, if W is CH, [ka] It is a double bond.

[0178] In a particular embodiment, if W is selected from the group consisting of O, OCH2, OCH, and CH2, [ka] It is a single bond.

[0179] In certain embodiments, the dsRNA contains at least 70% chemically modified nucleotides (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides).

[0180] In certain embodiments, the dsRNA is fully chemically modified. In certain embodiments, the dsRNA contains at least 60% 2'-O-methylnucleotide modification (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl modification).

[0181] In certain embodiments, the dsRNA contains approximately 80% to 90% 2'-O-methyl nucleotide modifications (e.g., approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% 2'-O-methyl nucleotide modifications). In certain embodiments, the dsRNA contains approximately 83% to 86% 2'-O-methyl modifications (e.g., approximately 83%, 84%, 85%, or 86% 2'-O-methyl modifications).

[0182] In certain embodiments, the dsRNA contains about 70% to about 80% of 2'-O-methyl nucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% of 2'-O-methyl nucleotide modifications). In certain embodiments, the dsRNA contains about 75% to about 78% of 2'-O-methyl modifications (e.g., about 75%, 76%, 77%, or 78% of 2'-O-methyl modifications).

[0183] In some embodiments of any one of the aforementioned aspects, the dsRNA contains about 60% to about 70% of 2'-O-methylnucleotide modifications (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of 2'-O-methylnucleotide modifications). In some embodiments, the dsRNA contains about 60% to about 65% of 2'-O-methylnucleotide modifications (e.g., about 60%, 61%, 62%, or 63% of 2'-O-methyl modifications).

[0184] In certain embodiments, the antisense strand comprises at least 70% chemically modified nucleotides (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides).

[0185] In certain embodiments, the antisense chain is fully chemically modified. In certain embodiments, the antisense chain contains at least 55% 2'-O-methylnucleotide modification (e.g., 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methylnucleotide modification). In some embodiments, the antisense chain contains approximately 55% to 90% 2'-O-methylnucleotide modifications (e.g., 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% 2'-O-methyl modifications).

[0186] In certain embodiments, the antisense strand contains about 70% to 90% 2'-O-methylnucleotide modifications (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% 2'-O-methyl modifications). In certain embodiments, the antisense strand contains about 85% to about 90% 2'-O-methyl modifications (e.g., about 85%, 86%, 87%, 88%, 89%, or 90% 2'-O-methyl modifications).

[0187] In certain embodiments, the antisense strand comprises about 75% to 85% 2'-O-methyl nucleotide modifications (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% 2'-O-methyl modifications).

[0188] In certain embodiments, the sense strand comprises at least 70% chemically modified nucleotides (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides).

[0189] In certain embodiments, the sense strand is fully chemically modified. In certain embodiments, the sense strand includes at least 55% 2'-O-methylnucleotide modification (e.g., 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl modification). In certain embodiments, the sense strand contains 100% 2'-O-methylnucleotide modification.

[0190] In certain embodiments, the sense strand contains about 70% to about 85% of 2'-O-methylnucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% of 2'-O-methylnucleotide modifications).

[0191] In certain embodiments, the sense strand contains about 65% to about 75% of 2'-O-methylnucleotide modifications (e.g., about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% of 2'-O-methylnucleotide modifications).

[0192] In certain embodiments, the sense strand contains about 67% to about 73% of 2'-O-methylnucleotide modifications (e.g., about 67%, 68%, 69%, 70%, 71%, 72%, or 73% of 2'-O-methylnucleotide modifications).

[0193] In some embodiments of any one of the aforementioned aspects, the sense strand contains about 55% to about 65% of 2'-O-methylnucleotide modifications (e.g., about 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65% of 2'-O-methylnucleotide modifications).

[0194] In certain embodiments, the sense strand contains one or more nucleotide mismatches between the antisense strand and the sense strand. In certain embodiments, the one or more nucleotide mismatches are located at positions 2, 6, and 12 from the 5' end of the sense strand. In certain embodiments, the nucleotide mismatches are located at positions 2, 6, and 12 from the 5' end of the sense strand.

[0195] In certain embodiments, the antisense chain includes a 5'-phosphate, a 5'-alkylphosphonate, a 5'-alkylenephosphonate, or a 5'-alkenylphosphonate.

[0196] In certain embodiments, the antisense chain comprises a 5' vinyl phosphonate.

[0197] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand alternately comprises 2'-methoxyribonucleotides and 2'-fluororibonucleotides; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand alternately comprises 2'-methoxyribonucleotides and 2'-fluororibonucleotides; (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0198] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295; and (2) the antisense strand has at least 55% 2'-O-methyl modification (e.g., 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%). (3) The nucleotide at position 14 from the 5' end of the antisense strand contains 2'-methoxy-ribonucleosaccharide (66%, 67%, 68%, 69%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl modification); (4) The nucleotide at position 14 from the 5' end of the antisense strand contains 2'-methoxy-ribonucleosaccharide (4) The nucleotides from positions 1-2 to 1-7 at the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) Part of the antisense strand is complementary to part of the sense strand; (6) The sense strand has at least 55% 2'-O-methyl modification (e.g., 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%) (7) Nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0199] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 85% 2'-O-methyl modifications; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains 100% 2'-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0200] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains 100% 2'-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0201] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains 100% 2'-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0202] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 85% 2'-O-methyl modifications (e.g., about 85% to about 90% 2'-O-methyl modifications); (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides (e.g., the nucleotides at positions 2 and 14 from the 5' end of the antisense strand may be 2'-fluoronucleotides); (4) the nucleotides at positions 1- from the 3' end of the antisense strand (5) Nucleotides from position 2 to 1-7 are linked to each other via phosphorothioate nucleotide linkages; (6) Part of the antisense strand is complementary to part of the sense strand; (7) The sense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (8) Nucleotides from the 3' end of the sense strand to positions 7, 10, and 11 are not 2'-methoxyribonucleotides (e.g., nucleotides from the 3' end of the sense strand to positions 7, 10, and 11 are 2'-fluoronucleotides); (9) Nucleotides from the 5' end of the sense strand to positions 1-2 are linked to each other via phosphorothioate nucleotide linkages.

[0203] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (3) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides (e.g., the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand may be 2'-fluoronucleotides); (4) the 3' end of the antisense strand (5) Nucleotides from positions 1-2 to 1-7 from the end are linked to each other via phosphorothioate nucleotide linkages; (6) Part of the antisense strand is complementary to part of the sense strand; (7) The sense strand contains at least 65% 2'-O-methyl modifications (e.g., about 65% to about 75% 2'-O-methyl modifications); (8) Nucleotides from positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides (e.g., nucleotides from positions 7, 9, 10, and 11 from the 3' end of the sense strand are 2'-fluoronucleotides); (9) Nucleotides from positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0204] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a sequence substantially complementary to one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 2, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) the nucleotides at the 3' end of the antisense strand are not 2'-methoxyribonucleotides. (5) Nucleotides from position 2 to 1 to 7 are linked to each other via phosphorothioate internucleotide linkages; (6) Part of the antisense strand is complementary to part of the sense strand; (7) The sense strand contains at least 80% 2'-O-methyl modifications; (8) Nucleotides from the 3' end of the sense strand to positions 7, 10, and 11 are not 2'-methoxyribonucleotides; (9) Nucleotides from the 5' end of the sense strand to positions 1 to 2 are linked to each other by phosphorothioate internucleotide linkages.

[0205] In certain embodiments, the functional portion is coupled to the 5' and / or 3' ends of the antisense chain. In certain embodiments, the functional portion is coupled to the 5' and / or 3' ends of the sense chain. In certain embodiments, the functional portion is coupled to the 3' end of the sense chain.

[0206] In certain embodiments, the functional portion includes a hydrophobic portion.

[0207] In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.

[0208] In certain embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).

[0209] In certain embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanic acid (DCA).

[0210] In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, their derivatives, and their metabolites.

[0211] In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

[0212] In certain embodiments, the functional components are linked to the antisense chain and / or sense chain by a linker.

[0213] In certain embodiments, the linker includes a divalent or trivalent linker.

[0214] In certain embodiments, the divalent or trivalent linker is selected from the group consisting of: [ka] (wherein n is 1, 2, 3, 4, or 5).

[0215] In certain embodiments, the linker includes ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphodiesters, phosphorothioates, phosphoramidates, amides, carbamates, or combinations thereof.

[0216] In certain embodiments, if the linker is a trivalent linker, the linker further links phosphodiesters or phosphodiester derivatives.

[0217] In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] (In the formula, X is O, S, or BH3).

[0218] In certain embodiments, the nucleotides at positions 1 and 2 from the 3' end of the sense strand, and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand, are linked to adjacent ribonucleotides via phosphorothioate linkages.

[0219] In one embodiment, the present disclosure provides a pharmaceutical composition for inhibiting the expression of a tau protein (MAPT) gene in an organism, comprising the above-mentioned dsRNA and a pharmaceutically acceptable carrier.

[0220] In certain embodiments, the dsRNA inhibits MAPT gene expression by at least 50%. In certain embodiments, the dsRNA inhibits MAPT gene expression by at least 80%.

[0221] In one embodiment, the present disclosure provides a method for inhibiting the expression of the MAPT gene in cells. The method comprises (a) introducing the above-mentioned double-stranded ribonucleic acid (dsRNA) into cells; and (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the MAPT gene, thereby inhibiting the expression of the MAPT gene in the cells.

[0222] In one embodiment, the present disclosure provides a method for treating or managing a neurodegenerative disease, comprising administering a therapeutically effective amount of the above-mentioned dsRNA to a patient in need of such treatment or management.

[0223] In certain embodiments, dsRNA is administered to the patient's brain.

[0224] In certain embodiments, dsRNA is administered by intracerebral / intraventricular (ICV) injection, intrastriatal (IS) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

[0225] In certain embodiments, administration of dsRNA causes a decrease in MAPT gene mRNA in one or more of the following: hippocampus, striatum, cortex, cerebellum, thalamus, hypothalamus, and spinal cord.

[0226] In certain embodiments, the dsRNA inhibits MAPT gene expression by at least 50%. In certain embodiments, the dsRNA inhibits MAPT gene expression by at least 80%.

[0227] In one embodiment, the disclosure provides a vector comprising a regulatory sequence operably ligated to a nucleotide sequence encoding an RNA molecule substantially complementary to any one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295.

[0228] In certain embodiments, the RNA molecule inhibits MAPT gene expression by at least 50%. In certain embodiments, the RNA molecule inhibits MAPT gene expression by at least 80%.

[0229] In certain embodiments, the RNA molecule includes ssRNA or dsRNA.

[0230] In certain embodiments, the dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a sequence substantially complementary to one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295.

[0231] In one embodiment, the present disclosure provides cells containing the above-mentioned vector.

[0232] In one embodiment, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising the above-mentioned vector and AAV capsid.

[0233] In one embodiment, the disclosure provides branched RNA compounds comprising two or more RNA molecules, for example, 14 to 40 nucleotides in length (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length), where each RNA molecule comprises a portion having a nucleic acid sequence substantially complementary to a segment of MAPT mRNA. In certain embodiments, the two RNA molecules may be linked to each other by one or more portions independently selected from linkers, spacers, and branching points.

[0234] In certain embodiments, the branched RNA molecule includes one or both of ssRNA and dsRNA.

[0235] In certain embodiments, the branched RNA molecule includes an antisense oligonucleotide.

[0236] In certain embodiments, each RNA molecule comprises a dsRNA including a sense strand and an antisense strand, each antisense strand independently containing a sequence substantially complementary to one of the MAPT nucleic acid sequences: SEQ ID NOs: 1-13, 292, and 295.

[0237] In certain embodiments, the branched RNA compound comprises two or more copies of any RNA molecule of any of the above embodiments or aspects of the Disclosure, covalently linked to one another (e.g., by linkers, spacers, or branching points).

[0238] In certain embodiments, the branched RNA compound includes a portion having a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295. For example, the branched RNA compound may include two or more dsRNA molecules, each containing an antisense strand covalently linked to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295, and each having an antisense strand complementary to at least 10, 11, 12, or 13 consecutive nucleotides of one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295. For example, in certain embodiments, the dsRNA may include a segment of consecutive nucleotides 10-25 of one of the nucleic acid sequences of SEQ ID NOs: 1-13 (e.g., a segment of consecutive nucleotides 10-25 of the nucleic acid sequence of SEQ ID NOs: 1, a segment of consecutive nucleotides 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of the nucleic acid sequence of SEQ ID NOs: 2, and a segment of consecutive nucleotides 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of the nucleic acid sequence of SEQ ID NOs: 3 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotide segments of the nucleic acid sequence of SEQ ID NO: 4, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotide segments of the nucleic acid sequence of SEQ ID NO: 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, or 25 consecutive nucleotide segments of the nucleic acid sequence of SEQ ID NO: 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotide segments of the nucleic acid sequence of SEQ ID NO: 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotide segments of the nucleic acid sequence of SEQ ID NO: 8 Segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the sequence, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the nucleic acid sequence of sequence number 9, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,Segments of 21, 22, 23, 24, or 25 consecutive nucleotides, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 11, segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 12, segments of 10, 11, 12, 13, 14, 15, 16, 17, It includes an antisense strand complementary to the following segments of consecutive nucleotides: 18, 19, 20, 21, 22, 23, 24, or 25; the following segments of consecutive nucleotides: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of the nucleic acid sequence of SEQ ID NO: 292; or the following segments of consecutive nucleotides: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of the nucleic acid sequence of SEQ ID NO: 295.

[0239] In certain embodiments, each dsRNA in the branched RNA compound includes an antisense strand complementary to a segment of 15-25 consecutive nucleotides (e.g., a segment of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides) of any one of the nucleic acid sequences of SEQ ID NOs. For example, the antisense strand may complement the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO. 1. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 2, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 3, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 4, specifically the segments 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 5, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 6, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 7, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 8, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 9, specifically the segments 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 10, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 11, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 12, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 13, specifically the segments of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides. In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 292, specifically the 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides.In certain embodiments, the antisense strand is complementary to the segments of the nucleic acid sequence of SEQ ID NO: 295, specifically the 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 consecutive nucleotides.

[0240] In certain embodiments, each dsRNA in the branched RNA compound includes an antisense strand having 3 or fewer mismatches with one of the MAPT nucleic acid sequences, SEQ ID NOs. 1-13, 292, and 295. For example, the antisense strand may have 0-3 mismatches (e.g., 0, 1, 2, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO. 1. In certain embodiments, the antisense strand may have 0-3 mismatches (e.g., 0, 1, 2, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO. 2. In certain embodiments, the antisense strand may have 0-3 mismatches (e.g., 0, 1, 2, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO. 3. In certain embodiments, the antisense strand may have 0-3 mismatches (e.g., 0, 1, 2, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO. 4. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 6. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 8. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 9.In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 10. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 11. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 12. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 13. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 292. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with respect to the nucleic acid sequence of SEQ ID NO: 295.

[0241] In certain embodiments, each dsRNA in the branched RNA compound includes an antisense strand that is perfectly complementary to one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295.

[0242] In certain embodiments, the branched RNA compound includes a portion having a nucleic acid sequence substantially complementary to one or more of the MAPT nucleic acid sequences of sequence numbers 14-33, 299, and 302.

[0243] In certain embodiments, the RNA molecule includes an antisense oligonucleotide.

[0244] In certain embodiments, each RNA molecule contains 14 to 35 nucleotides (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35).

[0245] In certain embodiments, the antisense and / or sense strands are approximately 13 to 35 nucleotides long. For example, in certain embodiments, the antisense and / or sense strands are 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides long. In some embodiments, the antisense strand is 14 nucleotides long. In some embodiments, the antisense strand is 15 nucleotides long. In some embodiments, the antisense strand is 16 nucleotides long. In some embodiments, the antisense strand is 17 nucleotides long. In some embodiments, the antisense strand is 18 nucleotides long. In some embodiments, the antisense strand is 19 nucleotides long. In certain embodiments, the antisense strand is 20 nucleotides long. In certain embodiments, the antisense strand is 21 nucleotides long. In certain embodiments, the antisense strand is 22 nucleotides long. In some embodiments, the antisense chain is 23 nucleotides long. In some embodiments, the antisense chain is 24 nucleotides long. In some embodiments, the antisense chain is 25 nucleotides long. In some embodiments, the antisense chain is 26 nucleotides long. In some embodiments, the antisense chain is 27 nucleotides long. In some embodiments, the antisense chain is 28 nucleotides long. In some embodiments, the antisense chain is 29 nucleotides long. In some embodiments, the antisense chain is 30 nucleotides long. In some embodiments, the antisense chain is 31 nucleotides long. In some embodiments, the antisense chain is 32 nucleotides long. In some embodiments, the antisense chain is 33 nucleotides long. In some embodiments, the antisense chain is 34 nucleotides long. In some embodiments, the antisense chain is 35 nucleotides long.

[0246] In some embodiments of any one of the aforementioned embodiments, the sense chain is 13 nucleotides long. In certain embodiments, the sense chain is 14 nucleotides long. In certain embodiments, the sense chain is 15 nucleotides long. In certain embodiments, the sense chain is 16 nucleotides long. In certain embodiments, the sense chain is 17 nucleotides long. In certain embodiments, the sense chain is 18 nucleotides long. In certain embodiments, the sense chain is 19 nucleotides long. In some embodiments, the sense chain is 20 nucleotides long. In some embodiments, the sense chain is 21 nucleotides long. In some embodiments, the sense chain is 22 nucleotides long. In some embodiments, the sense chain is 23 nucleotides long. In some embodiments, the sense chain is 24 nucleotides long. In some embodiments, the sense chain is 25 nucleotides long. In some embodiments, the sense chain is 26 nucleotides long. In some embodiments, the sense chain is 27 nucleotides long. In some embodiments, the sense chain is 28 nucleotides long. In some embodiments, the sense chain is 29 nucleotides long. In some embodiments, the sense chain is 30 nucleotides long. In some embodiments, the sense strand is 31 nucleotides long. In some embodiments, the sense strand is 32 nucleotides long. In some embodiments, the sense strand is 33 nucleotides long. In some embodiments, the sense strand is 34 nucleotides long. In some embodiments, the sense strand is 35 nucleotides long.

[0247] In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 14 nucleotides long.

[0248] In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 15 nucleotides long.

[0249] In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 16 nucleotides long.

[0250] In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 17 nucleotides in length.

[0251] In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 18 nucleotides in length.

[0252] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 14 nucleotides in length.

[0253] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 15 nucleotides in length.

[0254] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 16 nucleotides in length.

[0255] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 17 nucleotides in length.

[0256] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 18 nucleotides in length.

[0257] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 19 nucleotides in length.

[0258] In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 14 nucleotides in length.

[0259] In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 15 nucleotides in length.

[0260] In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 16 nucleotides in length.

[0261] In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 17 nucleotides long.

[0262] In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 18 nucleotides long.

[0263] In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 19 nucleotides long.

[0264] In some embodiments, the antisense strand is 20 nucleotides long, and the sense strand is 20 nucleotides long.

[0265] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 14 nucleotides long.

[0266] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 15 nucleotides long.

[0267] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 16 nucleotides long.

[0268] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 17 nucleotides long.

[0269] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 18 nucleotides long.

[0270] In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 19 nucleotides long.

[0271] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 20 nucleotides in length.

[0272] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 21 nucleotides in length.

[0273] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 14 nucleotides in length.

[0274] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 15 nucleotides in length.

[0275] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 16 nucleotides in length.

[0276] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 17 nucleotides in length.

[0277] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 18 nucleotides in length.

[0278] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 19 nucleotides in length.

[0279] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 20 nucleotides in length.

[0280] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 21 nucleotides in length.

[0281] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 22 nucleotides in length.

[0282] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 14 nucleotides long.

[0283] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 15 nucleotides long.

[0284] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 16 nucleotides long.

[0285] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 17 nucleotides long.

[0286] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 18 nucleotides long.

[0287] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 19 nucleotides long.

[0288] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 20 nucleotides long.

[0289] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long.

[0290] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 22 nucleotides long.

[0291] In some embodiments, the antisense strand is 23 nucleotides long, and the sense strand is 23 nucleotides long.

[0292] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 14 nucleotides long.

[0293] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 15 nucleotides long.

[0294] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 16 nucleotides long.

[0295] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 17 nucleotides long.

[0296] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 18 nucleotides long.

[0297] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 19 nucleotides long.

[0298] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 20 nucleotides long.

[0299] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 21 nucleotides long.

[0300] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 22 nucleotides long.

[0301] In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 23 nucleotides long.

[0302] In some embodiments, the antisense strand is 24 nucleotides long, and the sense strand is 24 nucleotides long.

[0303] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 14 nucleotides long.

[0304] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 15 nucleotides long.

[0305] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 16 nucleotides long.

[0306] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 17 nucleotides long.

[0307] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 18 nucleotides long.

[0308] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 19 nucleotides long.

[0309] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 20 nucleotides long.

[0310] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 21 nucleotides long.

[0311] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 22 nucleotides long.

[0312] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 23 nucleotides long.

[0313] In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 24 nucleotides long.

[0314] In some embodiments, the antisense strand is 25 nucleotides long, and the sense strand is 25 nucleotides long.

[0315] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 14 nucleotides long.

[0316] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 15 nucleotides long.

[0317] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 16 nucleotides long.

[0318] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 17 nucleotides long.

[0319] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 18 nucleotides long.

[0320] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 19 nucleotides long.

[0321] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 20 nucleotides long.

[0322] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 21 nucleotides long.

[0323] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 22 nucleotides long.

[0324] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 23 nucleotides long.

[0325] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 24 nucleotides long.

[0326] In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 25 nucleotides long.

[0327] In some embodiments, the antisense strand is 26 nucleotides long, and the sense strand is 26 nucleotides long.

[0328] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 14 nucleotides long.

[0329] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 15 nucleotides long.

[0330] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 16 nucleotides long.

[0331] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 17 nucleotides long.

[0332] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 18 nucleotides long.

[0333] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 19 nucleotides long.

[0334] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 20 nucleotides long.

[0335] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 21 nucleotides long.

[0336] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 22 nucleotides long.

[0337] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 23 nucleotides long.

[0338] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 24 nucleotides long.

[0339] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 25 nucleotides long.

[0340] In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 26 nucleotides long.

[0341] In some embodiments, the antisense strand is 27 nucleotides long, and the sense strand is 27 nucleotides long.

[0342] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 14 nucleotides long.

[0343] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 15 nucleotides long.

[0344] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 16 nucleotides long.

[0345] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 17 nucleotides long.

[0346] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 18 nucleotides long.

[0347] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 19 nucleotides long.

[0348] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 20 nucleotides long.

[0349] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 21 nucleotides long.

[0350] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 22 nucleotides long.

[0351] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 23 nucleotides long.

[0352] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 24 nucleotides long.

[0353] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 25 nucleotides long.

[0354] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 26 nucleotides long.

[0355] In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 27 nucleotides long.

[0356] In some embodiments, the antisense strand is 28 nucleotides long, and the sense strand is 28 nucleotides long.

[0357] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 14 nucleotides long.

[0358] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 15 nucleotides long.

[0359] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 16 nucleotides long.

[0360] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 17 nucleotides long.

[0361] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 18 nucleotides long.

[0362] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 19 nucleotides long.

[0363] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 20 nucleotides long.

[0364] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 21 nucleotides long.

[0365] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 22 nucleotides long.

[0366] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 23 nucleotides long.

[0367] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 24 nucleotides long.

[0368] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 25 nucleotides long.

[0369] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 26 nucleotides long.

[0370] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 27 nucleotides long.

[0371] In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 28 nucleotides long.

