Compositions and methods for modulating tau expression

Abstract

Microtubule-associated protein tau (MAPT) antisense oligonucleotides (ASOs) and MAPT ASO conjugates are described, as are methods of using the MAPT ASOs and MAPT ASO conjugates to treat neurodegenerative disorders such as Alzheimer’s disease.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 618,798, filed January 8, 2024, which is incorporated herein by reference.

[0002] sequence list The sequence list written in the file DNL-038-03-WO_SeqListing.xml is 237 kilobytes in size, created on January 6, 2025, and is hereby incorporated by reference. Background Technology

[0003] Alzheimer's disease is a progressive neurodegenerative disease, currently the seventh leading cause of death in the United States and the most common cause of dementia in the elderly. A prominent pathological feature of Alzheimer's disease and other neurodegenerative diseases is the abnormal aggregation and inclusion body formation of the microtubule-associated protein tau. The microtubule-associated protein tau (which encodes tau)... MAPT Mutations in the gene are associated with tau protein lesions related to neurodegeneration.

[0004] In vivo delivery of nucleic acid-based molecules (such as antisense oligonucleotides) often requires specific targeting to reach certain tissues or cell types. In particular, delivery to non-hepatic tissues remains a barrier and limits the use of such therapies. Delivery of oligonucleotides to the central nervous system (CNS) poses a significant problem due to the presence of the blood-brain barrier (BBB). One method of delivering oligonucleotides to the CNS is intrathecal delivery. However, intrathecal delivery is invasive, carries a higher risk of side effects, and often results in uneven distribution.

[0005] Therefore, there is a continued need for novel and improved methods for delivering nucleic acid-based therapeutics in vivo, particularly to CNS tissues. Summary of the Invention

[0006] This article describes a microtubule-associated protein tau (MAPT) antisense oligonucleotide (ASO) conjugate comprising at least one MAPT antisense oligonucleotide (ASO) and an Fc polypeptide dimer targeting the transferrin receptor (TfR), wherein the Fc polypeptide of the Fc polypeptide dimer is modified to bind to the TfR. TfR is highly expressed at the blood-brain barrier (BBB), and naturally transports transferrin from the blood to the brain. The described MAPT ASO conjugate binds to the TfR, transports it across the BBB, and transports the attached MAPT ASO across the BBB. In some embodiments, the TfR-targeting Fc polypeptide dimer further comprises at least one cysteine ​​substitution to facilitate MAPT ASO attachment. In some embodiments, the Fc dimer comprises a first Fc polypeptide and a second Fc polypeptide, wherein the second Fc polypeptide comprises a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) and comprises a sequence having at least 70% sequence identity with SEQ ID NO:12, wherein the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide.

[0007] The MAPT antisense oligonucleotide (ASO) is described, which contains the nucleic acid sequence: 5′ A L x d m C L x e m C L x f TTAp a Ap b Gp c TATTACTx g T L x h G L x i m C L 3′ (SEQ ID NO:111) or 5′ m C L x d T L x e G L x f Tp j Tp k Ap l Gp m ACATTCp n Ap o TTx g m C L xh T L x i m C L 3′ (SEQ ID NO:112), where A L , m C L G L and T L They are adenine-locked nucleoside, 5-methylcytosine-locked nucleoside, guanine-locked nucleoside, and thymine-locked nucleoside, respectively; A, C, G, and T are deoxyadenosine nucleoside, deoxycytidine nucleoside, deoxyguanosine nucleoside, and deoxythymidine nucleoside, respectively; each p is independently a phosphate thioester (PS) nucleoside linker or a stabilized nucleoside linker, and (P) a (P) b and (P) c At least two of them are not PS nucleoside links; each x is independently a PS nucleoside link, a phosphodiester (PO) nucleoside link, or a stabilized nucleoside link; and any nucleoside link that is not a stabilized nucleoside link is a PS nucleoside link or a phosphodiester nucleoside link. The stabilized nucleoside links are independently selected from the group consisting of: dithiophosphate (PS2) nucleoside links, phosphorylguanidine (PN) nucleoside links, methanesulfonylaminophosphate (MsPA) nucleoside links, and O-isopropylthiophosphate (OiPS) nucleoside links.

[0008] In some implementations, each p and x is a PS nucleotide linker.

[0009] In some implementation schemes, x g x h and x i Each is a PS nucleotide linker. In some implementations, x g For PS nucleoside linkage, and x h and x i Each is linked between PN nucleosides. In some implementations, x g x h and x i Each is linked between PN nucleosides. In some implementations, x g and x h Each is linked between PN nucleosides, and x i For PO nucleoside linkage. In some embodiments, x g and x i Each is linked between PN nucleosides, and x h For PO nucleoside linkage. In some embodiments, x h and x i Each is linked between PN nucleosides, and xg For PO nucleoside linkage. In some embodiments, x h and x i This is an internucleotide linking mechanism between PN nucleotides. In some implementations, x g x h and x i At least two of them are linked by PN nucleosides.

[0010] In some implementation schemes, x d x e and x f Each is a PS nucleotide linker. In some implementations, x d and x e This is an internucleotide linking mechanism between PN nucleotides. In some implementations, x d and x e It is a PN nucleoside linkage, and x f For PS nucleoside linkage. In some implementations, x d For PS nucleoside linkage, and x e and x f Each is a PS nucleotide linker. In some implementations, x d It is a PN nucleoside linkage, and x e and x f For PS nucleoside linkage. In some implementations, x d x e and x f Each is linked between PN nucleosides. In some implementations, x d and x e Each is linked between PN nucleosides, and x f For PO nucleoside linkage. In some embodiments, x d and x f Each is linked between PN nucleosides, and x e For PO nucleoside linkage. In some embodiments, x e and x f Each is linked between PN nucleosides, and x d It is a PO nucleoside linkage.

[0011] In some implementation schemes, x d x e x f x g x h and x i Each is a PS nucleotide linker. In some implementations, x d x e and x f Each is a PS nucleotide linker, x g For PS nucleoside linkage, and xh and x i Each is linked between PN nucleosides. In some implementations, x d x e and x f Each is a PS nucleotide linker, and x g x h and x i Each is linked between PN nucleosides. In some implementations, x d and x e For PN nucleoside linkage, x f For PS nucleoside linkage, and x g x h and x i Each is a PS nucleotide linker. In some implementations, x d and x e For PN nucleoside linkage, x f For PS nucleoside linkage, x g For PS nucleoside linkage, and x h and x i Each is linked between PN nucleosides. In some implementations, x d For PS nucleoside linkage, x e and x f Each is a PN nucleoside linker, x g For PS nucleoside linkage, and x h and x i Each is linked between PN nucleosides. In some implementations, x d It is a PN nucleoside linkage, and x e and x f Each is a PS nucleotide linker, x g For PS nucleoside linkage, and x h and x i Each is linked between PN nucleosides. In some implementations, x d x e x f x g x h and x i Each is linked between PN nucleosides. In some implementations, x d x e and x f Each is a PN nucleoside linker, x g and x h Each is linked between PN nucleosides, and x i For PO nucleoside linkage. In some embodiments, x d x e and x f Each is a PN nucleoside linker, xg and x i Each is linked between PN nucleosides, and x h For PO nucleoside linkage. In some embodiments, x d x e and x f Each is a PN nucleoside linker, x h and x i Each is linked between PN nucleosides, and x g For PO nucleoside linkage. In some embodiments, x d and x e Each is linked between PN nucleosides, and x f For PO nucleoside linkage, and x g x h and x i Each is linked between PN nucleosides. In some implementations, x d and x f Each is linked between PN nucleosides, and x e For PO nucleoside linkage, and x g x h and x i Each is linked between PN nucleosides. In some implementations, x e and x f Each is linked between PN nucleosides, and x d For PO nucleoside linkage, and x g x h and x i Each is linked between PN nucleosides. In some implementations, x h and x i or x g x h and x i It is a PN nucleoside linkage, and x d and x e or x d x e and x f It is a bond between PN nucleosides.

[0012] In some implementations, MAPT ASO includes SEQ ID NO:111, and: (a)p a p b and p c (b) p a To stabilize the internucleotide bond, and p b and p c For PS nucleoside linkage; (c)p b To stabilize the internucleotide bond, and p a and pc For PS nucleoside linkage; (d)p a and p b To stabilize the internucleotide bond, and p c For PS nucleoside linkage; (e)p b and p c To stabilize the internucleotide bond, and p a For PS nucleoside linkage; (f)p a p b and p c To stabilize internucleotide bonds; or (g)p a and p b It is a PS2 nucleoside linkage.

[0013] In some implementations, MAPT ASO includes SEQ ID NO:111, and: (a)p a For PS2 key binding, and p b and p c (b) p b For PS2 key binding, and p a and p c For PS nucleoside linkage; (c)p b It is a PN bond, and p a and p c For PS nucleoside linkage; (d)p a and p b It is a PN nucleoside linkage, and p c For PS nucleoside linkage; (e)p a and p b For PS2 nucleoside linkage, and p c For PS nucleoside linkage; (f)p a and p b For MsPA nucleoside linkage, and p c PS nucleoside linkage; (g)p a and p b For OiPS nucleoside linkage, and p c For PS nucleoside linkage; (h)p b and p c It is a PN nucleoside linkage, and p a For PS nucleoside linkage; (i)p a p b and p c For PN nucleoside linkage; or (j)p a p b and p c This is a nucleoside linker of MsPA.

[0014] In some implementations, MAPT ASO contains SEQ ID NO:111, and p a and p b For PS2 nucleoside linkage. In some embodiments, MAPT ASO comprises SEQ ID NO:111, and: (a)x h and x i or x g x h and x i For PN nucleoside linkage, x d and x e or x d x e and x f For PN nucleoside linkage, and p a and p b (b)x d x e x h and x i It is a PN nucleoside linkage, and p a and p b PS2 nucleoside linkage; (c)x d x e x f x g x h and x i It is a PN nucleoside linkage, and p a and p b PS2 nucleoside linkage; (d)x d x e x h and x i For PN nucleoside linkage, p a and p b PS2 nucleoside linkages, and all other nucleoside linkages are PS linkages; or (e)x d x e x f x g x h and x i For PN nucleoside linkage, p a and p b PS2 is the internucleotide linker, and all other internucleotide links are PS links.

[0015] In some implementations, MAPT ASO contains SEQ ID NO:111, and p b and p c For PN nucleoside linkage. In some embodiments, MAPT ASO comprises SEQ ID NO:111, and: (a)xh and x i or x g x h and x i For PN nucleoside linkage, x d and x e or x d x e and x f For PN nucleoside linkage, and p b and p c (b)x h and x i It is a PN nucleoside linkage, and p b and p c For PN nucleoside linkage; or (c)x h and x i For PN nucleoside linkage, p b and p c The PN nucleoside linkage is a PN nucleoside linkage, and all other nucleoside linkages are PS linkages.

[0016] In some implementations, MAPT ASO contains SEQ ID NO:111, and p a and p b For MsPA nucleoside linkage, or p a p b and p c For MsPA nucleoside linkage. In some embodiments, MAPT ASO comprises SEQ ID NO:111, and: (a)x h and x i or x g x h and x i For PN nucleoside linkage, x d and x e or x d x e and x f For PN nucleoside linkage, and p a and p b or p a p b and p c (b)x g x h and x i It is a PN nucleoside linkage, and p a p b and p c For MsPA nucleoside linkage; (c)x d x e x h and x iIt is a PN nucleoside linkage, and p a and p b For MsPA nucleoside linkage; (d)x g x h and x i For PN nucleoside linkage, p a p b and p c For MsPA nucleoside linkages, all other nucleoside linkages are PS linkages; or (e)x d x e x h and x i For PN nucleoside linkage, p a and p b The nucleoside linkages are MsPA, and all other nucleoside linkages are PS linkages.

[0017] In some implementations, MAPT ASO contains SEQ ID NO:111, and p a and p b For OiPS nucleoside linkage, or p a p b and p c For OiPS nucleoside linkage. In some embodiments, MAPT ASO comprises SEQ ID NO:111, and: (a)x h and x i or x g x h and x i For PN nucleoside linkage, x d and x e or x d x e and x f It is a PN nucleoside linkage, and p a and p b or p a p b and p c (b)x d x e x f x g x h and x i It is a PN nucleoside linkage, and p a and p b For OiPS nucleoside linkage; or (c)x d x e x f x g x h and x iFor PN nucleoside linkage, p a and p b The OiPS nucleoside linkage is a nucleotide-to-nucleotide linkage, and all other nucleoside linkages are PS linkages.

[0018] In some implementations, the MAPT ASO contains SEQ ID NO:111, and the modified MAPT ASO contains: (a) 5′ A L * m C L * m C L *T*T*A*A*G*T*A*T*T*A* m C*T*T L *G L * m C L 3′; (b) 5′ A L * m C L * m C L *T*T*A$A$G*T*A*T*T*A*C*T*T* L G L * m C L 3′; (c) 5′ A L * m C L * m C L *T*T*AnAnGnT*A*T*T*A*C*T*T L *G L * m C L 3′; (d) 5′ A L * m C L * m C L *T*T*A*AnGnT*A*T*T*A*C*T*T L nG L n m C L 3′; (e) 5′ A L * m C L * m C L *T*T*AnAnGnT*A*T*T*A*C*T*T L nG L nm C L 3′; (f) 5′ A L * m C L * m C L *T*T*AuAuGuT*A*T*T*A*C*TnT L nG L n m C L 3′; (g) 5′ A L n m C L * m C L *T*T*AnAnG*T*A*T*T*A*C*T*T L nG L n m C L 3′; (h) 5′ A L * m C L n m C L nT*T*AnAnG*T*A*T*T*A*C*T*T L nG L n m C L 3′; (i) 5′ A L n m C L n m C L *T*T*AnAnG*T*A*T*T*A*C*T*T L *G L * m C L 3′; (j) 5′ A L n m C L n m C L *T*T*A*AnGnT*A*T*T*A*C*T*T L *G L * m C L 3′; (k) 5′ A L n m C L n m C L*T*T*A*AnG*T*A*T*T*A*C*T*T L nG L n m C L 3′; (l) 5′ A L n m C L n m C L *T*T*AuAuG*T*A*T*T*A*C*T*T L nG L n m C L 3′; (m) 5′ A L n m C L n m C L *T*T*AtAtG*T*A*T*T*A*C*T*T L nG L n m C L 3′; (n) 5′ A L n m C L n m C L *T*T*A*AnGnT*A*T*T*A*C*T*T L nG L n m C L 3′; (o) 5′ A L n m C L n m C L nT*T*A*A*G*T*A*T*T*A*C*TnT L nG L n m C L 3′; (p) 5′ A L n m C L n m C L nT*T*AtA*G*T*A*T*T*A*C*TnT L nG L n m C L 3′; (q) 5′ A L nm C L n m C L nT*T*A*AtG*T*A*T*T*A*C*TnT L nG L n m C L 3′; (r) 5′ A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L 3′; (s) 5′ A L n m C L n m C L nT*T*AoAoG*T*A*T*T*A*C*TnT L nG L n m C L 3′; (t) 5′ A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TnT L nG L P m C L 3′; (u) 5′ A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TnT L PG L n m C L 3′; (v) 5′ A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TPT L nG L n m CL 3′; (w) 5′ A L n m C L n m CPT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L 3′; (x) 5′ A L n m C L P m CnT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L 3′; or (y) 5′ A L P m C L n m CnT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L 3′; Where A L , m C L T L and G L The four nucleotides are adenine-locked nucleic acid, 5-methylcytosine-locked nucleic acid, thymine-locked nucleic acid, and guanine-locked nucleic acid, respectively; A, C, T, and G are deoxyadenosine, deoxycytidine, deoxythymidine, and deoxyguanosine, respectively; s represents the PS nucleoside linkage; n represents the PN nucleoside linkage; t represents the PS2 nucleoside linkage; u represents the MsPA nucleoside linkage; and o represents the OiPS nucleoside linkage. The chemical structure of MAPT ASO is shown in [the diagram / image ... Figure 10 , Figure 11 , Figure 12 , Figure 13 and Figure 14 In the middle. MAPT ASO can also be used as... Figure 10 , Figure 11 , Figure 12 , Figure 13 and Figure 14 Salts of any structure shown in either of the above.

[0019] In some implementations, MAPT ASO includes SEQ ID NO:112, and: (a)p k p l p nand p o To stabilize internucleotide bonds; (b)p k p l p n and p o To stabilize the internucleotide bond, and p j and p m For PS nucleoside linkage; (c)p j p l and p o To stabilize internucleotide bonds; (d)p j p l and p o To stabilize the internucleotide bond, and p k p m and p n For PS nucleoside linkage; (e)p l p m p n and p o To stabilize internucleotide bonds; or (d)p l p m p n and p o To stabilize the internucleotide bond, and p j and p k It is a PS nucleoside linkage.

[0020] In some implementations, MAPT ASO contains SEQ ID NO:112, and (a)p k p l p n and p o (b) p k p l p n and p o It is a PN nucleoside linkage, and p j and p m For PS nucleoside linkage; (c)p j It is a PN bond, and p l and p o PS2 nucleoside linkage; (d)p j For PN bonding, p l and p o For PS2 nucleoside linkage, and p k p m and p n For PS nucleoside linkage; (e)p l and p m For PS2 nucleoside linkage, and p n and p oFor PN nucleoside linkage; or (d)p l and p m For PS2 nucleoside inter-linking, p n and p o It is a PN nucleoside linkage, and p j and p k It is a PS nucleoside linkage.

[0021] In some implementations, the MAPT ASO comprises SEQ ID NO:112, and the modified MAPT ASO comprises: (a) m C L nT L nG L *T*TnAnG*A*C*A*T*T*CnAnT*T* m C L *T L * m C L ; (b) m C L *T L *G L *T*TnAnG*A*C*A*T*T*CnAnT*T* m C L nT L n m C L ; (c) m C L nT L nG L nTnT*AtG*A*C*A*T*T*C*AtT*Tn m C L nT L n m C L ;or (d) m C L nT L nG L *T*T*AtGtA*C*A*T*T*CnAnT*T* m C L nT L n m C L ; in m C L T L and G LThey are 5-methylcytosine-locked nucleoside, thymidine-locked nucleoside, and guanosine-locked nucleoside, respectively; A, C, T, and G are deoxyadenosine, deoxycytidine, deoxythymidine, and deoxyguanosine, respectively; * represents PS nucleoside linkage; n represents PN nucleoside linkage; and t represents PS2 nucleoside linkage.

[0022] In some embodiments, the oligonucleotide contains a 5' terminal group, wherein the 5' terminal group may be, but is not limited to, a phosphodiester (PO) group, a thiophosphate (PS) group, a dithiophosphate (PS2) group, a methanesulfonyl-aminophosphate (MsPA) group, a cyclic phosphorylguanidine (PN) group, or... O - Isopropyl phosphate thioester (OiPS) group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' terminal group selected from the group consisting of PO, PS, and PN. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PO 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PN 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS2 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a MsPA 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains an OiPS 5' terminal group.

[0023] MAPT ASOs conjugated to Fc polypeptide dimers targeting the transferrin receptor (TfR) are described. Any of the described MAPT ASOs may be conjugated to Fc polypeptide dimers targeting TfR. In some embodiments, the TfR-targeting Fc polypeptide dimer comprises a first Fc polypeptide and a second Fc polypeptide, wherein the second Fc polypeptide comprises a modified constant domain that specifically binds to human transferrin receptor 1 (TfR), and wherein the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide. In some embodiments, the TfR-targeting Fc polypeptide dimer comprises a first Fc polypeptide lacking a modified constant domain that specifically binds to human transferrin receptor 1 (TfR), and a second Fc polypeptide comprising a modified constant domain that specifically binds to TfR, wherein the first Fc polypeptide and the second Fc polypeptide form an Fc dimer. In some embodiments, the first Fc polypeptide, the second Fc polypeptide, or both the first Fc polypeptide and the second Fc polypeptide are modified to reduce effector function. In some embodiments, the first and second Fc peptides are modified to reduce effector function. In some embodiments, the first Fc peptide comprises a cysteine ​​residue at position 239, an A residue at position 234, an A residue at position 235, and a serine residue at position 329, respectively, according to EU numbering. In some embodiments, the second Fc peptide comprises an A residue at position 234, an A residue at position 235, and a serine residue at position 329, respectively, and comprises a sequence having at least 90% sequence identity with SEQ ID NO:12.

[0024] In some embodiments, the TfR-targeting Fc polypeptide dimer further comprises a non-targeted Fab (NTF) fused to a first Fc polypeptide via a hinge region to form a first Fab-Fc fusion polypeptide. In some embodiments, the TfR-targeting Fc polypeptide dimer further comprises an NTF fused to a second Fc polypeptide via a hinge region to form a second Fab-Fc fusion polypeptide. In some embodiments, the TfR-targeting Fc polypeptide dimer comprises a non-targeted Fab (NTF) fused to a first Fc polypeptide via a hinge region to form a first Fab-Fc fusion polypeptide, and a non-targeted Fab (NTF) fused to a second Fc polypeptide via a hinge region to form a second Fab-Fc fusion polypeptide, wherein the first Fab-Fc fusion polypeptide and the second Fab-Fc fusion polypeptide form a Fab-Fc dimer. In some embodiments, the first NTF and the second NTF each comprise a heavy chain segment containing SEQ ID NO: 109 or 110 and a light chain containing SEQ ID NO: 108. In some embodiments, the first NTF and the second NTF each comprise a heavy chain segment containing SEQ ID NO: 130 or 131 and a light chain containing SEQ ID NO: 129. In some embodiments, the hinge regions each comprise SEQ ID NO: 121.

[0025] In some embodiments, the TfR-targeting Fc peptide dimer further includes a non-binding variable region (NBVR) fused with a first Fc peptide via a hinge region to form an NVBR-Fc fusion peptide. In some embodiments, the TfR-targeting Fc peptide dimer further includes an NVBR fused with a second Fc peptide via a hinge region to form an NBVR-Fc fusion peptide. In some embodiments, each NVBR includes a variable region of SEQ ID NO: 109 or 110. In some embodiments, each hinge region includes SEQ ID NO: 121.

[0026] MAPT ASO is linked to a TfR-targeting Fc polypeptide dimer via a linker. The linker can be any linker available in the art suitable for linking oligonucleotides to polypeptides. MAPT ASO can be linked to a first Fc polypeptide, a second Fc polypeptide, a first NTF (if present), and / or a second NTF (if present). In some embodiments, MAPT ASO is covalently linked to the first Fc polypeptide, the second Fc polypeptide, the first NTF (if present), and / or the second NTF (if present). In some embodiments, MAPT ASO is linked to the first Fc polypeptide at a cysteine ​​residue at position 239. In some embodiments, the first Fc polypeptide contains a CH1 domain, and MAPT ASO is linked to the first Fc polypeptide at a cysteine ​​residue at position 114 (according to Kabat numbering) or position 124 (according to EU numbering) (i.e., the CH1 domain contains an A114C substitution or an S124C substitution). In some embodiments, MAPT ASO is linked to the first Fc polypeptide via the 5' end of MAPT ASO. In some embodiments, the first Fc polypeptide is linked to MAPT ASO via a linker attached to a cysteine ​​residue at position 239 and the 5' end (or the 5' terminal group of MAPT ASO), wherein the linker comprises: , Among them, the "intersecting with bonds in the chemical structure" "" indicates the point where a wavy bond intersects with the rest of the molecule.

