Composition and method for regulating tau expression

Abstract

Microtubule-associated protein tau (MAPT) antisense oligonucleotide (ASO) and MAPT-ASO conjugates, and a method of using MAPT-ASO and MAPT-ASO conjugates to treat neurodegenerative disorders such as Alzheimer's disease are described.

Description

Technology Field

[0001] Cross-reference with related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 618,798 filed January 8, 2024, which is incorporated herein by reference.

[0003] Sequence list

[0004] The sequence list written in file DNL-038-03-WO_SeqListing.xml has a size of 237 kilobytes, was created on January 6, 2025, and is incorporated herein by reference. Background Technology

[0005] Alzheimer's disease is a progressive neurodegenerative disorder that is 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 in Alzheimer's disease and other neurodegenerative diseases is the abnormal aggregation and inclusion body formation of the microtubule-associated protein tau. Microtubule-associated protein tau encoding tau ( MAPT Mutations in the ) gene are associated with neurodegenerative tauopathy.

[0006] In vivo delivery of nucleic acid-based molecules, such as antisense oligonucleotides, often requires specific targeting to reach particular tissues or cell types. In particular, delivery to non-liver tissues remains a barrier and has limited the use of such therapies. Delivery of oligonucleotides to the central nervous system (CNS) presents distinct challenges due to the blood-brain barrier (BBB). One means of delivering oligonucleotides into the CNS is intrathecal delivery. However, intrathecal delivery is invasive, carries a higher risk of side effects, and often results in non-uniform distribution.

[0007] Therefore, there is a continuing need for new and improved methods to deliver nucleic acid-based therapeutics in vivo, particularly to CNS tissues.

[0008] The present invention describes a microtubule-associated protein tau (MAPT) antisense oligonucleotide (ASO) conjugate comprising at least one microtubule-associated protein tau (MAPT) antisense oligonucleotide (ASO) and a transferrin receptor (TfR)-targeting Fc polypeptide dimer, wherein the Fc polypeptide of the Fc polypeptide dimer is modified to bind to TfR. TfR is highly expressed at the blood-brain barrier (BBB), and TfR naturally transports transferrin from the blood into the brain. The described MAPT ASO conjugate binds to TfR, is transported 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 the attachment of MAPT ASO. 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 comprising a sequence that specifically binds to human transferrin receptor 1 (TfR) and has at least 70% sequence identity with SEQ ID NO. 12, and the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide.

[0009] Nucleic acid sequence: 5'-A L x d m C L x e m C L x f TTA p a A p b G p c TATTACT x g T L x h G L x im C L -3'(sequence number 111) or 5'- m C L x d T L x e G L x f T p j T p k A p l G p m ACATTC p n A p o TT x g m C L x h T L x i m C L A MAPT antisense oligonucleotide (ASO) containing -3' (SEQ No. 112) is described, wherein A L , m C L , G L and T L are adenine, 5-methylcytosine, guanine, and thymine lock nucleosides, respectively, and A, C, G, and T are deoxyadenosine, deoxycytidine, deoxyguanidine, and deoxythymidine nucleosides, respectively, and each p is independently a phosphorothioate (PS) nucleoside inter-nucleoside bond or a stabilized nucleoside inter-nucleoside bond, and (p) a , (p) b and (p) c At least two of them are not PS nucleoside bonds, and each xThe bonds are independently PS nucleoside bonds, phosphodiester (PO) nucleoside bonds, or stabilized nucleoside bonds, and any nucleoside bond that is not a stabilized nucleoside bond is a PS nucleoside bond or a phosphodiester nucleoside bond. The stabilized nucleoside bonds are independently selected from the group consisting of phosphorodithioate (PS2) nucleoside bonds, phosphorylguanidine (PN) nucleoside bonds, mesylphosphoramidate (MsPA) nucleoside bonds, and O-isopropylphosphorothioate (OiPS) nucleoside bonds.

[0010] In some embodiments, each p and x is a bond between PS nucleosides.

[0011] In some embodiments, x g , x h and x i Each is a PS nucleoside bond, and in some embodiments, x g is a PS internucleoside bond, and x h and x i Each is a PN nucleoside bond. In some embodiments, x g , x h and x i Each is a PN nucleoside bond. In some embodiments, x g and x h are respectively PN nucleoside interlinks, and x i is a PO nucleoside inter-nucleoside bond. In some embodiments, x g and x i are respectively PN nucleoside interlinks, and xh is a PO nucleoside inter-nucleoside bond. In some embodiments, x h and x i are respectively PN nucleoside interlinks, and x g is a PO nucleoside inter-nucleoside bond. In some embodiments, x h and x i is a PN internucleoside bond. In some embodiments, x g , x h and x i At least two of them are PN nucleoside bonds.