[0372] In some embodiments, the antisense strand is 29 nucleotides long, and the sense strand is 29 nucleotides long.

[0373] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 14 nucleotides long.

[0374] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 15 nucleotides long.

[0375] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 16 nucleotides long.

[0376] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 17 nucleotides long.

[0377] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 18 nucleotides long.

[0378] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 19 nucleotides long.

[0379] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 20 nucleotides long.

[0380] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 21 nucleotides long.

[0381] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 22 nucleotides long.

[0382] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 23 nucleotides long.

[0383] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 24 nucleotides long.

[0384] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 25 nucleotides long.

[0385] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 26 nucleotides long.

[0386] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 27 nucleotides long.

[0387] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 28 nucleotides long.

[0388] In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 29 nucleotides long.

[0389] In some embodiments, the antisense strand is 30 nucleotides long, and the sense strand is 30 nucleotides long.

[0390] In certain embodiments, the antisense strand is 20 nucleotides long, and the sense strand is 15 or 16 nucleotides long.

[0391] In certain embodiments, the antisense strand is 21 nucleotides long, and the sense strand is 15 or 16 nucleotides long.

[0392] In certain embodiments, the antisense strand is 20 or 21 nucleotides long, and the sense strand is 15 nucleotides long.

[0393] In certain embodiments, the antisense strand is 20 or 21 nucleotides long, and the sense strand is 16 nucleotides long.

[0394] In certain embodiments, the antisense strand is 20 nucleotides long and the sense strand is 15 nucleotides long.

[0395] In certain embodiments, the antisense strand is 21 nucleotides long and the sense strand is 16 nucleotides long.

[0396] In certain embodiments, the dsRNA contains a double-stranded region of 14 to 35 base pairs. In certain embodiments, the dsRNA contains a double-stranded region of 14 base pairs. In certain embodiments, the dsRNA contains a double-stranded region of 15 base pairs. In certain embodiments, the dsRNA contains a double-stranded region of 16 base pairs. In certain embodiments, the dsRNA contains a double-stranded region of 18 base pairs. In certain embodiments, the dsRNA contains a double-stranded region of 20 base pairs. In some embodiments, the dsRNA contains a double-stranded region of 21 base pairs. In some embodiments, the dsRNA contains a double-stranded region of 22 base pairs. In some embodiments, the dsRNA contains a double-stranded region of 23 base pairs. In some embodiments, the dsRNA contains a double-stranded region of 24 base pairs. In some embodiments, the dsRNA contains a double-stranded region of 25 base pairs. In some embodiments, the dsRNA contains a double-stranded region of 26 base pairs. In some embodiments, the dsRNA contains a 27-base pair double-stranded region. In some embodiments, the dsRNA contains a 28-base pair double-stranded region. In some embodiments, the dsRNA contains a 29-base pair double-stranded region. In some embodiments, the dsRNA contains a 30-base pair double-stranded region. In some embodiments, the dsRNA contains a 31-base pair double-stranded region. In some embodiments, the dsRNA contains a 32-base pair double-stranded region. In some embodiments, the dsRNA contains a 33-base pair double-stranded region. In some embodiments, the dsRNA contains a 34-base pair double-stranded region. In some embodiments, the dsRNA contains a 35-base pair double-stranded region.

[0397] In certain embodiments, the dsRNA contains blunt ends.

[0398] In certain embodiments, the dsRNA includes at least one single-stranded nucleotide overhang. In certain embodiments, the dsRNA includes a single-stranded nucleotide overhang of 2 to 5 nucleotides.

[0399] In certain embodiments, the dsRNA contains naturally occurring nucleotides.

[0400] In certain embodiments, the dsRNA includes at least one modified nucleotide.

[0401] In certain embodiments, the modified nucleotides include nucleotides containing 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramides, or non-natural bases.

[0402] In certain embodiments, the dsRNA includes at least one modified nucleotide linkage.

[0403] In certain embodiments, the modified nucleotide junctions include phosphorothioate nucleotide junctions. In certain embodiments, the branched RNA compound includes 4 to 16 phosphorothioate nucleotide junctions. In certain embodiments, the branched RNA compound includes 8 to 13 phosphorothioate nucleotide junctions.

[0404] In certain embodiments, the dsRNA includes at least one modified nucleotide linkage of formula I: [ka] (In the formula, B is the base pair portion; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH; X is a halo, hydroxy, and C 1-6 Selected from the group consisting of alkoxys, Y is O - OH, OR, NH - NH2, S - Selected from the group consisting of , and SH, Z is selected from the group consisting of O and CH2; R is a protecting group, [ka] (where is any double bond).

[0405] In a particular embodiment, if W is CH, [ka] It is a double bond.

[0406] In a particular embodiment, if W is selected from the group consisting of O, OCH2, OCH, and CH2, [ka] It is a single bond.

[0407] In certain embodiments, the dsRNA contains at least 70% chemically modified nucleotides (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides). In certain embodiments, the dsRNA is fully chemically modified. In certain embodiments, the dsRNA contains at least 60% 2'-O-methylnucleotide modifications (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl modifications).

[0408] In certain embodiments, the antisense strand comprises at least 70% chemically modified nucleotides (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides).

[0409] In certain embodiments, the antisense chain is fully chemically modified.

[0410] In certain embodiments, the antisense strand comprises at least 55% 2'-O-methylnucleotide modification (e.g., 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl modification). In certain embodiments, the antisense strand contains 70% to 90% 2'-O-methyl nucleotide modifications. In certain embodiments, the antisense strand contains about 85% to about 90% 2'-O-methyl modifications (e.g., about 85%, 86%, 87%, 88%, 89%, or 90% 2'-O-methyl modifications).

[0411] In certain embodiments, the antisense strand comprises about 75% to 85% 2'-O-methyl nucleotide modifications (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% 2'-O-methyl modifications).

[0412] In certain embodiments, the sense strand contains at least 70% chemically modified nucleotides (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides). In certain embodiments, the sense strand is fully chemically modified. In certain embodiments, the sense strand contains at least 55% 2'-O-methylnucleotide modification (e.g., 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl modification). In certain embodiments, the sense strand contains 100% 2'-O-methylnucleotide modification.

[0413] In certain embodiments, the sense strand contains one or more nucleotide mismatches between the antisense strand and the sense strand. In certain embodiments, the one or more nucleotide mismatches are located at positions 2, 6, and 12 from the 5' end of the sense strand. In certain embodiments, the nucleotide mismatches are located at positions 2, 6, and 12 from the 5' end of the sense strand.

[0414] In certain embodiments, the antisense chain comprises a 5'-phosphate, a 5'-alkylphosphonate, a 5'-alkylenephosphonate, a 5'-alkenylphosphonate, or a mixture thereof.

[0415] In certain embodiments, the antisense chain comprises a 5' vinyl phosphonate.

[0416] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand alternately comprises 2'-methoxyribonucleotides and 2'-fluororibonucleotides; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand alternately comprises 2'-methoxyribonucleotides and 2'-fluororibonucleotides; (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0417] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 70% of 2'-O-methyl modifications (e.g., about 75% to about 80%, or about 85% to about 90%); (3) the nucleotide from the 5' end to position 14 of the antisense strand is a 2'-methoxy-ribonucleo (4) The nucleotides from positions 1-2 to 1-7 at the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) Part of the antisense strand is complementary to part of the sense strand; (6) The sense strand contains at least 65% 2'-O-methyl modifications (e.g., about 65% to about 75%, or about 75% to about 80%); (7) The nucleotides from positions 1-2 at the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages.

[0418] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 85% 2'-O-methyl modifications; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains 100% 2'-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0419] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains 100% 2'-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0420] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 85% 2'-O-methyl modifications (e.g., about 85% to about 90% 2'-O-methyl modifications); (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides (e.g., the nucleotides at positions 2 and 14 from the 5' end of the antisense strand may be 2'-fluoronucleotides); (4) the nucleotides at positions 1- from the 3' end of the antisense strand (5) Nucleotides from position 2 to 1-7 are linked to each other via phosphorothioate nucleotide linkages; (6) Part of the antisense strand is complementary to part of the sense strand; (7) The sense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (8) Nucleotides from the 3' end of the sense strand to positions 7, 10, and 11 are not 2'-methoxyribonucleotides (e.g., nucleotides from the 3' end of the sense strand to positions 7, 10, and 11 are 2'-fluoronucleotides); (9) Nucleotides from the 5' end of the sense strand to positions 1-2 are linked to each other via phosphorothioate nucleotide linkages.

[0421] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); and (3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides (e.g., (4) The nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand may be 2'-fluoronucleotides; (5) The nucleotides at positions 1-2 to 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (6) Part of the antisense strand is complementary to part of the sense strand; (7) The sense strand contains 100% 2'-O-methyl modification; (8) The nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0422] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (3) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides (e.g., the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are 2'-methoxyribonucleotides). (4) The nucleotides from positions 1-2 to 1-7 of the antisense strand from the 3' end are linked to each other via phosphorothioate nucleotide linkages; (5) Part of the antisense strand is complementary to part of the sense strand; (6) The sense strand contains at least 65% 2'-O-methyl modifications (e.g., about 65% to about 75% 2'-O-methyl modifications); (7) The nucleotides from positions 7, 9, 10, and 11 of the sense strand from the 3' end are not 2'-methoxyribonucleotides; (8) The nucleotides from positions 1-2 of the sense strand from the 5' end are linked to each other via phosphorothioate nucleotide linkages.

[0423] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where (1) the antisense strand has a nucleic acid sequence substantially complementary to one of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 2, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) the 3' end of the antisense strand (5) Nucleotides at positions 1-2 through 1-7 are linked to each other via phosphorothioate internucleotide linkages; (6) Part of the antisense strand is complementary to part of the sense strand; (7) The sense strand contains at least 80% 2'-O-methyl modifications; (8) Nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides; (9) Nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0424] In certain embodiments, the functional portion is coupled to the 5' and / or 3' ends of the antisense chain. In certain embodiments, the functional portion is coupled to the 5' and / or 3' ends of the sense chain. In certain embodiments, the functional portion is coupled to the 3' end of the sense chain.

[0425] In certain embodiments, the functional portion includes a hydrophobic portion.

[0426] In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.

[0427] In certain embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).

[0428] In certain embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanic acid (DCA).

[0429] In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, their derivatives, and their metabolites.

[0430] In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

[0431] In certain embodiments, the functional components are linked to the antisense chain and / or sense chain by a linker.

[0432] In certain embodiments, the linker includes a divalent or trivalent linker.

[0433] In certain embodiments, the divalent or trivalent linker is selected from the group consisting of: [ka] (wherein n is 1, 2, 3, 4, or 5).

[0434] In certain embodiments, the linker includes ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphodiesters, phosphorothioates, phosphoramidates, amides, carbamates, or combinations thereof.

[0435] In certain embodiments, if the linker is a trivalent linker, the linker further links phosphodiesters or phosphodiester derivatives.

[0436] In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] (In the formula, X is O, S, or BH3).

[0437] In certain embodiments, the nucleotides at positions 1 and 2 from the 3' end of the sense strand, and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand, are linked to adjacent ribonucleotides via phosphorothioate linkages.

[0438] In one embodiment, the present disclosure provides a compound of formula (I): [ka] (In the formula: L comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramide, an ester, an amide, a triazole, or a combination thereof, and optionally, formula (I) further comprises one or more branch points B and one or more spacers S, in the formula, Each instance of B is independently a polyvalent organic species or a derivative thereof; S, independently of each occurrence, includes ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramides, esters, amides, triazoles, or combinations thereof; n is 2, 3, 4, 5, 6, 7, or 8; N is a double-stranded nucleic acid, such as a dsRNA molecule, in any of the above embodiments or aspects of this disclosure. In certain embodiments, each N is 15 to 40 nucleotides long. In a particular embodiment, each N includes a sense chain and an antisense chain: The antisense strand contains a sequence substantially complementary to one of the MAPT nucleic acid sequences 1-13, 292, and 295; The sense chain and antisense chain each independently contain one or more chemical modifications.

[0439] In certain embodiments, the compound comprises a structure selected from formulas (I-1) to (I-9): [Table 1]

[0440] In certain embodiments, the antisense chain includes a 5' terminal group R selected from the group consisting of: [ka]

[0441] In certain embodiments, the compound comprises the structure of formula (II): [ka] (In the formula, X is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each instance. Y is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each instance. - represents a phosphodiester nucleoside linkage; The symbol = represents a linkage between phosphorothioate nucleosides; --- represents a base pair interaction or mismatch, independently of each occurrence.

[0442] In certain embodiments, the compound includes the structure of formula (IV): [ka] (In the formula, X is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each instance. Y is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each instance. - represents a phosphodiester nucleoside linkage; The symbol = represents a linkage between phosphorothioate nucleosides; --- represents a base pair interaction or mismatch, independently of each occurrence.

[0443] In certain embodiments, L is of structure L1:

Chemical formula

[0444] In certain embodiments, R is R 3 and n is 2.

[0445] In certain embodiments, L is of structure L2:

Chemical formula

[0446] In certain embodiments, R is R 3 and n is 2.

[0447] In one aspect, the present disclosure provides a delivery system for a therapeutic nucleic acid having a structure of formula (VI):

Chemical formula

[0448] In a particular embodiment, the delivery system includes a structure selected from formulas (VI-1) to (VI-9): [Table 2]

[0449] In certain embodiments, each cNA independently comprises a chemically modified nucleotide.

[0450] In a particular embodiment, the delivery system further comprises n therapeutic nucleic acids (NAs), each NA hybridizing to at least one cNA.

[0451] In certain embodiments, each NA independently comprises at least 14 consecutive nucleotides (e.g., at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more consecutive nucleotides).

[0452] In certain embodiments, each NA independently contains 14 to 35 consecutive nucleotides. In some embodiments, each NA independently contains 14 consecutive nucleotides. In some embodiments, each NA independently contains 15 consecutive nucleotides. In some embodiments, each NA independently contains 16 consecutive nucleotides. In some embodiments, each NA independently contains 17 consecutive nucleotides. In some embodiments, each NA independently contains 18 consecutive nucleotides. In some embodiments, each NA independently contains 19 consecutive nucleotides. In some embodiments, each NA independently contains 20 consecutive nucleotides. In some embodiments, each NA independently contains 21 consecutive nucleotides. In some embodiments, each NA independently contains 22 consecutive nucleotides. In some embodiments, each NA independently contains 23 consecutive nucleotides. In some embodiments, each NA independently contains 24 consecutive nucleotides. In some embodiments, each NA independently contains 25 consecutive nucleotides. In some embodiments, each NA independently contains 26 consecutive nucleotides. In some embodiments, each NA independently contains 27 consecutive nucleotides. In some embodiments, each NA independently contains 28 consecutive nucleotides. In some embodiments, each NA independently contains 29 consecutive nucleotides. In some embodiments, each NA independently contains 30 consecutive nucleotides. In some embodiments, each NA independently contains 31 consecutive nucleotides. In some embodiments, each NA independently contains 32 consecutive nucleotides. In some embodiments, each NA independently contains 33 consecutive nucleotides. In some embodiments, each NA independently contains 34 consecutive nucleotides. In some embodiments, each NA independently contains 35 consecutive nucleotides.

[0453] In certain embodiments, each NA includes at least two unpaired overhangs of nucleotides.

[0454] In certain embodiments, the nucleotides in the overhangs are linked via phosphorothioate linkages.

[0455] In certain embodiments, each NA is independently selected from the group consisting of DNA, siRNA, antagomiR, miRNA, gapmer, mixedmer, and guide RNA.

[0456] In certain embodiments, each NA is substantially complementary to one of the MAPT nucleic acid sequences of sequence numbers 1-13, 292, and 295.

[0457] In one embodiment, the present disclosure provides a pharmaceutical composition for inhibiting the expression of a MAPT gene in an organism, comprising the above-mentioned compound or system and a pharmaceutically acceptable carrier.

[0458] In certain embodiments, the compound or system inhibits MAPT gene expression by at least 50%. In certain embodiments, the compound or system inhibits MAPT gene expression by at least 80%.

[0459] In one embodiment, the present disclosure provides a method for inhibiting the expression of the MAPT gene in cells. The method comprises (a) introducing the above compound or system into cells; and (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the MAPT gene mRNA transcript, thereby inhibiting the expression of the MAPT gene in the cells.

[0460] In one embodiment, the present disclosure provides a method for treating or managing a neurodegenerative disease, comprising administering a therapeutically effective amount of the above compound or system to a patient in need of such treatment or management.

[0461] In certain embodiments, dsRNA is administered to the patient's brain.

[0462] In certain embodiments, dsRNA is administered by intracerebral / intraventricular (ICV) injection, intrastriatal (IS) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

[0463] In certain embodiments, administration of dsRNA causes a decrease in MAPT gene mRNA in one or more of the following: hippocampus, striatum, cortex, cerebellum, thalamus, hypothalamus, and spinal cord.

[0464] In certain embodiments, the dsRNA inhibits MAPT gene expression by at least 50%. In certain embodiments, the dsRNA inhibits MAPT gene expression by at least 80%.

[0465] The above and other features and advantages of this disclosure will be better understood from the detailed description of the exemplary embodiments in conjunction with the accompanying drawings. This patent or application document includes at least one drawing drawn in color. Copies of this patent or patent application publication, including the color drawing, are available from the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0466] [Figure 1A] This figure shows the screening of siRNAs targeting human MAPT mRNA sequences in SH-SY5Y human neuroblastoma cells. It shows the screening of 12 sequences that identify MAPT1971, MAPT2051, and MAPT2012 as novel target regions. [Figure 1B] This figure shows the screening of siRNAs targeting the human MAPT mRNA sequence in SH-SY5Y human neuroblastoma cells. The 8-point dose-response curve obtained with MAPT1971(B)siRNA is shown. [Figure 1C] This figure shows the screening of siRNAs targeting human MAPT mRNA sequences in SH-SY5Y human neuroblastoma cells. The 8-point dose-response curve obtained with MAPT2051(C)siRNA is shown. [Figure 1D]This figure shows the screening of siRNAs targeting the human MAPT mRNA sequence in SH-SY5Y human neuroblastoma cells. The 8-point dose-response curve obtained with MAPT2012(D)siRNA is shown. [Figure 2A] This report describes a screening of siRNAs targeting human and mouse MAPT mRNA sequences in SH-SY5Y human neuroblastoma cells. It also presents a screening of 12 sequences that identify MAPT2034, MAPT2007, and MAPT2005 as novel target regions. [Figure 2B] This paper shows the screening of siRNAs targeting human and mouse MAPT mRNA sequences in SH-SY5Y human neuroblastoma cells. The 8-point dose-response curve obtained with MAPT2034(B)siRNA is shown. [Figure 2C] This report shows the screening of siRNAs targeting human and mouse MAPT mRNA sequences in SH-SY5Y human neuroblastoma cells. The 8-point dose-response curves obtained with MAPT2007(C)siRNA are shown. [Figure 2D] This report shows the screening of siRNAs targeting human and mouse MAPT mRNA sequences in SH-SY5Y human neuroblastoma cells. The 8-point dose-response curves obtained with MAPT2005(D)siRNA are shown. [Figure 3] The siRNA chemical scaffolds evaluated for MAPT are shown. [Figure 4A] This shows a screening of 48 sequences targeting MAPT using a smooth scaffold, which is the applied chemical scaffold. Hit sequences are shown in yellow. *, small amount of double stranding; **, not fully protected; red arrow: cell death induced. [Figure 4B] This shows a screening of 48 sequences targeting MAPT using P3 blunting and mismatching at positions 10 and 11 of the applied chemical scaffold, the sense chain scaffold. Hit sequences are shown in yellow. *, small amount of double stranding; **, not fully protected; red arrow: cell death induced. [Figure 4C]This shows a screening of 48 sequences targeting MAPT using the applied chemical scaffold, the P3 asymmetric scaffold. Hit sequences are shown in yellow. *, small amount of double stranding; **, not fully protected; red arrow: cell death induced. [Figure 4D] This shows a screening of 48 sequences targeting MAPT using the applied chemical scaffolds, P3 asymmetric and ribose sensed chain scaffolds. Hit sequences are shown in yellow. *, small amount of double stranding; **, not fully protected; red arrow: cell death induced. [Figure 4E] This shows a screening of 48 sequences targeting MAPT using the applied chemical scaffold, an OMe-rich asymmetric scaffold. Hit sequences are shown in yellow. *, small amount of double stranding; **, not fully protected; red arrow: cell death induced. [Figure 4F] This shows a screening of 48 MAPT-targeting sequences using the applied chemical scaffold, an OMe-rich asymmetric plus-ribose sense chain scaffold. Hit sequences are shown in yellow. *, small amount of double stranding; **, not fully protected; red arrow: cell death induced. [Figure 5A] This figure shows the concentration response of the active MAPT sequence (selected) MAPT357. [Figure 5B] This figure shows the concentration response of the active MAPT sequence (selected) MAPT2257. [Figure 5C] This figure shows the concentration response of the active MAPT sequence (selected) MAPT2378. [Figure 6] This figure shows the screening of siRNAs targeting human MAPT mRNA sequences in SH-SY5Y human neuroblastoma cells. [Figure 7A] This figure shows the screening of siRNAs targeting human MAPT mRNA sequences in SH-SY5Y human neuroblastoma cells. [Figure 7B] This figure shows the screening of siRNAs targeting mouse MAPT mRNA sequences in N2A mouse neuroblastoma cells. [Figure 8-1]This figure shows the dose response of selected MAPT target sequences in the P5 chemical modification pattern. [Figure 8-2] This figure shows the dose response of selected MAPT target sequences in the P5 chemical modification pattern. [Figure 9] This figure shows the dose response of selected MAPT target sequences in the P3 chemical modification pattern. [Figure 10] This figure shows further screening of siRNAs targeting various MAPT mRNA target sequences in the ORF and 3'UTR. Screening was performed in SH-SY5Y human neuroblastoma cells. Each siRNA was used at a concentration of 1.5 μM and incubated with cells for 72 hours before relative mRNA expression was quantified. [Figure 11] This figure shows further screening of siRNAs targeting various MAPT mRNA target sequences in ORFs. The targets are found in both human and mouse MAPT mRNA. Screening was performed in SH-SY5Y human neuroblastoma cells. Each siRNA was used at a concentration of 1.5 μM and incubated with cells for 72 hours before relative mRNA expression was quantified. [Figure 12A] This figure shows normalized MAPT mRNA expression levels in several mouse brain regions one month after intracerebral / intraventricular (ICV) injection. A 10 nmol dose was used with a 10 μl injection volume of siRNA targeting MAPT target sites designated as MAPT2005, MAPT3309, and MAPT3292. Tau protein levels were normalized against protein vinculin and gapdh. [Figure 12B] This figure shows normalized protein expression levels in several mouse brain regions one month after intracerebral / intraventricular (ICV) injection. A 10 nmol dose was used with a 10 μl injection volume of siRNA targeting MAPT target sites designated as MAPT2005, MAPT3309, and MAPT3292. Tau protein levels were normalized against protein vinculin and gapdh. [Modes for carrying out the invention]

[0467] Novel MAPT target sequences are provided. Novel RNA molecules, such as siRNAs and branched RNA compounds containing siRNAs, that target MAPT mRNA, including one or more of the target sequences of this disclosure.

[0468] Unless otherwise specified, the nomenclature used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization is well known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are carried out in accordance with conventional methods well known in the art, and unless otherwise specified, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out as described herein, in accordance with the manufacturer's specifications or as commonly achieved in the art. The terminology used herein in relation to analytical chemistry, synthetic organic chemistry and pharmaceutical / medical chemistry, as well as laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques are used for the chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0469] Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The use of "or" shall mean "and / or" unless otherwise stated. The use of the term "including," as well as other forms such as "include" and "included," is not limited to these.