[0027] In some embodiments, the modified constant domains of the second Fc polypeptide include: glutamic acid (E), leucine (L), serine (S), valine (V), tryptophan (W), or tyrosine (Y) at position 153; Y, phenylalanine (F), W, methionine (M), proline (P), or V at position 157; threonine (T), asparagine (N), or V at position 159; E, isoleucine (I), P, or V at position 160; W at position 161; and alanine (A) at position 162. I, V, serine (S) or T; N, S, arginine (R) or T at position 163; T, histidine (H) or S at position 186; E, S, aspartic acid (D), glycine (G), T, P, glutamine (Q) or R at position 188; E or R at position 189; Q at position 191; Q at position 192; F, H, lysine (K), Y or W at position 194; S, T or W at position 197; and S, C, P, M or W at position 199, and refer to SEQ ID NO:1.

[0028] In some embodiments, the modified constant domains of the second Fc polypeptide include: E, L, S, V, W, or Y at position 380 according to the EU number; Y, F, W, M, P, or V at position 384; T, N, or V at position 386; E, I, P, or V at position 387; W at position 388; A, I, V, S, or T at position 389; N, S, R, or T at position 390; T, H, or S at position 413; E, S, D, G, T, P, Q, or R at position 415; E or R at position 416; Q at position 418; Q at position 419; F, H, K, Y, or W at position 421; S, T, or W at position 424; and S, C, P, M, or W at position 426.

[0029] In some embodiments, the modified constant domains of the second Fc polypeptide include: E at position 153; Y at position 157; T at position 159; E at position 160; W at position 161; A at position 162; N at position 163; T at position 186; E at position 188; E at position 189; Q at position 191; Q at position 192; F at position 194; S at position 197; and S at position 199, and refer to SEQ ID NO:1.

[0030] In some embodiments, the modified constant domain of the second Fc polypeptide further includes E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, A at position 389, N at position 390, T at position 413, E at position 415, E at position 416, and F at position 421, according to EU numbering.

[0031] In some embodiments, the modified constant domains of the second Fc polypeptide include: E at position 380 according to EU number; Y at position 384; T at position 386; E at position 387; W at position 388; A at position 389; N at position 390; T at position 413; E at position 415; E at position 416; Q at position 418; Q at position 419; F at position 421; S at position 424; and S at position 426.

[0032] In some embodiments, the second Fc polypeptide further comprises W at position 366 according to EU designation, and the first Fc polypeptide further comprises serine at position 366, A at position 368, and valine at position 407 according to EU designation.

[0033] In some embodiments, the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO:63 or 80. In some embodiments, the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:12, and the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO:63 or 80.

[0034] In some implementations, the first NTF and the second NTF each include a heavy chain variable region containing SEQ ID NO:109 or 110 and a light chain variable region containing SEQ ID NO:108.

[0035] In some embodiments, the first Fab-Fc fusion polypeptide comprises SEQ ID NO: 100 or 101, and the second Fab-Fc fusion polypeptide comprises SEQ ID NO: 98 or 99.

[0036] Some implementations provide pharmaceutical compositions comprising a MAPT ASO conjugate as described herein and a pharmaceutically acceptable carrier or diluent.

[0037] Some embodiments provide a method for generating neuronal cells with reduced Tau expression, the method comprising delivering a MAPT ASO conjugate as described herein to the neuronal cells, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene. The neuronal cells may be, but are not limited to, brain cells, deep brain cells, or spinal cord cells.

[0038] Some embodiments provide a method for modifying neuronal cells to reduce Tau expression, the method comprising delivering a MAPT ASO conjugate as described herein to the neuronal cells, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene. The neuronal cells may be, but are not limited to, brain cells, deep brain cells, or spinal cord cells.

[0039] Some implementations provide a method for delivering MAPT ASO to the CNS or CNS cells of a human subject in need, the method comprising administering a MAPT ASO conjugate as described herein to the subject.

[0040] Delivery of MAPT ASO to neurons using the described MAPT ASO conjugate may be used to treat neurodegenerative diseases. Delivery of the described MAPT ASO to neurons may also be used to treat tau-related neurodegenerative diseases. Neurodegenerative diseases may include, but are not limited to, Alzheimer's disease.

[0041] Some implementations provide a method for reducing the expression or level of MAPT messenger ribonucleic acid (mRNA) in human subjects in need, the method comprising administering a MAPT ASO conjugate as described herein to the human subject. Attached Figure Description

[0042] Figure 1 The plasma pharmacokinetic curves of the modified MAPT ASO OTV are shown.

[0043] Figure 2 The brain ASO concentrations of a single dose of modified MAPT ASO OTV are shown.

[0044] Figure 3 The brain ASO concentrations of modified MAPT ASO OTV at multiple doses are shown.

[0045] Figure 4 The target MAPT was knocked down after four doses of modified MAPT ASO OTV.

[0046] Figure 5 The target MAPT was knocked down after 8 doses of modified MAPT ASO OTV.

[0047] Figure 6 The brain ASO concentrations of unmodified MAPT ASO OTV are shown.

[0048] Figure 7 The target MAPT knockdown is shown in the unmodified MAPT ASO OTV.

[0049] Figure 8 This demonstrates the effectiveness of MAPT ASO.

[0050] Figure 9 This demonstrates ASO stability in brain tissue.

[0051] Figure 10 The diagram shows the chemical structure of one embodiment of MAPT ASO.

[0052] Figure 11 The diagram shows the chemical structure of one embodiment of MAPT ASO.

[0053] Figure 12 The diagram shows the chemical structure of one embodiment of MAPT ASO.

[0054] Figure 13 The diagram shows the chemical structure of one embodiment of MAPT ASO.

[0055] Figure 14 The diagram shows the chemical structure of one embodiment of MAPT ASO. Detailed Implementation

[0056] I. Definition Before detailing the teachings herein, it should be understood that this disclosure is not limited to specific compositions or process steps, as these are subject to change. It should be noted that, as used in this specification and the appended claims, the singular forms “an,” “a,” and “described” include plural references unless the context clearly specifies otherwise. Thus, for example, a reference to “an oligonucleotide” includes multiple oligonucleotides, etc. The conjunction “or” should be interpreted inclusively, i.e., equivalent to “and / or,” unless such inclusiveness is unreasonable in the context.

[0057] Generally, the term "about" indicates that the quantity of the components in the composition has not changed significantly and will not have any significant effect on the activity or stability of the composition. When a specification discloses a specific value for a parameter, the specification should be understood to additionally disclose the parameter in the format of the value "about". When the terms "about" and "approximately" are used to modify a quantity specified in a numerical value or range, it indicates that the numerical value, as well as reasonable deviations from values ​​known to those skilled in the art (e.g., measurement standard error margin (SEM)) (e.g., ±20%, ±10%, or ±5%), are within the expected meaning of the value.

[0058] A composition or method that “contains” or “includes” one or more of the aforementioned elements may include other elements not specifically listed. For example, a composition that “contains” or “includes” an Fc polypeptide dimer may contain the Fc polypeptide dimer alone or in combination with other components.

[0059] The specification of a range of values ​​includes all integers within the range or defining the range, and all subranges defined by the integers within the range.

[0060] The use of the terms “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” is not intended to be limiting and may include other elements not specifically stated. For example, a composition that “comprises” or “includes” an Fc polypeptide dimer may contain the Fc polypeptide dimer alone or in combination with other ingredients. It should be understood that the foregoing general description and details are exemplary and illustrative only and are not intended to limit the teaching. If any material incorporated by reference is inconsistent with the express content of this disclosure, the express content shall prevail.

[0061] The specification of a range of values ​​includes all integers within the range or defining the range, and all subranges defined by the integers within the range.

[0062] As used herein, the term "antibody" refers to a protein having an immunoglobulin fold that specifically binds to an antigen via its variable region. The term encompasses complete polyclonal antibodies, complete monoclonal antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. As used herein, the term "antibody" also includes antibody fragments that retain antigen-binding specificity, including but not limited to Fab, F(ab')2, Fv, scFv, and bivalent scFv. Antibodies may contain light chains classified as κ (kappa) or λ (lambda). Antibodies may contain heavy chains classified as γ (gamma), μ (mu), α (alpha), δ (delta), or ε (epsilon), thereby defining the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0063] When referring to residues in the constant region of the antibody heavy chain, the “EU numbering scheme” is commonly used in the art. For SEQ ID NO:5 (clone CH3C.35.23.2), the EU numbering scheme is shown below: .

[0064] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The term "variable light chain" (V...) L ) and "variable heavy chain" (V HThese refer to the light chains and heavy chains, respectively.

[0065] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is derived from germline variable (V) genes, diversity (D) genes, or linker (J) genes (rather than from constant (Cμ and Cδ) gene segments), and that confers the specificity of the antibody's binding to its antigen. Typically, an antibody variable region contains four conserved "framework" regions, interspersed with three hypervariable "complementarity-determining regions."

[0066] The term "complementarity-determining region" or "CDR" refers to three hypervariable regions in each chain, which are interrupted by four framework regions established by the variable regions of the light and heavy chains. CDRs are primarily responsible for antibody-antigen epitope binding. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (numbered sequentially starting from the N-terminus) and are also identified by the chain in which the specific CDR is located. Therefore, V H CDR3 or CDR-H3 is located in the heavy chain variable region of the antibody in which it is present, while V L CDR1 or CDR-L1 is CDR1 derived from the light chain variable region of the antibody in which it is present.

[0067] The "frame regions" or "FRs" of different light or heavy chains are relatively conserved within a species. The frame regions of antibodies (i.e., the combined frame regions that make up the light and heavy chains) are used to locate and align CDRs in three-dimensional space. Frame sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBASE2" germline variable gene sequence database of human and mouse sequences.

[0068] The amino acid sequences of the CDR and frame region can be determined using various well-known definitions in the art, such as Kabat, Chothia, the International ImMunoGeneTics Database (IMGT), AbM, and observed antigen contact (“Contact”). In some embodiments, the CDR is determined according to the definition of Contact. See MacCallum et al. , J. Mol. Biol. 262:732-745, 1996. In some implementations, the CDR is determined based on a combination of the definitions of Kaba, Chothia, and / or Contact CDR.

[0069] The term "Fd moiety" refers to the N-terminal portion of the immunoglobulin heavy chain. Typically, the Fd moiety includes a variable (VH) region and a constant (CH1) region.

[0070] The term "Fab" refers to an antigen-binding fragment consisting of a light chain variable region, a light chain constant region, a heavy chain variable region, and a heavy chain CH1 constant region.

[0071] The term "single-chain variable fragment" or "scFv" refers to an antigen-binding fragment composed of heavy chain variable regions and light chain variable regions linked together via a polypeptide linker. The linker can connect V... H N-terminus and V L The C end can be connected, or the V end can be connected. L N-terminus and V H The C-terminal connection is missing in scFv.

[0072] The term "epitope" refers to an antigenic region that specifically binds to a molecule (e.g., the CDR of an antibody) and may comprise a portion of several amino acids, such as 5 or 6 or more, or 20 or more amino acids, or portions of those amino acids. In some cases, epitopes include non-protein components (e.g., from carbohydrates, nucleic acids, or lipids). In some cases, epitopes are three-dimensional. Thus, for example, when the target is a protein, an epitope may consist of continuous amino acids (e.g., a linear epitope) or amino acids from different portions of a protein that are close together due to protein folding (e.g., a discontinuous or conformational epitope).

[0073] As used herein, when referring to antibodies, the phrase “recognition epitope” means that the antibody CDR interacts with or specifically binds to an antigen on the epitope or a portion of an antigen containing the epitope.

[0074] The term "specific binding" refers to a molecule (e.g., a Fab or scFv) binding to an epitope or target in a sample with greater affinity, stronger binding force, and / or a longer duration of binding than it binds to another epitope or non-target compound (e.g., a structurally different antigen). In some embodiments, a Fab or scFv that specifically binds to an epitope or target is a Fab or scFv whose binding to the epitope or target is at least 5 times (e.g., at least 6, 7, 8, 9, 10, 25, 50, 100, 1000, 10,000, or higher affinity) for the epitope or target compared to its binding to other epitopes or non-target compounds. As used herein, the terms "specific binding," "specifically binding," or "specific to" a particular epitope or target can be, for example, defined by having an equilibrium dissociation constant K with which the epitope or target binds. D The molecular manifestations, for example, 10 -4 M or smaller, for example, 10 - 5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9M, 10 -10 M, 10 -11 M or 10 -12 M. Technicians will recognize that Fab or scFv that binds specifically to a target from a species can also bind specifically to an ortholog of the target.

[0075] The term "binding affinity" is used herein to refer to the strength of a non-covalent interaction between two molecules (e.g., between Fab or scFv and an antigen). Therefore, for example, the term may refer to a 1:1 interaction between Fab or scFv and an antigen, unless otherwise stated or the context clearly indicates otherwise. Binding affinity can be measured by measuring the equilibrium dissociation constant (K0). D ) and quantify, the equilibrium dissociation constant (K D ) refers to the dissociation rate constant (k d ,time -1 Divide by the association rate constant (k) a ,time -1 M -1 K D This can be determined by measuring the kinetics of complex formation and dissociation, for example, using surface plasmon resonance (SPR) methods, such as the Biacore™ system; or by kinetic exclusion assays, such as KinExA. ® ; and BioLayer interferometry (e.g., using ForteBio ® (Octet platform). As used in this article, "binding affinity" includes not only formal binding affinity, such as those reflecting a 1:1 interaction between Fab or scFv and the antigen, but also calculated K. D This may reflect the apparent affinity of the affinity binding.

[0076] Monoclonal antibodies and their fragments (including Fabs and conjugates) or other biological entities are typically provided in isolated form. Antibodies (or their fragments or conjugates) generally contain at least 50% w / w purified interfering protein and other contaminants generated during their production or purification, but the possibility of the antibody or Fab being combined with excess pharmaceutically acceptable carriers or other mediators intended to facilitate their use is not excluded. Antibodies (or their fragments or conjugates) may be at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% w / w purified from interfering protein and contaminants generated during production or purification. Isolated antibodies (or their fragments or conjugates) may contain the remaining major macromolecular species after purification.

[0077] As used herein, the term "Fc region" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by an Ig fold as a domain. Fc polypeptides typically contain a constant region sequence including at least a CH2 domain and / or a CH3 domain, and may contain at least a partial hinge region. Two Fc polypeptides dimerize to form an Fc region or Fc fragment.

[0078] The terms "polypeptide" and "peptide" refer to polymers of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as both naturally occurring and non-naturally occurring amino acid polymers. "Protein" can refer to a polypeptide, polypeptide dimer, or polypeptide multimer. Single-chain polypeptides in protein dimers or multimers can be linked by covalent bonds (e.g., disulfide bonds) or non-covalent interactions.

[0079] For the purpose of classifying amino acid substitutions as conserved or non-conserved, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues affecting chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions involve exchanging members of one class for members of another class.

[0080] In the context of two or more polypeptide sequences, the term "identity" or "identity percentage" refers to two or more identical or identical amino acid residues in a sequence or subsequence, for example, when compared and aligned within a comparison window or designated region to achieve maximum correspondence, as measured by a sequence comparison algorithm or by manual alignment and visual inspection, indicating at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or higher identity in a designated region. For polypeptide sequence comparison, typically an amino acid sequence is used as a reference sequence against which candidate sequences are compared. Alignment can be performed using various methods available to those skilled in the art, such as visual alignment or using publicly available software utilizing known algorithms to achieve maximum alignment. Such programs include BLAST, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). Those skilled in the art can determine the alignment parameters used to achieve maximum alignment. For sequence comparison of polypeptide sequences for the purposes of this application, the standard protein BLAST algorithm with default parameters, BLASTP, is used to compare two protein sequences.

[0081] The percentage of sequence identity is determined using antibody or Fc peptide sequences that have been aligned to the maximum extent possible using the Kabat numbering convention. After alignment, if the subject antibody region (e.g., the entire mature variable region of the heavy chain or light chain or Fc peptide) is compared with the same region of the reference antibody, the percentage of sequence identity between the subject antibody region and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acids in both regions by the total number of aligned positions in both regions (excluding gaps), and multiplying by 100 to convert it to a percentage.

[0082] Nucleic acid sequence identity can be determined by using algorithms (which use default gap parameters) (e.g., BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.) or by examining and aligning sequences with the best alignment (i.e., the one that results in the highest percentage of sequence similarity within the comparison window). The percentage of sequence identity is calculated by comparing two best-aligned sequences within the comparison window, determining the number of positions where the same residues appear in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of matching and non-matching positions (excluding gaps in the comparison window) (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise stated, the comparison window between two sequences is defined by the entire length of the shorter of the two sequences.

[0083] Modified internucleotide linkages are those other than naturally occurring phosphate ester linkages. The figure below shows a modified internucleotide linkage connecting the 5' and 3' glycosyl groups of the modified internucleotide chain (included in the structure).

[0084]

[0085] As used in this article, PN refers to structure (b).

[0086] As used herein, "stabilized internucleotide links" refers to modified internucleotide links specifically introduced into the oligonucleotide backbone of an oligonucleotide polypeptide conjugate to remove soft spots (e.g., nuclease-sensitive sites), increase the breakage resistance of the oligonucleotide, or otherwise modulate one or more pharmacokinetic or pharmacodynamic properties of the introduced oligonucleotide and / or oligonucleotide polypeptide conjugate. Stabilized internucleotide links do not include phosphate thioesters or phosphate diesters. Oligonucleotides and / or oligonucleotide polypeptide conjugates containing one or more stabilized internucleotide links (stabilized oligonucleotides) have improved properties compared to oligonucleotides or oligonucleotide polypeptide conjugates having the same oligonucleotide sequence but without one or more stabilized internucleotide links. Stabilized oligonucleotides and oligonucleotide polypeptide conjugates have improved properties compared to those containing only phosphate thioester internucleotide links or combinations of phosphate diester and phosphate thioester internucleotide links. In addition to stabilizing internucleotide links, stabilized oligonucleotides may also contain phosphate-thioester internucleotide links and / or phosphodiester internucleotide links. In some embodiments, one or more phosphate-thioester and / or phosphodiester internucleotide links in the oligonucleotide (e.g., ASO; e.g., gapmer) are replaced by stabilizing internucleotide links. In some embodiments, one or more phosphate-thioester and / or phosphodiester internucleotide links may be replaced by one or more stabilizing internucleotide links located at or near sites in the oligonucleotide that are identified as vulnerable to breakage (e.g., soft spots). In some embodiments, one or more phosphate-thioester and / or phosphodiester internucleotide links in the wing segment of the gapmer are replaced by stabilizing internucleotide links. In some embodiments, one or more phosphate-thioester and / or phosphodiester internucleotide links in the gap segment of the gapmer are replaced by stabilizing internucleotide links. Examples of modified nucleoside linkages that can be used to stabilize inter-nucleoside linkages include, but are not limited to, dithiophosphates, aminophosphates, methanesulfonylaminophosphates, phosphonates, triphosphates, and guanidine phosphates (see: Nucleic Acids Res., 47, 5465–5479 (2019); Proc. Natl. Acad. Sci. USA, 116, 1229–1234 (2019); Nucleic Acids Research, 50(10), 5401–5423 (2022); Vasquez G., Nucleic Acid Therapeutics, 32(1), 40–50 (2022); and Molecular Therapy: Nucleic Acids; 29: 176–188 (2022)).Other examples of modified nucleoside links that can be used to stabilize inter-nucleoside links include, but are not limited to, modified nucleoside links s1, s2, s3, s4, s5, s6, s7, s8, s8, s10, s11, s12, s13, s14, 15, s16, s17 or s18 as described in WO 2017210647, the disclosure of which is incorporated herein by reference in its entirety. Other examples of modified nucleoside links that can be used to stabilize inter-nucleoside links include, but are not limited to, modified nucleoside links n001, n002, n003, n004, n005, n006, n007, n008, n009, n010, n020, n025 or n026 as described in WO 2022099159. In some implementations, the stabilized nucleoside-to-nucleotide linkages are selected from the group consisting of PS2, MsPA, OiPS, and PN nucleoside-to-nucleotide linkages.

[0087] "LNA" refers to a bicyclic nucleoside analog (2' to 4' bicyclic nucleotide analog) containing a bridge between the 2' and 4' positions of the ribose ring, and is referred to as "locked nucleic acid" or "locked nucleoside". As used herein, "LNA oligonucleotide" refers to an oligonucleotide containing one or more of these bicyclic nucleoside analogs. Biochemistry , 43(42):13233-13240 (2004). In some embodiments, the LNA provided herein has the following structure, wherein a “base” is a nucleobase: .

[0088] "Expression" refers to the transcription and / or translation of endogenous genes, heterologous genes, or nucleic acid segments or transgenes in a cell. For example, expression can refer to the transcription and stable accumulation of sense RNA (mRNA) or functional RNA. Expression can also refer to the production of proteins.

[0089] The term "pharmaceuticalally acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other components of the formulation and is harmless to the recipient.

[0090] The terms “subject,” “individual,” and “patient” refer to mammals, including but not limited to humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cattle, pigs, horses, and other mammal species. In some embodiments, the subject is a human. The term “subject” includes human and other mammalian subjects receiving preventative or therapeutic treatment.

[0091] The term "disease" or "disorder" refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any symptom, disease, abnormality, pathology, ailment, disorder, or syndrome that impairs physiological function, regardless of its etiological nature.

[0092] The terms “treat” and “treatment” refer to methods or procedures taken to alleviate or reduce the number, severity, and / or frequency of one or more symptoms of a subject’s disease or ailment. Treatment generally refers to achieving the desired pharmacological and / or physiological effect. The effect may be preventative, but not necessarily preventative, in relation to the prevention or partial prevention of a disease, symptom, or ailment. The effect may be therapeutic in relation to the partial or complete cure of a disease, ailment, symptom, or adverse reaction caused by said disease, ailment, or ailment. The term treatment may include: (a) preventing a subject who may be susceptible to a disease but has not yet been diagnosed with said disease from developing the disease; (b) suppressing the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., reducing or improving the disease and / or its symptoms or ailment. Treatment may refer to therapeutic treatment only, preventative treatment only, or both therapeutic and preventative treatment. Those who require treatment (subjects in need) may include those who already have a disease or ailment or those who have a disease or ailment to prevent. Treatment may include suppressing a disease, symptom, or disorder, for example, by halting its progression; and alleviating a disease, symptom, or disorder, for example, by causing its remission. Treating a disease, symptom, or disorder may include improving at least one symptom of a particular disease, symptom, or disorder, even if the underlying pathophysiology is not affected, for example, treating the symptom without affecting or eliminating the underlying cause of the symptom. Treatment or improvement of symptoms may be based on objective or subjective parameters. Treatment effectiveness may be compared with untreated individuals or a pool of individuals, or with the same patient at different times before or during treatment.

[0093] "Pharmacologically effective amount," "therapeuticly effective amount," or simply "effective amount" refers to the amount (dosage) of the described active pharmaceutical ingredient or pharmaceutical composition that produces the intended pharmacological, therapeutic, or preventative results. "Effective amount" can also refer to the amount of, for example, an excipient in a pharmaceutical composition, sufficient to achieve the desired properties of the composition. Effective amounts can be administered once or multiple times, applied, or dosed.

[0094] II. Overview Oligonucleotide therapy for CNS disorders caused by gene abnormalities or increased protein accumulation is becoming an increasingly popular approach to modulating gene expression in these neurological conditions. The blood-brain barrier (BBB) ​​poses a challenge to the delivery of systemically administered oligonucleotides to their relevant sites of action within the CNS. Intrathecal (IT) delivery (where drugs are administered directly into the cerebrospinal fluid (CSF) space) can bypass the BBB. However, a limitation of this approach is that direct delivery of these oligonucleotide therapies to the CSF via the IT method cannot achieve uniform distribution throughout the CNS.