[0012] In some embodiments, x d , x e and x f Each is a PS nucleoside bond. In some embodiments, x d and x e is a PN internucleoside bond. In some embodiments, x d and x e is a PN internucleoside bond, and x f is a PS internucleoside bond. In some embodiments, x d is a PS internucleoside bond, and x e and x f Each is a PN nucleoside bond. In some embodiments, x d is a PN internucleoside bond, and x e and x f Each is a PS nucleoside bond. In some embodiments, x d ,x e and x f Each is a PN nucleoside bond. In some embodiments, x d and x e are respectively PN nucleoside interlinks, and x f is a PO nucleoside inter-nucleoside bond. In some embodiments, x d and x f are respectively PN nucleoside interlinks, and x e is a PO nucleoside inter-nucleoside bond. In some embodiments, x e and x f are respectively PN nucleoside interlinks, and x d PO is a bond between nucleosides.

[0013] In some embodiments, x d , x e , x f , x g , x h and x i Each is a PS nucleoside bond. In some embodiments, x d , x e and x f are each PS nucleoside bonds, and x g is a PS internucleoside bond, and x h and x i Each is a PN nucleoside bond. In some embodiments, x d , x e and x fare each PS nucleoside bonds, and x g , x h and x i Each is a PN nucleoside bond. In some embodiments, x d and x e is a PN internucleoside bond, and x f is a PS internucleoside bond, and x g , x h and x i Each is a PS nucleoside bond. In some embodiments, x d and x e is a PN internucleoside bond, and x f is a PS internucleoside bond, and x g is a PS internucleoside bond, and x h and x i Each is a PN nucleoside bond. In some embodiments, x d is a PS internucleoside bond, and x e and x f are respectively PN nucleoside interlinks, and x g is a PS internucleoside bond, and x h and x i Each is a PN nucleoside bond. In some embodiments, x d is a PN internucleoside bond, and x e and x f are each PS nucleoside bonds, and x gis a PS internucleoside bond, and x h and x i Each is a PN nucleoside bond. In some embodiments, x d , x e , x f , x g , x h and x i Each is a PN nucleoside bond. In some embodiments, x d , x e and x f are respectively PN nucleoside interlinks, and x g and x h are respectively PN nucleoside interlinks, and x i is a PO nucleoside inter-nucleoside bond. In some embodiments, x d , x e and x f are respectively PN nucleoside interlinks, and x g and x i are respectively PN nucleoside interlinks, and x h is a PO nucleoside inter-nucleoside bond. In some embodiments, x d , x e and x f are respectively PN nucleoside interlinks, and x h and x i are respectively PN nucleoside interlinks, and x g is a PO nucleoside inter-nucleoside bond. In some embodiments, xd and x e are respectively PN nucleoside interlinks, and x f is a PO internucleoside bond, and x g , x h and x i Each is a PN nucleoside bond. In some embodiments, x d and x f are respectively PN nucleoside interlinks, and x e is a PO internucleoside bond, and x g , x h and x i Each is a PN nucleoside bond. In some embodiments, x e and x f are respectively PN nucleoside interlinks, and x d is a PO internucleoside bond, and x g , x h and x i Each is a PN nucleoside bond. In some embodiments, x h and x i or x g , x h and x i is a PN internucleoside bond, and x d and x e or x d , x e and x f is a PN internucleoside bond.

[0014] In some embodiments, MAPT ASO comprises SEQ ID NO. 111 and (a) p a , p b and p c is a PS nucleoside bond, or (b) p a is a stabilized nucleoside-nucleoside bond, and p b and p c is a PS nucleoside bond, or (c) p b is a stabilized nucleoside-nucleoside bond, and p a and p c is a PS nucleoside bond, or (d) p a and p b is a stabilized internucleoside bond and p c is a PS nucleoside bond, or (e) p b and p c is a stabilized internucleoside bond and p a is a PS nucleoside bond, or (f) p a , p b and p c is a stabilized nucleoside bond, or (g) p a and p b is a PS2 nucleoside bond.