[0470] To make this disclosure easier to understand, certain terms are defined first.

[0471] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and N2,N2-dimethylguanosine (also known as "rare" nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups attached to the sugar moiety within an ester linkage. Exemplary nucleotides include nucleoside monophosphates, diphosphates, and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to polymers of nucleotides linked together by phosphodiester or phosphorothioate linkages between 5' and 3' carbon atoms.

[0472] The terms “RNA,” “RNA molecule,” or “ribonucleic acid molecule” refer to polymers of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The terms “DNA,” “DNA molecule,” or “deoxyribonucleic acid molecule” refer to polymers of deoxyribonucleotides. DNA and RNA can be synthesized spontaneously (e.g., by DNA replication or DNA transcription, respectively). RNA can be modified after transcription. DNA and RNA can also be synthesized chemically. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNA that identifies the amino acid sequence of one or more polypeptide chains. This information is converted during protein synthesis when ribosomes bind to mRNA.

[0473] As used herein, the term “small interfering RNA” (“siRNA”) (also known in the art as “short interfering RNA”) refers to RNA (or RNA analogues) containing about 10 to 50 nucleotides (or nucleotide analogues) that can direct or mediate RNA interference. In certain embodiments, an siRNA contains about 15 to 30 nucleotides or nucleotide analogues, or about 16 to 25 nucleotides (or nucleotide analogues), or about 18 to 23 nucleotides (or nucleotide analogues), or about 19 to 22 nucleotides (or nucleotide analogues) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogues). The term “short” siRNA refers to an siRNA containing about 21 nucleotides (or nucleotide analogues), e.g., 19, 20, 21, or 22 nucleotides. The term “long” siRNA refers to an siRNA containing about 24 to 25 nucleotides, e.g., 23, 24, 25, or 26 nucleotides. Short siRNAs may, in some cases, contain fewer than 19 nucleotides, e.g., 16, 17, or 18 nucleotides, provided that the short siRNA retains its ability to mediate RNAi. Similarly, long siRNAs may, in some cases, contain more than 26 nucleotides, provided that the longer siRNA retains its ability to mediate RNAi without further processing of the short siRNA, e.g., enzymatic processing.

[0474] The terms “nucleotide analog,” “modified nucleotide,” or “modified nucleotide” refer to non-standard nucleotides, such as ribonucleotides or deoxyribonucleotides, which do not exist in nature. Exemplary nucleotide analogs are modified at any position to alter the specific chemical properties of a nucleotide, while retaining the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine; the 6-position, e.g., 6-(2-amino)propyluridine; and the 8-position of adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. Furthermore, examples of nucleotide analogs include deazanucleotides, such as 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or those known in the art) nucleotides; and other heterocyclic modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug.10(4):297-310.

[0475] Nucleotide analogs may also involve modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be substituted with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or COOR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications are described in U.S. Patents No. 5,858,988 and No. 6,291,438.

[0476] The phosphate group of a nucleotide can also be modified, for example, by substituting one or more oxygen atoms of the phosphate group with sulfur (e.g., a phosphorothioate), or by making other substitutions so that the nucleotide can perform its intended function. For example, see Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5):317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Patent No. 5,684,143. The specific modifications referenced above (e.g., phosphate group modifications) reduce the hydrolysis rate of polynucleotides, including their analogs, for example, in vivo or in vitro.

[0477] The term "oligonucleotide" refers to a short polymer of nucleotides and / or nucleotide analogs.

[0478] The term “RNA analog” refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one modified or altered nucleotide compared to the corresponding unmodified or unaltered RNA, but retains the same or similar properties or functions as the corresponding unmodified or unaltered RNA. As described above, oligonucleotides may be linked by phosphodiester linkages that, compared to RNA molecules with phosphodiester linkages, result in a reduced hydrolysis rate of the RNA analog. For example, the nucleotides of an analog may include methylenediol, ethylenediol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoramidate, and / or phosphorothioate linkages. Some RNA analogs include sugar and / or skeletal-modified ribonucleotides and / or deoxyribonucleotides. Such alterations or alterations may further include the addition of non-nucleotide substances to the terminal(s) or internal(s) (one or more nucleotides) of the RNA. RNA analogs only need to be sufficiently similar to native RNA that has the ability to mediate RNA interference.

[0479] As used herein, the term “RNA interference” (“RNAi”) refers to the selective intracellular degradation of RNA. RNAi occurs naturally within cells and removes foreign RNA (e.g., viral RNA). Natural RNAi proceeds via fragments cleaved from free dsRNA, directing the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by humans, for example, to silence the expression of a target gene.

[0480] RNAi agents, such as RNA silencing agents, have a chain that is "sufficiently complementary to the target mRNA sequence in order to direct target-specific RNA interference (RNAi)." This means that the chain has a sequence sufficient to cause the destruction of the target mRNA by the RNAi mechanism or process.

[0481] As used herein, “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) refers to an RNA molecule that, if produced by recombinant technology, is substantially free of other cell material or culture medium, and, if chemically synthesized, is substantially free of chemical precursors or other chemicals.

[0482] As used herein, the term “RNA silencing” refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), querring, co-repression, and translational repression) that result in inhibition or “silencing” of the expression of the corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0483] The term “discriminative RNA silencing” refers to the ability of an RNA molecule to substantially inhibit the expression of a “first” or “target” polynucleotide sequence while not substantially inhibiting the expression of a “second” or “non-target” polynucleotide sequence, for example, when both polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene, and the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele, and the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is a DNA sequence encoding a regulatory region (e.g., a promoter or enhancer element) of the target gene. In other embodiments, the target polynucleotide sequence is the target mRNA encoded by the target gene.

[0484] The term "in vitro" has the meaning recognized in the art, including, for example, purified reagents or extracts, such as cell extracts. The term "in vivo" also has the meaning recognized by those skilled in the art, including, for example, living cells, such as immortalized cells, primary cells, cell lines, and / or cells within an organism.

[0485] As used herein, the term “transgene” refers to any nucleic acid molecule that is artificially inserted into a cell and becomes part of the genome of an organism that develops from the cell. Such a transgene may include a gene that is partially or completely heterologous (i.e., exotic) to the transgenic organism, or it may represent a gene that is homologous to the organism’s endogenous genes. “Transgene” also means a nucleic acid molecule containing one or more selected nucleic acid sequences, e.g., DNA, that encodes one or more manipulated RNA precursors expressed in a transgenic organism, e.g., an animal, which is either partially or completely heterologous (i.e., exogenous) to the transgenic animal, or homologous to the transgenic animal’s endogenous genes but designed to be inserted into the animal’s genome at a location different from that of the native genes. The transgene may include one or more promoters and any other DNA, e.g., introns, all of which are operably ligated to the selected nucleic acid sequence and may include enhancer sequences.

[0486] Genes “involved” in a disease or disorder include genes whose normal or abnormal expression or function affects or causes a disease or disorder, or at least one symptom of a disease or disorder.

[0487] As used herein, the term “gain-of-function mutation” refers to any mutation in a gene that causes or contributes to a disease or disorder by causing a protein encoded by that gene (i.e., a mutant protein) to acquire a function not normally associated with that protein (i.e., a wild-type protein). A gain-of-function mutation may be a deletion, addition, or substitution of nucleotides in a gene that causes a change in the function of the encoded protein. In one embodiment, a gain-of-function mutation alters the function of the mutant protein or causes an interaction with another protein. In another embodiment, a gain-of-function mutation causes, for example, a reduction or removal of the normal wild-type protein through interaction between the modified mutant protein and the normal wild-type protein.

[0488] As used herein, the term “target gene” refers to a gene whose expression is substantially inhibited or “silenced.” This silencing can be achieved, for example, by cleaving the mRNA of the target gene or by silencing the RNA by translational repression of the target gene. The term “non-target gene” refers to a gene whose expression is not substantially silenced. In one embodiment, the polynucleotide sequences of the target gene and the non-target gene (e.g., the mRNA encoded by the target gene and the non-target gene) may differ by one or more nucleotides. In another embodiment, the target and non-target genes may differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In yet another embodiment, the target and non-target genes may share less than 100% sequence identity. In yet another embodiment, the non-target gene may be a homolog (e.g., an ortholog or paralog) of the target gene.

[0489] A “target allele” is an allele (e.g., an SNP allele) whose expression is selectively inhibited or “silenced.” This silencing can be achieved by RNA silencing, for example, by cleaving the target gene or the mRNA of the target allele with siRNA. A “non-target allele” is an allele whose expression is substantially not silenced. In certain embodiments, the target allele and the non-target allele may correspond to the same target gene. In other embodiments, the target allele may correspond to or be associated with the target gene, and the non-target allele may correspond to or be associated with the non-target gene. In one embodiment, the polynucleotide sequences of the target allele and the non-target allele may differ by one or more nucleotides. In another embodiment, the target allele and the non-target allele may differ by one or more allele polymorphisms (e.g., one or more SNPs). In yet another embodiment, the target allele and the non-target allele may share less than 100% sequence identity.

[0490] As used herein, the term “polymorphism” refers to a variation in a gene sequence (e.g., one or more deletions, insertions, or substitutions) that is identified or detected when comparing the same gene sequence from different sources or subjects (but from the same organism). For example, polymorphisms can be identified when comparing the same gene sequence from different subjects. Identification of such polymorphisms is commonplace in the art, and the methodology is similar to that used, for example, to detect point mutations in breast cancer. Identification can be performed, for example, from DNA extracted from lymphocytes of the subject, and the polymorphic region can then be amplified using a specific primer for the polymorphic region. Alternatively, polymorphisms can be identified when comparing two alleles of the same gene. In certain embodiments, the polymorphism is a single nucleotide polymorphism (SNP).

[0491] A variation in the sequence between two alleles of the same gene within an organism is referred to herein as “allelic polymorphism.” In certain embodiments, allelic polymorphisms correspond to SNP alleles. For example, an allelic polymorphism may include a single-nucleotide variation between two alleles of an SNP. Polymorphisms may be located in nucleotides within a coding region, but due to the degeneracy of the genetic code, changes in the amino acid sequence are not encoded. Alternatively, a polymorphic sequence may encode different amino acids at a particular position, but the changes in amino acids do not affect the function of the protein. Polymorphic regions can also be found in the non-coding regions of a gene. In exemplary embodiments, polymorphisms are found in the coding region of a gene or in the uncoding region of a gene (e.g., 5'UTR or 3'UTR).

[0492] As used herein, the term “allele frequency” is a measure (e.g., a proportion or percentage) of the relative frequency of an allele (e.g., a SNP allele) at a single locus in an individual population. For example, if a population of individuals has n loci of a particular chromosomal locus (and the gene occupying that locus) within each somatic cell, the allele frequency of an allele is the proportion or percentage of the locus that the allele occupies in the population. In certain embodiments, the allele frequency of an allele (e.g., a SNP allele) is at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, or more) in the sample population.

[0493] As used herein, the term “sample population” refers to a group of individuals that includes a statistically significant number of individuals. For example, a sample population may include 50, 75, 100, 200, 500, 1000, or more individuals. In certain embodiments, a sample population may include individuals that share at least a common disease phenotype (e.g., gain-of-function disorder) or mutation (e.g., gain-of-function mutation).

[0494] As used herein, the term “heterozygous” refers to the proportion of individuals in any given population who are heterozygous (i.e., possess two or more different alleles) at a particular locus (e.g., a SNP). Heterozygousness can be calculated for a sample population using methods well known to those skilled in the art.

[0495] As used herein, the term “polyglutamine domain” refers to a protein segment or domain consisting of consecutive glutamine residues linked by peptide bonds. In one embodiment, the consecutive region includes at least five glutamine residues.

[0496] As described herein, MAPT refers to the gene encoding the microtubule-associated tau protein. The MAPT gene encoding the tau protein is located on chromosome 17q21 and contains 16 exons. The major tau protein in the human brain is encoded by 11 exons. Exons 2, 3, and 10 are alternatively spliced, resulting in the formation of six tau isoforms ranging in size from 352 to 441 amino acids. The tau protein can be subdivided into four domains: the N-terminal domain, the proline-rich domain, the microtubule-binding domain, and the C-terminal domain. The N-terminal domain plays a role in providing spacing between microtubules. The proline-rich domain plays a role in cellular signaling and interaction with protein kinases. The microtubule-binding domain is important for binding to microtubules. The C-terminal domain is important for regulating microtubule polymerization. Normally, tau is unfolded and phosphorylated. As seen in the brains of patients with primary tauopathy, in its abnormal form, tau protein is hyperphosphorylated and aggregates, containing a β-pleated sheet conformation. Tau binding to microtubules is regulated by the tau phosphorylation / dephosphorylation equilibrium. Tau hyperphosphorylation leads to a loss of tau interaction with microtubules, which results in microtubule dysfunction, impaired axonal transport, and tau fibrillation.

[0497] As described herein, the term tauopathy refers to a family of neurodegenerative diseases characterized by the aggregation of tau protein into neurofibrillary tangles or collagen tangles (NFTs) in the human brain. These tangles are formed by hyperphosphorylation of tau protein. Hyperphosphorylation causes tau protein to dissociate from microtubules, forming insoluble aggregates. These aggregates are sometimes called paired helix filaments. Examples of tauopathy include Alzheimer's disease, primary age-related tauopathy (PART) (senile dementia with a predominance of neurofibrillary tangles, similar to AD but without plaques), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), Ritico-Bodig disease (Guam's Parkinson-dementia complex), gliomas and gangliocytomas, meningeal hemangiomatosis, post-encephalitis parkinsonism, subacute sclerosing panencephalitis (SSPE), lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, and lipofuscinosis, Pick's disease, and corticobasal degeneration. Furthermore, patients with Huntington's disease exhibit tau inclusions aggregated within various brain structures. Tauopathies may overlap with synucleinopathy, such as Parkinson's disease, due to potential interactions between synuclein and tau proteins.

[0498] As used herein, the terms “elongated polyglutamine domain” or “elongated polyglutamine segment” refer to a segment or domain of protein containing at least 35 consecutive glutamine residues linked by peptide bonds. Such elongated segments are found in subjects suffering from polyglutamine disorders as described herein, regardless of whether the subjects exhibit symptoms.

[0499] As used herein, the terms “trinucleotide repeat” or “trinucleotide repeat region” refer to a segment of a nucleic acid sequence consisting of consecutive repeats of a particular trinucleotide sequence. In one embodiment, a trinucleotide repeat includes at least five consecutive trinucleotide sequences. Examples of trinucleotide sequences, but not limited to these, include CAG, CGG, GCC, GAA, CTG, and / or CGG.

[0500] As used herein, the term “trinucleotide repeat disorder” refers to any disorder or disability characterized by an elongated trinucleotide repeat region located within a gene, where the elongated trinucleotide repeat region is the cause of the disorder or disability. Examples of trinucleotide repeat disorders, but not limited to, include Huntington's disease (HD), spinocerebellar ataxia type 12, spinocerebellar ataxia type 8, fragile X syndrome, fragile XE intellectual disability, Friedreich’s ataxia, and myotonic dystrophy. Representative trinucleotide repeat disorders for treatment according to this disclosure are characterized by, or caused by, an elongated trinucleotide repeat region at the 5' end of the coding region of a gene, where the gene encodes a mutated protein, causing or being the cause of the disorder or disability. Certain trinucleotide disorders, such as fragile X syndrome, where the mutation is not related to the coding region, may not be suitable for treatment by the methodology of this disclosure because there is no suitable mRNA to target by RNAi. In contrast, diseases such as Friedreich's ataxia may be suitable for treatment using the methodology of this disclosure because the causative mutation is not located in the coding region (i.e., within an intron), but rather in, for example, the mRNA precursor (e.g., a press-plicated mRNA precursor).

[0501] The phrase "to examine the function of genes in cells or organisms" refers to examining or studying the resulting expression, activity, function, or phenotype.

[0502] As used herein, the term “RNA silencing agent” refers to RNA capable of inhibiting or “silencing” the expression of a target gene. In certain embodiments, RNA silencing agents can prevent the complete processing of mRNA molecules (e.g., complete translation and / or expression) through a post-transcriptional silencing mechanism. Examples of RNA silencing agents include small (<50 b.p.), non-coding RNA molecules, such as double-stranded RNAs containing paired strands, and precursor RNAs capable of generating such small non-coding RNAs. Exemplary RNA silencing agents include siRNA, miRNA, siRNA-like double-stranded RNAs, antisense oligonucleotides, GAPMER molecules, and dual-function oligonucleotides, as well as their precursors. In one embodiment, RNA silencing agents can induce RNA interference. In another embodiment, RNA silencing agents can mediate translational repression.

[0503] As used herein, the term “rare nucleotide” refers to naturally occurring nucleotides that occur infrequently, such as naturally occurring deoxyribonucleotides or ribonucleotides that do not occur infrequently, such as guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides, but not limited to, include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0504] The term "engineered" indicates that the precursor or molecule is not found in nature, in that all or part of the nucleic acid sequence of the precursor or molecule is created or selected by humans, as in the case of an engineered RNA precursor or engineered nucleic acid molecule. Once created or selected, the sequence is replicated, translated, transcribed, or otherwise processed by intracellular mechanisms. Therefore, an RNA precursor produced intracellularly from a transgene containing an engineered nucleic acid molecule is an engineered RNA precursor.

[0505] As used herein, the term “microRNA” (“miRNA”) is also known in the art as “small temporal RNA” (“stRNA”) and refers to a small (e.g., 10 to 50 nucleotides) RNA that is genetically encoded (e.g., by the genomes of viruses, mammals, or plants) and can direct or mediate RNA silencing. “miRNA disorder” refers to a disease or disorder characterized by abnormal expression or activity of miRNA.

[0506] As used herein, the term “dual-functional oligonucleotide” refers to an RNA silencing agent having the formula TL-μ, where T is the mRNA targeting portion, L is the ligation portion, and μ is the miRNA mobilization portion. As used herein, the terms “mRNA targeting portion,” “targeting portion,” “mRNA targeting portion,” or “targeting portion” refer to a domain, portion, or region of a dual-functional oligonucleotide that is sufficiently sized and sufficiently complementary to a portion or region of mRNA selected or targeted for silencing (i.e., the portion has a sequence sufficient to capture the target mRNA).

[0507] As used herein, the terms “linking moiety” or “linking portion” refer to a domain, part, or region of an RNA silencing agent that covalently joins or links mRNA.

[0508] As used herein, the term “antisense strand” of an RNA silencing agent, e.g., siRNA or RNA silencing agent, refers to a strand substantially complementary to a section of approximately 10–50 nucleotides, e.g., approximately 15–30 nucleotides, 16–25, 18–23, or 19–22 nucleotides, of the mRNA of the targeted gene for silencing. The antisense strand, or first strand, has a sequence that is sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., sufficiently complementary to cause disruption of the desired target mRNA by an RNAi mechanism or process (RNAi interference), or sufficiently complementary to cause translational repression of the desired target mRNA.

[0509] The terms “sense strand” or “second strand” of an RNA silencing agent, such as siRNA, refer to a strand complementary to the antisense strand or first strand. The antisense strand and sense strand can also be referred to as the first strand or second strand, where the first strand or second strand is complementary to the target sequence, and each second strand or first strand is complementary to the first strand or second strand. A miRNA double-stranded intermediate or siRNA-like double-stranded complex includes a miRNA strand that is sufficiently complementary to a section of approximately 10–50 nucleotides of the mRNA of the gene targeted for silencing, and a miRNA* strand that is sufficiently complementary to form a double-stranded complex with the miRNA strand.

[0510] As used herein, the term “guide strand” refers to the strand of RNA silencing agent, such as a double-stranded siRNA or antisense strand of an siRNA sequence, that enters the RISC complex and directs the cleavage of the target mRNA.

[0511] As used herein, the term “asymmetry” refers to an unevenness in the binding strength or base pair strength between the ends of an RNA silencing agent (e.g., between the terminal nucleotides on the first strand or stem portion and the terminal nucleotides on the opposing second strand or stem portion), such as in the asymmetry of the double-stranded region of the RNA silencing agent (e.g., the stem of shRNA). This results in the 5' end of one strand of the double helix being more frequently in a transient unpaired state, e.g., a single-stranded state, than the 5' end of the complementary strand. This structural difference determines that one strand of the double helix is ​​preferentially incorporated into the RISC complex. The strand whose 5' end is less firmly paired with the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

[0512] As used herein, the terms “bond strength” or “base pair strength” primarily refer to the strength of interactions between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide double-strand (e.g., an siRNA double-strand), and between these nucleotides (or nucleotide analogs), such as through H bonds and van der Waals interactions.

[0513] As used herein, "5' end" refers to the 5' terminal nucleotide, for example, the 1 to approximately 5 nucleotides at the 5' end of an antisense strand, such as the 5' end of an antisense strand. As used herein, "3' end" refers to the region complementary to the 5' terminal nucleotide of a complementary antisense strand, for example, the 1 to approximately 5 nucleotide region, such as the 3' end of a sense strand.

[0514] As used herein, the term “destabilized nucleotide” refers to a first nucleotide or nucleotide analog that can form a base pair with a second nucleotide or nucleotide analog such that the base pair has a lower binding strength than a conventional base pair (i.e., a Watson-Crick base pair). In certain embodiments, the destabilized nucleotide can form a mismatch base pair with the second nucleotide. In other embodiments, the destabilized nucleotide can form a fluctuation base pair with the second nucleotide. In yet another embodiment, the destabilized nucleotide can form an ambiguous base pair with the second nucleotide.

[0515] As used herein, the term “base pair” refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide double helix (e.g., a double helix formed by the strands of an RNA silencing agent and a target mRNA sequence), primarily through nucleotide (or nucleotide analog) H bonds, van der Waals interactions, and other interactions. As used herein, the terms “bond strength” or “base pair strength” refer to the strength of a base pair.

[0516] As used herein, the term "mismatched base pair" refers to a base pair consisting of a non-complementary or non-Watson-Crick base pair that is not a normal complementary G:C, A:T, or A:U base pair. As used herein, the term "ambiguous base pair" (also known as a non-recognized base pair) refers to a base pair formed by a universal nucleotide.

[0517] As used herein, the term “universal nucleotide” (also known as “neutral nucleotide”) includes nucleotides (e.g., certain destabilized nucleotides) that have bases that do not significantly distinguish between bases on a complementary polynucleotide when forming base pairs (“universal bases” or “neutral bases”). Universal nucleotides are primarily hydrophobic molecules and can efficiently pack into antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA) through stacking interactions. The base portion of a universal nucleotide typically contains a nitrogen-containing aromatic heterocyclic moiety.

[0518] As used herein, the terms “sufficient complementarity” or “sufficient degree of complementarity” mean that the RNA silencing agent has sequences (e.g., in the antisense strand, mRNA targeting portion, or miRNA recruiting portion) sufficient to bind to the desired target and induce RNA silencing of the target mRNA, respectively.

[0519] As used herein, the term “translational repression” refers to the selective inhibition of mRNA translation. Spontaneous translational repression proceeds via miRNA cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur spontaneously or can be initiated by humans, for example, to silence the expression of a target gene.