[0095] A MAPT ASO conjugate is described that can be administered intravenously to subjects and promotes the transport of MAPT ASO across the BBB and the delivery of MAPT ASO to CNS cells, wherein the MAPT ASO provides knockdown of MAPT expression in the brain and spinal cord. The MAPT ASO conjugate comprises an Fc polypeptide dimer targeting transferrin linked to MAPT ASO. The conjugation of MAPT ASO to the Fc polypeptide dimer can be used to increase the targeting efficacy of the molecule after administration to subjects. The Fc polypeptide dimer-cargo molecule conjugate exhibits increased targeting efficacy compared to the unconjugated molecule. When MAPT ASO is linked to the Fc polypeptide dimer targeting transferrin, the MAPT ASO contains modifications that reduce plasma clearance and / or degradation / cracking in vivo.

[0096] In some embodiments, the described MAPT ASO conjugate provides knockdown of MAPT expression throughout the CNS region (including the entire brain region, including deep brain regions as well as the frontal, parietal, temporal, occipital, and cerebellum). In some embodiments, the described MAPT ASO conjugate provides knockdown of MAPT expression in multiple CNS cell types (including endothelial cells, neurons, stellate cells, oligodendrocytes, and microglia). In some embodiments, the described MAPT ASO conjugate provides knockdown of MAPT expression in the spinal cord.

[0097] In some implementations, the described MAPT ASO conjugates provide knockdown of MAPT expression in skeletal muscle and cardiac muscle. Knockdown of MAPT expression in skeletal muscle, cardiac muscle, and diaphragm muscle can be used to treat neuromuscular disorders.

[0098] Upon systemic administration, the described MAPT ASO conjugates provide targeted delivery of therapeutic levels of MAPT ASO to various tissues, including the CNS. This targeted delivery allows for the use of lower doses of MAPT ASO compared to administration of MAPT ASO alone (i.e., non-targeted delivery).

[0099] The text describes a MAPT ASO conjugate comprising an Fc polypeptide dimer targeting the transferrin receptor (TfR) and a MAPT antisense oligonucleotide (ASO), wherein: (a) The Fc polypeptide dimer targeting TfR comprises: (i) A first Fc polypeptide, comprising cysteine ​​at position 239, A at position 234, A at position 235 and serine at position 329, respectively, according to EU numbering; (ii) A second Fc polypeptide comprising serine residues at positions 234, 235, and 329 according to EU designation, and a modified constant domain specifically binding to human transferrin receptor 1 (TfR), wherein the second Fc polypeptide comprises a sequence having at least 90% sequence identity with SEQ ID NO: 11 or 12, wherein the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide; and (iii) A first untargeted Fab (NTF) fused with the first Fc peptide via a first hinge region to form a first Fab-Fc fusion peptide, and a second NTF fused with the second Fc peptide via a second hinge region to form a second Fab-Fc fusion peptide, wherein the first NTF and the second NTF each comprise SEQ ID NO: 109 or 110, wherein each hinge region comprises SEQ ID NO: 121; and (b) The MAPT ASO includes 5' A L x d m C L x e m C L x f TTAp a Ap b Gp c TATTACTx g T L x h G L x i m C L 3′ (SEQ ID NO:111) in A L , m C L G L and T L These are adenine-locked nucleoside, 5-methylcytosine-locked nucleoside, guanosine-locked nucleoside, and thymine-locked nucleoside. A, C, G, and T represent deoxyadenosine nucleoside, deoxycytidine nucleoside, deoxyguanosine nucleoside, and deoxythymidine nucleoside, respectively. Each p is independently linked to a phosphate thioester (PS) nucleoside, a phosphate dithioester (PS2) nucleoside, or a methanesulfonyl aminophosphate (MsPA) nucleoside. O - Isopropyl phosphate thioester (OiPS) nucleoside linkage or phosphoryl guanidine (PN) nucleoside linkage Each x is independently a PN nucleoside linker, a PS nucleoside linker, or a phosphodiester (PO) nucleoside linker, and Any nucleoside link that is not a PO, PS2, MsPA, OiPS or PN nucleoside link is a PS nucleoside link; The first Fc polypeptide is linked to MAPT ASO via a linker.

[0100] In some implementation schemes, p a p b and p c At least one of them is not a PS nucleotide linker. In some implementations, p a p b and p c At least two of them are not PS nucleotides linked together.

[0101] In some implementation schemes, x g x h and x i At least two of them are linked between PN nucleosides.

[0102] In some embodiments, the modified constant domains of the second Fc polypeptide include: E at position 380 according to EU number; Y at position 384; T at position 386; E at position 387; W at position 388; A at position 389; N at position 390; T at position 413; E at position 415; E at position 416; Q at position 418; Q at position 419; F at position 421; S at position 424; and S at position 426.

[0103] In some embodiments, MAPT ASO is linked to a cysteine ​​residue at position 239 (EU designation) of the first Fc polypeptide. In some embodiments, the 5' end of MAPT ASO is linked to a cysteine ​​residue at position 239 of the first Fc polypeptide, wherein the linker is... , Among them, the "intersecting with bonds in the chemical structure" "" indicates the point where a wavy bond intersects with the rest of the molecule.

[0104] In some implementations, the NTF includes SEQ ID NO:109 or 110 and SEQ ID NO:108.

[0105] The text describes a MAPT ASO conjugate comprising an Fc polypeptide dimer targeting the transferrin receptor (TfR) and a MAPT antisense oligonucleotide (ASO), wherein: (a) The Fc polypeptide dimer targeting TfR comprises: (i) A first Fc polypeptide, comprising cysteine ​​at position 239, A at position 234, A at position 235 and serine at position 329, respectively, according to EU numbering; (ii) A second Fc polypeptide comprising serine residues at position 234, position 235, and position 329 respectively according to EU number, and a modified constant domain specifically binding to human transferrin receptor 1 (TfR), wherein the second Fc polypeptide comprises a sequence having at least 90% sequence identity with SEQ ID NO: 11 or 12, wherein the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide; (iii) A first untargeted Fab (NTF) fused with the first Fc peptide via a first hinge region to form a first Fab-Fc fusion peptide, and a second NTF fused with the second Fc peptide via a second hinge region to form a second Fab-Fc fusion peptide, wherein the first NTF and the second NTF each comprise SEQ ID NO: 109 or 110, wherein each hinge region comprises SEQ ID NO: 121; and (b) The MAPT ASO includes 5' C L x d T L x e G L x f Tp j Tp k Ap l Gp m ACATTCp n Ap o TTx g C L x h T L x i C L 3′ (SEQ ID NO:112) in m C L G L and T L These are 5-methylcytosine, guanosine, and thymine nucleoside, respectively. A, C, G, and T represent deoxyadenosine nucleoside, deoxycytidine nucleoside, deoxyguanosine nucleoside, and deoxythymidine nucleoside, respectively. Each p is independently linked to a phosphate thioester (PS) nucleoside, a phosphate dithioester (PS2) nucleoside, or a methanesulfonyl aminophosphate (MsPA) nucleoside.O - Isopropyl thiophosphate (OiPS) nucleoside linkage or phosphoryl guanidine (PN) nucleoside linkage, and p a p b and p c At least two of them are not PS nucleotide linkages. Each x is independently a PN nucleoside linker or a PS nucleoside linker, and x g x h and x i At least two of them are linked by PN nucleoside bonds, and Any nucleoside link that is not a PS2, MsPA, OiPS or PN nucleoside link is a PS nucleoside link; The first Fc polypeptide is linked to MAPT ASO via a linker.

[0106] In some embodiments, the modified constant domain of the second Fc polypeptide includes: E at position 380 (EU number); Y at position 384; T at position 386; E at position 387; W at position 388; A at position 389; N at position 390; T at position 413; E at position 415; E at position 416; Q at position 418; Q at position 419; F at position 421; S at position 424; and S at position 426. In some embodiments, MAPT ASO is linked to the cysteine ​​residue at position 239 (EU number) of the first Fc polypeptide. In some embodiments, the 5' end of MAPT ASO is linked to the cysteine ​​residue at position 239 of the first Fc polypeptide, wherein the linker is: , Among them, the "intersecting with bonds in the chemical structure" "" indicates the point where a wavy bond intersects with the rest of the molecule.

[0107] In some implementations, the NTF includes SEQ ID NO:109 or 110 and SEQ ID NO:108.

[0108] A. Fc polypeptide dimer targeting transferrin receptor (TfR) In some embodiments, the first Fc peptide or the first Fab-Fc fusion peptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with a human Fc peptide. In some embodiments, the first Fc peptide comprises a cysteine ​​residue at positions 239, 442, 330, and 289 according to EU designations (i.e., the Fc domain comprises an S239C substitution, S442C substitution, A330C substitution, K149C substitution, or T289C substitution). In some embodiments, the first Fc peptide comprises a cysteine ​​residue at position 239 according to EU designations. In some embodiments, the first Fc peptide further comprises a CH1 domain (i.e., the Fab-Fc fusion peptide), wherein the CH1 domain comprises a cysteine ​​residue at position 114 (according to Kabat designation) or position 124 (according to EU designation) (i.e., the first Fc peptide comprises an A114C substitution or an S124C substitution). In some embodiments, the first Fc polypeptide comprises a LALA-PS mutation (each of the A residues at position 234, position 235, and position 329 according to EU number). In some embodiments, the first Fc polypeptide comprises a cysteine ​​residue at position 239 according to EU number, a LALA-PS mutation, and a club or mortar mutation. In some embodiments, the first FC polypeptide comprises a LALA-PS mutation and a club or mortar mutation, and further comprises a CH1 domain, wherein the CH1 domain comprises a cysteine ​​residue at position 114 (according to Kabat number) or position 124 (according to EU number). In some embodiments, the first FC polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 47-48, 51-54, 61-63, 66, 73-77, and 80. In some embodiments, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 63 or 80. In some embodiments, the first Fc polypeptide comprises a cysteine ​​residue at position 239, an amino acid residue at position 234, an amino acid residue at position 235, and a serine residue at position 329 (each according to an EU designation), and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 63 or 80. In some embodiments, the first Fc polypeptide comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 63 or 80.

[0109] In some embodiments, the modified constant domains of the second Fc polypeptide include: E at position 380 according to EU number; Y at position 384; T at position 386; E at position 387; W at position 388; A at position 389; N at position 390; T at position 413; E at position 415; E at position 416; Q at position 418; Q at position 419; F at position 421; S at position 424; and S at position 426. In some embodiments, the second Fc polypeptide or Fab-Fc fusion polypeptide includes, as per SEQ ID NO:5 number, E at position 153, Y at position 157, T at position 159, E at position 160, W at position 161, A at position 162, N at position 163, T at position 186, E at position 188, E at position 189, and F at position 194. In some embodiments, the modified constant domains of the second Fc polypeptide include: E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; A at position 389; N at position 390; T at position 413; E at position 415; E at position 416; Q at position 418; Q at position 419; F at position 421; S at position 424; and S at position 426 (according to EU number), and have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4, 5, 11, or 12. In some embodiments, the second Fc polypeptide comprises E at position 153, Y at position 157, T at position 159, E at position 160, W at position 161, A at position 162, N at position 163, T at position 186, E at position 188, E at position 189, and F at position 194, as per SEQ ID NO: 5, and has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4, 5, 11, or 12. In some embodiments, the second Fc polypeptide comprises LALA-PS mutations (A at position 234, A at position 235, and serine at position 329, respectively, according to EU numbers). In some embodiments, the second Fc polypeptide comprises both LALA-PS mutations and pestle or mortar mutations.In some embodiments, the modified constant domains of the second Fc polypeptide include: E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; A at position 389; N at position 390; T at position 413; E at position 415; E at position 416; Q at position 418; Q at position 419; F at position 421; S at position 424; and S at position 426 (according to EU number), and have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 4-29, 36, and 43-44. In some embodiments, the second Fc polypeptide comprises E at position 153, Y at position 157, T at position 159, E at position 160, W at position 161, A at position 162, N at position 163, T at position 186, E at position 188, E at position 189, and F at position 194, as per reference to SEQ ID NO: 4-29, 36, and 43-44, and has 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 4-29, 36, and 43-44.

[0110] In some embodiments, the first FC polypeptide and / or the second FC polypeptide contains one or more mutations or groups of mutations selected from the group consisting of: pestle mutations (e.g., T139W as referred to in SEQ ID NO:5), mortis mutations (e.g., T139S, L141A, and Y180V as referred to in SEQ ID NO:5), one or more mutations regulating effector function (e.g., L7A, L8A, and / or P102G or P102S as referred to in SEQ ID NO:5; L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and / or one or more mutations increasing serum stability (e.g., (i) M25Y, S27T, and T29E as referred to in SEQ ID NO:5, or (ii) N207S as referred to in SEQ ID NO:5 and with or without M201L). If the first Fc polypeptide contains a pestle mutation, the second Fc polypeptide contains a mortis mutation. If the second Fc polypeptide contains a club-shaped mutation, then the first Fc polypeptide contains a mortar-shaped mutation.

[0111] In some embodiments, both the first Fc peptide and the second Fc peptide contain one or more mutations that reduce effector function. In some embodiments, both the first Fc peptide and the second Fc peptide contain an LALA mutation. In some embodiments, both the first Fc peptide and the second Fc peptide contain an LALA-PG mutation. In some embodiments, both the first Fc peptide and the second Fc peptide contain an LALA-PD mutation.

[0112] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a club-shaped mutation (e.g., T139W, as per SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a club-shaped mutation and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:5 or 6. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:5 or 6.

[0113] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a club-shaped mutation (e.g., as shown in SEQ ID NO:5, T139W) and one or more mutations regulating effector functions (e.g., as shown in SEQ ID NO:5, L7A, L8A, and / or P102G or P102S (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, the second Fc polypeptide contains a club-shaped mutation and one or more mutations regulating effector functions, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:7, 9, 11, 8, 10, or 12. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:7, 9, or 11. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:8, 10, or 12.

[0114] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a club-shaped mutation (e.g., T139W as per SEQ ID NO:5) and one or more mutations that increase serum stability (e.g., M25Y, S27T, and T29E as per SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a club-shaped mutation and one or more mutations that increase serum stability, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:13. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:13.

[0115] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a club-shaped mutation (e.g., T139W as per SEQ ID NO:5) and one or more mutations that increase serum stability (e.g., N207S and with or without M201L as per SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a club-shaped mutation and one or more mutations that increase serum stability, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:14. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:14.

[0116] In some embodiments, the first or second Fc polypeptide contains a club-shaped mutation (e.g., T139W as per SEQ ID NO:5), one or more mutations regulating effector functions (e.g., L7A, L8A, and / or P102G or P102S as per SEQ ID NO:5; e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and one or more mutations increasing serum stability (e.g., M25Y, S27T, and T29E as per SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a club-shaped mutation, one or more mutations regulating effector functions, one or more mutations increasing serum stability, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:15 or 16. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:15 or 16.

[0117] In some embodiments, the first or second Fc polypeptide contains a club-shaped mutation (e.g., T139W as per SEQ ID NO:5), one or more mutations regulating effector function (e.g., L7A, L8A, and / or P102G or P102S as per SEQ ID NO:5; e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), or one or more mutations increasing serum stability (e.g., N207S as per SEQ ID NO:5 and with or without M201L). In some embodiments, the second Fc polypeptide contains a club-shaped mutation, one or more mutations regulating effector function, one or more mutations increasing serum stability, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:17 or 18. In some embodiments, the second Fc polypeptide contains the sequence of SEQ ID NO:17 or 18.

[0118] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a mortar-like mutation (e.g., as shown by reference to T139S, L141A, and Y180V of SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a mortar-like mutation and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:19. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:19.

[0119] In some embodiments, the first or second Fc polypeptide contains a mortise mutation (e.g., as referred to in SEQ ID NO:5, T139S, L141A, and Y180V) and one or more mutations regulating effector functions (e.g., as referred to in SEQ ID NO:5, L7A, L8A, and / or P102G or P102S (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, the second Fc polypeptide may contain a mortise mutation and one or more mutations regulating effector functions, and have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:20 or 21. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:20 or 21.

[0120] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a mortar-related mutation (e.g., T139S, L141A, and Y180V as per SEQ ID NO:5) and one or more mutations that increase serum stability (e.g., M25Y, S27T, and T29E as per SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a mortar-related mutation and one or more mutations that increase serum stability, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:22. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:22.

[0121] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a mortar-like mutation (e.g., T139S, L141A, and Y180V as per SEQ ID NO:5) and one or more mutations that increase serum stability (e.g., N207S and with or without M201L as per SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a mortar-like mutation and one or more mutations that increase serum stability, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:23. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:23.

[0122] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a mortise mutation (e.g., as referred to in SEQ ID NO:5, T139S, L141A, and Y180V), one or more mutations that regulate effector function (e.g., as referred to in SEQ ID NO:5, L7A, L8A, and / or P102G or P102S (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and one or more mutations that increase serum stability (e.g., as referred to in SEQ ID NO:5, M25Y, S27T, and T29E). In some embodiments, the second Fc polypeptide contains a mortarsal mutation, one or more mutations regulating effector function, and one or more mutations increasing serum stability, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:24 or 25. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:24 or 25.

[0123] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a mortise mutation (e.g., as referred to in SEQ ID NO:5, T139S, L141A, and Y180V), one or more mutations that regulate effector function (e.g., as referred to in SEQ ID NO:5, L7A, L8A, and / or P102G or P102S (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and one or more mutations that increase serum stability (e.g., as referred to in SEQ ID NO:5, N207S, and with or without M201L). In some embodiments, the second Fc polypeptide contains a mortise mutation, a mutation regulating one or more effector functions, and one or more mutations increasing serum stability, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:26 or 27. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:26 or 27.

[0124] In some embodiments, the N-terminus of the second Fc polypeptide includes a hinge sequence or a portion thereof (e.g., SEQ ID NO: 49 or 50 of the second Fc polypeptide). In some embodiments, the N-terminus of the first Fc polypeptide is further conjugated to a CH1 region (e.g., SEQ ID NO: 86, 87 or 94) or an NTF heavy chain sequence.

[0125] In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO:4, 5, 6, 7, 9, 11, 50, 86, and 87, wherein the polypeptide comprises, for example, E at position 153, Y at position 157, T at position 159, E at position 160, W at position 161, A at position 162, N at position 163, T at position 186, E at position 188, E at position 189, and F at position 194 as per SEQ ID NO:5; and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises a sequence of any of SEQ ID NO:54, 63, 66, and 90. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises, as shown in SEQ ID NO:5, Q at position 192; Q at position 193; S at position 197; and S at position 199. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:7, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:54. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:9, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:66. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:11, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:63.

[0126] In some embodiments, the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide includes all or a portion of a hinge region (e.g., TCPPCP (SEQ ID NO: 121), DKTHTCP (SEQ ID NO: 91), or DKTHTCPPCP (SEQ ID NO: 92)). Therefore, in some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 50, and the first Fc polypeptide comprises the sequence of SEQ ID NO: 90.

[0127] In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 6, 8, 10, 12, 43, 44, 49, and 94, wherein the polypeptide comprises E at position 153, Y at position 157, T at position 159, E at position 160, W at position 161, A at position 162, N at position 163, T at position 186, E at position 188, E at position 189, and F at position 194, as per reference to SEQ ID NO: 5; and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises a sequence of any of SEQ ID NO: 48, 77, 78, 80, and 101. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises, as shown in reference SEQ ID NO:5, Q at position 192; Q at position 193; S at position 197; and S at position 199. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:8, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:77. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:10, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:78. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:12, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:80. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:49, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:48. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:94, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:101. In some embodiments, the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide includes a portion of a hinge region (e.g., DKTHTCP (SEQ ID NO:91) or DKTHTCPPCP (SEQ ID NO:92)).

[0128] In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 29, 36, and 44, wherein the polypeptide comprises E at position 153, Y at position 157, T at position 159, E at position 160, W at position 161, A at position 162, N at position 163, T at position 186, E at position 188, E at position 189, and F at position 194 (as per SEQ ID NO: 5) and Ser at position 239 (according to EU); and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises a sequence of any of SEQ ID NO: 47, 48, 51-54, 61-63, 66, 73-78, 80, and 90. In some embodiments, the second Fc peptide or the second Fab-Fc fusion peptide comprises, as shown in reference SEQ ID NO:5, Q at position 192; Q at position 193; S at position 197; and S at position 199. In some embodiments, the second peptide or the second Fab-Fc fusion peptide comprises the sequence of SEQ ID NO:29, and the first peptide or the first Fab-Fc fusion peptide comprises the sequence of SEQ ID NO:48, 53, 54, 74, or 77. In some embodiments, the second peptide or the second Fab-Fc fusion peptide comprises the sequence of SEQ ID NO:36, and the first peptide or the first Fab-Fc fusion peptide comprises the sequence of SEQ ID NO:61, 66, 75, or 78. In some embodiments, the second peptide or the second Fab-Fc fusion peptide comprises the sequence of SEQ ID NO:44, and the first peptide or the first Fab-Fc fusion peptide comprises the sequence of SEQ ID NO:74 or 77.

[0129] In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:44 or 49, wherein the polypeptide comprises, for example, E at position 153, Y at position 157, T at position 159, E at position 160, W at position 161, A at position 162, N at position 163, T at position 186, E at position 188, E at position 189, and F at position 194, as per reference to SEQ ID NO:5; and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of any one of SEQ ID NO:48, 73-75, and 77. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:44, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:77.

[0130] In some embodiments, the second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO:4, 5, 7, 9, 50, 86, and 87, wherein the polypeptide comprises, for example, E at position 153, Y at position 157, T at position 159, E at position 160, W at position 161, A at position 162, N at position 163, T at position 186, E at position 188, E at position 189, and F at position 194 as per SEQ ID NO:5; and the first Fab-Fc fusion polypeptide comprises a sequence of any of SEQ ID NO:52, 53, and 61.

[0131] In some embodiments, the second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 6, 8, 10, 43, 49, and 94, wherein the polypeptide comprises, for example, E at position 153, Y at position 157, T at position 159, E at position 160, W at position 161, A at position 162, N at position 163, T at position 186, E at position 188, E at position 189, and F at position 194 as per SEQ ID NO: 5; and the first Fab-Fc fusion polypeptide comprises a sequence of any of SEQ ID NO: 73-75.

[0132] In some embodiments, the second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 6, 8, 10, 43, 49, and 94, wherein the polypeptide comprises, for example, E at position 153, Y at position 157, T at position 159, E at position 160, W at position 161, A at position 162, N at position 163, T at position 186, E at position 188, E at position 189, and F at position 194 as per SEQ ID NO: 5; and the first Fab-Fc fusion polypeptide comprises a sequence of any of SEQ ID NO: 73-75.

[0133] In some embodiments, the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12. In some embodiments, the first Fc polypeptide comprises A at position 234, A at position 235, serine at position 329, E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, A at position 389, N at position 390, T at position 413, E at position 415, E at position 416, and F at position 421, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11 or 12. In some embodiments, the first Fc polypeptide comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 11 or 12.