[0015] In some embodiments, MAPT ASO comprises SEQ ID NO. 111, and (a) p a It is a PS2 combination, and p b and p c is a PS nucleoside bond, or (b) pb It is a PS2 combination, and p a and p c is a PS nucleoside bond, or (c) p b is a PN bond, and p a and p c is a PS nucleoside bond, or (d) p a and p b is a PN internucleoside bond and p c is a PS nucleoside bond, or (e) p a and p b is a PS2 internucleoside bond and p c is a PS nucleoside bond, or (f) p a and p b is an MsPA internucleoside bond and p c is a PS nucleoside bond, or (g) p a and p b is an OiPS internucleoside bond and p c is a PS nucleoside bond, or (h) p b and p c is a PN internucleoside bond and p a is a PS nucleoside bond, or (i) p a , p b and p c is a PN internucleoside bond, or (j) p a , p b and p cis a MsPA internucleoside bond.

[0016] In some embodiments, MAPT ASO comprises SEQ ID NO. 111, and p a and p b is a PS2 nucleoside interlink. 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 is a PN internucleoside bond, and x d and x e or x d , x e and x f is a PN internucleoside bond and p a and p b is a PS2 nucleoside bond, or (b) x d , x e , x h and x i is a PN internucleoside bond and p a and p b is a PS2 nucleoside interlink, or (c) x d , x e , x f , x g , x h and x i is a PN internucleoside bond and p a and pb is a PS2 nucleoside interlink, or (d) x d , x e , x h and x i is a PN internucleoside bond and p a and p b is a PS2 nucleoside bond, and all other nucleoside bonds are PS bonds, or (e) x d , x e , x f , x g , x h and x i is a PN internucleoside bond, and p a and p b PS2 is a nucleoside bond, and all other nucleoside bonds are PS bonds.

[0017] In some embodiments, MAPT ASO comprises SEQ ID NO. 111, and p b and p c is a PN nucleoside interlink. 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 is a PN internucleoside bond, and x d and x e or x d , x e and x fis a PN internucleoside bond, and p b and p c is a PN nucleoside interlink, or (b) x h and x i is a PN internucleoside bond, and p b and p c is a PN nucleoside interlink, or (c) x h and x i is a PN internucleoside bond, and p b and p c PN is a PN-nucleoside bond, and all other nucleoside bonds are PS bonds.

[0018] In some embodiments, MAPT ASO comprises SEQ ID NO. 111, and p a and p b is an MsPA internucleoside bond or p a , p b and p c is an MsPA nucleoside interlink. 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 is a PN internucleoside bond, and x d and x e or x d , x e and x f is a PN internucleoside bond andp a and p b or p a , p b and p c is an MsPA nucleoside bond, or (b) x g , x h and x i is a PN internucleoside bond, and p a , p b and p c is an MsPA nucleoside bond, or (c) x d , x e , x h and x i is a PN internucleoside bond and p a and p b is an MsPA nucleoside interlink, or (d) x g , x h and x i is a PN internucleoside bond, and p a , p b and p c is an MsPA internucleoside bond, and all other internucleoside bonds are PS bonds, or (e) x d , x e , x h and x i is a PN internucleoside bond, and p a and p bMsPA is an internucleoside bond, and all other internucleoside bonds are PS bonds.

[0019] In some embodiments, MAPT ASO comprises SEQ ID NO. 111, and p a and p b is an OiPS internucleoside bond, or p a , p b and p c is an OiPS internucleoside bond. 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 is a PN internucleoside bond and x d and x e or x d , x e and x f is a PN internucleoside bond and p a and p b or p a , p b and p c is an OiPS internucleoside bond, or (b) x d , x e , x f , x g , x h and x i is a PN internucleoside bond and p a andp b is an OiPS internucleoside bond, or (c) x d , x e , x f , x g , x h and x i is a PN internucleoside bond, and p a and p b OiPS is an internucleoside bond, and all other internucleoside bonds are PS bonds.

[0020] In some embodiments, MAPT ASO comprises SEQ ID NO. 111, and modified MAPT ASO comprises the following.

[0021] (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';

[0022] (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';

[0023] (c) 5' A L * m C L * m C L *T*T*A n A n G n T*A*T*T*A*C*T*TL *G L * m C L 3';

[0024] (d) 5' A L * m C L * m C L *T*T*A*A n G n T*A*T*T*A*C*T*T L n G L n m C L 3';

[0025] (e) 5' A L * m C L * m C L *T*T*A n A n G n T*A*T*T*A*C*T*T L n G L n m C L 3';

[0026] (f) 5' A L * m C L * m C L *T*T*A u A u G u T*A*T*T*A*C*T n T L n G L n m C L 3';

[0027] (g) 5' A L n m C L * m C L *T*T*A n A n G*T*A*T*T*A*C*T*T L n GL n m C L 3';

[0028] (h) 5' A L * m C L n m C L n T*T*A n A n G*T*A*T*T*A*C*T*T L n G L n m C L 3';