[0520] Various methodologies of this disclosure include a step of comparing values, levels, features, properties, etc., with a “preferred control,” which is interchangeably referred to herein as “appropriate control.” A “preferred control” or “appropriate control” is any control or standard well known to those skilled in the art and useful for comparison purposes. In one embodiment, a “preferred control” or “appropriate control” is a value, level, feature, property, etc., determined before performing the RNAi methodology as described herein. For example, transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristics or properties, genotype, phenotype, etc., can be determined before introducing the RNA silencing agent of this disclosure into cells or organisms. In another embodiment, a “preferred control” or “appropriate control” is a value, level, feature, property, etc., determined in cells or organisms, e.g., a control or, e.g., normal cells or organisms exhibiting normal traits. In yet another embodiment, a “preferred control” or “appropriate control” is a predefined value, level, feature, property, etc.

[0521] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Similar or equivalent methods and materials may be used in the implementation or testing of this disclosure, but suitable methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification shall prevail, including definitions. In addition, materials, methods, and examples are illustrative and not intended to be limiting.

[0522] Various aspects of this disclosure are described in further detail in the following subsections.

[0523] I. Novel target sequences In certain exemplary embodiments, the RNA silencing agent of the Disclosure may target one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295, as listed in Tables 4-6. In certain exemplary embodiments, the RNA silencing agent of the Disclosure may target one or more MAPT nucleic acid sequences selected from the group consisting of SEQ ID NOs: 14-33, 299, and 302, as listed in Tables 7-8.

[0524] The genomic sequences of each target sequence can be found, for example, in publicly available databases maintained by NCBI.

[0525] II. Design of AVA In some embodiments, the siRNA is designed as follows: First, a portion of the target gene (e.g., the MAPT gene), for example, one or more target sequences shown in Tables 4-6, are selected. The cleavage of mRNA at these sites eliminates the need for translation of the corresponding protein. The antisense strand is designed based on the target sequence, and the sense strand is designed to be complementary to the antisense strand. Hybridization of the antisense and sense strands forms the siRNA double helix. The antisense strand contains approximately 19-25 nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides. In other embodiments, the antisense strand contains 20, 21, 22, or 23 nucleotides. The sense strand contains approximately 14-25 nucleotides, e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In other embodiments, the sense strand is 15 nucleotides. In certain embodiments, the sense strand is 18 nucleotides. In other embodiments, the sense strand is 20 nucleotides. However, those skilled in the art will understand that siRNAs having a length of less than 19 nucleotides or a length of more than 25 nucleotides can also function to mediate RNAi. Thus, siRNAs of such lengths are also within the scope of this disclosure, insofar as they retain the ability to mediate RNAi. Longer RNAi agents have been demonstrated to induce interferon or PKR responses in certain mammalian cells, which may be undesirable. In certain embodiments, the RNAi agents of this disclosure do not induce a PKR response (i.e., are sufficiently short in length). However, longer RNAi agents may be useful, for example, in cell types that cannot produce a PKR response, or in situations where the PKR response is downregulated or suppressed by alternative means.

[0526] Sense strand sequences can be designed so that the target sequence is essentially located in the center of the strand. By shifting the target sequence to an off-center position, the efficiency of siRNA cleavage may, in some cases, be reduced. Such compositions, i.e., less efficient compositions, may be desirable for use when off-silencing of wild-type mRNA is detected.

[0527] The antisense strand may be the same length as the sense strand and may contain complementary nucleotides. In one embodiment, the strands are perfectly complementary; that is, they have blunt ends when aligned or annealed. In another embodiment, the strands are aligned or annealed such that a 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide overhang is generated; that is, the 3' end of the sense strand is 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides longer than the 5' end of the antisense strand, and / or the 3' end of the antisense strand is 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides longer than the 5' end of the sense strand. The overhang may contain (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhang may contain (or consist of) deoxyribonucleotides, e.g., dT, or nucleotide analogs, or other suitable non-nucleotide substances.

[0528] To facilitate the entry of the antisense strand into RISC (and thus increase or improve the efficiency of target cleavage and silencing), the base pair strength between the 5' end of the sense strand and the 3' end of the antisense strand may be modified (decreased or reduced). These are described in detail below and are incorporated by this reference into U.S. Patents 7,459,547, 7,772,203 and 7,732,593, titled “Methods and Compositions for Controlling Efficacy of RNA Silencing” (filed June 2, 2003) and U.S. Patents 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705, titled “Methods and Compositions for Enhancing the Efficacy and Specificity of RNAi” (filed June 2, 2003). In one embodiment of these aspects of the Disclosure, the base pair strength is low because there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the second or sense strand than there are between the 3' end of the first or antisense strand and the 5' end of the second or sense strand. In another embodiment, the base pair strength is low due to at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In certain exemplary embodiments, the mismatched base pair is selected from the group consisting of: G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In another embodiment, the base pair strength is low due to at least one fluctuating base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In another embodiment, the base pair strength is low due to at least one base pair containing a rare nucleotide, e.g., inosine(I). In certain exemplary embodiments, the base pairs are selected from the group consisting of I:A, I:U, and I:C. In yet another embodiment, the base pair strength is lower due to at least one base pair containing a modified nucleotide.In certain exemplary embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

[0529] The designs of siRNAs suitable for targeting the MAPT target sequences shown in Tables 4-6 are described in detail below. siRNAs can be designed for any other target sequences found in the MAPT gene according to the exemplary teachings above. Furthermore, this technique is applicable to targeting any other target sequences, such as non-disease-causing target sequences.

[0530] To verify the effectiveness of siRNA in disrupting mRNA (e.g., MAPT mRNA), siRNA can be incubated with cDNA (e.g., MAPT cDNA) in an in vitro mRNA expression system based on Drosophila melanogaster. 32 Newly synthesized mRNA (e.g., MAPT mRNA) radiolabeled with 3P is detected by autoradiography on an agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include the omission of siRNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA but lacks significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to confirm that the negative control lacks homology to any other gene in the appropriate genome. Furthermore, negative control siRNA can be designed by introducing one or more base mismatches into the sequence. The siRNA-mRNA complementation site that yields optimal mRNA specificity and maximum mRNA cleavage is selected.

[0531] III. RNAi agents This disclosure includes RNAi molecules, such as siRNA molecules designed as described above. The siRNA molecules of this disclosure can be chemically synthesized, or transcribed in vitro from a DNA template, or in vivo from shRNA, for example, or by cleaving a transcribed dsRNA template in vitro using recombinant human DICER enzyme to form a pool of 20, 21, or 23 bp double-stranded RNA-mediated RNAi. The siRNA molecules can be designed using any method known in the art.

[0532] In one embodiment, instead of the RNAi agent being an interfering ribonucleic acid, such as the siRNA or shRNA described above, the RNAi agent may encode an interfering ribonucleic acid, such as the shRNA described above. In other words, the RNAi agent may serve as a transcription template for an interfering ribonucleic acid. Therefore, the RNAi agents of this disclosure may also include small hairpin RNA (shRNA) and expression constructs engineered to express shRNA. The transcription of shRNA is thought to be initiated by the polymerase III (pol III) promoter and terminated at position 2 of the 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with a 3' UU overhang. Subsequently, the ends of these shRNAs are processed, converting them into siRNA-like molecules of approximately 21-23 nucleotides (Brummelkamp et al., 2002; Lee et al., 2002, above; Miyagishi et al., 2002; Paddison et al., 2002, above; Paul et al., 2002, above; Sui et al., 2002, above; Yu et al., 2002, above. Further information on shRNA design and use can be found on the internet at the following addresses: katandin.cshl.org:9331 / RNAi / docs / BseRI-BamHI_Strategy.pdf and katandin.cshl.org:9331 / RNAi / docs / Web_version_of_PCR_strategy1.pdf).

[0533] Expression constructs in this disclosure include, but are not limited to, any constructs suitable for use in a suitable expression system, as are known in the art, retroviral vectors, linear expression cassettes, plasmids, and viruses or virus-derived vectors. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems, e.g., the U6 snRNA promoter or the H1 RNA polymerase III promoter, or other promoters known in the art. A construct may contain one or both strands of siRNA. An expression construct expressing both strands may also contain a loop structure linking both strands, or each strand may be transcribed separately from separate promoters within the same construct. Each strand may be transcribed from a separate expression construct (Tuschl, T., 2002, above).

[0534] Synthetic siRNA can be delivered to cells by methods known in the art, such as cationic liposome transfection and electroporation. One or more siRNAs can be expressed intracellularly from recombinant DNA constructs to obtain long-term suppression of target genes (e.g., MAPT genes) and to facilitate delivery under specific circumstances. Such methods for expressing double-stranded siRNAs intracellularly from recombinant DNA constructs to enable longer-term target gene suppression in cells are known in the art, such as mammalian Pol III promoter systems capable of expressing functional double-stranded siRNA (e.g., H1 or U6 / snRNA promoter systems (Tuschl, T., 2002, above) (Bagella et al., 1998; Lee et al., 2002, above; Miyagishi et al., 2002, above; Paul et al., 2002, above; Yu et al., 2002, above; Sui et al., 2002, above)). PolIII-mediated transcription termination occurs at a sequence of four consecutive T residues within the DNA template, providing a mechanism to terminate siRNA transcripts at specific sequences. siRNA is complementary to the target gene sequence in the 5'-3' and 3'-5' directions, and the two strands of siRNA can be expressed in the same construct or separate constructs. Hairpin siRNAs, driven by H1 or U6 snRNA promoters and expressed intracellularly, can inhibit the expression of target genes (Bagella et al., 1998; Lee et al., 2002, above; Miyagishi et al., 2002, above; Paul et al., 2002, above; Yu et al., 2002), above; Sui et al., 2002, above). Constructs containing siRNA sequences under the control of the T7 promoter also produce functional siRNA when co-transfected into cells with a vector expressing T7 RNA polymerase (Jacque et al., 2002, above).A single construct may include multiple sequences encoding siRNA, such as multiple regions of a MAPT-encoding gene targeting the same gene or multiple genes, and may be driven, for example, by another PolIII promoter site.

[0535] Animal cells express a series of non-coding RNAs called microRNAs (miRNAs), each approximately 22 nucleotides long. These can regulate gene expression at the post-transcriptional or translational level during animal development. One common characteristic of miRNAs is that they are entirely cleaved from a precursor RNA stem-loop of approximately 70 nucleotides, likely by the RNase III enzyme Dicer or its homolog. By substituting the stem sequence of the miRNA precursor with a sequence complementary to the target mRNA, vector constructs expressing the engineered precursor can be used to produce siRNA and initiate RNAi against specific mRNA targets in mammalian cells (Zeng et al., above, 2002). When expressed by a DNA vector containing a polymerase III promoter, microRNA-designed hairpins can perform gene expression silencing (McManus et al., 2002, above). MicroRNAs targeting polymorphisms may also be useful for blocking the translation of mutant proteins in the absence of siRNA-mediated gene silencing. Such applications may be useful, for example, in situations where a designed siRNA causes off-target silencing of wild-type proteins.

[0536] Viral delivery mechanisms can also be used to induce specific silencing of targeted genes by generating recombinant adenoviruses containing siRNA, for example, under the transcriptional regulation of the RNA Pol II promoter, via siRNA expression (Xia et al., 2002, see above). Infection of HeLa cells with these recombinant adenoviruses can reduce the expression of endogenous target genes. Injecting recombinant adenovirus vectors into transgenic mice expressing siRNA target genes results in in vivo reduction of target gene expression. Ibid. In animal models, synthetic siRNA can be efficiently delivered to transplanted mouse embryos by whole-embryonic electroporation (Calegari et al., 2002). In adult mice, efficient delivery of siRNA can be achieved by “high pressure” delivery techniques, rapidly injecting a large volume of siRNA-containing solution into the animal’s tail vein (within 5 seconds) (Liu et al., 1999, above; McCaffrey et al., 2002, above; Lewis et al., 2002). siRNA can also be delivered to animals using nanoparticles and liposomes. In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs to cells, such as nerve cells (e.g., brain cells) (U.S. Patent Applications Nos. 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542, and 2005 / 0220766).

[0537] The nucleic acid compositions of this disclosure include both unmodified siRNA and modified siRNA, such as cross-linked siRNA derivatives or derivatives having non-nucleotide moieties linked to their 3' or 5' ends. By modifying siRNA derivatives in this way, it is possible to improve cell uptake or enhance the cell targeting activity of the resulting siRNA derivatives compared to the corresponding siRNA, and it is also useful for tracking siRNA derivatives within cells or improving the stability of siRNA derivatives compared to the corresponding siRNA.

[0538] As described herein, an engineered RNA precursor introduced into a cell or organism leads to the production of a desired siRNA molecule. Such siRNA molecule then associates with endogenous protein components of the RNAi pathway, binding to a specific mRNA sequence and targeting it for cleavage and disruption. In this way, the mRNA targeted by the siRNA produced from the engineered RNA precursor is depleted from the cell or organism, resulting in a decrease in the concentration of the protein encoded by that mRNA in the cell or organism. The RNA precursor is typically a nucleic acid molecule that either codes for one strand of dsRNA individually or for the entire nucleotide sequence of an RNA hairpin loop structure.

[0539] The nucleic acid compositions of this disclosure may be conjugated or conjugated to another portion such as nanoparticles, thereby improving the properties of the compositions, such as pharmacokinetic parameters such as absorption, efficacy, bioavailability and / or half-life. Conjugation can be achieved, for example, using the methods known in this technology, or the following methods: Lambert et al., Drug Deliv. Rev.:47(1),99-112(2001) (described on nucleic acids attached to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43(1998) (described on nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8(1994) (described on nucleic acids linked to intercalating agents, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10(1995) (described on nucleic acids linked to nanoparticles).

[0540] The nucleic acid molecules of this disclosure can also be labeled using any method known in the art. For example, nucleic acid compositions can be labeled with fluorophores, such as Cy3, fluorescein, or rhodamine. Labeling can be carried out using a kit, such as the SILENCER® siRNA labeling kit (Ambion). Furthermore, siRNA can be labeled, for example, 3 H, 32 It can be radiolabeled using phosphate (P) or another suitable isotope.

[0541] Furthermore, since RNAi is thought to proceed via at least one single-stranded RNA intermediate, those skilled in the art will understand that ss-siRNA (e.g., the antisense strand of ds-siRNA) can also be designed (e.g., for chemosynthesis), generated (e.g., enzymatically), or expressed (e.g., from a vector or plasmid) as described herein, and utilized according to the claimed methodology. Furthermore, in invertebrates, RNAi can be effectively induced by long dsRNAs (e.g., dsRNAs of approximately 100–1000 nucleotides in length, e.g., approximately 200–500, e.g., approximately 250, 300, 350, 400, or 450 nucleotides in length) acting as effectors of RNAi (Brondani et al., Proc Natl Acad Sci USA. 2001 Dec. 4; 98(25): 14428-33. Epub 2001 Nov. 27.).

[0542] IV. Anti-MAPT RNA silencing agents In certain embodiments, the disclosure provides novel anti-MAPT RNA silencing agents (e.g., siRNA, shRNA, and antisense oligonucleotides), methods for constructing RNA silencing agents, and methods for using improved RNA silencing agents (or parts thereof) for RNA silencing of MAPT proteins (e.g., research and / or therapeutic methods). The RNA silencing agent comprises an antisense strand (or part thereof), the antisense strand having sufficient complementarity to the target MAPT mRNA to mediate an RNA-mediated silencing mechanism (e.g., RNAi).

[0543] In certain embodiments, siRNA compounds having one or any combination of the following properties are provided: (1) completely chemically stabilized (i.e., no unmodified 2'-OH residues); (2) asymmetric; (3) 11-20 base pair double hedges; (4) more than 50% 2'-methoxy modification, e.g., 70-100% 2'-methoxy modification, alternating patterns of chemically modified nucleotides (e.g., 2'-fluoro and 2'-methoxy modification) are also intended; and (5) single-stranded, with a fully phosphorothiolated 5-8 base tail. In certain embodiments, the number of phosphorothioate modifications varies from a total of 4 to 16. In certain embodiments, the number of phosphorothioate modifications varies from a total of 8 to 13.

[0544] In certain embodiments, the siRNA compounds described herein can be conjugated to a variety of targeting agents, including, but are not limited to, cholesterol, docosahexaenoic acid (DHA), phenyltropane, cortisol, vitamin A, vitamin D, N-acetylgalactosamine (GalNac), and gangliosides. The cholesterol-modified form showed a 5- to 10-fold improvement in efficacy in vitro across a wide range of cell types (e.g., HeLa, neurons, hepatocytes, trophoblasts) compared to previously used chemical stabilization patterns (e.g., all purines modified instead of pyrimidines).

[0545] Certain compounds of this disclosure having the structural properties described above and herein may be referred to as “hsiRNA-ASP” (hydrophobically modified small interfering RNA characterized by a highly stabilized pattern). Furthermore, this hsiRNA-ASP pattern exhibits dramatically improved distribution via delivery from the brain and spinal cord to the liver, placenta, kidneys, spleen, and several other tissues, making it available for therapeutic intervention.

[0546] The compounds of this disclosure can be described in the following aspects and embodiments.

[0547] In a first embodiment, a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand is provided herein, each strand comprising at least 14 consecutive nucleotides having a 5' end and a 3' end. (1) The antisense strand contains a sequence substantially complementary to one of the MAPT nucleic acid sequences of sequence numbers 1-13, 292, and 295; (2) The antisense chain consists of alternating 2'-methoxyribonucleotides and 2'-fluororibonucleotides; (3) The nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from positions 1-2 to 1-7 of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense strand consists of alternating 2'-methoxyribonucleotides and 2'-fluororibonucleotides; (7) The nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0548] In a second embodiment, a dsRNA comprising an antisense strand and a sense strand is provided herein, each strand comprising at least 14 consecutive nucleotides having a 5' end and a 3' end, (1) The antisense strand contains a sequence substantially complementary to one of the MAPT nucleic acid sequences of sequence numbers 1-13, 292, and 295; (2) The antisense chain contains at least 70% 2'-O-methyl modification; (3) The nucleotide at position 14 from the 5' end of the antisense strand is not a 2'-methoxyribonucleotide; (4) The nucleotides from positions 1-2 to 1-7 of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains at least 70% 2'-O-methyl modification; (7) The nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0549] In a third aspect, a dsRNA comprising an antisense strand and a sense strand is provided herein, each strand comprising at least 14 consecutive nucleotides having a 5' end and a 3' end, (1) The antisense strand contains a sequence substantially complementary to one of the MAPT nucleic acid sequences of sequence numbers 1-13, 292, and 295; (2) The antisense chain contains at least 85% 2'-O-methyl modification; (3) The nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from positions 1-2 to 1-7 of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains 100% 2'-O-methyl modification; (7) The nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0550] In a fourth aspect, a dsRNA comprising an antisense strand and a sense strand is provided herein, each strand comprising at least 14 consecutive nucleotides having a 5' end and a 3' end, (1) The antisense strand contains a sequence substantially complementary to one of the MAPT nucleic acid sequences of sequence numbers 1-13, 292, and 295; (2) The antisense chain contains at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from positions 1-2 to 1-7 of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains 100% 2'-O-methyl modification; (7) The nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0551] In a fifth aspect, a dsRNA comprising an antisense strand and a sense strand is provided herein, each strand comprising at least 14 consecutive nucleotides having a 5' end and a 3' end, (1) The antisense strand contains a sequence substantially complementary to one of the MAPT nucleic acid sequences of sequence numbers 1-13, 292, and 295; (2) The antisense chain contains at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from positions 1-2 to 1-7 of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains 100% 2'-O-methyl modification; (7) The nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0552] In a sixth aspect, a dsRNA comprising an antisense strand and a sense strand is provided herein, each strand comprising at least 14 consecutive nucleotides having a 5' end and a 3' end, (1) The antisense strand contains a sequence substantially complementary to one of the MAPT nucleic acid sequences of sequence numbers 1-13, 292, and 295; (2) The antisense chain contains at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from positions 1-2 to 1-7 of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains at least 70% 2'-O-methyl modification; (7) The nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides; (8) The nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0553] In a seventh aspect, a dsRNA comprising an antisense strand and a sense strand is provided herein, each strand comprising at least 14 consecutive nucleotides having a 5' end and a 3' end, (1) The antisense strand contains a sequence substantially complementary to one of the MAPT nucleic acid sequences of sequence numbers 1-13, 292, and 295; (2) The antisense chain contains at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from positions 1-2 to 1-7 of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains at least 80% 2'-O-methyl modification; (7) The nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides; (8) The nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

[0554] a) Design of anti-MAPT siRNA molecules The siRNA molecule of this application is a double helix composed of a sense strand and a complementary antisense strand, the antisense strand being sufficiently complementary to MAPT mRNA for mediating RNAi. In certain embodiments, the siRNA molecule has a nucleotide length of about 10 to 50 or more, i.e., each strand contains 10 to 50 nucleotides (or nucleotide analogs). In other embodiments, the siRNA molecule has a nucleotide length of about 15 to 30 in each strand, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, where one of the strands is sufficiently complementary to the target region. In certain embodiments, the strands are arranged such that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases are present at the ends of the strands that do not align (i.e., no complementary bases are produced in the opposing strands). This results in an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues occurring at one or both ends of the double helix when the chain is annealed.

[0555] Typically, siRNA can be designed using any method known in the art, for example, using the following protocol:

[0556] 1. The siRNA shall be specific to the target sequence, for example, the target sequence described in the Examples. The first strand shall be complementary to the target sequence, and the other strand shall be substantially complementary to the first strand. (See Examples for exemplary sense and antisense strands.) Exemplary target sequences are selected from any region of the target gene that results in potent gene silencing. Regions of the target gene include, but are not limited to, the 5' untranslated region (5'-UTR) of the target gene, the 3' untranslated region (3'-UTR) of the target gene, the exon of the target gene, or the intron of the target gene. Cleavage of mRNA at these sites eliminates the need for translation of the corresponding MAPT protein. Target sequences from other regions of the MAPT gene are also suitable for targeting. The sense strand is designed based on the target sequence.

[0557] 2. The sense strand of the siRNA is designed based on the sequence of the selected target site. In certain embodiments, the sense strand contains approximately 15–25 nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In certain embodiments, the sense strand contains 15, 16, 17, 18, 19, or 20 nucleotides. In certain embodiments, the sense strand is 15 nucleotides long. In certain embodiments, the sense strand is 18 nucleotides long. In certain embodiments, the sense strand is 20 nucleotides long. However, those skilled in the art will understand that siRNAs having lengths less than 15 nucleotides or longer than 25 nucleotides can also function to mediate RNAi. Thus, siRNAs of such lengths are also within the scope of this disclosure, insofar as they retain the ability to mediate RNAi. Longer RNA silencing agents have been demonstrated to induce interferon or protein kinase R (PKR) responses in certain mammalian cells, which may be undesirable. In certain embodiments, the RNA silencing agents of this disclosure do not induce a PKR response (i.e., are sufficiently short in length). However, longer RNA silencing agents may be useful, for example, in cell types that cannot produce a PKR response, or in situations where the PKR response is downregulated or suppressed by alternative means.

[0558] The siRNA molecules of this disclosure have sufficient complementarity with the target sequence so that the siRNA can mediate RNAi. Generally, siRNA containing a nucleotide sequence sufficiently complementary to the target sequence portion of the target gene is intended to result in RISC-mediated cleavage of the target gene. Therefore, in certain embodiments, the antisense strand of the siRNA is designed to have a sequence sufficiently complementary to a portion of the target. For example, the antisense strand may have 100% complementarity with respect to the target site. However, 100% complementarity is not required. A greater than 80% identity between the antisense strand and the target RNA sequence is intended, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% complementarity. This invention has the advantage of allowing for specific sequence modifications to enhance the efficiency and specificity of RNAi. In one embodiment, the antisense strand has 4, 3, 2, 1, or 0 mismatched nucleotides in a target region, such as a target region, where at least one base pair differs between the wild-type allele and the mutant allele. For example, the target region may contain a gain-of-function mutation, and the other strand may be identical or substantially identical to the first strand. Furthermore, siRNA sequences with small insertions or deletions of 1 or 2 nucleotides may also be effective in mediating RNAi. Alternatively, siRNA sequences with substitutions or insertions of nucleotide analogs may be effective in inhibition.