[0134] B. Non-targeted Fab Untargeted Fab (NTF) comprises a light chain and a heavy chain, wherein the light chain contains V L The heavy chain contains V and a light chain constant region (CL). H The NTF binds to the CH1 constant region of the heavy chain. In some embodiments, the NTF does not bind specifically to naturally occurring epitopes in the subject. In some embodiments, the NTF does not bind specifically to antigens expressed in a given mammal, mammalian tissue, or mammalian cell type. The antigen may be a mammalian antigen or an antigen found in mammals, such as antigens from infectious organisms such as viruses, bacteria, fungi, or parasites. Mammals may be, but are not limited to, non-human primates, humans, or rodents (e.g., mice).

[0135] The specific binding of an antibody to an antigen refers to an affinity of at least 10. 6 M -1Specific binding is detectable at higher levels and can be distinguished from nonspecific binding occurring on at least one unrelated target. Nonspecific binding is typically a result of van der Waals forces. Nonspecific binding does not mean that the NTF does not bind to any antigen with any affinity. Rather, in some embodiments, the NTF does not exhibit specific binding to: (a) any protein or epitope in mammalian cells, mammalian tissues, or mammals; (b) any surface-accessible protein or epitope on mammalian cells or mammalian tissues; or (c) any serum-accessible protein or epitope in mammalian tissues or mammals.

[0136] The NTF comprises three light chain CDRs and three heavy chain CDRs. In some embodiments, the heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H1) comprise SEQ ID NO: 105, 106 or 128 and 107, respectively, and the light chain CDRs (CDR-L1, CDR-L2, and CDR-L1) comprise SEQ ID NO: 102, 103, and 104, respectively. In some embodiments, the heavy chain CDRs (CDR-L1, CDR-L2, and CDR-L1) comprise SEQ ID NO: 125, 126 or 128 and 127, respectively, and the light chain CDRs (CDR-L1, CDR-L2, and CDR-L1) comprise SEQ ID NO: 122, 123, and 124, respectively.

[0137] In some embodiments, the NTF comprises a heavy chain containing an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or 100% identity with the amino acid sequence of SEQ ID NO: 109 or 110; and a light chain containing an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or 100% identity with the amino acid sequence of SEQ ID NO: 108.

[0138] In some embodiments, the NTF comprises a heavy chain containing an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or 100% identity with the amino acid sequence of SEQ ID NO: 130 or 131; and a light chain containing an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or 100% identity with the amino acid sequence of SEQ ID NO: 129.

[0139] In some embodiments, the NTF comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises SEQ ID NO: 109 or 110, and the light chain sequence comprises SEQ ID NO: 108. In some embodiments, the NTF comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises SEQ ID NO: 109, and the light chain sequence comprises SEQ ID NO: 108. In some embodiments, the NTF comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises SEQ ID NO: 110, and the light chain sequence comprises SEQ ID NO: 108.

[0140] In some embodiments, the NTF comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises SEQ ID NO: 130 or 131, and the light chain sequence comprises SEQ ID NO: 129. In some embodiments, the NTF comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises SEQ ID NO: 130, and the light chain sequence comprises SEQ ID NO: 129. In some embodiments, the NTF comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises SEQ ID NO: 131, and the light chain sequence comprises SEQ ID NO: 129.

[0141] In some embodiments, the NTF comprises a light chain containing at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence identical to that of SEQ ID NO:108, and a heavy chain containing at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence identical to that of SEQ ID NO:109 or 110, and contains CDR sequences of SEQ ID NO:102 (CDR-L1), SEQ ID NO:103 (CDR-L2), SEQ ID NO:104 (CDR-L3), SEQ ID NO:105 (CDR-H1), SEQ ID NO:106 (CDR-H2), and SEQ ID NO:107 (CDR-H3) and maintains the non-targeting nature of the NTF containing the light chain of SEQ ID NO:108 and the heavy chain of SEQ ID NO:109 or 110.

[0142] In some embodiments, the NTF comprises a light chain containing at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence identical to that of SEQ ID NO:129, and a heavy chain containing at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence identical to that of SEQ ID NO:130 or 131, and contains CDR sequences of SEQ ID NO:122 (CDR-L1), SEQ ID NO:123 (CDR-L2), SEQ ID NO:124 (CDR-L3), SEQ ID NO:125 (CDR-H1), SEQ ID NO:126 (CDR-H2), and SEQ ID NO:127 (CDR-H3) and maintains the non-targeting nature of the NTF containing the light chain of SEQ ID NO:129 and the heavy chain of SEQ ID NO:130 or 131.

[0143] The NTF light and / or heavy chains may contain one or more modifications that facilitate conjugation to one or more cargo molecules. These modifications may be amino acid substitutions or insertions. The amino acid substitutions may be, but are not limited to, lysine at position 149 (EU number) of the light chain being replaced with cysteine, serine at position 156 (EU number) of the light chain being replaced with cysteine, A at position 118 (EU number (according to position 114 of the Kabat number)) of the heavy chain being replaced with cysteine, or serine at position 124 (EU number) of the heavy chain being replaced with cysteine, or combinations thereof.

[0144] In some embodiments, the first Fab-Fc fusion polypeptide comprises a cysteine ​​residue at position 239, an A residue at position 234, an A residue at position 235, and a serine residue at position 329 (each according to EU designation), and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 100 or 101. In some embodiments, the first Fab-Fc fusion polypeptide comprises a cysteine ​​residue at position 114 (according to Kbat designation) or position 124 (according to EU designation), and an A residue at position 234, an A residue at position 235, and a serine residue at position 329 (according to EU designation). In some embodiments, the second Fab-Fc fusion polypeptide comprises A at position 234, A at position 235, serine at position 329, E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, A at position 389, N at position 390, T at position 413, E at position 415, E at position 416, and F at position 421 according to EU numbers (each according to EU number), and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 98 or 99. In some embodiments, the first Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 100 or 101. In some embodiments, the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 98 or 99. In some embodiments, the first Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 100 or 101, and the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 98 or 99. In some embodiments, the first Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 100, and the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 98. In some embodiments, the first Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 100, and the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 99. In some embodiments, the first Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 101, and the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 98. In some embodiments, the first Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 101, and the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 99.

[0145] C. MAPT ASO In some embodiments, the nucleobase sequence of MAPT ASO comprises the nucleobase sequence of SEQ ID NO:111 (5'ACCTTAAGTATTACTTGC 3'). In some embodiments, the nucleobase sequence of MAPT ASO consists of the nucleobase sequence of SEQ ID NO:111.

[0146] In some embodiments, the nucleobase sequence of MAPT ASO comprises the nucleobase sequence of SEQ ID NO:112 (5'CTGTTAGACATTCATTCTC 3'). In some embodiments, the nucleobase sequence of MAPT ASO consists of the nucleobase sequence of SEQ ID NO:112.

[0147] In some implementations, MAPT ASO is linked to a delivery medium (e.g., an Fc polypeptide dimer targeting TfR) via the 5' end of an oligonucleotide (e.g., the delivery medium is linked to the 5' end of MAPT ASO).

[0148] MAPT ASO comprising SEQ ID NO: 111 or 112 is described, and when MAPT ASO is linked to a peptide delivery medium (e.g., an Fc polypeptide dimer targeting TfR), it has modifications that reduce nonspecific plasma clearance and / or degradation / cracking of MAPT ASO. In some embodiments, MAPT ASO is a nicked polymer. In some embodiments, the MAPT ASO sequence consists of the nucleobase sequence of SEQ ID NO: 111 or 112.

[0149] In some embodiments, the MAPT nick polymer comprises a 5' wing segment having 3, 4, or 5 nucleotides. In some embodiments, the MAPT nick polymer comprises a 3' wing segment having 3, 4, or 5 nucleotides. In some embodiments, the MAPT nick polymer comprises a 5' wing segment having 3, 4, or 5 nucleotides and a 3' wing segment having 3, 4, or 5 nucleotides.

[0150] In some embodiments, the MAPT nick polymer comprises a 5' wing segment having three nucleosides. In some embodiments, the MAPT nick polymer comprises a 3' wing segment having three nucleosides. In some embodiments, the MAPT nick polymer comprises both a 5' wing segment having three nucleosides and a 3' wing segment having three nucleosides.

[0151] In some embodiments, the MAPT nick polymer comprises a nick segment containing 8-16 nucleotides. In some embodiments, the MAPT nick polymer comprises a nick segment containing 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides. In some embodiments, the MAPT nick polymer comprises a nick segment containing 10-14 nucleotides. In some embodiments, the MAPT nick polymer comprises a nick segment containing 12 nucleotides. In some embodiments, the MAPT nick polymer comprises a nick segment containing 13 nucleotides. In some embodiments, the MAPT nick polymer comprises a nick segment containing 14 nucleotides. In some embodiments, each nucleotide in the nick segment comprises 2'-deoxyribose.

[0152] In some implementations, the MAPT notched polymer comprises: X a -Y b -Z c Where a is an integer from 2 to 5, b is an integer from 8 to 16, and c is an integer from 2 to 5, each X nucleotide contains a modified sugar, each Y nucleotide has a 2' deoxyribose, and each Z nucleotide contains a modified sugar. In some embodiments, the MAPT nicked polymer contains a 3-12-3 nicked polymer. In some embodiments, the MAPT nicked polymer contains a 3-13-3 nicked polymer. In some embodiments, a and c are each 3, b is 12, and each X and Z nucleotide is LNA.

[0153] In some embodiments, the winged nucleotides of the MAPT nick polymer comprise at least one nucleotide having a modified sugar (modified ribose). In some embodiments, each nucleotide in the 5' wing segment, 3' wing segment, or both the 5' and 3' wing segments of the MAPT nick polymer comprises a modified sugar. The modified sugar may be, but is not limited to, a 2'-O-methoxyethyl (MOE) sugar moiety or a bicyclic sugar moiety comprising a 2'-4' bridge. The 2'-4' bridge may be, but is not limited to, -O-CH2- (locked nucleic acid or locked nucleoside (LNA)) or -O-CH(CH)- (locked ethyl (cEt)). In some embodiments, each winged nucleotide of the MAPT nick polymer comprises a bicyclic sugar moiety comprising an -O-CH2-2'-4' bridge (LNA).

[0154] i. Nucleoside-to-nucleotide linkage: In some implementations, each nucleoside link in MAPT ASO comprises a modified link (i.e., a link other than naturally occurring phosphate ester links).

[0155] In some embodiments, the MAPT ASO contains at least two stabilizing internucleotide links at the end of the MAPT ASO opposite to the end connected to the delivery medium. If the delivery medium is connected to the 5' end of the MAPT ASO, then at least two internucleotide links at the 3' end of the MAPT ASO contain stabilizing internucleotide links. In some embodiments, the MAPT ASO also contains stabilizing internucleotide links at the 5' end. In some embodiments, the stabilizing internucleotide links have increased stability or nuclease resistance compared to PS internucleotide links. In some embodiments, the stabilizing internucleotide links are PN internucleotide links.

[0156] In some embodiments, MAPT ASO includes at least two PN links at the 3' end (i.e., a PN link between the nucleoside at position n-2 (n-2) and n-1 and a PN link between the nucleoside at position n-1 and n, where n is the 3' terminal nucleoside). In some embodiments, MAPT ASO also includes at least two PN links at the 5' end (i.e., a PN link between the nucleoside at positions 1 and 2 and a PN link between the nucleoside at positions 2 and 3).

[0157] In some embodiments, MAPT ASO includes at least three PN bonds at the 3' end (i.e., a PN bond between the nucleosides at positions n-3 and n-2, a PN bond between the nucleosides at positions n-2 and n-1, and a PN bond between the nucleosides at positions n-1 and n, where n is the 3' terminal nucleoside). In some embodiments, MAPT ASO also includes at least three PN bonds at the 5' end (i.e., a PN bond between the nucleosides at positions 1 and 2, a PN bond between the nucleosides at positions 2 and 3, and a PN bond between the nucleosides at positions 3 and 4).

[0158] In some embodiments, MAPT ASO comprises at least two stable inter-nucleoside links between nucleosides at positions 6-9. In some embodiments, the stable inter-nucleoside links have increased stability or nuclease resistance compared to PS inter-nucleoside links. In some embodiments, the stable inter-nucleoside links are located at and / or near sites identified as soft spots or catabolism sites of MAPT ASO. The stable inter-nucleoside links may be, but are not limited to, PS2 links, MsPA links, OiPS links, or PN links.

[0159] In some embodiments, MAPT ASO contains at least two PS2 internucleotide links between the nucleosides at positions 6-9. In some embodiments, MAPT ASO contains PS2 internucleotide links between the nucleosides at positions 6 and 7 and between the nucleosides at positions 7 and 8.

[0160] In some embodiments, MAPT ASO contains at least two MsPA nucleoside links between the nucleosides at positions 6-9. In some embodiments, MAPT ASO contains MsPA nucleoside links between the nucleosides at positions 6 and 7 and between the nucleosides at positions 7 and 8. In some embodiments, MAPT ASO contains MsPA nucleoside links between the nucleosides at positions 6 and 7, 7 and 8, and 8 and 9.

[0161] In some embodiments, MAPT ASO contains at least two OiPS nucleoside links between the nucleosides at positions 6-9. In some embodiments, MAPT ASO contains OiPS nucleoside links between the nucleosides at positions 6 and 7 and between the nucleosides at positions 7 and 8. In some embodiments, MAPT ASO contains OiPS nucleoside links between the nucleosides at positions 6 and 7, 7 and 8, and 8 and 9.

[0162] In some embodiments, MAPT ASO contains at least two PN nucleoside links between the nucleosides at positions 6-9. In some embodiments, MAPT ASO contains PN nucleoside links between the nucleosides at positions 7 and 8 and between the nucleosides at positions 8 and 9. In some embodiments, MAPT ASO contains PN nucleoside links between the nucleosides at positions 6 and 7, between the nucleosides at positions 7 and 8, and between the nucleosides at positions 8 and 9.

[0163] In some implementations, MAPT ASO includes: X a Y b Z c , in (a) a is an integer from 3 to 5, b is an integer from 8 to 16, and c is an integer from 3 to 5; (b) Each X is a nucleoside containing a modified sugar, each Y is a nucleoside having a 2' deoxyribose, and each Z is a nucleoside containing a modified sugar; (c) Each internucleotide link between X nucleosides contains a PS link or a PN link, and each internucleotide link between Z nucleosides contains a PN link; (d) Nucleoside linkages between X and Y nucleosides and between Y and Z nucleosides independently include PS linkages or PN linkages; (e) Each internucleotide link between Y nucleotides comprises a modified link, wherein Yb comprises two to three adjacent PS2 links, two to three adjacent MsPA links, two to three adjacent OiPS links, or two to three adjacent PN links, and wherein other internucleotide links between Y nucleotides comprise PS links.

[0164] In some embodiments, a and c are each 3. In some embodiments, a and c are each 3 and b is 10-14. In some embodiments, a and c are each 3 and b is 12. In some embodiments, a and c are each 3 and b is 13.

[0165] In some implementations, a and c are each 3 and b is 12-13, and each inter-nucleoside link between X nucleosides includes a PS link.

[0166] In some implementations, a and c are each 3 and b is 12-13, and each inter-nucleoside link between X nucleosides includes a PN link.

[0167] In some embodiments, a and c are each 3 and b is 12-13, each internucleotide link between X nucleosides includes a PS link, the internucleotide link between X and Y nucleosides includes a PS link, and the internucleotide link between Y and Z nucleosides includes a PS link.

[0168] In some embodiments, a and c are each 3 and b is 12-13, each internucleotide link between X nucleosides comprises a PS link, the internucleotide link between X and Y nucleosides comprises a PS link, and the internucleotide link between Y and Z nucleosides comprises a PN link.

[0169] In some embodiments, a and c are each 3 and b is 12-13, each internucleotide link between X nucleosides comprises a PN link, the internucleotide link between X and Y nucleosides comprises a PS link, and the internucleotide link between Y and Z nucleosides comprises a PS link.

[0170] In some embodiments, a and c are each 3 and b is 12-13, and each inter-nucleoside link between X nucleosides includes a PN link, and the inter-nucleoside link between X and Y nucleosides includes a PN link, and the inter-nucleoside link between Y and Z nucleosides includes a PN link.

[0171] Notched polymers with PS2, MsPA, OiPS, or PN bonds in the notch In some embodiments, a and c are each 3 and b is 12-13, and Yb contains two to three adjacent PS2 bonds. In some embodiments, Yb contains two adjacent PS2 bonds. In some embodiments, the two adjacent PS2 bonds are between the 3rd and 4th nucleosides and between the 4th and 5th nucleosides of Yb. In some embodiments, Yb contains three adjacent PS2 bonds.

[0172] In some embodiments, a and c are each 3 and b is 12-13, and Yb contains two to three adjacent MsPA links. In some embodiments, Yb contains two adjacent MsPA links. In some embodiments, the two adjacent MsPA links are between the 3rd and 4th nucleosides and between the 4th and 5th nucleosides of Yb. In some embodiments, Yb contains three adjacent MsPA links. In some embodiments, the three adjacent MsPA links are between the 3rd and 4th nucleosides, between the 4th and 5th nucleosides, and between the 5th and 6th nucleosides of Yb.

[0173] In some embodiments, a and c are each 3 and b is 12-13, and Yb contains two to three adjacent OiPS bonds. In some embodiments, Yb contains two adjacent OiPS bonds. In some embodiments, the two adjacent OiPS bonds are between the 3rd and 4th nucleosides and between the 4th and 5th nucleosides of Yb. In some embodiments, Yb contains three adjacent OiPS bonds. In some embodiments, the three adjacent OiPS bonds are between the 3rd and 4th nucleosides, between the 4th and 5th nucleosides, and between the 5th and 6th nucleosides of Yb.

[0174] In some embodiments, a and c are each 3 and b is 12-13, and Yb contains two to three adjacent PN bonds. In some embodiments, Yb contains two adjacent PN bonds. In some embodiments, the two adjacent PN bonds are located between the 4th and 5th nucleotides and between the 5th and 6th nucleotides of Yb. In some embodiments, Yb contains three adjacent PN bonds.

[0175] In some embodiments, a and c are each 3 and b is 12-13, and Yb contains two to four stable nucleoside links. In some embodiments, Yb contains PN links between the second and third nucleosides of Yb, between the third and fourth nucleosides, between the tenth and eleventh nucleosides, and between the eleventh and twelfth nucleosides. In some embodiments, Yb contains two different stable nucleoside links. In some embodiments, Yb contains a PN link between the first and second nucleosides of Yb and PS2 links between the third and fourth nucleosides and between the eleventh and twelfth nucleosides. In some embodiments, Yb contains two adjacent PS2 links and two adjacent PN links. In some embodiments, two adjacent PS2 bonds are linked between the 3rd and 4th nucleosides and between the 4th and 5th nucleosides of Yb, and two adjacent PN bonds are linked between the 10th and 11th nucleosides and between the 11th and 12th nucleosides of Yb.

[0176] In some implementation schemes, X a or Z c At least one C-nucleotide (if present) contains 5-methylcytosine. In some embodiments, X a or Z c Any C-nucleotide (if present) contains 5-methylcytosine.

[0177] ii. Specific combinations In some embodiments, a and c are each 3 and b is 12-13, each internucleotide link between X nucleosides comprises a PS link, the internucleotide link between X and Y nucleosides comprises a PS link, the internucleotide link between Y and Z nucleosides comprises a PS link, the internucleotide links connecting the 4th and 5th and 5th and 6th nucleosides of Yb comprise a PN link, and X a or Z c Any C-nucleotide (if present) contains 5-methylcytosine.

[0178] In some implementations, MAPT ASO includes: 5′ A L * m C L * m C L *T*T*A*AnGnT*A*T*T*A*C*T*T L nG L n m C L 3′ (SEQ ID NO:111) in: A L It is an adenine-locked nucleic acid; m C L It is a 5-methylcytosine-locked nucleic acid; T L It is a thymine-locked nucleic acid; G L Guanine locks in nucleic acids; A represents deoxyadenosine; C is deoxycytidine; T stands for deoxythymidine; G stands for deoxyguanosine; * indicates a PS nucleotide inter-bonding; and n represents the inter-nucleotide bond between PN (see...) Figure 10 ).

[0179] In some embodiments, a and c are each 3 and b is 12, each internucleotide link between X nucleosides comprises a PN link, the internucleotide link between X and Y nucleosides comprises a PN link, the internucleotide link between Y and Z nucleosides comprises a PN link, the internucleotide links connecting the 3rd and 4th nucleosides and the 4th and 5th nucleosides of Yb comprise PS2 links, and X a or Z c Any C-nucleotide (if present) contains 5-methylcytosine.

[0180] In some implementations, MAPT ASO includes: 5′ A L n m C L n m C L nT*T*As2As2G*T*A*T*T*A*C*TnT L nG L n m C L 3′ (SEQ ID NO:111) in: A L It is an adenine-locked nucleic acid; m C L It is a 5-methylcytosine-locked nucleic acid; T L It is a thymine-locked nucleic acid; G L Guanine locks in nucleic acids; A represents deoxyadenosine; C is deoxycytidine; T stands for deoxythymidine; G stands for deoxyguanosine; * indicates a PS nucleotide inter-linking; s2 is a PS2 nucleotide inter-bond; and n represents the inter-nucleotide bond between PN (see...) Figure 11 ).

[0181] In some embodiments, a and c are each 3 and b is 12, each internucleotide link between X nucleosides comprises a PN link, the internucleotide link between X and Y nucleosides comprises a PS link, the internucleotide link between Y and Z nucleosides comprises a PS link, the internucleotide links connecting the 3rd and 4th nucleosides and the 4th and 5th nucleosides of Yb comprise PS2 links, and X a or Z c Any C-nucleotide (if present) contains 5-methylcytosine.

[0182] In some implementations, MAPT ASO includes: 5′ A L n m C L n m C L *T*T*As2As2G*T*A*T*T*A*C*T*T L nG L n m C L 3′ (SEQ ID NO:111) in: A L It is an adenine-locked nucleic acid; m C L It is a 5-methylcytosine-locked nucleic acid; T L It is a thymine-locked nucleic acid; G L Guanine locks in nucleic acids; A represents deoxyadenosine; C is deoxycytidine; T stands for deoxythymidine; G stands for deoxyguanosine; * indicates a PS nucleotide inter-linking; s2 is a PS2 nucleotide inter-bond; and n represents the inter-nucleotide bond between PN (see...) Figure 12 ).

[0183] In some embodiments, a and c are each 3 and b is 12, each internucleotide link between X nucleosides comprises a PS link, the internucleotide link between X and Y nucleosides comprises a PS link, the internucleotide link between Y and Z nucleosides comprises a PN link, the internucleotide links connecting the 3rd and 4th nucleosides, the 4th and 5th nucleosides, and the 5th and 6th nucleosides of Yb comprise MsPA links, and Xa or Z c Any C-nucleotide (if present) contains 5-methylcytosine.

[0184] In some implementations, MAPT ASO includes: 5′ A L s m C L s m C L *T*T*AuAuGuT*A*T*T*A*C*TnT L nG L n m C L 3′ (SEQ ID NO:111) in: A L It is an adenine-locked nucleic acid; m C L It is a 5-methylcytosine-locked nucleic acid; T L It is a thymine-locked nucleic acid; G L Guanine locks in nucleic acids; A represents deoxyadenosine; C is deoxycytidine; T stands for deoxythymidine; G stands for deoxyguanosine; * indicates a PS nucleotide inter-linking; u is a nucleoside linker to MsPA; and n represents the inter-nucleotide bond between PN (see...) Figure 13 ).