[0559] Sequence identity can be determined by sequence comparison and alignment algorithms known in the art. To determine the percentage of identity between two nucleic acid sequences (or two amino acid sequences), the sequences are aligned for optimal comparison (for example, gaps can be introduced into the first or second sequence for optimal alignment). Then, the nucleotides (or amino acid residues) at the corresponding nucleotide (or amino acid) positions are compared. If a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences corresponds to the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), and optionally, a penalty is given to the score for the number and / or length of introduced gaps.

[0560] The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. In one embodiment, alignment is generated in specific parts of aligned sequences that have sufficient identity, but not in parts with a low degree of identity (i.e., local alignment). A non-restrictive example of a local alignment algorithm used for sequence comparison is the algorithm of Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 87:2264-68, revised Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-77. Such an algorithm is incorporated into the BLAST program (version 2.0), Altschul, et al. (1990) J.Mol.Biol.215:403-10.

[0561] In another embodiment, the alignment is optimized by introducing appropriate gaps, and percentage identity is determined over the length of the aligned sequence (i.e., the gapped alignment). To obtain a gap alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps, and percentage identity is determined over the entire length of the aligned sequence (i.e., global alignment). A non-restrictive example of a mathematical algorithm used for global sequence comparison is the algorithm by Myers and Miller, CABIOS (1989). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, 12 gap length penalties, and 4 gap penalties can be used.

[0562] 3. The antisense or guide strand of an siRNA is routinely the same length as the sense strand and contains complementary nucleotides. In one embodiment, the guide and sense strands are perfectly complementary; that is, the strands are blunt-ended when aligned or annealed. In another embodiment, the siRNA strands may be paired to have 1-7 (e.g., 2, 3, 4, 5, 6, or 7) or 1-4 3' overhangs, e.g., 2, 3, or 4 nucleotides. The overhangs may contain (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhangs may contain (or consist of) deoxyribonucleotides, e.g., dT, or nucleotide analogs, or other suitable non-nucleotide substances. Thus, in another embodiment, the nucleic acid molecule may have a 2-nucleotide 3' overhang, such as TT. The overhang nucleotides may be either RNA or DNA. As described above, it is desirable to select a target region where the mutant:wild-type mismatch is a purine:purine mismatch.

[0563] 4. By using any method known in the art, potential targets can be compared to appropriate genome databases (human, mouse, rat, etc.) to eliminate the need to consider any target sequences that have significant homology with other coding sequences. One such sequence homology search method is known as BLAST and is available on the National Center for Biotechnology Information website.

[0564] 5. Select one or more sequences that meet the evaluation criteria.

[0565] General information regarding the design and use of siRNA can be found in "The siRNA User Guide," available on the website of The Max-Plank-Institut fur Biophysikalische Chemie.

[0566] Alternatively, siRNA can be functionally defined as a nucleotide sequence (or oligonucleotide sequence) that can hybridize with a target sequence (e.g., hybridize with 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM MEDTA, at 50°C or 70°C for 12–16 hours, followed by washing). Additional hybridization conditions include hybridization with 1xSSC at 70°C or 1xSSC at 50°C with 50% formamide, followed by washing with 0.3xSSC at 70°C, or hybridization with 4xSSC at 70°C or 4xSSC at 50°C with 50% formamide, followed by washing with 1xSSC at 67°C. The hybridization temperature for hybrids expected to be less than 50 base pairs in length is the melting temperature (T) of the hybrid. m ) should be 5-10°C lower. Here, T m This is determined according to the following formula. For hybrids with a length of less than 18 base pairs, T m (°C) = 2(A + T base number #) + 4(G + C base number #). For hybrids with a length of 18-49 base pairs, T m (°C) = 81.5 + 16.6 (log10[Na+]) + 0.41 (%G+C) - (600 / N) (wherein N is the number of bases in the hybridization buffer and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] of 1xSSC) + ]=0.165M). Examples of additional stringency conditions for polynucleotide hybridization are described in Sambrook, J., EFFritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, FMAusubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, which are incorporated herein by reference.

[0567] Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but lack significant sequence complementarity with the appropriate genome. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. Homology searches can be performed to confirm that the negative control lacks homology to any other gene in the appropriate genome. Furthermore, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.

[0568] 6. To verify the effectiveness of siRNA in disrupting mRNA (e.g., wild-type or mutant MAPT mRNA), siRNA can be incubated with target cDNA (e.g., MAPT cDNA) in an in vitro mRNA expression system based on Drosophila melanogaster. 32 A newly synthesized target mRNA (e.g., MAPT mRNA) radiolabeled with 3P is detected by autoradiography on an agarose gel. The presence of cleaved target mRNA indicates mRNA nuclease activity. Suitable controls include the omission of siRNA and the use of non-target cDNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA but does not have significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. Homology searches can be performed to confirm that the negative control lacks homology to any other gene in the appropriate genome. Furthermore, negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0569] Anti-MAPT siRNA can be designed to target any of the target sequences described above. siRNA comprises an antisense strand that is sufficiently complementary to the target sequence to mediate the silencing of the target sequence. In certain embodiments, the RNA silencing agent is siRNA.

[0570] In certain embodiments, the siRNA comprises a sense strand containing the sequences shown in Tables 7 and 8, and an antisense strand containing the sequences shown in Tables 7 and 8.

[0571] The siRNA-mRNA complementation site that provides optimal mRNA specificity and maximum mRNA cleavage is selected.

[0572] b) siRNA-like molecule The siRNA-like molecules of this disclosure have a sequence that is "sufficiently complementary" to the target sequence of MAPT mRNA in order to direct gene silencing by RNAi or translational repression (i.e., they have a sequence-containing strand). The siRNA-like molecules are designed in the same manner as siRNA molecules, but the degree of sequence identity between the sense strand and the target RNA approximates that is observed between miRNA and its target. Generally, when the degree of sequence identity between the miRNA sequence and the corresponding target gene sequence decreases, the tendency for post-transcriptional gene silencing to be mediated by translational repression rather than RNAi increases. Therefore, in another embodiment where post-transcriptional gene silencing by translational repression of the target gene is desired, the miRNA sequence has partial complementarity with the target gene sequence. In certain embodiments, the miRNA sequence has partial complementarity with one or more short sequences (complementary sites) dispersed within the target mRNA (e.g., within the 3'UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003). Because the translational repression mechanism is cooperative, in certain embodiments, multiple complementary sites (e.g., 2, 3, 4, 5, or 6) may be targeted.

[0573] The ability of an siRNA-like double-stranded molecule to mediate RNAi or translational repression can be predicted by the distribution of non-identical nucleotides between the target gene sequence and the nucleotide sequence of the silencing agent in the complementary region. In one embodiment where gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central part of the complementary region such that the double helix formed by the miRNA guide strand and the target mRNA contains a central "bulge" (Doench JG et al., Genes & Dev., 2003). In another embodiment, 2, 3, 4, 5, or 6 consecutive or non-identical nucleotides are introduced. The non-identical nucleotides may be selected to form fluctuating base pairs (e.g., G:U) or mismatch base pairs (G:A, C:A, C:U, G:G, A:A, C:C, U:U). In a further embodiment, the "bulge" is centered on the nucleotides at positions 12 and 13 from the 5' end of the miRNA molecule.

[0574] c) Short hairpin RNA (shRNA) molecule In certain characteristic embodiments, the present disclosure provides shRNAs capable of mediating highly selective RNA silencing of MAPT target sequences. In contrast to siRNAs, shRNAs mimic the natural precursors of microRNAs (miRNAs) and enter the top of the gene silencing pathway. For this reason, shRNAs are thought to more efficiently mediate gene silencing by being supplied throughout the entire natural gene silencing pathway.

[0575] miRNAs are non-coding RNAs of approximately 22 nucleotides that can regulate gene expression at the post-transcriptional or translational level during plant and animal development. One common characteristic of miRNAs is that they are all cleaved from a precursor RNA stem-loop of approximately 70 nucleotides, called pre-miRNA, likely by the RNase type III enzyme Dicer or its homolog. Naturally occurring miRNA precursors (pre-miRNAs) generally have a single strand that forms a double-stranded stem containing two complementary parts, and a loop connecting the two parts of the stem. In a typical pre-miRNA, the stem contains one or more bulges, e.g., extra nucleotides that create a single nucleotide “loop” within a part of the stem, and / or one or more unpaired nucleotides that create a gap in the mutual hybridization of the two parts of the stem. The short hairpin RNAs or engineered RNA precursors of this application are artificial constructs based on these naturally occurring pre-miRNAs but have been engineered to deliver a desired RNA silencing agent (e.g., siRNA of this disclosure). shRNA is formed by substituting the pre-miRNA stem sequence with a sequence complementary to the target mRNA. shRNA is processed throughout the cell's gene silencing pathway, thereby efficiently mediating RNAi.

[0576] The necessary elements of an shRNA molecule include a first and a second portion that have sufficient complementarity to anneal or hybridize to form a double-stranded or double-stranded stem portion. The two portions do not need to be completely or perfectly complementary. The first and second “stem” portions are joined by a portion having sequences that are not sufficiently sequence-complementary to anneal or hybridize to the other portion of the shRNA. This latter portion is called the “loop” portion within the shRNA molecule. The shRNA molecule is processed to produce siRNA. The shRNA may also contain one or more bulges, i.e., extra nucleotides that create small nucleotide “loops,” e.g., 1, 2, or 3 nucleotide loops, within the stem portion. The stem portions may be of the same length, or some may contain overhangs of, for example, 1 to 5 nucleotides. Overhang nucleotides may include, for example, uracil (U), e.g., all U. Such U is specifically encoded by thymidine (T) in the shRNA coding DNA, which signals the termination of transcription.

[0577] In the shRNA (or engineered precursor RNA) of this disclosure, a portion of the double-stranded stem is a nucleic acid sequence complementary (or antisense) to the MAPT target sequence. In certain embodiments, one strand of the shRNA stem portion is sufficiently complementary (e.g., antisense) to the target RNA (e.g., mRNA) sequence and mediates the degradation or cleavage of the target RNA via RNA interference (RNAi). Thus, the engineered RNA precursor comprises a double stem having two portions and a loop connecting the two stem portions. The antisense portion may be at the 5' or 3' end of the stem. The shRNA stem portion is about 15 to about 50 nucleotides long. In certain embodiments, the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides long. In certain embodiments, the length of the stem portion is 21 nucleotides or more. When used in mammalian cells, the stem length should be less than approximately 30 nucleotides to avoid inducing nonspecific responses such as the interferon pathway. In non-mammalian cells, the stem may exceed 30 nucleotides. In fact, the stem may contain much larger sections complementary to the target mRNA (up to the entire mRNA, and including the entire mRNA).

[0578] The two parts of a double stem must be sufficiently complementary to hybridize and form a double stem. Therefore, the two parts may, but do not, be completely or perfectly complementary. Furthermore, the two stem parts may be the same length, or one part may contain an overhang of 1, 2, 3, or 4 nucleotides. The overhanging nucleotide may contain, for example, uracil (U), or all U. Loops in shRNA or engineered RNA precursors may differ from the native pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or by replacing all or part of the loop sequence with a tetraloop or other loop sequence. Therefore, loops in shRNA or engineered RNA precursors may have 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotide lengths, for example, 15 or 20 or more.

[0579] Loops within shRNA or engineered RNA precursors may differ from the native pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or by replacing all or part of the loop sequence with a tetraloop or other loop sequence. Therefore, the loop portion within shRNA may be approximately 2 to approximately 20 nucleotides long, i.e., approximately 2, 3, 4, 5, 6, 7, 8, 9, or more, for example, 15 or 20 nucleotides, or more. In certain embodiments, the loop consists of or includes a “tetraloop” sequence. Exemplary tetraloop sequences, but not limited to these, include the sequences GNRA (wherein N is any nucleotide and R is a purine nucleotide), GGGG, and UUUU.

[0580] In certain embodiments, the shRNA of the present application comprises the sequence of the desired siRNA molecule described above. In other embodiments, the sequence of the antisense portion of the shRNA can be designed essentially as described above, or more generally, by selecting a sequence of 18, 19, 20, 21 nucleotides, or longer, from a region of 100-200 or 300 nucleotides upstream or downstream of the translation start site within the target RNA (e.g., MAPT mRNA). Generally, the sequence can be selected from any portion of the target RNA (e.g., mRNA), such as the 5'UTR (untranslated region), coding sequence, or 3'UTR. This sequence can optionally follow immediately after a region of the target gene containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence can be selected to be UU. Using this approximately 21 nucleotide sequence, a portion of the double-stranded stem of the shRNA is constructed. This sequence can, for example, enzymatically replace the stem portion of the wild-type pre-miRNA sequence, or it can be included in the complete sequence that is synthesized. For example, DNA oligonucleotides can be synthesized that encode the entire stem-loop manipulated RNA precursor, or only the portion that is inserted into the double-stranded stem of the precursor, and a manipulated RNA precursor construct can be constructed from, for example, wild-type pre-miRNA using restriction enzymes.

[0581] The engineered RNA precursor contains approximately 21-22 nucleotide sequences of siRNA or siRNA-like double helix that are desired to be produced in vivo within the double-stranded stem. Therefore, the stem portion of the engineered RNA precursor contains at least 18 or 19 nucleotide pairs corresponding to the sequence of the exon portion of the gene whose expression is reduced or inhibited. Two 3' nucleotides adjacent to this region of the stem are selected to maximize siRNA production from the engineered RNA precursor and to maximize the effectiveness of the resulting siRNA when targeting the corresponding mRNA for RNAi-mediated translational repression or disruption in vivo and in vitro.

[0582] In certain embodiments, the shRNAs of this disclosure include miRNA sequences, optionally more preferably terminally modified miRNA sequences, to enhance entry into RISC. The miRNA sequences may be similar to or identical to any naturally occurring miRNA sequence (e.g., The miRNA Registry; Griffiths-Jones S, Nuc. Acids Res., 2004). To date, more than 1,000 naturally occurring miRNAs have been identified, and together they are thought to represent about 1% of all predicted genes in the genome. Many naturally occurring miRNAs are clustered together in the introns of pre-mRNA and can be identified in silico using homology-based searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms that predict the ability of candidate miRNA genes to form stem-loop structures with pre-mRNA (e.g., MiRScan, MiRSeeker) (Grad et al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai EC et al., Genome Bio., 2003). Online registries provide searchable databases of all publicly available miRNA sequences (The miRNA Registry, Sanger Institute website; Griffiths-Jones S, Nuc. Acids Res., 2004).Exemplary natural miRNAs include lin-4, let-7, miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other natural miRNAs of human origin and those of specific model organisms, such as Drosophila melanogaster, Caenorhabditis elegans, zebrafish, Arabidopsis thalania, Mus musculus, and Rattus norvegicus (described in PCT International Publication No. WO03 / 029459).

[0583] Naturally occurring miRNAs are expressed in vivo by endogenous genes and processed from hairpin or stem-loop precursors (pre-miRNA or pri-miRNA) by Dicer or other RNAses (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003; Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can exist transiently in vivo as double-stranded double helixes, but only one strand is incorporated into the RISC complex to direct gene silencing. Certain miRNAs, such as plant miRNAs, have complete or near-complete complementarity with their target mRNAs and therefore directly cleave the target mRNA. Other miRNAs have incomplete complementarity with their target mRNAs and therefore directly repress the translation of the target mRNA. The degree of complementarity between the miRNA and its target mRNA is thought to determine its mechanism of action. For example, complete or near-complete complementarity between the miRNA and its target mRNA predicts a cleavage mechanism (Yekta et al., Science, 2004), while incomplete complementarity predicts a translation repression mechanism. In certain embodiments, the miRNA sequence is a naturally occurring miRNA sequence whose abnormal expression or activity correlates with miRNA dysfunction.

[0584] d) Dual-function oligonucleotide tethering factors In other embodiments, the RNA silencing agents of this disclosure include dual-function oligonucleotide tethering factors useful for intercellular recruitment of miRNAs. Animal cells express a set of miRNAs, which are non-coding RNAs of about 22 nucleotides that can regulate gene expression at the post-transcriptional or translational level. Dual-function oligonucleotide tethering factors can suppress the expression of genes involved in processes such as atherosclerosis by binding miRNAs bound to RISC and recruiting them to target mRNAs. The use of oligonucleotide tethering factors offers several advantages over existing techniques for suppressing the expression of specific genes. First, the methods described herein allow endogenous molecules (often abundant), miRNAs, to mediate RNA silencing. Thus, the methods described herein eliminate the need to introduce exogenous molecules (e.g., siRNA) to mediate RNA silencing. Second, the RNA silencing agents and their binding portions (e.g., oligonucleotides, e.g., 2'-O-methyloligonucleotides) can be stabilized and made resistant to nuclease activity. As a result, the tethering factors of this disclosure can be designed for direct delivery, eliminating the need for indirect delivery (e.g., viruses) of precursor molecules or plasmids designed to produce the desired agent within the cell. Thirdly, the tethering factors and their respective portions can be designed to fit specific mRNA sites and specific miRNAs. The design may be cell and gene product specific. Fourthly, in the methods disclosed herein, the mRNA remains intact, allowing those skilled in the art to use the cell's own mechanisms to block protein synthesis with short pulses. Consequently, these RNA silencing methods are highly regulated.

[0585] The dual-function oligonucleotide tethering factors ("tethering factors") of this disclosure are designed to recruit miRNAs (e.g., endogenous cellular miRNAs) to target mRNAs to induce regulation of a gene of interest. In certain embodiments, the tethering factor has the formula TL-μ, where T is an mRNA targeting portion, L is a ligation portion, and μ is a miRNA recruitment portion. Any one or more portions may be double-stranded. In certain embodiments, each portion is single-stranded.

[0586] The portions within the tethering element can be positioned or ligated as shown in formula TL-μ (5' to 3' direction) (i.e., the 3' end of the targeting portion ligated to the 5' end of the ligation portion and the 3' end of the ligation portion ligated to the 5' end of the miRNA mobilization portion). Alternatively, these portions can be positioned or ligated within the tethering element as follows: μ-TL (i.e., the 3' end of the miRNA mobilization portion ligated to the 5' end of the ligation portion, and the 3' end of the ligation portion ligated to the 5' end of the targeting portion).

[0587] The mRNA targeting moiety described above can capture a specific target mRNA. According to this disclosure, since the expression of the target mRNA is undesirable, translational repression of the mRNA is desirable. The mRNA targeting moiety is sized to effectively bind to the target mRNA. The length of the targeting moiety varies considerably, partly depending on the length of the target mRNA and the degree of complementarity between the target mRNA and the targeting moiety. In various embodiments, the targeting moiety is less than about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides long. In certain embodiments, the targeting moiety is about 15 to about 25 nucleotides long.

[0588] As described above, the miRNA recruitment portion can associate with a miRNA. According to the present invention, the miRNA can be any miRNA capable of repressing a target mRNA. Mammals have been reported to have more than 250 endogenous miRNAs (Lagos-Quintana et al. (2002) Current Biol. 12:735-739; Lagos-Quintana et al. (2001) Science 294:858-862; and Lim et al. (2003) Science 299:1540). In various embodiments, the miRNA can be any miRNA recognized in the art.

[0589] The ligation site is any agent that can ligate the targeting site so as to maintain the activity of the targeting site. The ligation site may be an oligonucleotide moiety containing a sufficient number of nucleotides so that the targeting agent can adequately interact with its respective target. The ligation site has little or no sequence homology to the cellular mRNA or miRNA sequence. Exemplary ligation sites include one or more 2'-O-methylnucleotides, such as 2'-β-methyladenosine, 2'-O-methylthymidine, 2'-O-methylguanosine, or 2'-O-methyluridine.

[0590] e) Gene silencing oligonucleotides In certain exemplary embodiments, gene expression (i.e., MAPT gene expression) can be regulated using oligonucleotide-based compounds comprising two or more single-stranded antisense oligonucleotides linked via their 5' ends, allowing for the presence of two or more accessible 3' ends, in order to effectively inhibit or reduce MAPT gene expression. Such linked oligonucleotides are also known as gene silencing oligonucleotides (GSOs) (see, for example, U.S. Patent No. 8,431,544, assigned to Idera Pharmaceuticals, Inc., which is incorporated herein by reference in its entirety for all purposes).

[0591] Linking at the 5' end of GSO is independent of other oligonucleotide linking and can occur directly via the 5', 3', or 2' hydroxyl group, or indirectly via a non-nucleotide linker or nucleoside, using either the 2' or 3' hydroxyl position of the nucleoside. Linking can also utilize a functionalized sugar or nucleic acid base of the 5' terminal nucleotide.

[0592] GSOs can contain two identical or different sequences conjugated at their 5'-5' ends via phosphodiesters, phosphorothioates, or non-nucleoside linkers. Such compounds may contain 15-27 nucleotides complementary to a specific portion of the target mRNA for downregulation of antisense of a gene product. GSOs containing identical sequences can bind to specific mRNAs via Watson-Crick hydrogen bond interactions and inhibit protein expression. GSOs containing different sequences can bind to two or more different regions of one or more mRNA targets and inhibit protein expression. Such compounds consist of heteronucleotide sequences complementary to the target mRNA, forming a stable double-strand structure via Watson-Crick hydrogen bonds. Under certain conditions, GSOs containing two free 3' ends (5'-5' attached antisense) can be more potent gene expression inhibitors than those with a single free 3' end or those without a free 3' end.

[0593] In some embodiments, the non-nucleotide linker is glycerol or formula HO-(CH2) o -CH(OH)-(CH2) p -OH is a glycerol homologue, where o and p are integers independently between 1 and about 6, 1 and about 4, or 1 and about 3. In some other embodiments, the non-nucleotide linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO--(CH2)m--C(O)NH--CH2--CH(OH)--CH2--NHC(O)--(CH2) m--It has an OH group, where m is an integer between 0 and approximately 10, 0 and approximately 6, 2 and approximately 6, or 2 and approximately 4.

[0594] Some non-nucleotide linkers allow for the attachment of three or more GSO components. For example, the non-nucleotide linker glycerol has three hydroxyl groups to which GSO components can be covalently attached. Thus, some oligonucleotide-based compounds of this disclosure contain two or more oligonucleotides linked to a nucleotide or non-nucleotide linker. Such oligonucleotides according to this disclosure are referred to as "branched".

[0595] In certain embodiments, the GSO is at least 14 nucleotides long. In certain exemplary embodiments, the GSO is 15–40 nucleotides long or 20–30 nucleotides long. Thus, the component oligonucleotides of the GSO can independently be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long.

[0596] These oligonucleotides can be prepared by methods recognized in the art, such as phosphoramidate or H-phosphonate chemistry, which can be carried out by manual or automated synthesizers. These oligonucleotides can also be modified in multiple ways without impairing their ability to hybridize to mRNA. Such modifications may include at least one internucleotide linkage of an oligonucleotide between the 5' end of one nucleotide and the 3' end of another nucleotide, which is an alkylphosphonate, phosphorothioate, phosphorodithioate, methylphosphonate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate hydroxyl, acetamidate, carboxymethyl ester, or a combination of these and other internucleotide linkages, where the phosphodiester linkage of the 5' nucleotide is substituted with any number of chemical groups.

[0597] V. Modified anti-MAPT RNA silencing agents In certain aspects of this disclosure, the RNA silencing agents of this application (or any part thereof) may be modified to further improve the activity of the agent as described above. For example, the RNA silencing agents described in Section II above may be modified by any of the modifications described below. The modifications may act in part to further improve target recognition, to improve the stability of the agent (e.g., to prevent degradation), to promote cell uptake, to improve targeting efficiency, to improve the effectiveness of binding (e.g., to the target), to improve patient tolerance to the agent, and / or to reduce toxicity.

[0598] 1) Modifications to improve target identification In certain embodiments, the RNA silencing agents of this application may be substituted with destabilized nucleotides to improve single-nucleotide target recognition (see U.S. Patent Application No. 11 / 698,689, filed January 25, 2007, and U.S. Provisional Application No. 60 / 762,225, filed January 25, 2006, both of which are incorporated herein by reference). Such modifications may be sufficient to neutralize the specificity of the RNA silencing agent to non-target mRNA (e.g., wild-type mRNA) without any apparent effect on the specificity of the RNA silencing agent to target mRNA (e.g., gain-of-function mutant mRNA).

[0599] In certain embodiments, the RNA silencing agent of the present invention is modified by introducing at least one universal nucleotide into its antisense strand. The universal nucleotide comprises a base moiety that can indiscriminately base-pair with any of the four conventional nucleotide bases (e.g., A, G, C, U). The universal nucleotide is intended to have only a relatively small effect on the stability of the RNA double helix, or the stability of the double helix formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotides include those having an inosine base moiety or inosine-like base moiety selected from the group consisting of deoxyinosine (e.g., 2'-deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. In certain embodiments, the universal nucleotide is an inosine residue or a natural analog thereof.

[0600] In certain embodiments, the RNA silencing agents of this disclosure are modified by introducing at least one destabilizing nucleotide within 5 nucleotides of a specificity-determining nucleotide (i.e., a nucleotide that recognizes a disease-associated polymorphism). For example, the destabilizing nucleotide may be introduced at a position within 5, 4, 3, 2, or 1 nucleotide from the specificity-determining nucleotide. In exemplary embodiments, the destabilizing nucleotide is introduced at a position 3 nucleotides away from the specificity-determining nucleotide (i.e., so that two stabilizing nucleotides are present between the destabilizing nucleotide and the specificity-determining nucleotide). In RNA silencing agents having two strands or strand portions (e.g., siRNA and shRNA), the destabilizing nucleotide may be introduced into a strand or strand portion that does not contain the specificity-determining nucleotide. In certain embodiments, the destabilizing nucleotide is introduced into the same strand or strand portion that contains the specificity-determining nucleotide.

[0601] 2) Modifications to enhance efficacy and specificity In certain embodiments, the RNA silencing agents of this disclosure may be modified to easily enhance their efficacy and specificity in mediating RNAi in accordance with asymmetric design rules (see U.S. Patents 8,309,704, 7,750,144, 8,304,530, 8,329,892, and 8,309,705). Such modifications facilitate the entry of the antisense strand of siRNA (e.g., siRNA designed using the method of the present invention, or siRNA produced from shRNA) into RISC in a manner favorable to the sense strand. This allows the antisense strand to preferentially induce cleavage or translational repression of the target mRNA, thereby increasing or improving the efficiency of target cleavage and silencing. In certain embodiments, the asymmetry of the RNA silencing agent is enhanced by reducing the base pair strength between the antisense 5' end (AS5') and the sense 3' end (S3') of the RNA silencing agent, relative to the binding strength or base pair strength between the antisense 3' end (AS3') and the sense 5' end (S'5) of the RNA silencing agent.

[0602] In one embodiment, the asymmetry of the RNA silencing agent of the present application may be enhanced such that the number of G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand portion is less than the number of G:C base pairs between the 3' end of the first or antisense strand and the 5' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the present disclosure may be enhanced such that there is at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In a particular embodiment, the mismatched base pair is selected from the group consisting of: G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In another embodiment, the asymmetry of the RNA silencing agent of the present disclosure may be enhanced such that there is at least one fluctuation base pair, for example, G:U, between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the Disclosure may be enhanced to include at least one base pair containing a rare nucleotide, such as inosine (I). In a particular embodiment, the base pair is selected from the group consisting of I:A, I:U, and I:C. In yet another embodiment, the asymmetry of the RNA silencing agent of the Disclosure may be enhanced to include at least one base pair containing a modified nucleotide. In a particular embodiment, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

[0603] 3) RNA silencing agents with improved stability The RNA silencing agent of this invention may be modified to improve its stability in serum or growth medium for cell culture. To improve stability, the 3'-residue may be stabilized against degradation and may be selected to consist of a purine nucleotide such as adenosine or guanosine nucleotide, for example. Alternatively, substitution of pyrimidine nucleotides with modification analogs, such as substitution of uridine with 2'-deoxythymidine, is acceptable and does not affect the efficiency of RNA interference.

[0604] In one embodiment, the present application features an RNA silencing agent comprising first and second strands, wherein the second strand and / or the first strand are modified by substituting internal nucleotides with modified nucleotides to improve in vivo stability compared to the corresponding unmodified RNA silencing agent. As defined herein, “internal” nucleotides are those located at any position other than the 5' or 3' end of a nucleic acid molecule, polynucleotide, or oligonucleotide. Internal nucleotides may be located within a single-stranded molecule or within a double-stranded or double-stranded molecule. In one embodiment, the sense strand and / or antisense strand are modified by the substitution of at least one internal nucleotide. In another embodiment, the sense strand and / or antisense strand are modified by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more internal nucleotides. In another embodiment, the sense strand and / or antisense strand are modified by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more internal nucleotides. In yet another embodiment, the sense strand and / or antisense strand are modified by all substitutions of internal nucleotides.

[0605] In one embodiment, the present application features an RNA silencing agent in which at least 80% are chemically modified. In certain embodiments, the RNA silencing agent may be fully chemically modified, i.e., 100% of the nucleotides are chemically modified. In another embodiment, the present application features an RNA silencing agent comprising at least 80% chemically modified 2'-OH ribose groups. In certain embodiments, the RNA silencing agent comprises 2'-OH ribose groups that are chemically modified by about 80%, 85%, 90%, 95%, or 100%.

[0606] In certain embodiments, the RNA silencing agent may comprise at least one modified nucleotide analog. The nucleotide analog may be positioned in a location where target-specific silencing activity, such as RNAi-mediated activity or translational repression activity, is substantially unaffected, for example, in a region at the 5' and / or 3' ends of the siRNA molecule. Furthermore, the incorporation of the modified nucleotide analog may stabilize the ends.

[0607] Exemplary nucleotide analogs include sugar- and / or skeletal-modified ribonucleotides (i.e., modifications to the phosphate sugar backbone). For example, phosphodiester linkages in native RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary skeletal-modified ribonucleotides, the phosphate ester group attached to an adjacent ribonucleotide is replaced by a modifying group, for example, a phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2'OH- group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is a C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0608] In certain embodiments, modifications are 2'-fluoro, 2'-amino, and / or 2'-thio modifications. Examples of modifications include 2'-fluorocytidine, 2'-fluorouridine, 2'-fluoroadenosine, 2'-fluoroguanosine, 2'-aminocytidine, 2'-aminouridine, 2'-aminoadenosine, 2'-aminoguanosine, 2,6-diaminopurine, 4-thiouridine, and / or 5-aminoallyluridine. In certain embodiments, the 2'-fluororibonucleotide is any uridine or cytidine. Additional exemplary modifications include 5-bromouridine, 5-iodouridine, 5-methylcytidine, ribothymidine, 2-aminopurine, 2'-aminobutyrylpyreneuridine, 5-fluorocytidine, and 5-fluorouridine. 2'-deoxynucleotides and 2'-Omenucleotides can also be used within the modified RNA silencing agent moiety of this disclosure. Additional modified residues include deoxy debases, inosine, N3-methyluridine, N6,N6-dimethyladenosine, pseudouridine, purine ribonucleosides, and ribavirin. In certain embodiments, the 2' portion is a methyl group, such that the linking portion is a 2'-O-methyl oligonucleotide.

[0609] In certain embodiments, the RNA silencing agent of the present invention comprises locked nucleic acid (LNA). The LNA contains sugar-modified nucleotides that are resistant to nuclease activity (highly stable) and have single-nucleotide recognition for mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1):439-447; Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). These molecules have modifiable 2'-O, 4'-C-ethylene-bridged nucleic acids, such as 2'-deoxy-2''-fluorouridine. Furthermore, the LNA increases the specificity of oligonucleotides by restricting the sugar moiety to a 3'-endoconformation, thereby pre-organizing the nucleotides for base pairing and raising the melting temperature of the oligonucleotides by about 10°C per nucleotide.

[0610] In another exemplary embodiment, the RNA silencing agent of the present invention comprises peptide nucleic acid (PNA). The PNA comprises modified nucleotides in which the sugar-phosphate moiety of the nucleotide is replaced with a neutral 2-aminoethylglycine moiety capable of forming a polyamide skeleton, thereby providing high resistance to nuclease digestion and conferring improved binding specificity to molecules (Nielsen, et al., Science, (2001), 254:1497-1500).

[0611] Nucleic acid base-modified ribonucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleic acid base instead of naturally occurring ones, are also considered. The bases can be modified to block the activity of adenosine deaminase. Examples of modified nucleic acid bases, though not limited to these, include, but are preferred, uridine and / or cytidine modified at position 5, e.g., 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at position 8, e.g., 8-bromoguanosine; deazanucleotides, e.g., 7-deaza-adenosine; and O- and N-alkylated nucleotides, e.g., N6-methyladenosine. It should be noted that combinations of the above modifications are also possible.

[0612] In other embodiments, crosslinking can be used to alter the pharmacokinetics of RNA silencing agents, for example, to extend their half-life in the body. Therefore, this application includes RNA silencing agents having two complementary strands of nucleic acid, where the two strands are crosslinked. Thus, this application includes RNA silencing agents that are conjugated (e.g., at their 3' end) to another portion (e.g., a non-nucleic acid portion such as a peptide) or an organic compound (e.g., a dye). Modifying siRNA derivatives in this way can improve cellular uptake or enhance the cellular targeting activity of the resulting siRNA derivatives compared to the corresponding siRNA, and is useful for tracking siRNA derivatives within cells or improving the stability of siRNA derivatives compared to the corresponding siRNA.

[0613] Other exemplary modifications include: (a) modifications at the 2' position, e.g., providing a 2'OMe portion on U in a sense or antisense chain, particularly in the sense chain, or providing a 2'OMe portion in the 3' overhang, e.g., at the 3' terminus (where the 3' terminus means the 3' atom or at most 3' portion of the molecule, e.g., the most 3' P or 2' position as indicated by the context); (b) modifications of the phosphate skeleton, e.g., by substitution of O with S, e.g., providing phosphorothioate modifications to U or A or both in an antisense chain, e.g., by substitution of O with S; (c) substitution of U with the C5 aminolinker; (d) substitution of A with G (the sequence change may, in certain embodiments, be located in the sense chain rather than the antisense chain); and (d) modifications at the 2', 6', 7', or 8' position. Exemplary embodiments are embodiments in which one or more of these modifications are present on the sense but not on the antisense chain, or in embodiments in which the antisense chain has few such modifications. Further exemplary modifications include the use of methylated P in the 3' overhang, for example at the 3' terminus; combinations of 2' modifications, for example providing a 2'OMe moiety and modifying the skeleton, for example substituting O with S, for example providing a phosphorothioate modification; or the use of methylated P in the 3' overhang, for example at the 3' terminus; modification with a 3' alkyl group; modification with debasal pyrrolidone in the 3' overhang, for example at the 3' terminus; or modification with naproxen, ibuprofen, or other moieties that inhibit degradation at the 3' terminus.

[0614] Highly modified RNA silencing agents In certain embodiments, the RNA silencing agent contains at least 80% chemically modified nucleotides. In certain embodiments, the RNA silencing agent is fully chemically modified, i.e., 100% of the nucleotides are chemically modified.

[0615] In certain embodiments, the RNA silencing agent is 2'-O-methyl rich, i.e., contains more than 50% 2'-O-methyl. In certain embodiments, the RNA silencing agent contains at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 2'-O-methyl nucleotide content. In certain embodiments, the RNA silencing agent contains at least about 70% 2'-O-methyl nucleotide modification. In certain embodiments, the RNA silencing agent contains about 70% to about 90% 2'-O-methyl nucleotide modification. In certain embodiments, the RNA silencing agent is dsRNA including an antisense strand and a sense strand. In certain embodiments, the antisense strand contains at least about 70% 2'-O-methyl nucleotide modification. In certain embodiments, the antisense strand contains about 70% to about 90% 2'-O-methyl nucleotide modification. In certain embodiments, the sense strand contains at least about 70% 2'-O-methylnucleotide modifications. In certain embodiments, the sense strand contains about 70% to about 90% 2'-O-methylnucleotide modifications. In certain embodiments, the sense strand contains 100% 2'-O-methylnucleotide modifications.

[0616] 2'-O-methyl-rich RNA silencing agents and specific chemical modification patterns are further described in USSN 16 / 550,076 (filed August 23, 2019) and USSN 62 / 891,185 (filed August 23, 2019), respectively, which are incorporated herein by reference.

[0617] Internucleotide ligation modification In certain embodiments, at least one nucleotide linkage, subunit linkage, or nucleotide backbone is modified in the RNA silencing agent. In certain embodiments, all nucleotide linkages in the RNA silencing agent are modified. In certain embodiments, the modified nucleotide linkages include phosphorothioate nucleotide linkages. In certain embodiments, the RNA silencing agent includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 phosphorothioate nucleotide linkages. In certain embodiments, the RNA silencing agent includes 4 to 16 phosphorothioate nucleotide linkages. In certain embodiments, the RNA silencing agent includes 8 to 13 phosphorothioate nucleotide linkages. In certain embodiments, the RNA silencing agent is a dsRNA including an antisense strand and a sense strand, each including a 5' end and a 3' end. In certain embodiments, the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to adjacent ribonucleotides via phosphorothioate nucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 3' end of the sense strand are linked to adjacent ribonucleotides via phosphorothioate nucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate nucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 and 1-8 from the 3' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate nucleotide linkages. In certain embodiments, the nucleotides at positions 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, or 1-8 from the 3' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate nucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 and 1-7 from the 3' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate nucleotide linkages.

[0618] In one embodiment, the disclosure provides a modified oligonucleotide having a 5' end and a 3' end complementary to a target, and the oligonucleotide comprising sense and antisense strands and a linkage between at least one modified subunit of formula (I): [ka] (In the formula, B is the base pair portion; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH; X is a halo, hydroxy, and C 1-6 Selected from the group consisting of alkoxys, Y is O - OH, OR, NH - NH2, S - Selected from the group consisting of , and SH, Z is selected from the group consisting of O and CH2; R is a protecting group, [ka] (where is any double bond).

[0619] In the embodiment of formula (I), when W is CH, [ka] It is a double bond.

[0620] In the embodiment of formula (I), when W is selected from the group consisting of O, OCH2, OCH, and CH2, [ka] It is a single bond.

[0621] In the embodiment of formula (I), Y is O - If that is the case, then either Z or W is not O.

[0622] In the embodiment of formula (I), Z is CH2 and W is CH2. In another embodiment, the modified subunit connection of formula (I) is the modified subunit connection of formula (II): [ka]

[0623] In one embodiment of formula (I), Z is CH2 and W is O. In another embodiment, the modifying subunit linkage of formula (I) is the modifying subunit linkage of formula (III): [ka]

[0624] In the embodiment of formula (I), Z is O and W is CH2. In another embodiment, the modified subunit linkage of formula (I) is the modified subunit linkage of formula (IV): [ka]

[0625] In one embodiment of formula (I), Z is O and W is CH. In another embodiment, the modifying subunit linkage of formula (I) is the modifying subunit linkage of formula V: [ka]

[0626] In an embodiment of formula (I), Z is O and W is OCH2. In another embodiment, the modified subunit linkage of formula (I) is the modified subunit linkage of formula VI: [ka]

[0627] In one embodiment of formula (I), Z is CH2 and W is CH. In another embodiment, the modifying subunit linkage of formula (I) is the modifying subunit linkage of formula VII: [ka]

[0628] In the embodiment of formula (I), the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0629] In one embodiment, the modified oligonucleotide is incorporated into the siRNA, the modified siRNA has 5' and 3' ends complementary to the target, is complementary to the target, and the siRNA includes a sense strand and an antisense strand, as well as at least one modified subunit linkage from one or more of formulas (I), (II), (III), (IV), (V), (VI), or (VII).

[0630] In one embodiment, a modified oligonucleotide is incorporated into an siRNA, the modified siRNA having a 5' end, a 3' end, being complementary to the target, and comprising sense and antisense strands, the siRNA comprising at least one modified subunit linkage, and having formula VIII: [ka] (In the formula, D is selected from the group consisting of O, OCH2, OCH, CH2, and CH; C is O - , OH, OR 1 NH - NH2, S - Selected from the group consisting of , and SH, A is selected from the group consisting of O and CH2; R 1 is a protecting group; [ka] is any double bond; The subunits are bridged by two optionally modified nucleosides.

[0631] In one embodiment, C is O - If that is the case, then either A or D is not O.

[0632] In one embodiment, D is CH2. In another embodiment, the modified subunit linkage of formula VIII is the modified subunit linkage of formula (IX): [ka]

[0633] In one embodiment, D is O. In another embodiment, the modified subunit linkage of formula VIII is the modified subunit linkage of formula (X): [ka]

[0634] In one embodiment, D is CH2. In another embodiment, the modified subunit linkage of formula (VIII) is the modified subunit linkage of formula (XI): [ka]

[0635] In one embodiment, D is CH. In another embodiment, the modified subunit linkage of formula VIII is the modified subunit linkage of formula (XII): [ka]

[0636] In another embodiment, the modified subunit linkage of formula (VII) is the modified subunit linkage of formula (XIV): [ka]

[0637] In one embodiment, D is OCH2. In another embodiment, the modified subunit linkage of formula (VII) is the modified subunit linkage of formula (XIII): [ka]

[0638] In another embodiment, the modified subunit linkage of formula (VII) is the modified subunit linkage of formula (XXa): [ka]

[0639] In one embodiment of modified siRNA linkage, each optionally modified nucleoside is independently selected from the group consisting of adenosine, guanosine, cytidine, and uridine, with each occurrence being.

[0640] In one exemplary embodiment of formula (I), W is O. In another embodiment, W is CH2. In yet another embodiment, W is CH.

[0641] In a particular exemplary embodiment of formula (I), X is OH. In another embodiment, X is OCH3. In yet another embodiment, X is halo.

[0642] In certain embodiments of formula (I), the modified siRNA does not contain a 2'-fluoro substituent.

[0643] In the embodiment of formula (I), Y is O -In another embodiment, Y is OH. In yet another embodiment, Y is OR. In yet another embodiment, Y is NH - In one embodiment, Y is NH2. In another embodiment, Y is S - In yet another embodiment, Y is SH.

[0644] In one embodiment of formula (I), Z is O. In another embodiment, Z is CH2.

[0645] In one embodiment, the modified subunit linkage is inserted at positions 1-2 of the antisense chain. In another embodiment, the modified subunit linkage is inserted at positions 6-7 of the antisense chain. In yet another embodiment, the modified subunit linkage is inserted at positions 10-11 of the antisense chain. In yet another embodiment, the modified subunit linkage is inserted at positions 19-20 of the antisense chain. In one embodiment, the modified subunit linkage is inserted at positions 5-6 and 18-19 of the antisense chain.

[0646] In an exemplary embodiment of the modified siRNA linkage of formula (VIII), C is O - In another embodiment, C is OH. In yet another embodiment, C is OR 1 In yet another embodiment, C is NH - In one embodiment, C is NH2. In another embodiment, C is S - In yet another embodiment, C is SH.

[0647] In an exemplary embodiment of the modified siRNA linkage of formula (VIII), A is O. In another embodiment, A is CH2. In yet another embodiment, C is OR 1 In yet another embodiment, C is NH - In one embodiment, C is NH2. In another embodiment, C is S - In yet another embodiment, C is SH.

[0648] In certain embodiments of the modified siRNA linker of formula (VIII), the optionally modified nucleoside is adenosine. In another embodiment of the modified siRNA linker of formula (VIII), the optionally modified nucleoside is guanosine. In certain embodiments of the modified siRNA linker of formula (VIII), the optionally modified nucleoside is cytidine. In certain embodiments of the modified siRNA linker of formula (VIII), the optionally modified nucleoside is uridine.

[0649] In one embodiment of the modified siRNA linker, the linker is inserted at positions 1-2 of the antisense strand. In another embodiment, the linker is inserted at positions 6-7 of the antisense strand. In yet another embodiment, the linker is inserted at positions 10-11 of the antisense strand. In yet another embodiment, the linker is inserted at positions 19-20 of the antisense strand. In one embodiment, the linker is inserted at positions 5-6 and 18-19 of the antisense strand.

[0650] In certain embodiments, base pair portion B is adenine. In certain embodiments of formula (I), base pair portion B is guanine. In certain embodiments of formula (I), base pair portion B is cytosine. In certain embodiments of formula (I), base pair portion B is uracil.

[0651] In one embodiment of formula (I), W is O. In one embodiment of formula (I), W is CH2. In the embodiment of formula (I), W is CH.

[0652] In the embodiment of formula (I), X is OH. In the embodiment of formula (I), X is OCH3. In the embodiment of formula (I), X is halo.

[0653] In exemplary embodiments of formula (I), the modified oligonucleotide does not contain a 2'-fluoro substituent.

[0654] In the embodiment of formula (I), Y is O -In the embodiment of formula (I), Y is OH. In the embodiment of formula (I), Y is OR. In the embodiment of formula (I), Y is NH. - In the embodiment of formula (I), Y is NH2. In the embodiment of formula (I), Y is S - Therefore, in the embodiment of equation (I), Y is SH.

[0655] In the embodiment of formula (I), Z is O. In one embodiment of formula (I), Z is CH2.

[0656] In an embodiment of formula (I), the linkage is inserted at positions 1-2 of the antisense chain. In another embodiment of formula (I), the linkage is inserted at positions 6-7 of the antisense chain. In yet another embodiment of formula (I), the linkage is inserted at positions 10-11 of the antisense chain. In yet another embodiment of formula (I), the linkage is inserted at positions 19-20 of the antisense chain. In an embodiment of formula (I), the linkage is inserted at positions 5-6 and 18-19 of the antisense chain.

[0657] Interconnections between modifying subunits are further described in USSN62 / 824,136 (filed March 26, 2019), USSN62 / 826,454 (filed March 29, 2019), and USSN62 / 864,792 (filed June 21, 2019), which are incorporated herein by reference, respectively.

[0658] 4) Conjugate function In other embodiments, the RNA silencing agent may be modified with one or more functional moieties. The functional moieties are molecules that confer one or more additional activities to the RNA silencing agent. In certain embodiments, the functional moieties enhance cellular uptake by target cells (e.g., nerve cells). Accordingly, the disclosure includes RNA silencing agents that are conjugated or unconjugated (e.g., at their 5' and / or 3' ends) to other moieties (e.g., non-nucleic acid moieties such as peptides) or organic compounds (e.g., dyes), etc. Conjugation can be achieved, for example, using the methods known in this technology, or the following methods: Lambert et al., Drug Deliv. Rev.:47(1),99-112(2001) (described on nucleic acids attached to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43(1998) (described on nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8(1994) (described on nucleic acids linked to intercalating agents, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10(1995) (described on nucleic acids linked to nanoparticles).