[0185] In some embodiments, a and c are each 3 and b is 12, each internucleotide link between X nucleosides comprises a PN link, the internucleotide link between X and Y nucleosides comprises a PS link, the internucleotide link between Y and Z nucleosides comprises a PS link, the internucleotide links connecting the 3rd and 4th nucleosides and the 4th and 5th nucleosides of Yb comprise MsPA links, and X a or Z c Any C-nucleotide (if present) contains 5-methylcytosine.

[0186] In some implementations, MAPT ASO includes: 5′ A L n m C L n m C L *T*T*AuAuG*T*A*T*T*A*C*T*T L nGL n m C L 3′ (SEQ ID NO:111) in: A L It is an adenine-locked nucleic acid; m C L It is a 5-methylcytosine-locked nucleic acid; T L It is a thymine-locked nucleic acid; G L Guanine locks in nucleic acids; A represents deoxyadenosine; C is deoxycytidine; T stands for deoxythymidine; G stands for deoxyguanosine; * indicates a PS nucleotide inter-linking; u is a nucleoside linker to MsPA; and n represents the inter-nucleotide bond between PN (see...) Figure 14 ).

[0187] D. Connecting base MAPT ASO can be linked to a first Fc peptide, a second Fc peptide, a first NTF, and / or a second NTF. In some embodiments, MAPT ASO is covalently linked to the first Fc peptide, the second Fc peptide, the first NTF, and / or the second NTF. The first Fc peptide, the second Fc peptide, the first NTF, and / or the second NTF may contain amino acid substitutions (e.g., cysteine ​​substitutions) to facilitate the attachment of MAPT ASO.

[0188] In some embodiments, MAPT ASO is linked to the first Fc polypeptide. In some embodiments, MAPT ASO is linked to cysteine ​​residues in the first Fc polypeptide. In some embodiments, the cysteine ​​residues in the first Fc polypeptide include a cysteine ​​substitution at position 239 (according to EU designation). In some embodiments, the first Fc polypeptide includes cysteine ​​residues at positions 239, 442, 330, and 289 according to EU designation. In some embodiments, the cysteine ​​residues in the first Fab-Fc fusion polypeptide include a cysteine ​​substitution at position 114 (according to Kabat designation) or position 124 (according to EU designation).

[0189] MAPT ASO can be linked to a first Fc polypeptide, a second Fc polypeptide, or a first NTF and / or a second NTF via any linker available in the art suitable for linking oligonucleotides (e.g., ASO, such as nicked polymers) to polypeptides (e.g., Fc polypeptides or Fab polypeptides). In some embodiments, the linker comprises any known linker that is a bifunctional adapter capable of covalently linking oligonucleotides to polypeptides.

[0190] Oligonucleotide peptide conjugates can be generated using well-known chemical crosslinking agents and schemes, which covalently link oligonucleotides and peptides via linkers. For example, a wide range of chemical crosslinking agents are known to those skilled in the art for use in crosslinking proteins with agents of interest. For instance, crosslinking agents can be heterobifunctional crosslinking agents, which can be used to link molecules in a stepwise manner. Heterobifunctional crosslinking agents provide the ability to design more specific coupling methods to conjugate proteins, thereby reducing the occurrence of unwanted side reactions (e.g., homologous-protein polymers). A wide variety of heterobifunctional crosslinking agents are known in the art, including but not limited to N-hydroxysuccinimide (NHS) or its water-soluble analogues such as N-hydroxysulfosuccinimide (sulfon-NHS), succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate (SMCC), m-maleimide benzoyl-N-hydroxysuccinimide ester (MBS); N-succinimide-4-iodoacetyl)aminobenzoate (SIAB), succinimide... Crosslinking agents include 4-(p-maleimide phenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), 4-succinimide oxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimide 3-(2-pyridyldithio)propionate (SPDP), and succinimide 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Those crosslinking agents with an N-hydroxysuccinimide moiety can be obtained as N-hydroxysulfosuccinimide analogs, which generally have high water solubility. Furthermore, crosslinking agents with disulfide bridges within the linker chain can be synthesized as alkyl derivatives to reduce the amount of linker breakage in bulk. Besides heterobifunctional crosslinking agents, many other crosslinking agents exist, including homobifunctional crosslinking agents and photoreactive crosslinking agents. Disuccinimidyl octanoate (DSS), bismaleimide hexane (BMH), and dimethyl heptamethinyl ester .2HCl (DMP) are examples of useful homologous bifunctional crosslinking agents, and bis-[B-(4-azidosylsalinomyl)ethyl] disulfide (BASED) and N-succinimidyl-6-(4'-azido-2'-nitrophenyl-amino)hexanoate (SANPAH) are examples of useful photoreactive crosslinking agents.

[0191] Non-limiting examples of splice joints include those described in WO2023 / 279099 and WO2023 / 056388, each of which is incorporated herein by reference in its entirety. In some embodiments, the connector base comprises a val-cit connector as described in US 6,214,345, which is incorporated herein by reference in its entirety. In some embodiments, the connector base comprises those described in WO2020 / 028840 and WO2022 / 212886, each of which is incorporated herein by reference in its entirety. Other connectors may be included in Bioconjugate Chemistry 2023. 34 Those described in (11), 2096-2111.

[0192] Linkers can attach to any region of the polypeptide (e.g., to the N-terminal region, to the C-terminal region, or to an amino acid within the protein (e.g., a cysteine ​​residue or a glutamine residue), as long as the oligonucleotide does not prevent the second Fc polypeptide from binding to the TfR. Similarly, linkers can attach to any region of the oligonucleotide (e.g., the 5' end, the 3' end, or a nucleic acid residue within the molecule), as long as the polypeptide does not interfere with the functionality of the oligonucleotide (e.g., complementary binding to the target nucleic acid). For example, linkers can attach to the oligonucleotide at any number of synthetically feasible sites throughout the oligonucleotide (e.g., at the 3' or 5' terminal residues; at the sugar moiety; at the base moiety; or at residues within the backbone). In some embodiments, the linker attaches to the oligonucleotide at the 5' terminal residue.

[0193] In some embodiments, the linker comprises a spacer. The spacer may be, but is not limited to, a hydrophilic spacer. The hydrophilic spacer may be, but is not limited to, polyethylene glycol (PEG).

[0194] The linker can be a cleavable linker or a non-cleavable linker. In some embodiments, the linker is cleavable. The cleavable linker contains a cleavable adapter. Cleavable adapters include, but are not limited to, nuclease-cleavable adapters, acid-labile adapters, peptidase-sensitive adapters, light-labile adapters, dimethyl adapters, and disulfide-containing adapters (Chari et al.). Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020.

[0195] In some embodiments, the linker comprises maleimide. In some embodiments, the linker has the following structure: , Wherein L' is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are optionally replaced by (-O-), (-NH-), (-S-), amino acids, hydrazones (-C(R')=N=N(R')-), nucleotides, or 3-12-membered divalent heterocycles, wherein the chain and any 3-12-membered divalent heterocycle are optionally replaced by one or more (e.g., 1, 2, 3 or 4) substituents, wherein the substituents are independently selected from the following composition The group comprises: (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halogen, hydroxyl, oxo (=O), hydrazone (=N=N(R')-), carboxyl, aryl, aryloxy, heteroaryl, and heteroaryloxy; wherein each R' is independently H or (C1-C6)alkyl; and wherein the valence state marked * is attached to the polypeptide and the valence state marked ** is attached to MAPT ASO (or the 5' terminal group of MAPT ASO). In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated chain having 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises one or more disulfide bonds. In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated chain having 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain contains one or more hydrazone groups in or attached to a carbon atom of the chain. In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain contains one or more amino acids in the chain. In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain contains a dipeptide in the chain. In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain contains the dipeptide Val-Cit in the chain. In another embodiment, L' comprises one or more nucleotides. In another embodiment, L' comprises two or more nucleotides. In another embodiment, L' comprises a trinucleotide group. In yet another embodiment, L' comprises one or more nucleotides having unmodified bases, unmodified sugar groups, and / or unmodified phosphate groups.

[0196] In some implementations, the linker includes the following structure: , Among them, the "intersecting with bonds in the chemical structure" The symbol * indicates the attachment point of a wavy bond intersecting with the rest of the molecule in the chemical structure. In some embodiments, the valence state marked * is attached to the Fc polypeptide dimer targeting TfR, and the valence state marked ** is attached to MAPT ASO. In some embodiments, the valence state marked ** is attached to MAPT ASO via the 5' terminal group of MAPT ASO. In some embodiments, the valence state marked ** is attached to MAPT ASO via the 5' terminal PO group of MAPT ASO. In some embodiments, the valence state marked ** is attached to MAPT ASO via the 5' terminal PS group of MAPT ASO. In some embodiments, the valence state marked ** is attached to MAPT ASO via the 5' terminal PS2 group of MAPT ASO. In some embodiments, the valence state marked ** is attached to MAPT ASO via the 5' terminal PN group of MAPT ASO. In some embodiments, the valence state marked ** is attached to MAPT ASO via the 5' terminal MsPA group of MAPT ASO. In some embodiments, the valence state marked ** is attached to MAPT ASO via MAPT... The 5' terminal OiPS group of ASO is attached to MAPT ASO.

[0197] In some implementations, the linker base includes: , The wavy lines intersecting with bonds in the chemical structure "" indicates the point where a wavy bond intersects with the rest of the molecule.

[0198] In some embodiments, the stabilized oligonucleotide peptide conjugate comprises: , Where P is a polypeptide, and the phosphodiester (PO) group is the 5' terminal group of the oligonucleotide.

[0199] In some embodiments, the stabilized oligonucleotide peptide conjugate comprises: , Where P is a polypeptide, and the thiophosphate (PS) group is the 5' terminal group of the oligonucleotide.

[0200] In some embodiments, the stabilized oligonucleotide peptide conjugate comprises: , Where P is a polypeptide, and the cyclic phosphoryl guanidine (PN) group is the 5' terminal group of the oligonucleotide.

[0201] Nucleic acid This document describes nucleic acids encoding any heavy and light chains of any described Fc polypeptide, Fc polypeptide dimer, NTF, and / or Fab-Fc fusion polypeptide. Optionally, such nucleic acids further encode a signal peptide. The coding sequence of the nucleic acid may be operatively linked to one or more regulatory sequences to promote expression of the coding sequence in a host cell. Such regulatory sequences include, but are not limited to, promoters, enhancers, ribosome binding sites, transcription termination signals, etc. The nucleic acids encoding the heavy and light chains may exist in isolated form or may be cloned into one or more vectors. The nucleic acids may be synthesized, for example, by solid-state synthesis or by PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains may be synthesized as an adjacent nucleic acid (e.g., within an expression vector) or may be separate (e.g., each cloned into its specific expression vector).

[0202] In some embodiments, the nucleic acid encoding an Fc polypeptide, Fc polypeptide dimer, NTF, or Fab-Fc fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of any of the described Fc polypeptide, Fc polypeptide dimer, NTF, and / or Fab-Fc fusion polypeptide. The nucleic acid sequence may be provided in an expression vector to facilitate the expression of the heavy chain and / or light chain in host cells. The nucleic acid or an expression vector containing said nucleic acid is transformed into host cells. The host cells can then be used to generate the heavy chain and / or light chain of NTF.

[0203] In some embodiments, the nucleic acid encoding the Fc polypeptide dimer targeting TfR comprises a first nucleic acid sequence encoding a first Fc fusion polypeptide and a second nucleic acid sequence encoding a second Fab-Fc fusion polypeptide. In some embodiments, the first nucleic acid sequence encodes any one of SEQ ID NO: 1, 47-48, 51-54, 61-63, 66, 73-78, 80, and 90; and the second nucleic acid sequence encodes any one of SEQ ID NO: 4-29, 36, 43-44, and 49-50.

[0204] In some embodiments, the nucleic acid encoding the Fc polypeptide dimer targeting TfR comprises a first nucleic acid sequence encoding a first Fab-Fc fusion polypeptide, a second nucleic acid sequence encoding a second Fab-Fc fusion polypeptide, and a third nucleic acid sequence encoding the NTF light chain. In some embodiments, the first nucleic acid sequence encodes any one of SEQ ID NO: 100-101 and 130-131; the second nucleic acid sequence encodes any one of SEQ ID NO: 98-99; and the third nucleic acid sequence encodes any one of SEQ ID NO: 108 and 129.

[0205] In some embodiments, the first nucleic acid sequence comprises SEQ ID NO:133 or a sequence having at least 75% identity with SEQ ID NO:133; the second nucleic acid sequence comprises SEQ ID NO:132 or a sequence having at least 75% identity with SEQ ID NO:132; and the third nucleic acid sequence comprises SEQ ID NO:134 or a sequence having at least 75% identity with SEQ ID NO:134. In some embodiments, the first nucleic acid sequence comprises SEQ ID NO:133; the second nucleic acid sequence comprises SEQ ID NO:132; and the third nucleic acid sequence comprises SEQ ID NO:134.

[0206] Methods for preparing the described Fc peptides, Fc peptide dimers, NTF, Fab-Fc fusion peptides, or antibodies include, but are not limited to, expressing one or more nucleic acids encoding the heavy and light chains of the Fc peptide, Fc peptide dimer, NTF, Fab-Fc fusion peptide, or NTF in multiple host cells; proliferating cells under conditions suitable for expression of the heavy and light chains of the Fc peptide, Fc peptide dimer, NTF, Fab-Fc fusion peptide, or NTF in cells; and purifying the heavy and light chains of the Fc peptide, Fc peptide dimer, NTF, Fab-Fc fusion peptide, or NTF. The Fc peptide, Fc peptide dimer, Fab-Fc fusion peptide, and purified NTF may be conjugated to one or more of the described MAPT ASOs.

[0207] This describes a cell containing nucleic acid encoding the described Fc polypeptide, Fc polypeptide dimer, NTF, Fab-Fc fusion polypeptide, or the heavy and light chains of Fab. The cell can be a bacterial cell, yeast cell, insect cell, or mammalian cell. The cell can be used to express the Fc polypeptide, Fc polypeptide dimer, NTF, or Fc polypeptide-NTF heavy chain fusion polypeptide. The expressed polypeptide can then be isolated from the cell and optionally purified.

[0208] How to use The MAPT ASO conjugates described herein can be used for a variety of purposes, including therapeutic indications.

[0209] In some embodiments, the MAPT ASO conjugate is used to deliver MAPT ASO to target cell types expressing transferrin receptors. In some embodiments, the MAPT ASO conjugate can be used to transport MAPT ASO across the endothelium (e.g., the blood-brain barrier) for brain uptake.

[0210] In some embodiments, a method for reducing MAPT gene expression in a subject is described, the method comprising administering to the subject an effective amount of a MAPT ASO conjugate or a combination thereof as described herein. In some embodiments, a MAPT ASO conjugate or a combination thereof is provided for reducing MAPT gene expression in the cells of a subject. In some embodiments, MAPT ASO binds to the MAPT transcript and recruits ribonuclease H, which degrades the transcript. In some embodiments, administration of the described MAPT ASO conjugate or a combination thereof to cells or a subject reduces MAPT gene expression in the cells or the subject. Compared to expression in a control (e.g., cells or subjects not administered MAPT ASO, a MAPT ASO conjugate, or a combination thereof as described herein), or compared to MAPT expression levels in cells or the subject prior to administration of MAPT ASO, expression may be reduced by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%.

[0211] For example, some embodiments provide a method for transendothelial transport of MAPT ASO, the method comprising contacting the endothelium (e.g., the blood-brain barrier (BBB)) with a MAPT ASO conjugate as described herein. Thus, some embodiments provide a method for transporting MAPT ASO across the BBB of a subject in need, the method comprising administering a MAPT ASO conjugate as described herein to the subject. In some embodiments, a MAPT ASO conjugate as described herein is provided for transporting MAPT ASO across the BBB of a subject in need. In some embodiments, a method for delivering MAPT ASO to the CNS is provided herein. In some embodiments, a method for delivering MAPT ASO to deep brain regions (e.g., the cortex, brainstem, hippocampus, striatum, cerebellum, thalamus, caudate putamen, and substantia nigra) is provided herein. In some embodiments, a method for delivering MAPT ASO to deep brain regions and the spinal cord (e.g., the cervical spinal cord, lumbar spinal cord) is provided herein. In some embodiments, a method for delivering MAPT ASO to the CNS and muscles (e.g., the heart and bones) is provided herein. In some implementations, this document provides methods for delivering MAPT ASO to the CNS, peripheral nerves (e.g., retina, sciatic nerve), muscles (e.g., quadriceps), and other peripheral organs (e.g., heart, diaphragm, spleen, intestine, lung, liver, and kidney).

[0212] Some embodiments also provide a method for modulating MAPT expression in a subject with a need, the method comprising administering to the subject an effective amount of a MAPT ASO conjugate as described herein. In some embodiments, the MAPT ASO conjugate as described herein is used to modulate MAPT expression in a subject. In some embodiments, MAPT is expressed in the brain cells of the subject.

[0213] A therapeutically effective dose or dosage of MAPT ASO or MAPT ASO conjugates as described herein may be administered to the subject. However, the dosage may vary depending on several factors, including the chosen route of administration, the formulation of the composition, patient response, severity of the condition, the subject's weight, and the prescribing physician's judgment. The dosage may be increased or decreased over time if requested by the individual patient.

[0214] In several embodiments, MAPT ASO or its conjugates as described herein are administered parenterally. In some embodiments, MAPT ASO or its conjugates are administered intravenously. Intravenous administration can be performed by infusion, for example, over a period of about 10 to about 30 minutes, or over a period of at least 1 hour, 2 hours, or 3 hours. In some embodiments, MAPT ASO or its conjugates are administered by intravenous bolus injection. A combination of infusion and bolus administration may also be used.

[0215] In some parenteral embodiments, MAPT ASO or its conjugates are administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, MAPT ASO or its conjugates are administered intradermally or intramuscularly. In some embodiments, MAPT ASO or its conjugates are administered intrathecally, for example, via epidural administration or intraventricular administration.

[0216] In other embodiments, MAPT ASO or MAPT ASO conjugates as described herein can be administered orally, via the lungs, intranasally, intraocularly, or topically. Lung administration is also possible, for example, by using an inhaler or nebulizer and in combination with a nebulizer.

[0217] The MAPT ASO and MAPT ASO conjugates described herein can also be used to treat, prevent, or improve Tau-related diseases, conditions, and disorders. Therefore, in some embodiments, this document provides methods for treating, preventing, or improving Tau-related diseases, conditions, and disorders in subjects of need. In some embodiments, tau-related diseases are tau-related neurodegenerative disorders. In some embodiments, tau-related diseases include, but are not limited to, Tau protein disorders, Alzheimer's disease, frontotemporal dementia (FTD), FTDP-17, progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), epilepsy, and Dravet syndrome.

[0218] Therefore, certain implementations provide a method for treating tau-related neurodegenerative diseases in subjects of need, the method comprising administering to the subject a MAPT ASO, MAPT ASO conjugate, or composition as described herein.

[0219] In some embodiments, tau-related neurodegenerative syndromes are Alzheimer's disease. Therefore, some embodiments provide a method for treating Alzheimer's disease, the method comprising administering MAPT ASO, MAPT ASO conjugates, or compositions as described herein to a subject in need. The subject may have been diagnosed with Alzheimer's disease, diagnosed with one or more symptoms of Alzheimer's disease, or be at risk of developing Alzheimer's disease or one or more symptoms associated with Alzheimer's disease.

[0220] A method for targeted delivery of MAPT ASO to CNS tissues in a patient is described, the method comprising administering any of the described MAPT ASO conjugates or pharmaceutical compositions to a subject. In some embodiments, MAPT ASO is distributed throughout the CNS. In some embodiments, MAPT ASO is distributed in brain regions. Brain regions include, but are not limited to, the frontal lobe, parietal lobe, temporal lobe, occipital lobe, and cerebellum. In some embodiments, MAPT ASO is distributed in deep brain regions. In some embodiments, MAPT ASO is distributed in the spinal cord. In some embodiments, MAPT ASO modulates the expression of a target gene. In some embodiments, the regulation of target gene expression is a suppression of gene expression (i.e., gene knockdown).

[0221] In some implementations, the subjects are human subjects.

[0222] Pharmaceutical Compositions and Kits In some embodiments, pharmaceutical compositions and kits comprising MAPT ASO conjugates as described herein are provided.

[0223] A. Pharmaceutical Composition Instructions for preparing formulations for the purposes described herein are available in any number of pharmaceutical preparation and formulation manuals known to those skilled in the art.

[0224] In some embodiments, the pharmaceutical composition comprises a MAPT ASO conjugate as described herein, and also comprises one or more pharmaceutically acceptable carriers and / or excipients.

[0225] In some embodiments, the composition comprises a variety of MAPT ASO conjugates as described herein, which may be the same or different (e.g., a mixture of different conjugates).

[0226] In some embodiments, the MAPT ASO conjugates described herein comprise 1 to 8 (1, 2, 3, 4, 5, 6, 7, or 8) MAPT ASOs linked to one or more amino acids of the MAPT ASO conjugate. In some embodiments, the MAPT ASO conjugate comprises one (1) MAPT ASO linked to an amino acid of the MAPT ASO conjugate. In some embodiments, the MAPT ASO conjugate comprises two (2) MAPT ASOs linked to two amino acids of the MAPT ASO conjugate. In some embodiments, the MAPT ASO conjugate comprises two (2) MAPT ASOs linked to one amino acid of the MAPT ASO conjugate. In some embodiments, the MAPT ASO conjugate comprises four (4) MAPT ASOs linked to four amino acids of the MAPT ASO conjugate. In some embodiments, the MAPT ASO conjugate comprises four (4) MAPT ASOs linked to two amino acids of the MAPT ASO conjugate (two MAPT ASOs linked to each of the two amino acids). In some embodiments, the MAPT ASO conjugate comprises four (4) MAPT ASO molecules linked to one amino acid of the MAPT ASO conjugate.

[0227] In some embodiments, a single MAPT ASO is attached to a TfR-targeting Fc dimer or Fab-Fc dimer. In some embodiments, two or more MAPT ASOs are attached to a TfR-targeting Fc dimer or Fab-Fc dimer. The two or more MAPT ASOs may be linked to a single linker, or the two or more MAPT ASOs may be linked to the TfR-targeting Fc dimer or Fab-Fc dimer via two or more linkers. The two or more linkers may be the same or different. In some embodiments, two MAPT ASOs are attached to a TfR-targeting Fc dimer or Fab-Fc dimer. In some embodiments, four MAPT ASOs are attached to a TfR-targeting Fc dimer or Fab-Fc dimer.

[0228] In some embodiments, one MAPT ASO is attached to a single linker (L). In some embodiments, two MAPT ASOs are attached to a single linker (L). In some embodiments, two or more MAPT ASOs are connected in series with a single linker (L). For series connection, L may be connected to the 5' end of the first MAPT ASO, and the second MAPT ASO may be connected to the 3' end of the first MAPT ASO. Alternatively, for series connection, L may be connected to the 3' end of the first MAPT ASO, and the second MAPT ASO may be connected to the 5' end of the first MAPT ASO. The first and second MAPT ASOs may be connected to each other via nucleic acid linkers or non-nucleic acid cleavable linkers.

[0229] In some implementations, two or more MAPT ASOs are attached to a single branched linker (L). The linker may be a branched linker, wherein two or more oligonucleotides are attached to a single linker (L) (i.e., y is 2 or greater).