[0659] In certain embodiments, the functional portion is a hydrophobic portion. In certain embodiments, the hydrophobic portion is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In certain embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA). In certain embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanic acid (DCA). In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, their derivatives, and their metabolites. In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

[0660] In certain embodiments, the RNA silencing agent of the disclosure is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand containing a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of the siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, and cationic dyes (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic components include cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0661] In certain embodiments, the functional portion may include one or more ligands tethered to the RNA silencing agent to improve stability, hybridization thermodynamics with target nucleic acids, targeting to specific tissues or cell types, or cell permeability, for example, by endocytosis-dependent or independent mechanisms. The ligands and associated modifications may also enhance sequence specificity, thereby reducing off-site targeting. The tethering ligand may include one or more modified bases or sugars that can function as intercalators. These may also be located within internal regions, such as within the bulge of the RNA silencing agent / target double helix. The intercalators may be aromatic, e.g., polycyclic aromatic or heterocyclic aromatic compounds. Polycyclic intercalators may have stacking capabilities and may include systems having two, three, or four fused rings. The universal bases described herein may be included as ligands. In one embodiment, the ligand may include cleavage groups that contribute to the inhibition of the target gene by cleaving the target nucleic acid. The cleavage group may be, for example, bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), polyamine, tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. Examples of metal ion chelating groups include Lu(III) or EU(III) macrocyclic complexes, Zn(II) 2,9-dimethylphenanthroline derivatives, Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the bulge region by free metal ions such as Lu(III). In some embodiments, the peptide ligand can be tethered to an RNA silencing agent to promote the cleavage of target RNA, for example, at the bulge region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (Cycram) can be conjugated into peptides (e.g., by amino acid derivatives) to facilitate target RNA cleavage.The tethering ligand can be an aminoglycoside ligand, which can impart improved hybridization properties or improved sequence specificity to the RNA silencing agent. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. The use of acridine analogs can enhance sequence specificity. For example, neomycin B has high affinity for RNA compared to DNA, but lower sequence specificity. The acridine analog neo-5-acridine has high affinity for HIV Rev-response elements (RREs). In some embodiments, guanidine analogs (guanidinoglycosides) of aminoglycoside ligands are tethered to the RNA silencing agent. Within guanidinoglycosides, the amine group on the amino acid is replaced with a guanidine group. The attachment of a guanidine analog can increase the cellular permeability of RNA silencing agents. The tethering ligand may be a polyarginine peptide, peptoid, or peptide mimetic that can increase the cellular uptake of oligonucleotide agents.

[0662] Exemplary ligands are coupled to ligand-conjugate carriers, either directly or indirectly via intervening tethering factors. In certain embodiments, the coupling is via covalent bonding. In certain embodiments, the ligand is attached to the carrier via intervening tethering factors. In certain embodiments, the ligand alters the distribution, targeting, or lifetime of the RNA silencing agent into which it is incorporated. In certain embodiments, the ligand provides, for example, higher affinity to selected targets, such as molecules, cells or cell types, compartments, such as cell or organ compartments, tissues, organs, or regions of the body, compared to species in which such ligands are absent.

[0663] Exemplary ligands can improve transport, hybridization, and specificity properties, and may also improve the nuclease resistance of polymer molecules and / or natural or modified ribonucleotides, including the resulting natural or modified RNA silencing agents or any combination of monomers described herein. Ligands can generally include, for example, therapeutic modifiers to increase uptake, e.g., diagnostic compounds or reporter groups to monitor distribution; crosslinkers; nuclease resistance-constituting moieties; and natural or aberrant nucleic acid bases. Common examples include lipophilic substances, lipids, steroids (e.g., ubaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friederin, epifriederanol-deranolic acid-derived lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binders, integrin target molecules, polycations, peptides, polyamines, and peptide mimetic compounds. Ligands may include naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); amino acids, or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, such as 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-coglycolated) 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 polyphosphatidine.Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0664] Ligands can also include target groups that bind to specific cell types, such as kidney cells, e.g., cell or tissue targeting agents, e.g., lectins, glycoproteins, lipids or proteins, e.g., antibodies. Target groups can also be thyroid-stimulating hormone, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine (GalNAc) or its derivatives, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, biotin, or RGD peptides or RGD peptide mimetic compounds. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridines), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidium aminoglycosides, artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol and its thio analogues), cholic acid, cholanic acid, lithocholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., C) 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C18 , C 19 or C 20 fatty acids) and their ethers, for example, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 Alkyl groups (e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antexols). Napedia peptides, Tat peptides), alkylating agents, phosphates, amino acids, mercaptos, PEGs (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu tetraazal macrorings) 3+ Examples include complexes, dinitrophenyl, HRP, or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof.

[0665] Ligands can be proteins such as glycoproteins, peptides such as molecules that have a specific affinity for the coligand, or antibodies such as antibodies that bind to specific cell types such as cancer cells, endothelial cells, or osteocytes. Examples of ligands include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Examples of ligands include lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0666] A ligand can be a substance, such as a drug, that can increase the uptake of RNA silencing agents into cells by, for example, disrupting the cytoskeleton of a cell, such as by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. Drugs may include, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latranculine A, phalloidin, swinholide A, indanosine, or myoservin. Ligands can also increase the uptake of RNA silencing agents into cells by, for example, activating an inflammatory response. Exemplary ligands with such effects include tumor necrosis factor alpha (TNF-1), interleukin-1 beta, or gamma interferon. In one embodiment, the ligand is a lipid or lipid system molecule. Such a lipid or lipid system molecule can bind to a serum protein, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of conjugates to target tissues, such as non-renal target tissues of the body. For example, the target tissue could be the liver, such as hepatic parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipids or lipid ligands can be used to (a) improve the degradation tolerance of the conjugate, (b) increase targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, such as HSA. Lipid ligands can be used to modulate, for example, control the binding of the conjugate to target tissues. For example, lipids or lipid ligands that bind more strongly to HSA are less likely to target the kidney and therefore less likely to be excreted from the body. Lipids or lipid ligands that do not bind less strongly to HSA can be used to target the conjugate to the kidney. In certain embodiments, the lipid ligand binds to HSA. The lipid ligand can bind to HSA with sufficient affinity so that the conjugate is distributed to tissues other than the kidney.However, the affinity is not intended to be strong enough to reverse HSA-ligand binding. In another embodiment, the lipid ligand binds weakly to HSA or not at all, so the conjugate is distributed to the kidney. Other parts that target kidney cells can also be used instead of, or in addition to, the lipid ligand.

[0667] In another embodiment, the ligand is a portion taken up by target cells, such as proliferating cells, such as a vitamin. These may be particularly useful in treating undesirable cell proliferation, such as disorders characterized by malignant or non-malignant forms, such as cancer cells. Representative vitamins include vitamins A, E, and K. Examples of other vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Similarly included are HSA and low-density lipoprotein (LDL).

[0668] In another embodiment, the ligand is a cell permeabilizer, such as a helical cell permeator. In certain embodiments, the agent is amphiphilic. Exemplary agents are peptides such as tat or antennopedia. If the agent is a peptide, it may be modified, including the use of peptidyl mimicry, inverted isomers, non-peptide or pseudopeptide bonds, and D-amino acids. The helical agent may be an alpha-helical agent that may have a lipophilic phase and an oleophobic phase.

[0669] The ligand may be a peptide or a peptide mimetic. A peptide mimetic (also referred to herein as an oligopeptide mimetic) is a molecule that can be folded into a specific three-dimensional structure similar to that of a natural peptide. The attachment of peptides and peptide mimetics to oligonucleotide agents can affect the pharmacokinetic distribution of RNA silencing agents, for example, by improving cellular recognition and absorption. The peptide or peptide mimetic moiety may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptide mimetic may be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (for example, mainly consisting of Tyr, Trp, or Phe). The peptide moiety may be a dendrimeric peptide, a restrictive peptide, or a cross-linked peptide. The peptide moiety may be an L-peptide or a D-peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane translocation sequence (MTS). Peptides or peptide mimetic compounds can be encoded by random DNA sequences, such as peptides identified from phage presentation libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, the peptide or peptide mimetic compound tethered to the RNA silencing agent via an incorporated monomer unit is a cell-targeted peptide, such as an arginine-glycine-aspartate (RGD) peptide or RGD mimic. Some peptide moieties can range in length from about 5 to about 40 amino acids. Peptide moieties can have structural modifications, such as improving stability or directing conformation. Any of the structural modifications described below may be used.

[0670] In certain embodiments, the functional portion is ligated to the 5' and / or 3' ends of the RNA silencing agent of the disclosure. In certain embodiments, the functional portion is ligated to the 5' and / or 3' ends of the antisense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional portion is ligated to the 5' and / or 3' ends of the sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional portion is ligated to the 3' end of the sense strand of the RNA silencing agent of the disclosure.

[0671] In certain embodiments, the functional portion is linked to the RNA silencing agent by a linker. In certain embodiments, the functional portion is linked to the antisense strand and / or sense strand by a linker. In certain embodiments, the functional portion is linked to the 3' end of the sense strand by a linker. In certain embodiments, the linker includes a divalent or trivalent linker. In certain embodiments, the linker includes an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof. In certain embodiments, the divalent or trivalent linker is selected from the following: [ka] (wherein n is 1, 2, 3, 4, or 5).

[0672] In certain embodiments, the linker further comprises a phosphodiester or a phosphodiester derivative. In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] (In the formula, X is O, S, or BH3).

[0673] Various functional components of this disclosure and means for conjugating them with RNA silencing agents are described in further detail in WO2017 / 030973A1 and WO2018 / 031933A2, which are incorporated herein by reference.

[0674] VI. Branched Oligonucleotides Two or more RNA silencing agents disclosed above, such as oligonucleotide constructs including anti-MAPT siRNA, can be linked together by one or more moieties independently selected from linkers, spacers, and branching points to form a branched oligonucleotide RNA silencing agent. In certain embodiments, the branched oligonucleotide RNA silencing agent consists of two siRNAs to form a di-branched siRNA ("di-siRNA") scaffold for delivering two siRNAs. In typical embodiments, the nucleic acids of the branched oligonucleotide each include an antisense strand (or a portion thereof), the antisense strand having sufficient complementarity to the target mRNA (e.g., MAPT mRNA) and mediating an RNA-mediated silencing mechanism (e.g., RNAi).

[0675] In exemplary embodiments, the branched oligonucleotide may have 2 to 8 RNA silencing agents attached via a linker. The linker may be hydrophobic. In one embodiment, the branched oligonucleotide of the present application has 2 to 3 oligonucleotides. In one embodiment, the oligonucleotides independently have substantial chemical stability (e.g., at least 40% of the constituent bases are chemically modified). In exemplary embodiments, the oligonucleotides have complete chemical stability (i.e., all constituent bases are chemically modified). In some embodiments, the branched oligonucleotide comprises one or more single-stranded phosphorothioate tails, each independently having 2 to 20 nucleotides. In non-limiting embodiments, each single-stranded tail has 2 to 10 nucleotides.

[0676] In certain embodiments, branched oligonucleotides are characterized by three properties: (1) a branched structure, (2) complete metabolic stabilization, and (3) the presence of a single-stranded tail containing a phosphorothioate linker. In certain embodiments, branched oligonucleotides have two or three branches. It is thought that an increase in the overall size of the branched structure promotes increased uptake. Also, although not bound by any particular theory regarding activity, it is thought that multiple adjacent branches (e.g., two or three) allow each branch to act in coordination, and thus dramatically improve the rates of internalization, transport, and release.

[0677] Branched oligonucleotides are provided in a variety of structurally diverse embodiments. In some embodiments, the nucleic acid attached at the branching point is single-stranded or double-stranded and consists of a miRNA inhibitor, gapmer, micmer, SSO, PMO, or PNA. These single strands can be attached to the 3' or 5' end. Combinations of siRNA and single-stranded oligonucleotides can also be used for dual function. In another embodiment, short nucleic acids complementary to gapmers, micmers, miRNA inhibitors, SSO, PMO, and PNA are used to carry these active single-stranded nucleic acids and enhance their distribution and intracellular integration. The short double-stranded region has a low melting temperature (Tm approximately 37°C) to rapidly dissociate when the branched structure is taken up into the cell.

[0678] Di-siRNA branched oligonucleotides may contain chemically diverse conjugates, such as the functional moieties described above. The conjugated bioactive ligands may be used to enhance cell specificity and promote membrane binding, internalization, and serum protein binding. Examples of bioactive moieties used for conjugation include DHA, GalNAc, and cholesterol. These moieties can be attached to Di-siRNA via connecting linkers or spacers, or added via additional linkers or spacers attached to another free siRNA end.

[0679] The presence of a branched structure improves tissue retention levels in the brain by more than 100 times compared to unbranched compounds of the same chemical composition, suggesting a novel mechanism of cell retention and distribution. Branched oligonucleotides are unexpectedly uniformly distributed throughout the spinal cord and brain. Furthermore, branched oligonucleotides exhibit unexpectedly efficient systemic delivery to various tissues and very high levels of tissue accumulation.

[0680] Branched oligonucleotides include a variety of therapeutic nucleic acids, such as siRNA, ASO, miRNA, miRNA inhibitors, splice switching, PMO, and PNA. In some embodiments, the branched oligonucleotides further include a conjugated hydrophobic moiety, exhibiting unprecedented silencing and effects in vitro and in vivo.

[0681] Linker In one embodiment of a branched oligonucleotide, each linker is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramides, esters, amides, triazoles, and combinations thereof; where any carbon or oxygen atom of the linker is optionally substituted with a nitrogen atom, harbors a hydroxyl substituent, or harbors an oxo substituent. In one embodiment, each linker is an ethylene glycol chain. In another embodiment, each linker is an alkyl chain. In another embodiment, each linker is a peptide. In another embodiment, each linker is RNA. In another embodiment, each linker is DNA. In another embodiment, each linker is a phosphate. In another embodiment, each linker is a phosphonate. In another embodiment, each linker is a phosphoramide. In another embodiment, each linker is an ester. In another embodiment, each linker is an amide. In another embodiment, each linker is a triazole.

[0682] VII. Compounds of formula (I): In another embodiment, branched oligonucleotide compounds of formula (I) are provided herein: [ka] (wherein L is selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, and combinations thereof; formula (I) optionally further comprises one or more branching points B and one or more spacers S; where B is independently a polyvalent organic species or a derivative thereof in each instance, and S is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, and combinations thereof)

[0683] Part N is an RNA double helix comprising a sense strand and an antisense strand; n is 2, 3, 4, 5, 6, 7, or 8. In one embodiment, the antisense strand of N contains a sequence substantially complementary to any one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295 listed in Tables 4-6. In a further embodiment, N contains a strand capable of targeting one or more MAPT nucleic acid sequences selected from the group consisting of SEQ ID NOs: 14-33, 299, and 302 listed in Tables 7-8. The sense strand and antisense strand may each independently contain one or more chemical modifications.

[0684] In one embodiment, the compound of formula (I) has a structure selected from formulas (I-1) to (I-9) in Table 1. [Table 3]

[0685] In one embodiment, the compound of formula (I) is formula (I-1). In another embodiment, the compound of formula (I) is formula (I-2). In another embodiment, the compound of formula (I) is formula (I-3). In another embodiment, the compound of formula (I) is formula (I-4). In another embodiment, the compound of formula (I) is formula (I-5). In another embodiment, the compound of formula (I) is formula (I-6). In another embodiment, the compound of formula (I) is formula (I-7). In another embodiment, the compound of formula (I) is formula (I-8). In another embodiment, the compound of formula (I) is formula (I-9).

[0686] In embodiments of the compound of formula (I), each linker is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramides, esters, amides, triazoles, and combinations thereof; where any carbon or oxygen atom of the linker is optionally substituted with a nitrogen atom, has a hydroxyl substituent, or has an oxo substituent. In one embodiment of the compound of formula (I), each linker is an ethylene glycol chain. In another embodiment, each linker is an alkyl chain. In yet another embodiment of the compound of formula (I), each linker is a peptide. In yet another embodiment of the compound of formula (I), each linker is RNA. In yet another embodiment of the compound of formula (I), each linker is DNA. In yet another embodiment of the compound of formula (I), each linker is a phosphate. In yet another embodiment, each linker is a phosphonate. In yet another embodiment of the compound of formula (I), each linker is a phosphoramide. In another embodiment of the compound of formula (I), each linker is an ester. In another embodiment of the compound of formula (I), each linker is an amide. In another embodiment of the compound of formula (I), each linker is a triazole.

[0687] In one embodiment of the compound of formula (I), B is a polyvalent organic species. In another embodiment of the compound of formula (I), B is a derivative of a polyvalent organic species. In one embodiment of the compound of formula (I), B is a triol or tetrol derivative. In another embodiment, B is a tri or tetracarboxylic acid derivative. In another embodiment, B is an amine derivative. In another embodiment, B is a tri or tetraamine derivative. In another embodiment, B is an amino acid derivative. In another embodiment of the compound of formula (I), B is selected from the following formulas: [ka]

[0688] A polyvalent organic species is a carbon atom with a moiety containing three or more valencies (i.e., an attachment site to a moiety such as S, L, or N as defined above). Non-limiting examples of polyvalent organic species include triols (e.g., glycerol, phloroglucinol), tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene), tricarboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimesic acid), tetracarboxylic acids (e.g., ethylenediaminetetraacetic acid, pyromellitic acid), tertiary amines (e.g., tripropargylamine, triethanolamine), triamines (e.g., diethylenetriamine), tetramines, and species containing combinations of hydroxyl, thiol, amino, and / or carboxyl moieties (e.g., amino acids such as lysine, serine, and cysteine).

[0689] In embodiments of the compound of formula (I), each nucleic acid comprises one or more chemically modified nucleotides. In embodiments of the compound of formula (I), each nucleic acid consists of chemically modified nucleotides. In certain embodiments of the compound of formula (I), more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of each nucleic acid comprises chemically modified nucleotides.

[0690] In one embodiment, each antisense chain independently contains a 5' terminal group R selected from the group in Table 2. [Table 4]

[0691] In one embodiment, R is R1. In another embodiment, R is R2. In another embodiment, R is R3. In another embodiment, R is R4. In another embodiment, R is R5. In another embodiment, R is R6. In another embodiment, R is R7. In another embodiment, R is R8.

[0692] Structure of equation (II) In one embodiment, the compound of formula (I) has the structure of formula (II): [ka] (In the formula, X is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each occurrence; Y is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each occurrence; - represents a phosphodiester nucleoside bond; = represents a phosphorothioate nucleoside bond; and --- represents a base-pair interaction or mismatch, individually for each occurrence).

[0693] In certain embodiments, the structure of formula (II) does not contain mismatches. In one embodiment, the structure of formula (II) contains one mismatch. In another embodiment, the compound of formula (II) contains two mismatches. In another embodiment, the compound of formula (II) contains three mismatches. In yet another embodiment, the compound of formula (II) contains four mismatches. In one embodiment, each nucleic acid consists of a chemically modified nucleotide.

[0694] In certain embodiments, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of X' in the structure of formula (II) are chemically modified nucleotides. In other embodiments, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of X' in the structure of formula (II) are chemically modified nucleotides.

[0695] Structure of equation (III) In one embodiment, the compound of formula (I) has the structure of formula (III): [ka]

[0696] Here, X is a nucleotide containing a 2'-deoxy-2'-fluoro modification independently of each occurrence; X is a nucleotide containing a 2'-O-methyl modification independently of each occurrence; Y is a nucleotide containing a 2'-deoxy-2'-fluoro modification independently of each occurrence; Y is a nucleotide containing a 2'-O-methyl modification independently of each occurrence.

[0697] In one embodiment, X is selected from the group consisting of 2'-deoxy-2'-fluoro-modified adenosine, guanosine, uridine, or cytidine. In one embodiment, X is selected from the group consisting of 2'-O-methyl-modified adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is...

Claims

1. A double-stranded (dsRNA) molecule containing a sense strand and an antisense strand, The molecule wherein the antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295.

2. The dsRNA according to claim 1, wherein the antisense strand comprises a sequence substantially complementary to the MAPT nucleic acid sequence described in any one of SEQ ID NOs: 14-33, 299, and 302.

3. The dsRNA according to claim 1, comprising complementarity to at least 10, 11, 12, or 13 consecutive nucleotides of the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295.

4. The dsRNA according to claim 1 or 3, comprising three or fewer mismatches with the MAPT nucleic acid sequences of SEQ ID NOs. 1 to 13, 292, and 295.

5. The dsRNA according to claim 1, comprising complete complementarity to the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295.

6. The dsRNA according to any one of claims 1 to 5, wherein the antisense strand comprises about 15 to 25 nucleotides in length.

7. The dsRNA according to any one of claims 1 to 6, wherein the sense strand comprises a length of approximately 15 to 25 nucleotides.

8. The dsRNA according to any one of claims 1 to 7, wherein the antisense strand is 20 nucleotides long.

9. The dsRNA according to any one of claims 1 to 7, wherein the antisense strand is 21 nucleotides long.

10. The dsRNA according to any one of claims 1 to 7, wherein the antisense strand is 22 nucleotides long.

11. The dsRNA according to any one of claims 1 to 10, wherein the sense strand is 15 nucleotides long.

12. The dsRNA according to any one of claims 1 to 10, wherein the sense strand is 16 nucleotides long.

13. The dsRNA according to any one of claims 1 to 10, wherein the sense strand is 18 nucleotides long.

14. The dsRNA according to any one of claims 1 to 10, wherein the sense strand is 20 nucleotides long.

15. The dsRNA according to any one of claims 1 to 14, comprising a double-stranded region of 15 to 20 base pairs.

16. The dsRNA according to any one of claims 1 to 15, comprising a 15-base-pair double-stranded region.

17. The dsRNA according to any one of claims 1 to 15, comprising a 16-base-pair double-stranded region.

18. The dsRNA according to any one of claims 1 to 15, comprising an 18-base-pair double-stranded region.

19. The dsRNA according to any one of claims 1 to 15, comprising a 20-base-pair double-stranded region.

20. The dsRNA according to any one of claims 1 to 19, wherein the dsRNA includes a blunt end.

21. The dsRNA according to any one of claims 1 to 20, wherein the dsRNA comprises at least one single-stranded nucleotide overhang.

22. The dsRNA according to claim 21, wherein the dsRNA includes a single-stranded nucleotide overhang of about 2 to 5 nucleotides.

23. The dsRNA according to claim 21, wherein the dsRNA includes a single-stranded nucleotide overhang of two nucleotides.

24. The dsRNA according to claim 21, wherein the dsRNA includes a single-stranded nucleotide overhang of 5 nucleotides.

25. The dsRNA according to any one of claims 1 to 24, wherein the dsRNA comprises naturally occurring nucleotides.

26. The dsRNA according to any one of claims 1 to 24, wherein the dsRNA comprises at least one modified nucleotide.

27. The dsRNA according to claim 26, wherein the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramide, a non-natural base including a nucleotide, or a mixture thereof.

28. The dsRNA according to any one of claims 1 to 27, wherein the dsRNA includes at least one modified nucleotide linkage.

29. The dsRNA according to claim 28, wherein the modified nucleotide linkage includes a phosphorothioate nucleotide linkage.

30. The dsRNA according to any one of claims 1 to 29, comprising 4 to 16 phosphorothioate nucleotide linkages.

31. The dsRNA according to any one of claims 1 to 29, comprising 8 to 13 phosphorothioate nucleotide linkages.

32. The dsRNA according to any one of claims 1 to 28, wherein the dsRNA comprises at least one modified nucleotide linkage of formula I: 【Chemistry 1】 (In the formula, B is the base pair portion; W is O, OCH ２ , OCH, CH ２ Selected from the group consisting of , and CH; X is a halo, hydroxy, and C １－６ Selected from the group consisting of alkoxys, Y is O － OH, OR, NH － NH ２ S － Selected from the group consisting of , and SH, Z is O and CH ２ Selected from the group consisting of; R is a protecting group, 【Chemistry 2】 (where is any double bond).