[0230] In some embodiments, the ratio of oligonucleotides to proteins in the composition is from about 1:1 to about 4:1. In some embodiments, the ratio of oligonucleotides to proteins in the composition is from about 1:1 to about 2:1. In some embodiments, the ratio of oligonucleotides to proteins in the composition is about 1.23. In some embodiments, the ratio of oligonucleotides to proteins in the composition is from about 2:1 to about 3:1. In some embodiments, the ratio of oligonucleotides to proteins in the composition is about 2.5.

[0231] As used herein, the term pharmaceutically acceptable carrier includes any solvent, dispersion medium, or coating that is physiologically compatible and preferably does not interfere with or otherwise inhibit the activity of the active agent. Various pharmaceutically acceptable excipients are well known. In some embodiments, the carrier is suitable for intravenous, intrathecal, intraventricular, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. Pharmaceutically acceptable carriers may contain one or more physiologically acceptable compounds that serve, for example, to stabilize the composition or to increase or decrease the absorption of MAPT ASO conjugates. Physiologically acceptable compounds may include, for example, carbohydrates (e.g., glucose, sucrose, dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, compositions that reduce the scavenging or hydrolysis of the active agent, or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and formulations thereof are also available in the art.

[0232] The pharmaceutical compositions described herein can be manufactured in a manner known to those skilled in the art, for example, by conventional processes of mixing, dissolving, granulation, forming into sugar-coated pellets, emulsifying, encapsulating, embedding, or lyophilizing. The following methods and excipients are merely exemplary and are not intended to limit the scope in any way.

[0233] For administration to subjects, formulations can be made by combining the MAPT ASO conjugate as described herein with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compound to be formulated as tablets, pills, sugar-coated pills, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc. Pharmaceutical formulations for administration to subjects can be obtained by mixing the MAPT ASO conjugate with a solid excipient, optionally milling the resulting mixture, and processing the granular mixture after adding suitable adjuvants (if necessary) to obtain tablets or sugar-coated pill cores. Suitable excipients include, for example, fillers, such as sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If necessary, disintegrants (e.g., croscarmellose, agar, or alginate) or salts thereof (e.g., sodium alginate) can be added.

[0234] As disclosed above, the MAPT ASO conjugates described herein can be formulated for parenteral administration by injection (e.g., by bolus or continuous infusion). For injection, the MAPT ASO conjugates can be formulated by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent (e.g., vegetable oil or other similar oil, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol); and, where necessary, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the MAPT ASO conjugates can be formulated in an aqueous solution, preferably in a physiologically compatible buffer (e.g., Hank's solution, Ringer's solution, or physiological saline buffer). Formulations for injection can be presented in single-dose form (e.g., in ampoules or in multi-dose containers) with added preservatives. The composition may take the form of a suspension, solution or emulsion, for example, in an oily or aqueous medium, and may contain formulations (e.g., suspending agents, stabilizers and / or dispersants).

[0235] Typically, pharmaceutical compositions intended for internal administration are sterile. Sterilization can be performed using methods known in the art, such as heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0236] The dosage and desired drug concentration of the pharmaceutical compositions described herein may be varied depending on the intended specific use. Determining the appropriate dosage or route of administration is entirely within the skill of those skilled in the art. Appropriate dosages are also as described above.

[0237] B. Medicine box In some embodiments, a kit is provided comprising a MAPT ASO conjugate as described herein. In some embodiments, the kit is used to regulate the expression of a target gene or sequence (e.g., a target gene expressed in the brain or central nervous system (CNS)). In some embodiments, the kit is used to regulate the expression of a target gene.

[0238] In some embodiments, the kit also contains one or more additional therapeutic agents. For example, in some embodiments, the kit contains a MAPT ASO conjugate as described herein, and also contains one or more additional therapeutic agents. In some embodiments, the kit also contains instruction material containing instructions (i.e., instructions) for practicing the methods described herein (e.g., instructions for using the kit to administer the composition across the blood-brain barrier). While instruction material typically includes written or printed material, it is not limited thereto. Any medium capable of storing such instructions and communicating them to the end user is contemplated herein. Such media includes, but is not limited to, electronic storage media (e.g., disks, magnetic tapes, cassette tapes, chips), optical media (e.g., CD-ROMs), etc. Such media may include URLs providing such instruction material.

[0239] Table 1. Sequence

[0240] Example Example 1. Construction of OTV:MAPT ASO conjugate i. Design and cloning of Fab-Fc dimer fusions. A Fab-Fc dimer fusion was designed comprising: a first Fc polypeptide containing an S239C cysteine ​​substitution and a β and LALAPS mutation (SEQ ID NO: 63); and a second Fc polypeptide forming an Fc dimer with the first Fc polypeptide and containing a modified constant domain that specifically binds to the transferrin receptor (35.23.2), β, and the LALAPS mutation (SEQ ID NO: 11), wherein the first and second Fc polypeptides are each fused to untargeted Fab (SEQ ID NO: 108 and 109). Therefore, the first Fab-Fc polypeptide fusion has the sequence of SEQ ID NO: 100, and the second Fab-Fc polypeptide fusion has the sequence of SEQ ID NO: 98.

[0241] Additionally, as described herein, the C-terminal lysine residues of the Fc peptide can be completely or partially removed during protein production via cellular mechanisms. Therefore, the first Fc peptide may contain SEQ ID NO:80, and the second Fc peptide may contain SEQ ID NO:12. Similarly, the first Fab-Fc peptide fusion may have the sequence of SEQ ID NO:101, and the second Fab-Fc peptide fusion may have the sequence of SEQ ID NO:99.

[0242] The construct was cloned into the mammalian expression vector pRK5 via gene synthesis and Gibson assembly.

[0243] ii. Expression and purification of Fab-Fc dimer fusion protein. The vector and the corresponding light chain vector were co-transfected into Expi293 cells at a ratio of 1:1:2 (potassium:mortar:light chain). The expressed protein was purified from conditioned medium by loading the supernatant onto a protein A column. The column was washed with 10 column volumes of PBS (pH 7.4). The protein was eluted with 50 mM sodium citrate (pH 3.0) containing 150 mM NaCl and immediately neutralized with 200 mM arginine and 137 mM succinate (pH 5.0). The protein was further purified by size exclusion chromatography (SEC) (GE Superdex 200) using 200 mM arginine and 137 mM succinate (pH 5.0) as the electrophoresis buffer. The purified protein was confirmed by intact-weight LC / MS and its purity was >95% by SDS-PAGE and analytical HPLC-SEC.

[0244] iii. Synthesis of ASO modified with 5'-maleimide Preparation of ASO modified with 5'-amino group solid-phase oligonucleotide synthesisOligonucleotides were synthesized at a scale of 100 μmol using a MerMade 12 (LGC) DNA / RNA synthesizer, following a standard solid-phase oligonucleotide synthesis protocol. All locked nucleic acids (LNAs) and deoxyribonucleic acid (DNA) phosphoridamides were purchased from Hongene Biotech Corporation, including LNA-A(Bz), LNA-5MeC(Bz), LNA-T, LNA-G(dmf), and dA(Bz), dC(Ac), dT, and dG(dmf). LNA-5MeC(Bz) was dissolved in a mixed solvent of DCM / acetonitrile (1:1, v / v), while all other phosphoridamides were dissolved in acetonitrile, and molecular sieves (3 Å) were added. First, the parent antisense oligonucleotide (ASO) sequence was assembled on a UnyLinker CPG solid support, followed by attachment of an amino linker at the 5' end using 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)phosphamide (CAS: 133975-85-6; Glen Research catalog #10-1916). The synthetic cycle for adding a single nucleotide (or non-nucleic acid) unit consisted of four separate steps: detritylation, coupling, oxidation (or sulfidation), and end-capping. 5-Ethylthio-1H-tetrazole (ETT, 0.25 M in acetonitrile) was used as the activator solution. A 0.2 M solution of PADS (phenylacetyl disulfide) in 50% pyridine / 50% acetonitrile was used to introduce thiophosphate linkages. Detailed protocols for the 100 μmol-scale ASO synthesis are summarized in Tables 2A to 2D below.

[0245] Table 2A. Typical synthesis parameters for 100 μmol-scale ASO

[0246] Table 2B. Exemplary synthesis of oligonucleotides containing MsPA backbone modification

[0247] Table 2C. Exemplary synthesis of oligonucleotides containing PN backbone modification

[0248] Table 2D. Exemplary synthesis of oligonucleotides containing PS2 and PN backbone modifications

[0249] Table 2E. Exemplary synthesis of oligonucleotides containing OiPr-PS and PN backbone modifications

[0250] Manipulation after synthesis. After solid-phase oligonucleotide synthesis, the phosphate protecting group (2-cyanoethyl) was removed by reacting diethylamine (DEA) in 20% acetonitrile for 1 hour. Nucleotide fragmentation and deprotection (C and D) from the solid support were then carried out at 45°C in NH4OH / EtOH (3:1) for 20 hours. The crude oligonucleotide solution was concentrated by centrifugation under reduced pressure, and the solid residue was reconstituted in water for preparative HPLC purification.

[0251] 5 was purified by preparative HPLC. ' -Amino ASO. Crude 5'-aminoASO was purified by ion-pair reversed-phase HPLC. Detailed parameters are summarized in Table 3. Appropriate fractions were collected and lyophilized to obtain purified 5'-aminoASO.

[0252] Table 3. Preparative HPLC purification conditions

[0253] Synthesis of ASO modified with 5'-amino group. The 5'-amino-modified ASOs shown in Tables 4 and 5 below were synthesized according to the general method described above.

[0254] After 5 ' - Preparation of maleimide-modified ASO. General procedure: Add a solution of 3-maleimide propionic acid N-hydroxysuccinimide ester (MCOSu, 10 eq.) in DMF (2.5 mL) to a solution of ASO amine TEA salt (30 mg, based on OD) in PBS buffer (TEKNOVA 10× PBS stock solution, pH 6.0, 2.5 mL) at room temperature. Shake the resulting solution at room temperature for 18 hours. After completion, desalt the solution by passing it through a Sephadex G25 column (2.5 × 40 cm) on AKTA-pure 25 M, eluting with Milli Q water at 3 mL / min, monitored by UV 260 / 280 and conductivity. Collect the appropriate fractions and lyophilize to obtain the desired 5'-maleimide ASO.

[0255] iv. Biological conjoints.First, the Fab-Fc dimer fusion containing the S239C cysteine-modified derivative used for conjugation, generated above, was reduced at 37°C for 1 hour using 30 molar equivalents of TCEP and 2 mM EDTA. Reduction was confirmed by LC / MS. After reduction, residual TCEP (purified by dialysis, for example) was removed by dialysis with 1× PBS (pH 6.8) containing 2 mM EDTA, and the Fab-Fc dimer fusion was reoxidized with 50 molar equivalents of dHAA at room temperature for 3 hours. Oxidation was confirmed by LC / MS of dHAA. For bioconjugation, 1.2 molar equivalents of the 5'-maleimide-modified ASO generated above were added to the oxidized Fab-Fc dimer fusion at room temperature for 1 hour. The obtained MAPT ASO conjugate was purified by anion exchange chromatography using a Resource Q column (equilibration buffer: 50 mM Tris, pH 7.5; elution buffer: 50 mM Tris, pH 7.5 + 2 M NaCl) to remove unwanted and unconjugated products. The purity of the MAPT ASO conjugate was determined by LC / MS and SEC. The resulting conjugate was referred to as OTV or OTV:MAPT.

[0256] Example 2. In vitro screening of ASOs targeting the MAPT gene The selection of ASO sequences that are specific to a given target sequence is based on the analysis of the selected target sequence and the determination of many factors, including in vitro and in vivo potency and hepatotoxicity profile.

[0257] Preliminary MAPT ASO screening. Based on computer predictions of target binding, specificity, efficacy, and safety, 393 MAPT ASO sequences that are 100% complementary to MAPT pre-mRNA (Ensembl ENST0000034429) and / or MAPT mRNA (refseq NM_001123066) SEQ ID NO:115 were preliminarily identified.

[0258] The knockdown efficacy of MAPT-specific ASO in human MiaPaCa-2 and HDLM2 cells was tested. Cells were treated with 5 µM MAPT-specific ASO or control oligonucleotides without transfection. Cells lysed after three days of treatment. MAPT and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher). MAPT expression values ​​were normalized relative to HPRT1 values, and the degree of knockdown compared to simulated treatment cells was determined.

[0259] Dual concentration screening.Fifty-five MAPT-specific ASOs with >99.9% (5µM) knockdown efficacy in both cell lines were tested in a dual-concentration screening (100 nM, 1000 nM) trial in MiaPaCa-2 cells. Cells were lysed after three days of treatment. MAPT and HPRT1 mRNA expression was analyzed using a QuantiGene Singleplex assay (ThermoFisher). MAPT expression values ​​were normalized relative to HPRT1 values, and the degree of knockdown compared to simulated treatment cells was determined. Twenty-two ASOs were selected to investigate the TLR9 activation capacity and concentration-response relationship (IC50). 50 (Measurement).

[0260] Study on the activation ability of TLR9. The TLR9-dependent pro-inflammatory potential of 22 ASOs was tested. Reporter cell lines (HEK-Blue-hTLR9 cells, Invivogen) were treated with MAPT-specific ASOs, positive controls, and negative controls, without the use of 5 µM transfection reagent. After 20 h, QUANTI-Blue™ solution (Invivogen) was added to the cells. Optical density was measured 2 hours later to determine the TLR9 activation capacity of the tested ASOs.

[0261] Example 3. In vivo knockdown screening The ASO comprising SEQ ID NO:111 was synthesized according to the procedure described below for in vivo administration. The ASO was diluted in sterile saline and administered to hTau mice (mTau- / -) via intraventricular (ICV) injection at a single dose (total volume 10 μL) of 50 μg or 100 μg per mouse. The 50 μg or 100 μg dose was determined based on in vivo tolerability.

[0262] Two weeks after administration, tissues (brain and spinal cord) and peripheral blood were collected. MAPT expression in the brain and spinal cord was measured as described below. A piece of frontal lobe and cervical spinal cord was homogenized using a bead homogenizer from Trizol for bulk RNA isolation. Briefly, the homogenized tissue was incubated with chloroform for 3–5 minutes to allow phase separation after centrifugation. The aqueous phase was then incubated with isopropanol for 10 minutes to allow RNA precipitation, followed by washing with 75% ethanol and resuspending in nuclease-free water. MAPT expression was then measured by qPCR using the Express One-Step Superscript Kit and normalized relative to the expression of the housekeeping gene Gapdh. A set of glial activation markers (Aif1, Gfap, Tlr9, Itgax) were also quantified by qPCR using the Express One-Step Superscript Kit to determine whether ASO triggers a chronic neuroinflammatory response.

[0263] At a dose of 50 μg, ASO6 knocked down human tau expression by 76%.

[0264] Example 4. In vivo hepatotoxicity screening Assess the hepatic safety of ASO6. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) are abundant enzymes in the liver. When hepatocytes are damaged, they release these enzymes into the bloodstream. Therefore, serum ALT, AST, and LDH levels were measured to determine whether ASO induces liver injury.

[0265] ASO was diluted in sterile saline and administered subcutaneously to wild-type mice at a dose of 20 mpk daily for five consecutive days. Mice were weighed daily for 12 days, and blood was collected on days 5, 8, and 12. Serum was prepared by clotting the blood samples and then centrifuging. ALT, AST, and LDH levels in the serum samples were then measured. After administration of ASO6, ALT, AST, and LDH levels were low. Body weight did not change significantly.

[0266] To determine whether internal cytosine methylation (i.e., methylation of cytosine in the notch region of ASO) affects the hepatic safety profile of ASO, 5-methylcytosine was used instead of internal cytosine in ASO 6. Hepatic safety was analyzed using the same protocol as described above. The addition of 5-methylcytosine had only a slight beneficial effect on the hepatic safety profile of ASO 6; that is, a slight increase in liver enzyme levels was mitigated by cytosine methylation.

[0267] Example 5. In vivo ED50 screening ASO6 was diluted in sterile saline and injected via ICV into hTau (mTau) at several doses (ranges including 5 and 15 μg (using data from previous studies of 0 and 50 μg)). - / - The final volume of the drug administered to mice was 10 μL.

[0268] Two weeks after drug administration, tissues were collected, and MAPT expression in the brain and spinal cord was measured by batch RNA isolation according to the above protocol, followed by qPCR for MAPT and Gapdh. ED was observed in vivo. 50 The values ​​were 17.7 μg and 13.1 μg.

[0269] Example 6. In vivo ASO half-life determination ASO6 was diluted in sterile saline and administered via ICV injection at a moderate dose of 25 μg (total volume 10 μL) to hTau (mTau). - / - Mice. Tissue samples were collected at 5 and 9 weeks post-drug administration to determine the duration of tau knockdown. MAPT expression in the brain and spinal cord was measured by batch RNA isolation followed by qPCR for MAPT and Gapdh, according to the above protocol. MAPT knockdown in the brain and spinal cord was maintained for at least 9 weeks.

[0270] Example 7. In vivo rat nephrotoxicity assay Rats are considered the most sensitive species for detecting ASO-mediated nephrotoxicity and the preferred species for risk assessment because they can over-predict human nephrotoxicity. The aim of this study was to determine the effects (gross and histological evaluation) on renal function and morphology (if any) following repeated doses of five ASO6s in a 2-week rat study.

[0271] Five male Wistar Hannover rats (7-8 weeks old) were administered two subcutaneous injections of 40 mg / kg of control ASO A, ASO6, A, or saline (mediator control) on days 1 and 8; the animals were euthanized on day 15. Body weight was measured on days 1 and 8 before administration, and on days 5, 10, and 15. Clinical observation was conducted on days 1 and 8 after administration. Blood samples were collected on days 5, 10, and 15 to measure blood urea nitrogen (BUN) and creatinine as markers of renal function. On day 15, kidneys were collected for microscopic evaluation, and urine was collected to measure the KIM-1:creatinine ratio as an indicator of renal injury.

[0272] ASO6 was well tolerated in Wistar Hannover rats, with no changes in clinical signs or body weight throughout the treatment period. Serum BUN and creatinine levels were within the normal historical range in all treatment groups and were comparable to those in the mediator control. On day 15, changes in the urinary KIM-1:creatinine ratio were considered below the critical limit and the magnitude of change was similar to that commonly observed in rats under similar study conditions.

[0273] Minimal to mild proximal tubular epithelial degeneration with minimal regeneration and minimal to mild interstitial, perivascular, and / or periglomerular mononuclear cell infiltration were observed in the kidneys. Based on the minimal to mild severity and the absence of associated functional changes in clinical signs, body weight, and serum and urinary renal biomarkers (BUN, creatinine, and KIM-1), these microscopic changes were considered non-adverse. In conclusion, no evidence of adverse effects on renal function or histopathology was detected in Wistar Han rats.

[0274] Table 4

[0275] Example 8: In vivo potency and stability assessment of MAPT ASO The ASO SEQ ID NO:111 is further modified as shown in Table 5 below.

[0276] Table 5

[0277] ASO was diluted in sterile saline and administered via intraventricular injection (ICV) to hTau mice (mTau mice) at a single dose of 10 or 25 μg (total volume 10 μL). - / - (According to Table 6 below). Brain tissue was collected 10–14 days after administration (according to Table 6 below) to measure relative MAPT RNA levels (according to the tissue homogenization and qPCR procedure below) and total ASO concentration (according to the tissue homogenization and total ASO determination procedure below).

[0278] Table 6

[0279] The results are shown in Figure 8 (relative to MAPT RNA levels) and Figure 9 (Total ASO). These results indicate that the ASO base sequence is more potent than the existing control MAPT ASO, and the modified ASOs (e.g., ASOM4 and ASOM5) exhibit enhanced tissue stability compared to the unmodified versions (ASOM_unmod), which should prolong the duration of action of the modified ASOs in vivo.

[0280] Example 9: Modified OTV:MAPT followed by in vivo OTV brain uptake and MAPT knockdown Peripheral administration. The five modified ASOs selected for efficacy as described above were synthesized according to the above procedure and bioconjugated with TV proteins to create five MAPT OTV molecules (OTVM1, OTVM2, OTVM3, OTVM4, OTVM5) for evaluation of tau knockdown in the brain after peripheral intravenous administration.

[0281] To TfR ms / hu Knock-in mice (see U.S. Patent No. 10,143,187, which is incorporated herein by reference) were given each of the OTVs once at 25 mpk. MAPT OTVs were diluted in sterile saline and administered intravenously. Mice were weighed prior to each dose to determine the appropriate dosage. Plasma was collected at 4, 24, and 72 hours post-administration, and peripheral plasma and tissue samples (i.e., brain and liver) were collected one week post-administration to determine the level of total full-length ASO deposited in the brain according to the procedure described below. Figure 1 and Figure 2 ).

[0282] Additionally, TfR ms / hu Knock-in mice (see U.S. Patent No. 10,143,187, which is incorporated herein by reference) and mice with hTau + / - (human Tau) and mTau - / - (Tau knockout) transgenic mice were mated, and the resulting mice were administered either 4 doses (day 0, week 1, week 2, week 3) or 8 doses (day 0, day 3, day 7, day 10, day 14, day 17, day 21, day 24) of each of the MAPT OTVs described above. The MAPT OTVs were diluted in sterile saline and administered intravenously to the mice. Anti-CD4 was administered before the initial week of administration and before week 3 to prevent anti-drug antibody responses in the mice. Mice were weighed before each dose to determine the appropriate dosage. Brain samples were collected one week after the final dose to determine the ASO concentration in the brain using the total ASO assay described below according to the procedure below. Figure 3 ), and the level of human Tau knockdown relative to Gapdh and saline-treated mice ( Figure 4 and Figure 5 ).

[0283] These values ​​were then compared with separate studies using the same mouse lines to assess brain uptake and target knockdown of OTV molecules conjugated with unmodified ASO. In this study, mice were administered one dose (day 0) or four doses (day 0, week 1, week 2, week 3) of unmodified MAPT OTV. MAPT OTV was diluted in sterile saline and administered intravenously to mice. Anti-CD4 was administered before the initial week of administration and before week 3 to prevent anti-drug antibody responses in mice. Mice were weighed before each dose to determine the appropriate dosage. Brain samples were collected one week after the final dose to determine the ASO concentration in the brain using total ASO assays according to the procedure below. Figure 6 ), and human Tau knockdown levels relative to Gapdh and saline-treated mice ( Figure 7 ).

[0284] In summary, these results demonstrate that modified MAPT OTV exhibits superior ASO brain uptake and target knockdown compared to unmodified MAPT OTV. We observed 20–27 nM ASO brain uptake one week after a single 25 mpk IV dose (modified sequence) ( Figure 1 and Figure 2 (and 18 nM (unmodified)) Figure 6 Compared to 4 doses of 25 mpk IV, 33-53 nMASO brain uptake one week later (modified sequence) Figure 3 (and 18 nM (unmodified)) Figure 6 Compared to previous results, 53-75% of MAPT in the brain was knocked down (with modified sequences) one week after four doses of 25mpk IV. Figure 4 (and 21% (unmodified)) Figure 7 Compared to 84% MAPT knockdown, brain uptake of 137 nM ASO was significantly reduced one week after eight doses of 25 mpk IV. Figure 3 and Figure 5 ).

[0285] In vivo methods method Mouse processing and tissue collectionTherapeutic treatment was administered peripherally to mice via intravenous (IV) tail vein injection (approximately 200 μL total volume). For live plasma collection, blood was collected via submental puncture and transferred to EDTA-coated tubes. The tubes were then centrifuged at 12,700 rpm for 7 min at 4°C, and the top plasma layer was collected. For tissue collection, animals were anesthetized with tribromoethanol, and whole blood was collected via cardiac puncture into EDTA-coated tubes for plasma drug concentration assessment. After transferring the whole blood to EDTA-coated tubes, the tubes were centrifuged at 12,700 rpm for 7 min at 4°C, and the top plasma layer was collected. Mice were then perfused with ice-cold PBS via the heart at a rate of 5 mL / min for 5 min. For biochemical analysis, tissues were collected, weighed, rapidly frozen on dry ice, and then stored at -80°C.

[0286] Intraventricular embolization (ICV) surgery The procedure was performed as described in DeVos SL (“Direct intraventricular delivery of drugs to the rodent central nervous system” J Vis Exp 2013 May 12:(75):e50326; which is incorporated herein by reference), and is briefly summarized below. The surgical area was sterilized with 70% ethanol in preparation for the operation. Mice were anesthetized with 4% isoflurane. The hair between the shoulders and eyes of the mice was shaved, and the mice were placed on a tactile surface. An incision was made from the base of the neck to the eyes at a maintenance level of 2% isoflurane. After cleaning with hydrogen peroxide, the needle was slowly inserted through the skull at a rate of 1 mm / s. After a 2–3 min period of sealing the brain around the needle, a dose of 10 µL ASO was administered at a rate of 1 µL / s. A swab was held against the skull at the base of the needle, and the needle was lifted at a rate of 1 mm / s. The swab was held at the injection site for 1 min to limit drug leakage. Following ICV injection, the incision was sutured and treated with antibiotic ointment. The mice were then transferred to a heated recovery pad and observed for complete recovery. The mice were monitored daily post-surgery for pain, discomfort, or infection.

[0287] Tissue homogenization for drug concentration measurement and protein assayWeighed frozen tissue samples were processed for biochemical assays by adding 10 volumes of cooled 1% NP40 + PBS homogenization buffer containing added cOmplete protease inhibitor (Roche #04693132001) and PhosStop (Roche 04906837001) phosphatase inhibitor. Samples were homogenized using 3 mm tungsten carbide beads in 1.5 mL Eppendorf tubes and vortexed (2 × 3 min, 27 Hz) using a Qiagen TISsueLyzer II (catalog number / ID: 85300). For protein assays, samples were then centrifuged at 17000 × g for 15 min, and the supernatant was collected for assays.

[0288] Tissue homogenization for RNA measurements Weighed frozen tissue samples were processed for RNA assay by adding 10 volumes of Qiazol reagent. Samples were homogenized using 5 mm tungsten carbide beads in 2 mL Eppendorf tubes and vortexed (2 × 3 min, 27 Hz) using a Qiagen TisseLyzer II (catalog number / ID: 85300). After dissolution, the samples were incubated at room temperature for 5 min, followed by the addition of chloroform. The samples were vortexed, incubated at room temperature for 3 min, and then centrifuged at 12000 × g for 15 min at 4 °C. The aqueous phase was then isolated. RNA was then isolated by adding isopropanol, vortexing, incubating at room temperature for 10 min, and then centrifuging at 12000 × g for 10 min at 4 °C. The resulting pellets were then resuspended in 75% ethanol, vortexed, and centrifuged at 7500 × g for 5 min at 4 °C. The final pellets were resuspended in water.

[0289] huIgG assayThe quantification of humanized antibodies in mouse plasma and tissue lysates was measured using a universal electrochemiluminescence immunoassay (ECLIA). Briefly, biotinylated goat anti-human IgG polyclonal primary antibody (Southern Biotech, Birmingham, AL) at a working concentration prepared in the assay diluent was added to the wells of a streptavidin-coated microtiter plate (Meso Scale Discovery, Rockville, MD) of MSD GOLD 96-well plate and incubated for approximately 1 hour. Following this incubation and plate washing step, the prepared test sample (with sample pre-dilution where appropriate) and relevant standards were added to the assay plate and incubated for approximately 1 hour. After the test sample incubation and plate washing step, a working concentration of secondary ruthenium-plated (SULFO-TAG) goat anti-human IgG antibody (Meso Scale Discovery, Rockville, MD) in the assay diluent was added to the assay plate and incubated for approximately 1 hour. After plate washing, 1× MSD ReadBuffer T (Meso Scale Discovery, Rockville, MD) was added to generate an electrochemiluminescence (ECL) measurement signal, which was then expressed in ECL units (ECLU). All assay reaction steps were performed at ambient temperature and oscillated on a plate shaker (where appropriate); and all test samples were pre-diluted 1:20 with the assay MRD before analysis in the assay plate. The sample ECLU signals generated during the assay were then processed into concentrations by back-calculating the assay calibration (CS) curve. The assay CS curve was fitted with a weighted four-parameter nonlinear logistic regression to calculate the concentrations of unknown / test samples.

[0290] Total ASO determinationTotal ASO (conjugated and free forms) in mouse plasma and tissue homogenates was quantified using hybridization-based electrochemiluminescence immunoassay (ECLIA). Briefly, a working concentration of a custom biotinylated and digoxigenin-conjugated antisense probe (synthesized by Integrated DNA Technologies, Coralville, IA) was combined with a test sample (with sample pre-dilution, where appropriate) prepared in TE buffer (10 mM Tris-HCl containing 1 mM EDTA) and relevant standards. The prepared sample in TE buffer was added 1:1 to 1× SSC buffer (Sigma-Aldrich, St. Luois, MO) containing a working concentration of recombinant proteinase K (ThermoFisher, Waltham, MA). The hybridization / enzyme mixture was then digested, denatured, bound, and cooled in a thermal cycler. After incubation of the hybrid product, samples were added to the wells of a streptavidin-coated microtiter plate (Meso Scale Discovery, Rockville, MD) in 96 wells of an MSD GOLD microtiter plate and incubated for approximately 30 min. Following incubation and plate washing, a working concentration of secondary ruthenium-modified (SULFO-TAG) sheep anti-digoxin antibody (Novus Biologicals, Littleton, CO) in the assay diluent was added to the plate and incubated for approximately 30 min. After plate washing, 1× MSD Read Buffer T (Meso Scale Discovery, Rockville, MD) was added to generate an electrochemiluminescence (ECL) assay signal, which is then expressed in ECL units (ECLU). All assay steps were performed at ambient temperature and oscillated on a plate shaker (where appropriate); and all test samples were pre-diluted 1:20 with the assay MRD before analysis in the assay plate. The sample ECLU signals generated during the assay were then processed into concentrations by back-calculating the assay calibration (CS) curve. The CS curve was fitted using a weighted four-parameter nonlinear logistic regression to calculate the concentration of unknown / test samples.

[0291] qPCR analysisTo evaluate target mRNA levels, qRT-PCR was performed on RNA extracted from tissue lysates. Target mRNA levels were evaluated using Taqman probes (hMAPT, mGapdh) and the Express One-Step kit. For each sample, hMAPT mRNA levels were normalized relative to the housekeeping gene Gapdh. qRT-PCR was performed using a QuantStudio 6 Flex system (Applied Biosystems), and the mean CT value for each probe was measured using a double-replica technique. The delta delta CT (ΔΔCT) value relative to the non-ASO treatment group was then calculated and plotted as a relative expression level.

[0292] Example 10. Soft spot identification and measurement To determine the catabolism and biotransformation of oligonucleotides (ASOs) and to further characterize the oligonucleotides and oligonucleotide peptide conjugates described herein, a stability assay must be developed to identify potential catabolism sites (cleavage sites) of ASOs (referred to herein as “soft spots”). The catabolism assays detailed below will be used to identify soft spots of potential modifications in order to design and deliver more stable molecules. For example, a previous in vitro assay for oligonucleotides was described by Basiri et al. in Molecular Therapy: Nucleic Acids; 21:725-736 (2020).

[0293] Tissue homogenization and incubation—Frozen mouse livers were homogenized at a tissue concentration of 200 mg / ml in PBS buffer (pH 7.4) containing 1% NP-40. 1–2 mM ASO was incorporated into 200 μL of the homogenate, and the mixture was incubated at 37°C for 48 hours (with shaking at 300 g).

[0294] Sample preparation—Add 200 mL of 10% phosphate to 200 mL of incubated tissue homogenate and vortex for 5 min. Add 600 mL of Clarity OTX dissolution loading buffer (Phenomenex, PN AL0-8579) to each tube and vortex for 5 min, then centrifuge at 3200 rpm for 10 min at 4 °C. Use Clarity OTX SPE plates. Table 5 shows the detailed procedure.

[0295] Table 7. Sample Preparation

[0296] Sample analysis was performed using liquid chromatography and mass spectrometry.

[0297] Liquid chromatography (LC): ASO separation was performed using a Waters BEH oligonucleotide 2.1 × 50 mm column at 70°C via ion-pairing chromatography. Mobile phase buffers A and B were water and a mixture of 100 mM hexafluoroisopropanol (HFIP) and 15 mM N,N-diisopropylethylamine (DIEA). Chromatography was performed at 0.3 mL / min under the following gradient conditions (min-%B): 0-5, 1-5, 6-50, 6.1-95, 7.1-95, 7.2-5, 10-5. The total run time was 10 min, and the LC output was transferred to waste at 0-1 min and 7-10 min.

[0298] Mass spectrometry (MS): High-resolution MS using IDA was employed to identify catabolites. Identified soft spots—The table below shows the soft spots identified according to the methods described herein. Once one or more soft spots were identified, the associated internucleotide bonds were replaced with PS2, MsPA, OiPS, or PN groups. More than one internucleotide bond adjacent to a soft spot may be replaced. To improve the stability of ASO, the procedure may be repeated as needed after stability testing.

[0299] Table 8. Soft spots identified in exemplary ASO sequences

[0300] Example 11. In vivo plasma pharmacokinetics The ASO SEQ ID NO:111 and 112 are further modified as shown in Table 9 below.

[0301] Table 9. Stabilized ASO sequences

[0302] To evaluate the pharmacokinetics of the additional oligonucleotide peptide conjugates (see Table 7B), the additional conjugates were administered intravenously to Sprague Dawley rats at a concentration of 10 mg / kg, as described above. All oligonucleotide peptide conjugates were administered at a dose volume of 2 mL / kg. Plasma was collected at 0.25, 4, 24, 48, and 72 h post-injection. The concentrations of huIgG and ASO in the plasma were measured as described above.

[0303] The results are shown in Table 10. These results demonstrate that adding stabilizing modifications at various locations and in various forms consistently improves huIgG clearance, and that modifying soft spots or nearby areas with backbone modifications can modulate ASO clearance by protecting or shifting cleavage sites.

[0304] Table 10. Rat PK data including ASO CL and IgG CL (both conjugated to the S239C site).

[0305] A single dose (10 μL total volume) was injected per mouse via intraventricular (ICV) into hTau mice (mTau mice). - / - ASO (unconjugated) was administered. Brain tissue was collected to measure relative MAPT RNA levels. Results demonstrating significant knockdown of MAPT expression across the tested ASO are shown in [the table / image / etc.]. Figure 12 middle.

Claims

1. A microtubule-associated protein tau (MAPT) antisense oligonucleotide (ASO) conjugate, said MAPT ASO conjugate comprising an Fc polypeptide dimer targeting the transferrin receptor (TfR) and MAPT ASO, wherein: (a) The TfR-targeting Fc polypeptide dimer contains (i) First Fc polypeptide; (ii) A second Fc polypeptide, wherein the second Fc polypeptide comprises a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) and comprises a sequence having at least 90% sequence identity with SEQ ID NO:12, wherein the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide. (b) The MAPT ASO comprises: 5' A L x d m C L x e m C L x f TTAp a Ap b Gp c TATTACTx g T L x h G L x i m C L 3' (SEQ ID NO: 111), or 5′ C L x d T L x e Gx f Tp j Tp k Ap l Gp m A*C*A*T*T*Cp n Ap o T*Tx g C L x h T L x i C L 3′ (SEQ ID NO:112) in A L , m C L G L and T L These are adenine-locked nucleoside, 5-methylcytosine-locked nucleoside, guanine-locked nucleoside, and thymine-locked nucleoside; A, C, G and T are deoxyadenosine nucleoside, deoxycytidine nucleoside or 5-methyldeoxycytidine nucleoside, deoxyguanosine nucleoside and deoxythymidine nucleoside, respectively; Each p is independently a phosphate thioester (PS) nucleoside linker or a stabilized nucleoside linker; Each x is independently a PS nucleoside link, a phosphodiester (PO) nucleoside link, or a stabilized nucleoside link; and Any nucleoside linkage that is not a stabilizing nucleoside linkage is a PS nucleoside linkage or a phosphodiester nucleoside linkage; and The first Fc polypeptide is linked to the MAPT ASO.

2. The MAPT ASO conjugate of claim 1, wherein the stabilized internucleotide linking is independently selected from the group consisting of: dithiophosphate (PS2) internucleotide linking, phosphoryl guanidine (PN) internucleotide linking, methanesulfonyl aminophosphate (MsPA) internucleotide linking, and O-isopropyl thiophosphate (OiPS) internucleotide linking.

3. The MAPT ASO conjugate of claim 1, wherein each p and x is a PS nucleoside linker.

4. The MAPT ASO conjugate as described in claim 1 or 2, wherein... (a) x g x h and x i Each is a PS nucleotide linker; (b) x g For PS nucleoside linkage, and x h and x i Each is an internucleotide linker of PN; (c) x g x h and x i Each is an internucleotide linker of PN; (d) x g and x h Each is linked between PN nucleosides, and x i PO is linked between nucleosides; (e) x g and x i Each is linked between PN nucleosides, and x h For PO nucleoside linkage; or (f) x h and x i Each is linked between PN nucleosides, and x g It is a PO nucleoside linkage.

5. The MAPT ASO conjugate according to any one of claims 1, 2, and 4, wherein: (a) x d x e and x f Each is a PS nucleotide linker; (b) x d and x e It is a PN nucleoside linkage, and x f For PS nucleoside linkage; (c) x d For PS nucleoside linkage, and x e and x f Each is an internucleotide linker of PN; (d) x d It is a PN nucleoside linkage, and x e and x f Each is a PS nucleotide linker; (e) x d x e and x f Each is an internucleotide linker of PN; (d) x d and x e Each is linked between PN nucleosides, and x f PO is linked between nucleosides; (e) x d and x f Each is linked between PN nucleosides, and x e For PO nucleoside linkage; or (f) x e and x f Each is linked between PN nucleosides, and x d It is a PO nucleoside linkage.

6. The MAPT ASO conjugate as described in claim 4 or 5, wherein: (a) x d x e x f x g x h and x i Each is a PS nucleotide interlink (b) x d x e and x f Each is a PS nucleotide linker, x g For PS nucleoside linkage, and x h and x i Each is an internucleotide linker of PN; (c) x d x e and x f Each is a PS nucleotide linker, and x g x h and x i Each is an internucleotide linker of PN; (d) x d and x e For PN nucleoside linkage, x f For PS nucleoside linkage, and x g x h and x i Each is a PS nucleotide linker; (e) x d and x e For PN nucleoside linkage, x f For PS nucleoside linkage, x g For PS nucleoside linkage, and x h and x i Each is an internucleotide linker of PN; (f) x d For PS nucleoside linkage, x e and x f Each is a PN nucleoside linker, x g For PS nucleoside linkage, and x h and x i Each is an internucleotide linker of PN; (g) x d It is a PN nucleoside linkage, and x e and x f Each is a PS nucleotide linker, x g For PS nucleoside linkage, and x h and x i Each is an internucleotide linker of PN; (h) x d x e x f x g x h and x i Each is an internucleotide linker of PN; (i) x d x e and x f Each is a PN nucleoside linker, x g and x h Each is linked between PN nucleosides, and x i PO is linked between nucleosides; (j) x d x e and x f Each is a PN nucleoside linker, x g and x i Each is linked between PN nucleosides, and x h PO is linked between nucleosides; (k) x d x e and x f Each is a PN nucleoside linker, x h and x i Each is linked between PN nucleosides, and x g PO is linked between nucleosides; (l) x d and x e Each is linked between PN nucleosides, and x f For PO nucleoside linkage, and x g x h and x i Each is an internucleotide linker of PN; (m) x d and x f Each is linked between PN nucleosides, and x e For PO nucleoside linkage, and x g x h and x i Each is linked between PN nucleosides; or (n) x e and x f Each is linked between PN nucleosides, and x d For PO nucleoside linkage, and x g x h and x i Each is linked between PN nucleosides.

7. The MAPT ASO conjugate according to any one of claims 1-2 and 4-6, wherein the MAPT ASO comprises SEQ ID NO: 111, and (a) p a p b and p c For PS nucleoside linkage; (b) p a To stabilize the internucleotide bond, and p b and p c For PS nucleoside linkage; (c) p b To stabilize the internucleotide bond, and p a and p c For PS nucleoside linkage; (d) p a and p b To stabilize the internucleotide bond, and p c For PS nucleoside linkage; (e) p b and p c To stabilize the internucleotide bond, and p a For PS nucleoside linkage; or (f) p a p b and p c To stabilize the internucleotide bonds.

8. The MAPT ASO conjugate as described in claim 7, wherein... (a) p a For PS2 key binding, and p b and p c For PS nucleoside linkage; (b) p b For PS2 key binding, and p a and p c For PS nucleoside linkage; (c) p b It is a PN bond, and p a and p c For PS nucleoside linkage; (d) p a and p b It is a PN nucleoside linkage, and p c For PS nucleoside linkage; (e) p a and p b For PS2 nucleoside linkage, and p c For PS nucleoside linkage; (f) p a and p b For MsPA nucleoside linkage, and p c For PS nucleoside linkage; (g) p a and p b It is an internucleotide linking of OiPS, and p c For PS nucleoside linkage; (h) p b and p c It is a PN nucleoside linkage, and p a For PS nucleoside linkage; (i) p a p b and p c For PN nucleoside linkage; or (j) p a p b and p c This is a nucleoside linker of MsPA.

9. The MAPT ASO conjugate of claim 1, wherein the MAPT ASO comprises the following oligonucleotides: (a) A L * m C L * m C L *T*T*A*A*G*T*A*T*T*A* m C*T*T L *G L * m C L ; (b) A L * m C L * m C L *T*T*A$A$G*T*A*T*T*A*C*T*T* L G L * m C L ; (c) A L * m C L * m C L *T*T*AnAnGnT*A*T*T*A*C*T*T L *G L * m C L ; (d) A L * m C L * m C L *T*T*A*AnGnT*A*T*T*A*C*T*T L nG L n m C L ; (e) A L * m C L * m C L *T*T*AnAnGnT*A*T*T*A*C*T*T L nG L n m C L ; (f) A L * m C L * m C L *T*T*AuAuGuT*A*T*T*A*C*TnT L nG L n m C L ; (g) A L n m C L * m C L *T*T*AnAnG*T*A*T*T*A*C*T*T L nG L n m C L ; (h) A L * m C L n m C L nT*T*AnAnG*T*A*T*T*A*C*T*T L nG L n m C L ; (i) A L n m C L n m C L *T*T*AnAnG*T*A*T*T*A*C*T*T L *G L * m C L ; (j) A L n m C L n m C L *T*T*A*AnGnT*A*T*T*A*C*T*T L *G L * m C L ; (k) A L n m C L n m C L *T*T*A*AnG*T*A*T*T*A*C*T*T L nG L n m C L ; (l) A L n m C L n m C L *T*T*AuAuG*T*A*T*T*A*C*T*T L nG L n m C L ; (m) A L n m C L n m C L *T*T*AtAtG*T*A*T*T*A*C*T*T L nG L n m C L ; (n) A L n m C L n m C L *T*T*A*AuGuT*A*T*T*A*C*T*T L nG L n m C L ; (o) A L n m C L n m C L nT*T*A*A*G*T*A*T*T*A*C*TnT L nG L n m C L ; (p) A L n m C L n m C L nT*T*AtA*G*T*A*T*T*A*C*TnT L nG L n m C L ; (q) A L n m C L n m C L nT*T*A*AtG*T*A*T*T*A*C*TnT L nG L n m C L ; (r) A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L ; (s) A L n m C L n m C L nT*T*AoAoG*T*A*T*T*A*C*TnT L nG L n m C L ; (t) A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TnT L nG L P m C L ; (u) A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TnT L PG L n m C L ; (v) A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TPT L nG L n m C L ; (w) A L n m C L n m CPT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L ; (x) A L n m C L P m CnT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L ; or (y) A L P m C L n m CnT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L ; in: A L It is adenosine monophosphate; m C L It is a 5-methylcytosine-locked nucleoside; T L It is thymine-locked nucleoside; G L It is guanine nucleoside; A represents deoxyadenosine; C is deoxycytidine; m C is 5-methyldeoxycytidine; T stands for deoxythymidine; G stands for deoxyguanosine; * indicates a PS nucleotide inter-linking; n represents the inter-nucleotide linkage between PN nucleotides; t represents the internucleotide linkage of PS2; u represents the internucleotide linkage of MsPA; o represents the internucleotide linking of OiPS; and P represents the PO nucleotide inter-bonding.

10. The MAPT ASO conjugate according to any one of claims 1-2 and 4-6, wherein the MAPT ASO comprises SEQ ID NO: 112, and (a) p k p l p n and p o To stabilize the internucleotide bonds; (b) p k p l p n and p o To stabilize the internucleotide bond, and p j and p m For PS nucleoside linkage; (c) p j p l and p o To stabilize the internucleotide bonds; (d) p j p l and p o To stabilize the internucleotide bond, and p k p m and p n For PS nucleoside linkage; (e) p l p m p n and p o To stabilize internucleotide bonds; or (d) p l p m p n and p o To stabilize the internucleotide bond, and p j and p k It is a PS nucleoside linkage.

11. The MAPT ASO conjugate of claim 10, wherein... (a) p k p l p n and p o For PN nucleoside linkage; (b) p k p l p n and p o It is a PN nucleoside linkage, and p j and p m For PS nucleoside linkage; (c) p j It is a PN bond, and p l and p o For PS2 nucleoside linkage; (d) p j For PN bonding, p l and p o For PS2 nucleoside linkage, and p k p m and p n For PS nucleoside linkage; (e) p l and p m For PS2 nucleoside linkage, and p n and p o For PN nucleoside linkage; or (d) p l and p m For PS2 nucleoside inter-linking, p n and p o It is a PN nucleoside linkage, and p j and p k It is a PS nucleoside linkage.

12. The MAPT ASO conjugate of claim 1, wherein the MAPT ASO comprises the following oligonucleotides: (a) m C L nT L nG L *T*TnAnG*A*C*A*T*T*CnAnT*T* m C L *T L * m C L ; (b) m C L *T L *G L *T*TnAnG*A*C*A*T*T*CnAnT*T* m C L nT L n m C L ; (c) m C L nT L nG L nTnT*AtG*A*C*A*T*T*C*AtT*Tn m C L nT L n m C L ; or (d) m C L nT L nG L *T*T*AtGtA*C*A*T*T*CnAnT*T* m C L nT L n m C L in: m C L It is a 5-methylcytosine-locked nucleoside; T L It is thymine-locked nucleoside; G L It is guanosine-locked nucleoside; A represents deoxyadenosine; C is deoxycytidine; T stands for deoxythymidine; G stands for deoxyguanosine; * indicates a PS nucleotide inter-linking; n represents the internucleotide link between PN; and t represents the internucleotide linkage of PS2.