33. The dsRNA according to any one of claims 1 to 32, wherein the dsRNA comprises at least 80% chemically modified nucleotides.

34. The dsRNA according to any one of claims 1 to 33, wherein the dsRNA is fully chemically modified.

35. The dsRNA according to any one of claims 1 to 33, wherein the dsRNA comprises at least 70% 2'-O-methylnucleotide modifications.

36. The dsRNA according to any one of claims 1 to 33, wherein the antisense strand comprises at least 70% 2'-O-methylnucleotide modification.

37. The dsRNA according to claim 36, wherein the antisense strand comprises 70% to 90% 2'-O-methylnucleotide modifications.

38. The dsRNA according to any one of claims 1 to 33, wherein the sense strand comprises at least 65% 2'-O-methylnucleotide modifications.

39. The dsRNA according to claim 38, wherein the sense strand comprises 100% 2'-O-methylnucleotide modification.

40. The dsRNA according to any one of claims 1 to 39, wherein the sense strand contains one or more nucleotide mismatches between the antisense strand and the sense strand.

41. The dsRNA according to claim 40, wherein one or more nucleotide mismatches are located at positions 2, 6, and 12 from the 5' end of the sense strand.

42. The dsRNA according to claim 40, wherein the nucleotide mismatches are located at positions 2, 6, and 12 from the 5' end of the sense strand.

43. The dsRNA according to any one of claims 1 to 42, wherein the antisense strand comprises a 5'-phosphate, a 5'-alkylphosphonate, a 5'-alkylenephosphonate, or a 5'-alkenylphosphonate.

44. The dsRNA according to claim 43, wherein the antisense strand comprises a 5' vinyl phosphonate.

45. The dsRNA according to claim 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end, (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises alternating 2'-methoxyribonucleotides and 2'-fluororibonucleotides; (3) The nucleotides at the 2 and 14 positions from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain comprises alternating 2'-methoxyribonucleotides and 2'-fluororibonucleotides; (7) The dsRNA wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

46. The dsRNA according to claim 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end, (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 70% 2'-O-methyl modification; (3) The nucleotide at the 5' end to the 14th position of the antisense strand is not a 2'-methoxyribonucleotide; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain comprises at least 70% 2'-O-methyl modification; (7) The dsRNA wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

47. The dsRNA according to claim 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end, (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 85% 2'-O-methyl modification; (3) The nucleotides at the 2 and 14 positions from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains 100% 2'-O-methyl modification; (7) The dsRNA wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

48. The dsRNA according to claim 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end, (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 4, 5, 6 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains 100% 2'-O-methyl modification; (7) The dsRNA wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

49. The dsRNA according to claim 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end, (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 4, 5, 6 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains 100% 2'-O-methyl modification; (7) The dsRNA wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

50. The dsRNA according to claim 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end, (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain comprises at least 65% 2'-O-methyl modification; (7) The nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides; (8) The dsRNA wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

51. The dsRNA according to claim 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end, (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 75% 2'-O-methyl modification; (3) The nucleotides at the 2 and 14 positions from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain comprises at least 75% 2'-O-methyl modification; (7) The nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides; (8) The dsRNA wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

52. The dsRNA according to any one of claims 1 to 51, wherein the functional portion is ligated to the 5' end and / or 3' end of the antisense strand.

53. The dsRNA according to any one of claims 1 to 51, wherein the functional portion is ligated to the 5' end and / or 3' end of the sense strand.

54. The dsRNA according to any one of claims 1 to 51, wherein the functional portion is ligated to the 3' end of the sense strand.

55. The dsRNA according to any one of claims 52 to 54, wherein the functional portion includes a hydrophobic portion.

56. The dsRNA according to claim 55, wherein the hydrophobic portion is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.

57. The dsRNA according to claim 56, wherein the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).

58. The dsRNA according to claim 56, wherein the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanic acid (DCA).

59. The dsRNA according to claim 56, wherein the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, derivatives thereof, and metabolites thereof.

60. The dsRNA according to claim 59, wherein the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

61. The dsRNA according to any one of claims 54 to 60, wherein the functional portion is linked to the antisense strand and / or sense strand by a linker.

62. The dsRNA according to claim 61, wherein the linker comprises a divalent or trivalent linker.

63. The aforementioned divalent or trivalent linker, 【Transformation 3】 The dsRNA according to claim 62, selected from (wherein n is 1, 2, 3, 4, or 5).

64. The dsRNA according to claim 61 or 62, wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

65. The dsRNA according to claim 62 or 63, wherein, if the linker is a trivalent linker, the linker further links a phosphodiester or a phosphodiester derivative.

66. The dsRNA according to claim 65, wherein the phosphodiester or phosphodiester derivative is selected from the group consisting of the following: 【Chemistry 4】 (wherein X is O, S or BH ３ ).

67. The dsRNA according to any one of claims 1 to 66, wherein the nucleotides at positions 1 and 2 from the 3' end of the sense strand, and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand are bound to adjacent ribonucleotides via phosphorothioate bonds.

68. A pharmaceutical composition for inhibiting the expression of a tau protein (MAPT) gene in a living organism, comprising a dsRNA according to any one of claims 1 to 67 and a pharmaceutically acceptable carrier.

69. The pharmaceutical composition according to claim 68, wherein the dsRNA inhibits the expression of the MAPT gene by at least 50%.

70. The pharmaceutical composition according to claim 68, wherein the dsRNA inhibits the expression of the MAPT gene by at least 80%.

71. A method for regulating the expression of the MAPT gene in cells, (a) the step of introducing a double-stranded ribonucleic acid (dsRNA) according to any one of claims 1 to 67 into the cells; (b) The method comprising maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the MAPT gene, thereby inhibiting the expression of the MAPT gene in the cells.

72. A method for treating or managing a neurodegenerative disease, comprising administering a therapeutically effective amount of the dsRNA described in any one of claims 1 to 67 to a patient in need of such treatment or management.

73. The method according to claim 72, wherein the dsRNA is administered to the brain of the patient.

74. The method according to claim 72, wherein the dsRNA is administered by intracerebral or intraventricular (ICV) injection, intrastriatal (IS) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

75. The method according to claim 72, wherein administration of the dsRNA causes a decrease in MAPT gene mRNA in one or more of the hippocampus, striatum, cortex, cerebellum, thalamus, hypothalamus, and spinal cord.

76. The method according to any one of claims 71 to 75, wherein the dsRNA inhibits the expression of the MAPT gene by at least 50%.

77. The method according to any one of claims 71 to 75, wherein the dsRNA inhibits the expression of the MAPT gene by at least 80%.

78. A vector comprising regulatory sequences operably ligated to nucleotide sequences encoding dsRNA molecules substantially complementary to the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295.

79. The vector according to claim 78, wherein the RNA molecule inhibits the expression of the MAPT gene by at least 30%.

80. The vector according to claim 78, wherein the RNA molecule inhibits the expression of the MAPT gene by at least 50%.

81. The vector according to claim 78, wherein the RNA molecule inhibits the expression of the MAPT gene by at least 80%.

82. The vector according to claim 78, wherein the dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a sequence substantially complementary to the MAPT nucleic acid sequences of SEQ ID NOs: 1-13, 292, and 295.

83. A cell comprising the vector according to any one of claims 78 to 82.

84. Recombinant adeno-associated virus (rAAV), comprising the vector and AAV capsid described in any one of claims 78 to 82.

85. A branched RNA compound comprising two or more dsRNA molecules according to any one of claims 1 to 67, which are covalently linked to each other.

86. The branched RNA compound according to claim 85, wherein the dsRNA molecules are covalently linked to each other by linkers, spacers, or branching points.

87. Two or more RNA molecules containing 15 to 35 nucleotides in length, and MAPT mRNA contains a sequence that is substantially complementary to it. A branched RNA compound in which the two RNA molecules are linked to each other by one or more parts independently selected from linkers, spacers, and branching points.

88. The branched RNA compound according to claim 87, comprising a sequence substantially complementary to any one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295.

89. The branched RNA compound according to claim 87, comprising a nucleic acid sequence substantially complementary to one or more of the MAPT nucleic acid sequences of sequence numbers 14-33, 299, and 302.

90. The branched RNA compound according to any one of claims 87 to 89, wherein the RNA molecule comprises one or both of ssRNA and dsRNA.

91. The branched RNA compound according to any one of claims 87 to 89, wherein the RNA molecule comprises an antisense oligonucleotide.

92. A branched RNA compound according to any one of claims 87 to 91, wherein each RNA molecule contains a length of 15 to 25 nucleotides.

93. The branched RNA compound according to any one of claims 87 to 89, wherein each RNA molecule comprises a dsRNA including a sense strand and an antisense strand, and each antisense strand independently comprises a sequence substantially complementary to one of the MAPT nucleic acid sequences SEQ ID NOs: 1 to 13, 292, and 295.

94. The branched RNA compound according to claim 93, comprising complementarity to at least 10, 11, 12, or 13 consecutive nucleotides of any one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295.

95. The branched RNA compound according to claim 93, wherein each RNA molecule contains three or fewer mismatches with one of the MAPT nucleic acid sequences SEQ ID NOs. 1-13, 292, and 295.

96. The branched RNA compound according to claim 93, comprising substantially complete complementarity to any one of the MAPT nucleic acid sequences SEQ ID NOs: 1-13, 292, and 295.

97. The branched RNA compound according to any one of claims 93 to 96, wherein the antisense strand includes a portion having one of the nucleic acid sequences of sequence numbers 34 to 46.

98. The branched RNA compound according to any one of claims 93 to 97, wherein the antisense strand and / or the sense strand comprises about 15 to 25 nucleotides in length.

99. The branched RNA compound according to any one of claims 93 to 98, wherein the antisense strand is 20 nucleotides long.

100. The branched RNA compound according to any one of claims 93 to 98, wherein the antisense strand is 21 nucleotides long.

101. The branched RNA compound according to any one of claims 93 to 98, wherein the antisense strand is 22 nucleotides long.

102. The branched RNA compound according to any one of claims 93 to 101, wherein the sense strand is 15 nucleotides long.

103. The branched RNA compound according to any one of claims 93 to 101, wherein the sense strand is 16 nucleotides long.

104. The branched RNA compound according to any one of claims 93 to 101, wherein the sense strand is 18 nucleotides long.

105. The branched RNA compound according to any one of claims 93 to 101, wherein the sense strand is 20 nucleotides long.

106. The branched RNA compound according to any one of claims 90 to 105, wherein the dsRNA includes a double-stranded region of 15 to 20 base pairs.

107. The branched RNA compound according to any one of claims 90 to 106, wherein the dsRNA includes a 15-base-pair double-stranded region.

108. The branched RNA compound according to any one of claims 90 to 106, wherein the dsRNA includes a 16-base-pair double-stranded region.

109. The branched RNA compound according to any one of claims 90 to 106, wherein the dsRNA includes an 18-base-pair double-stranded region.

110. The branched RNA compound according to any one of claims 90 to 106, wherein the dsRNA includes a 20-base-pair double-stranded region.

111. The branched RNA compound according to any one of claims 90 to 110, wherein the dsRNA includes a blunt end.

112. The branched RNA compound according to any one of claims 90 to 110, wherein the dsRNA comprises at least one single-stranded nucleotide overhang.

113. The branched RNA compound according to any one of claims 90 to 112, wherein the dsRNA comprises a single-stranded nucleotide overhang of 2 to 5 nucleotides.

114. The branched RNA compound according to any one of claims 90 to 113, wherein the dsRNA comprises naturally occurring nucleotides.

115. The branched RNA compound according to any one of claims 90 to 114, wherein the dsRNA comprises at least one modified nucleotide.

116. The branched RNA compound according to claim 115, wherein the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramide, or a non-natural base containing a nucleotide.

117. The branched RNA compound according to any one of claims 90 to 116, wherein the dsRNA comprises at least one modified nucleotide linkage.

118. The branched RNA compound according to claim 117, wherein the modified nucleotide linkage includes a phosphorothioate nucleotide linkage.

119. A branched RNA compound according to any one of claims 90 to 118, comprising 4 to 16 phosphorothioate nucleotide linkages.

120. A branched RNA compound according to any one of claims 90 to 118, comprising 8 to 13 phosphorothioate nucleotide linkages.

121. The branched RNA compound according to any one of claims 90 to 117, wherein the dsRNA comprises at least one modified nucleotide linkage of formula (I): 【Transformation 5】 (In the formula, B is the base pair portion; W is O, OCH ２ , OCH, CH ２ Selected from the group consisting of , and CH; X is a halo, hydroxy, and C １－６ Selected from the group consisting of alkoxys, Y is O － OH, OR, NH － NH ２ S － Selected from the group consisting of , and SH, Z is O and CH ２ Selected from the group consisting of; R is a protecting group, 【Transformation 6】 (where is any double bond).

122. The branched RNA compound according to any one of claims 90 to 121, wherein the dsRNA comprises at least 75% chemically modified nucleotides.

123. The branched RNA compound according to any one of claims 90 to 122, wherein the dsRNA is completely chemically modified.

124. The branched RNA compound according to any one of claims 90 to 123, wherein the dsRNA comprises at least 70% 2'-O-methylnucleotide modification.

125. The branched RNA compound according to any one of claims 90 to 124, wherein the antisense strand comprises at least 70% 2'-O-methylnucleotide modification.

126. The branched RNA compound according to claim 125, wherein the antisense strand comprises 70% to 90% 2'-O-methylnucleotide modifications.

127. The branched RNA compound according to any one of claims 91 to 124, wherein the sense strand comprises at least 65% 2'-O-methylnucleotide modification.

128. The branched RNA compound according to claim 127, wherein the sense strand comprises 100% 2'-O-methylnucleotide modification.

129. The branched RNA compound according to any one of claims 93 to 128, wherein the sense strand contains one or more nucleotide mismatches between the antisense strand and the sense strand.

130. The branched RNA compound according to claim 129, wherein one or more nucleotide mismatches are located at the 2, 6, and 12 positions from the 5' end of the sense strand.

131. The branched RNA compound according to claim 129, wherein the nucleotide mismatch is located at positions 2, 6, and 12 from the 5' end of the sense strand.

132. The branched RNA compound according to any one of claims 93 to 131, wherein the antisense strand comprises a 5'-phosphate, a 5'-alkylphosphonate, a 5'-alkylenephosphonate, a 5'-alkenylphosphonate, or a mixture thereof.

133. The branched RNA compound according to claim 132, wherein the antisense strand comprises a 5' vinyl phosphonate.

134. The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where: (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises alternating 2'-methoxyribonucleotides and 2'-fluororibonucleotides; (3) The nucleotides at the 2 and 14 positions from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain comprises alternating 2'-methoxyribonucleotides and 2'-fluororibonucleotides; (7) The branched RNA compound according to claim 90, wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

135. The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where: (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 70% 2'-O-methyl modification; (3) The nucleotide at the 5' end to the 14th position of the antisense strand is not a 2'-methoxyribonucleotide; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain comprises at least 70% 2'-O-methyl modification; (7) The branched RNA compound according to claim 90, wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

136. The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where: (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 85% 2'-O-methyl modification; (3) The nucleotides at the 2 and 14 positions from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains 100% 2'-O-methyl modification; (7) The branched RNA compound according to claim 90, wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

137. The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where: (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 4, 5, 6 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains 100% 2'-O-methyl modification; (7) The branched RNA compound according to claim 90, wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

138. The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where: (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 4, 5, 6 and 14 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain contains 100% 2'-O-methyl modification; (7) The branched RNA compound according to claim 90, wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

139. The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where: (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain comprises at least 65% 2'-O-methyl modification; (7) The nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides; (8) The branched RNA compound according to claim 90, wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

140. The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where: (1) The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; (2) The antisense chain comprises at least 75% 2'-O-methyl modification; (3) The nucleotides at the 2 and 14 positions from the 5' end of the antisense strand are not 2'-methoxyribonucleotides; (4) The nucleotides from the 1-2 to the 1-7 positions from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide linkages; (5) A portion of the antisense chain is complementary to a portion of the sense chain; (6) The sense chain comprises at least 75% 2'-O-methyl modification; (7) The nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides; (8) The branched RNA compound according to claim 90, wherein the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide linkages.

141. The branched RNA compound according to any one of claims 93 to 140, wherein the functional portion is ligated to the 5' end and / or 3' end of the antisense strand.

142. A branched RNA compound according to any one of claims 93 to 140, wherein the functional portion is ligated to the 5' end and / or 3' end of the sense strand.

143. A branched RNA compound according to any one of claims 93 to 140, wherein the functional portion is ligated to the 3' end of the sense strand.

144. The branched RNA compound according to any one of claims 141 to 143, wherein the functional portion includes a hydrophobic portion.

145. The branched RNA compound according to claim 144, wherein the hydrophobic portion is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.

146. The branched RNA compound according to claim 145, wherein the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).

147. The branched RNA compound according to claim 145, wherein the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanic acid (DCA).

148. The branched RNA compound according to claim 145, wherein the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, derivatives thereof, and metabolites thereof.

149. The branched RNA compound according to claim 145, wherein the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

150. The branched RNA compound according to any one of claims 141 to 149, wherein the functional portion is linked to the antisense strand and / or sense strand by a linker.

151. The branched RNA compound according to claim 150, wherein the linker comprises a divalent or trivalent linker.

152. The aforementioned divalent or trivalent linker, 【Transformation 7】 A branched RNA compound according to claim 151, selected from the group consisting of (wherein n is 1, 2, 3, 4, or 5).

153. The branched RNA compound according to claim 150 or 151, wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

154. The branched RNA compound according to claim 150, wherein, if the linker is a trivalent linker, the linker further links a phosphodiester or a phosphodiester derivative.

155. The branched RNA compound according to claim 154, wherein the phosphodiester or phosphodiester derivative is selected from the group consisting of the following: 【Transformation 8】 (In the formula, X is O, S, or BH) ３ (That is the case.)

156. The branched RNA compound according to any one of claims 93 to 155, wherein the nucleotides at positions 1 and 2 from the 3' end of the sense strand, and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand, are bound to adjacent ribonucleotides via phosphorothioate bonds.

157. Compound of formula (I): 【Chemistry 9】 (In the formula: L comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramide, an ester, an amide, a triazole, or a combination thereof, and formula (I) further comprises one or more branch points B and one or more spacers S, in the formula, Each instance of B is independently a polyvalent organic species or a derivative thereof; S, independently of each occurrence, comprises ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramides, esters, amides, triazoles, or combinations thereof; N is a double-stranded nucleic acid containing a sense strand and an antisense strand, with a length of 15 to 35 bases; where The antisense strand comprises a sequence substantially complementary to any one of the MAPT nucleic acid sequences 1-13, 292, and 295; The sense chain and the antisense chain each independently include one or more chemical modifications; n is 2, 3, 4, 5, 6, 7, or 8).

158. The compound according to claim 157, having a structure selected from formulas (I-1) to (I-9): Table 1

159. The compound according to claim 157, wherein the antisense chain comprises a 5' terminal group R selected from the group consisting of: 【Chemistry 10】

160. The compound according to claim 157, having the structure of formula (II): 【Chemistry 11】 (In the formula, X is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each instance. Y is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives in each instance. The dash (-) represents a phosphodiester nucleoside linkage; The equals sign (=) represents a linkage between phosphorothioate nucleosides; The dashes (---) represent, independently, base pair interactions or mismatches.

161. The compound according to claim 157, having the structure of formula (IV): 【Chemistry 12】 (In the formula, X is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each instance. Y is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives in each instance. The dash (-) represents a phosphodiester nucleoside linkage; The equals sign (=) represents a linkage between phosphorothioate nucleosides; The dashes (---) represent, independently, base pair interactions or mismatches.

162. A compound according to any one of claims 157 to 161, wherein L is structure L1: 【Chemistry 13】

163. R is R ３ The compound according to claim 164, wherein n is 2.

164. A compound according to any one of claims 157 to 161, wherein L is structure L2: 【Chemistry 14】

165. R is R ３ The compound according to claim 164, wherein n is 2.

166. Delivery system for therapeutic nucleic acids having the structure of formula (VI): 【Chemistry 15】 (In the formula, L comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramide, an ester, an amide, a triazole, or a combination thereof, and formula (VI) further comprises one or more branch points B and one or more spacers S, in the formula, Each instance of B independently includes a polyvalent organic species or its derivatives; S, independently of each occurrence, comprises ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramides, esters, amides, triazoles, or combinations thereof; Each cNA is an independent carrier nucleic acid containing one or more chemical modifications. Each cNA independently comprises at least 15 consecutive nucleotides from one of the MAPT nucleic acid sequences of sequence numbers 1-13, 292, and 295; n is 2, 3, 4, 5, 6, 7, or 8).

167. A delivery system according to claim 166, having a structure selected from formulas (VI-1) to (VI-9): Table 2

168. The delivery system according to claim 166, wherein each cNA independently comprises a chemically modified nucleotide.

169. The delivery system according to claim 166, further comprising n therapeutic nucleic acids (NAs), each NA being hybridized to at least one cNA.

170. The delivery system according to claim 169, wherein each NA independently comprises at least 16 consecutive nucleotides.

171. The delivery system according to claim 170, wherein each NA independently comprises at least 16 to 20 consecutive nucleotides.

172. The delivery system according to claim 169, wherein each NA includes at least two unpaired overhangs of nucleotides.

173. The delivery system according to claim 172, wherein the nucleotides of the overhang are linked via phosphorothioate linkage.

174. The delivery system according to claim 169, wherein each NA is independently selected from the group consisting of DNA, siRNA, antagomiR, miRNA, gapmer, mixmer, and guide RNA.

175. The delivery system according to claim 169, wherein each NA is substantially complementary to any one of the MAPT nucleic acid sequences of sequence numbers 1-13, 292, and 295.

176. A pharmaceutical composition for inhibiting the expression of the MAPT gene in a living organism, comprising a compound according to any one of claims 85 to 165 or a system according to any one of claims 166 to 75, and a pharmaceutically acceptable carrier.

177. The pharmaceutical composition according to claim 176, wherein the compound or system inhibits the expression of the MAPT gene by at least 50%.

178. The pharmaceutical composition according to claim 176, wherein the compound or system inhibits the expression of the MAPT gene by at least 80%.

179. A method for inhibiting the expression of the MAPT gene in cells, (a) the step of introducing the compound according to any one of claims 85 to 162 or the system according to any one of claims 166 to 175 into the cells; (b) The method comprising maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the MAPT gene, thereby inhibiting the expression of the MAPT gene in the cells.

180. A method for treating or managing a neurodegenerative disease, comprising administering to a patient in need of such treatment or management a therapeutically effective amount of the compound according to any one of claims 85 to 165 or the system according to any one of claims 166 to 175.

181. The method according to claim 180, wherein the dsRNA is administered to the brain of the patient.

182. The method according to claim 180, wherein the dsRNA is administered by intracerebral or intraventricular (ICV) injection, intrastriatal (IS) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

183. The method according to claim 180, wherein administering the dsRNA causes a decrease in the MAPT gene mRNA in one or more of the hippocampus, striatum, cortex, cerebellum, thalamus, hypothalamus, and spinal cord.

184. The method according to any one of claims 179 to 183, wherein the dsRNA inhibits the expression of the MAPT gene by at least 50%.

185. The method according to any one of claims 179 to 183, wherein the dsRNA inhibits the expression of the MAPT gene by at least 80%.

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