13. The MAPT ASO conjugate according to any one of claims 1-12, wherein the modified constant domain specifically binding to TfR comprises glutamic acid at position 380, Y at position 384, T at position 386, glutamic acid at position 387, tryptophan at position 388, alanine at position 389, N at position 390, T at position 413, glutamic acid at position 415, glutamic acid at position 416, and phenylalanine at position 421.

14. The MAPT ASO conjugate of any one of claims 1-13, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises at least one cysteine ​​substitution.

15. The MAPT ASO conjugate of claim 14, wherein at least one cysteine ​​substitution is selected from the group consisting of S239C substitution, S442C substitution, A330C substitution, K149C substitution or T289C substitution.

16. The MAPT ASO conjugate of claim 15, wherein the first Fc polypeptide comprises at least one cysteine ​​substitution.

17. The MAPT ASO of any one of claims 1-16, wherein the MAPT ASO comprises a 5' terminal group selected from the group consisting of: phosphodiester group, thiophosphate group, dithiophosphate group, methanesulfonylaminophosphate group, cyclophosphoguanine group, and O -Isopropyl thiophosphate group.

18. The MAPT ASO conjugate according to any one of claims 14-17, wherein the Fc dimer is connected to the MAPT ASO via a linker attached to the at least one cysteine ​​substitution and the 5' end of the MAPT ASO, wherein the linker is ， The valence state marked with * is attached to the Fc polypeptide dimer targeting TfR, and the valence state marked with ** is attached to the MAPT ASO.

19. The MAPT ASO conjugate of any one of claims 1-18, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprises mutations that regulate the function of one or more effectors.

20. The MAPT ASO conjugate of claim 19, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises alanine at position 234, alanine at position 235, and serine or glycine at position 329, respectively, according to EU designation.

21. The MAPT ASO conjugate of any one of claims 1-20, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprises one or more mutations that increase serum stability.

22. The MAPT ASO conjugate according to any one of claims 1-21, wherein the second Fc polypeptide further comprises tryptophan at position 366 according to EU number.

23. The MAPT ASO conjugate of claim 22, wherein the first Fc polypeptide further comprises serine at position 366, alanine at position 368, and valine at position 407 according to EU numbering.

24. The MAPT ASO conjugate according to any one of claims 1-21, wherein the second Fc polypeptide comprises serine at position 366, alanine at position 368, and valine at position 407 according to EU numbering.

25. The MAPT ASO conjugate of claim 24, wherein the first Fc polypeptide further comprises tryptophan at position 366 according to EU number.

26. The MAPT ASO conjugate according to any one of claims 1-12, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1, 47-48, 51-54, 61-63, 66, 73-78, 80 and 90; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 4-29, 36, 43-44, 49 and 50.

27. The MAPT ASO conjugate of claim 26, wherein the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 63 or 80; and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12.

28. The MAPT ASO conjugate of any one of claims 1-27, wherein the first Fc polypeptide is fused to a first untargeted Fab (NTF) via a first hinge region to form a first Fab-Fc fusion polypeptide.

29. The MAPT ASO conjugate of any one of claims 1-27, wherein the second Fc polypeptide is fused to the second NTF via the second hinge region to form the second Fab-Fc fusion polypeptide.

30. The MAPT ASO conjugate of any one of claims 1-27, wherein the first Fc polypeptide is fused with an NTF via a first hinge region to form a first Fab-Fc fusion polypeptide, and the second Fc polypeptide is fused with a second NTF via a second hinge region to form a second Fab-Fc fusion polypeptide.

31. The MAPT ASO conjugate of any one of claims 28-30, wherein the first Fab-Fc fusion polypeptide and / or the second Fab-Fc fusion polypeptide comprises at least one cysteine ​​substitution, optionally wherein the cysteine ​​substitution is selected from the group consisting of: position 114 of the heavy chain (according to Kabat number), position 124 of the heavy chain (according to EU number); position 149 of the light chain (according to EU number), or position 156 of the light chain (according to EU number).

32. The MAPT ASO conjugate of claim 30 or 31, wherein the first NTF and the second NTF each comprise a heavy chain containing SEQ ID NO: 109 or 130 and a light chain containing SEQ ID NO: 108 or 129; optionally, wherein the heavy chain further comprises a hinge region containing SEQ ID NO: 91, 92, 93 or 121.

33. The MAPT ASO conjugate of claim 30 or 31, wherein the first Fab-Fc fusion polypeptide comprises SEQ ID NO: 100 or 101, and the second Fab-Fc fusion polypeptide comprises SEQ ID NO: 98 or 99.

34. A MAPT ASO conjugate comprising a TfR-targeting Fc polypeptide dimer and at least one MAPT ASO, wherein: (a) The TfR-targeting Fc polypeptide dimer contains (i) First Fc polypeptide, (ii) A second Fc polypeptide, wherein the second Fc polypeptide comprises alanine at position 234, alanine at position 235, and serine at position 329 according to EU numbering, and a modified constant domain that specifically binds to human transferrin receptor 1 (TfR), and comprises a sequence having at least 90% sequence identity with SEQ ID NO:12, wherein the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide. (iii) A first untargeted Fab (NTF) is formed by fusing with the first Fc peptide via a first hinge region to form a first Fab-Fc fusion peptide, and a second NTF is formed by fusing with the second Fc peptide via a second hinge region to form a second Fab-Fc fusion peptide. (b) The at least one MAPT ASO includes 5' A L x d m C L x e m C L x f TTAp a Ap b Gp c TATTACTx g T L x h G L x i m C L 3' (SEQ ID NO: 111), or 5′ C L x d T L x e Gx f Tp j Tp k Ap l Gp m A*C*A*T*T*Cp n Ap o T*Tx g C L x h T L x i C L 3′ (SEQ ID NO:112) in A L , m C L G L and T L These are adenine-locked nucleoside, 5-methylcytosine-locked nucleoside, guanine-locked nucleoside, and thymine-locked nucleoside; A, C, G and T are deoxyadenosine nucleoside, deoxycytidine nucleoside or 5-methyldeoxycytidine nucleoside, deoxyguanosine nucleoside and deoxythymidine nucleoside, respectively; Each p is independently a phosphate thioester (PS) nucleoside linker or a stabilized nucleoside linker; Each x is independently a PS nucleoside link, a phosphodiester (PO) nucleoside link, or a stabilized nucleoside link; and Any nucleoside linkage that is not a stabilizing nucleoside linkage is a PS nucleoside linkage or a phosphodiester nucleoside linkage; and The first Fab-Fc fusion peptide and / or the second Fab-Fc fusion peptide contain at least one cysteine ​​substitution, and The at least one MAPT ASO is conjugated to the at least one cysteine ​​substitute via a linker to the first Fab-Fc fusion peptide and / or the second Fab-Fc fusion peptide.

35. A MAPT ASO, said MAPT ASO comprising 5' A L x d m C L x e m C L x f TTAp a Ap b Gp c TATTACTx g T L x h G L x i m C L 3' (SEQ ID NO: 111), or 5′ C L x d T L x e Gx f Tp j Tp k Ap l Gp m A*C*A*T*T*Cp n Ap o T*Tx g C L x h T L x i C L 3′ (SEQ ID NO:112) in A L , m C L G L and T L These are adenine-locked nucleoside, 5-methylcytosine-locked nucleoside, guanine-locked nucleoside, and thymine-locked nucleoside; A, C, G and T are deoxyadenosine nucleoside, deoxycytidine nucleoside or 5-methyldeoxycytidine nucleoside, deoxyguanosine nucleoside and deoxythymidine nucleoside, respectively; Each p is independently a phosphate thioester (PS) nucleoside linker or a stabilized nucleoside linker; Each x is independently a PS nucleoside link, a phosphodiester (PO) nucleoside link, or a stabilized nucleoside link; and Any nucleoside linkage that is not a stabilizing nucleoside linkage is a PS nucleoside linkage or a phosphodiester nucleoside linkage, and The first Fc polypeptide is linked to the MAPT ASO.

36. The MAPT ASO of claim 29, wherein the stabilized internucleotide linking is independently selected from the group consisting of: dithiophosphate (PS2) internucleotide linking, phosphoryl guanidine (PN) internucleotide linking, methanesulfonyl aminophosphate (MsPA) internucleotide linking, and O-isopropyl thiophosphate (OiPS) internucleotide linking.

37. The MAPT ASO of claim 35, wherein each p and x is a PS nucleoside linker.

38. The MAPT ASO as described in claim 35 or 36, wherein... (a) x g x h and x i Each is a PS nucleotide linker; (b) x g For PS nucleoside linkage, and x h and x i Each is an internucleotide linker of PN; (c) x g x h xi and PN are each linked by an internucleotide bond; (d) x g and x h Each is linked between PN nucleosides, and x i PO is linked between nucleosides; (e) x g and x i Each is linked between PN nucleosides, and x h For PO nucleoside linkage; or (f) x h and x i Each is linked between PN nucleosides, and x g It is a PO nucleoside linkage.

39. MAPT ASO as claimed in any one of claims 35, 36, and 38, wherein: (a) x d x e and x f Each is a PS nucleotide linker; (b) x d and x e It is a PN nucleoside linkage, and x f For PS nucleoside linkage; (c) x d For PS nucleoside linkage, and x e and x f Each is an internucleotide linker of PN; (d) x d It is a PN nucleoside linkage, and x e and x f Each is a PS nucleotide linker; (e) x d x e and x f Each is an internucleotide linker of PN; (d) x d and x e Each is linked between PN nucleosides, and x f PO is linked between nucleosides; (e) x d and x f Each is linked between PN nucleosides, and x e For PO nucleoside linkage; or (f) x e and x f Each is linked between PN nucleosides, and x d It is a PO nucleoside linkage.

40. The MAPT ASO as described in claim 38 or 39, wherein: (a) x d x e x f x g x h and x i Each is a PS nucleotide interlink (b) x d x e and x f Each is a PS nucleotide linker, x g For PS nucleoside linkage, and x h and x i Each is an internucleotide linker of PN; (c) x d x e and x f Each is a PS nucleotide linker, and x g x h and x i Each is an internucleotide linker of PN; (d) x d and x e For PN nucleoside linkage, x f For PS nucleoside linkage, and x g x h and x i Each is a PS nucleotide linker; (e) x d and x e For PN nucleoside linkage, x f For PS nucleoside linkage, x g For PS nucleoside linkage, and x h and x i Each is an internucleotide linker of PN; (f) x d For PS nucleoside linkage, x e and x f Each is a PN nucleoside linker, x g For PS nucleoside linkage, and x h and x i Each is an internucleotide linker of PN; (g) x d It is a PN nucleoside linkage, and x e and x f Each is a PS nucleotide linker, x g For PS nucleoside linkage, and x h and x i Each is an internucleotide linker of PN; (h) x d x e x f x g x h and x i Each is an internucleotide linker of PN; (i) x d x e and x f Each is a PN nucleoside linker, x g and x h Each is linked between PN nucleosides, and x i PO is linked between nucleosides; (j) x d x e and x f Each is a PN nucleoside linker, x g and x i Each is linked between PN nucleosides, and x h PO is linked between nucleosides; (k) x d x e and x f Each is a PN nucleoside linker, x h and x i Each is linked between PN nucleosides, and x g PO is linked between nucleosides; (l) x d and x e Each is linked between PN nucleosides, and x f For PO nucleoside linkage, and x g x h and x i Each is an internucleotide linker of PN; (m) x d and x f Each is linked between PN nucleosides, and x e For PO nucleoside linkage, and x g x h and x i Each is linked between PN nucleosides; or (n) x e and x f Each is linked between PN nucleosides, and x d For PO nucleoside linkage, and x g x h and x i Each is linked between PN nucleosides.

41. The MAPT ASO as claimed in any one of claims 35-36 and 38-40, wherein the MAPT ASO comprises SEQ ID NO: 111, and (a) p a p b and p c For PS nucleoside linkage; (b) p a To stabilize the internucleotide bond, and p b and p c For PS nucleoside linkage; (c) p b To stabilize the internucleotide bond, and p a and p c For PS nucleoside linkage; (d) p a and p b To stabilize the internucleotide bond, and p c For PS nucleoside linkage; (e) p b and p c To stabilize the internucleotide bond, and p a For PS nucleoside linkage; (f) p a p b and p c To stabilize the internucleotide bonds.

42. The MAPT ASO as claimed in any one of claims 41, wherein (a) p a For PS2 key binding, and p b and p c For PS nucleoside linkage; (b) p b For PS2 key binding, and p a and p c For PS nucleoside linkage; (c) p b It is a PN bond, and p a and p c For PS nucleoside linkage; (d) p a and p b It is a PN nucleoside linkage, and p c For PS nucleoside linkage; (e) p a and p b For PS2 nucleoside linkage, and p c For PS nucleoside linkage; (f) p a and p b For MsPA nucleoside linkage, and p c For PS nucleoside linkage; (g) p a and p b For OiPS nucleoside linkage, and p c For PS nucleoside linkage; (h) p b and p c It is a PN nucleoside linkage, and p a For PS nucleoside linkage; (i) p a p b and p c For PN nucleoside linkage; or (j) p a p b and p c This is a nucleoside linker of MsPA.

43. The MAPT ASO of claim 35, wherein the MAPT ASO comprises the following oligonucleotides: (a) A L * m C L * m C L *T*T*A*A*G*T*A*T*T*A* m C*T*T L *G L * m C L ; (b) A L * m C L * m C L *T*T*A$A$G*T*A*T*T*A*C*T*T* L G L * m C L ; (c) A L * m C L * m C L *T*T*AnAnGnT*A*T*T*A*C*T*T L *G L * m C L ; (d) A L * m C L * m C L *T*T*A*AnGnT*A*T*T*A*C*T*T L nG L n m C L ; (e) A L * m C L * m C L *T*T*AnAnGnT*A*T*T*A*C*T*T L nG L n m C L ; (f) A L * m C L * m C L *T*T*AuAuGuT*A*T*T*A*C*TnT L nG L n m C L ; (g) A L n m C L * m C L *T*T*AnAnG*T*A*T*T*A*C*T*T L nG L n m C L ; (h) A L * m C L n m C L nT*T*AnAnG*T*A*T*T*A*C*T*T L nG L n m C L ; (i) A L n m C L n m C L *T*T*AnAnG*T*A*T*T*A*C*T*T L *G L * m C L ; (j) A L n m C L n m C L *T*T*A*AnGnT*A*T*T*A*C*T*T L *G L * m C L ; (k) A L n m C L n m C L *T*T*A*AnG*T*A*T*T*A*C*T*T L nG L n m C L ; (l) A L n m C L n m C L *T*T*AuAuG*T*A*T*T*A*C*T*T L nG L n m C L ; (m) A L n m C L n m C L *T*T*AtAtG*T*A*T*T*A*C*T*T L nG L n m C L ; (n) A L n m C L n m C L *T*T*A*AnGnT*A*T*T*A*C*T*T L nG L n m C L ; (o) A L n m C L n m C L nT*T*A*A*G*T*A*T*T*A*C*TnT L nG L n m C L ; (p) A L n m C L n m C L nT*T*AtA*G*T*A*T*T*A*C*TnT L nG L n m C L ; (q) A L n m C L n m C L nT*T*A*AtG*T*A*T*T*A*C*TnT L nG L n m C L ; (r) A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L ; (s) A L n m C L n m C L nT*T*AoAoG*T*A*T*T*A*C*TnT L nG L n m C L ; (t) A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TnT L nG L P m C L ; (u) A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TnT L PG L n m C L ; (v) A L n m C L n m C L nT*T*AtAtG*T*A*T*T*A*C*TPT L nG L n m C L ; (w) A L n m C L n m CPT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L ; (x) A L n m C L P m CnT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L ; or (y) A L P m C L n m CnT*T*AtAtG*T*A*T*T*A*C*TnT L nG L n m C L ; in: A L It is an adenosine-locked nucleic acid; m C L It is a 5-methylcytosine-locked nucleic acid; T L It is a thymidine-locked nucleic acid; G L It is a guanosine-locked nucleic acid; A represents deoxyadenosine; C is deoxycytidine; m C is 5-methyldeoxycytidine; T stands for deoxythymidine; G stands for deoxyguanosine; * indicates a PS nucleotide inter-linking; n represents the inter-nucleotide linkage between PN nucleotides; t represents the internucleotide linkage of PS2; u represents the internucleotide linkage of MsPA; o represents the internucleotide linking of OiPS; and P represents the PO nucleotide inter-bonding.

44. The MAPT ASO conjugate according to any one of claims 35-36 and 38-40, wherein the MAPT ASO comprises SEQ ID NO:112, and (a) p k p l p n and p o To stabilize the internucleotide bonds; (b) p k p l p n and p o To stabilize the internucleotide bond, and p j and p m For PS nucleoside linkage; (c) p j p l and p o To stabilize the internucleotide bonds; (d) p j p l and p o To stabilize the internucleotide bond, and p k p m and p n For PS nucleoside linkage; (e) p l p m p n and p o To stabilize internucleotide bonds; or (d) p l p m p n and p o To stabilize the internucleotide bond, and p j and p k It is a PS nucleoside linkage.

45. The MAPT ASO conjugate of claim 44, wherein... (a) p k p l p n and p o For PN nucleoside linkage; (b) p k p l p n and p o It is a PN nucleoside linkage, and p j and p m For PS nucleoside linkage; (c) p j It is a PN bond, and p l and p o For PS2 nucleoside linkage; (d) p j For PN bonding, p l and p o For PS2 nucleoside linkage, and p k p m and p n For PS nucleoside linkage; (e) p l and p m For PS2 nucleoside linkage, and p n and p o For PN nucleoside linkage; or (d) p l and p m For PS2 nucleoside inter-linking, p n and p o It is a PN nucleoside linkage, and p j and p k It is a PS nucleoside linkage.

46. ​​The MAPT ASO conjugate of claim 35, wherein the MAPT ASO comprises the following oligonucleotides: (a) m C L nT L nG L *T*TnAnG*A*C*A*T*T*CnAnT*T* m C L *T L m *C L ; (b) m C L *T L *G L *T*TnAnG*A*C*A*T*T*CnAnT*T* m C L nT L n m C L ; (c) m C L nT L nG L nTnT*AtG*A*C*A*T*T*C*AtT*Tn m C L nT L n m C L ; or (d) m C L nT L nG L *T*T*AtGtA*C*A*T*T*CnAnT*T* m C L nT L n m C L in: m C L It is a 5-methylcytosine-locked nucleoside; T L It is thymine-locked nucleoside; G L It is guanosine-locked nucleoside; A represents deoxyadenosine; C is deoxycytidine; T stands for deoxythymidine; G stands for deoxyguanosine; * indicates a PS nucleotide inter-linking; n represents the internucleotide link between PN; and t represents the internucleotide linkage of PS2.

47. A pharmaceutical composition comprising the MAPT ASO conjugate of any one of claims 1-34 or the MAPT ASO of any one of claims 35-46, and a pharmaceutically acceptable carrier or excipient.

48. A method for generating neuronal cells with reduced tau expression, the method comprising delivering to the neuronal cells a MAPT ASO conjugate of any one of claims 1-34, a MAPT ASO of any one of claims 35-46, or a pharmaceutical composition of claim 47, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene in the neuronal cells.

49. A method for modifying neuronal cells to reduce tau expression, the method comprising delivering to the neuronal cells a MAPT ASO conjugate of any one of claims 1-34, a MAPTASO of any one of claims 35-46, or a pharmaceutical composition of claim 47, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene.

50. A method for modifying neuronal cells to reduce tau expression, the method comprising delivering to the neuronal cells a MAPT ASO conjugate of any one of claims 1-34, a MAPTASO of any one of claims 35-46, or a pharmaceutical composition of claim 47, wherein the MAPT ASO specifically reduces the expression level of the MAPT transcript in the cells.

51. The method of claim 50, wherein reducing the expression level of endogenous MAPT comprises specifically reducing the expression level of MAPT transcripts in the cells.

52. A method for reducing tau expression in spinal cord cells of a subject, the method comprising administering, by intrathecal administration, the MAPT ASO conjugate of any one of claims 1-34, the MAPTASO of any one of claims 35-46, or the pharmaceutical composition of claim 47.

53. A method for reducing tau expression in a subject, the method comprising administering to the subject the MAPT ASO conjugate of any one of claims 1-34, the MAPT ASO of any one of claims 35-46, or the pharmaceutical composition of claim 47.

54. The method of claim 53, wherein tau expression in the subject's CNS is reduced.

55. The method of claim 53, wherein the MAPT ASO conjugate, the MAPT ASO, or the pharmaceutical composition is administered to the subject via intrathecal administration, intravenous injection, or intravenous infusion.

56. The method of any one of claims 48-55, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene or the level of MAPT mRNA transcript by at least about 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the level at which the pharmaceutical composition was not administered.

57. The method of claim 56, wherein the expression of the endogenous MAPT gene or the level of the MAPT transcript is reduced by at least about 50%.

58. The method of claim 56, wherein the expression of the endogenous MAPT gene or the level of the MAPT transcript is reduced by at least about 70%.

59. A method for treating tau-related neurodegenerative disease in a human subject in need, the method comprising administering to the human subject the MAPT ASO conjugate of any one of claims 1-34, the MAPT ASO of any one of claims 35-46, or the pharmaceutical composition of claim 47.

60. The method of claim 59, wherein the tau-related neurodegenerative syndrome is Alzheimer's disease.

61. A method for treating Alzheimer's disease, the method comprising administering to a human subject in need the MAPT ASO conjugate of any one of claims 1-34, the MAPT ASO of any one of claims 35-46, or the pharmaceutical composition of claim 47.

62. A method for reducing the expression of MAPT messenger ribonucleic acid (mRNA) in a human subject in need, the method comprising administering to the human subject the MAPT ASO conjugate of any one of claims 1-34, the MAPT ASO of any one of claims 35-46, or the pharmaceutical composition of claim 47.

63. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is used to deliver MAPT ASO to the CNS of a human subject in need, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene.

64. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is used to treat tau-related neurodegenerative diseases in human subjects in need.

65. The pharmaceutical composition of claim 64, wherein the tau-related neurodegenerative condition is Alzheimer's disease.

66. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is used to reduce MAPT mRNA expression in human subjects in need.

67. A nucleic acid sequence encoding an Fc polypeptide dimer, said nucleic acid sequence comprising a first nucleic acid encoding any one of SEQ ID NO:1, 47-48, 51-54, 61-63, 66, 73-78, 80 and 90; and a second nucleic acid sequence encoding any one of SEQ ID NO:4-29, 36, 43-44 and 49-50.

68. The nucleic acid sequence of claim 67, wherein the first nucleic acid sequence encodes a first Fab-Fc fusion polypeptide, and the second nucleic acid sequence encodes a second Fab-Fc fusion polypeptide.

69. The nucleic acid sequence of claim 68, wherein the first nucleic acid sequence encodes any one of SEQ ID NO: 100-101 and 130-131; the second nucleic acid sequence encodes any one of SEQ ID NO: 98-99; and wherein the nucleic acid sequence further comprises a third nucleic acid sequence encoding SEQ ID NO: 108 or 129.

70. The nucleic acid sequence of claim 69, wherein the first nucleic acid sequence comprises SEQ ID NO:133 or a sequence having at least 75% identity with SEQ ID NO:133; the second nucleic acid sequence comprises SEQ ID NO:132 or a sequence having at least 75% identity with SEQ ID NO:132; and the third nucleic acid sequence comprises SEQ ID NO:134 or a sequence having at least 75% identity with SEQ ID NO:134.

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