[0029] (i) 5' A L n m C L n m C L *T*T*A n A n G*T*A*T*T*A*C*T*T L *G L * m C L 3';

[0030] (j) 5' A L n m C L n m C L *T*T*A*A n G n T*A*T*T*A*C*T*T L *G L * m C L 3';

[0031] (k) 5' A L n m C L n m C L *T*T*A*A n G*T*A*T*T*A*C*T*T L n G Ln m C L 3';

[0032] (l) 5' A L n m C L n m C L *T*T*A u A u G*T*A*T*T*A*C*T*T L n G L n m C L 3';

[0033] (m) 5' A L n m C L n m C L *T*T*A t A t G*T*A*T*T*A*C*T*T L n G L n m C L 3';

[0034] (n) 5' A L n m C L n m C L *T*T*A*A n G n T*A*T*T*A*C*T*T L n G L n m C L 3';

[0035] (o) 5' A L n m C L n m C L n T*T*A*A*G*T*A*T*T*A*C*T n TL n G L n m C L 3';

[0036] (p) 5' A L n m C L n m C L n T*T*A t A*G*T*A*T*T*A*C*T n T L n G L n m C L 3';

[0037] (q) 5' A L n m C L n m C L n T*T*A*A t G*T*A*T*T*A*C*T n T L n G L n m C L 3';

[0038] (r) 5' A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L 3';

[0039] (s) 5' A L n m C Ln m C L n T*T*A o A o G*T*A*T*T*A*C*T n T L n G L n m C L 3';

[0040] (t) 5' A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L P m C L 3';

[0041] (u) 5' A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T n T L P G L n m C L 3';

[0042] (v) 5' A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T P T L n GL n m C L 3';

[0043] (w) 5' A L n m C L n m C P T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L 3';

[0044] (x) 5' A L n m C L P m C n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L 3'; 또는

[0045] (y) 5' A L P m C L n m C n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L 3'

[0046] 여기서 A L , m C L , T L 및 G Lare adenine lock nucleic acid, 5-methylcytosine lock nucleic acid, thymine lock nucleic acid; and guanine lock nucleic acid, respectively; A, C, T, and G are deoxyadenosine, deoxycytidine, deoxythymidine, and deoxyguanosine, respectively; and s is a PS nucleoside interlink, and n is a PN internucleoside bond, and t is a PS2 internucleoside bond, and u is an MsPA internucleoside bond, and o OiPS is a nucleoside interlink. The chemical structure representing MAPT ASO is shown in FIGS. 10, 11, 12, 13, and 14. MAPT ASO may also be a salt of any structure shown in FIGS. 10, 11, 12, 13, and 14.

[0047] In some embodiments, MAPT ASO comprises SEQ ID NO. 112, and (a) p k , p l , p n and p o is a stabilized nucleoside bond, or (b) p k , p l , p n and p o is a stabilized internucleoside bond and p j and p m is a PS nucleoside bond, or (c) p j , p l and p o is a stabilized nucleoside bond, or (d) p j , p l and p o is a stabilized internucleoside bond and p k ,p m and p n is a PS nucleoside bond, or (e) p l , p m , p n and p o is a stabilized nucleoside bond, or (d) p l , p m , p n and p o is a stabilized internucleoside bond and p j and p k is a bond between PS nucleosides.

[0048] In some embodiments, MAPT ASO comprises SEQ ID NO. 112, and (a) p k , p l , p n and p o is a PN nucleoside interlink, or (b) p k , p l , p n and p o is a PN internucleoside bond and p j and p m is a PS nucleoside bond, or (c) p j is a PN bond and p l and p o is a PS2 nucleoside interlink, or (d) p j is a PN bond, and p l and p ois a PS2 internucleoside bond and p k , p m and p n is a PS nucleoside bond, or (e) p l and p m is a PS2 internucleoside bond and p n and p o is a PN nucleoside interlink, or (d) p l and p m is a PS2 internucleoside bond and p n and p o is a PN internucleoside bond and p j and p k is a bond between PS nucleosides.

[0049] In some embodiments, MAPT ASO comprises SEQ ID NO. 112, and modified MAPT ASO comprises the following.

[0050] (a) m C L n T L n G L *T*T n A n G*A*C*A*T*T*C n A n T*T* m C L *T L * m C L ;

[0051] (b) m C L *T L *G L *T*T n A n G*A*C*A*T*T*C n A n T*T* m CL n T L n m C L ;

[0052] (c) m C L n T L n G L n T n T*A t G*A*C*A*T*T*C*A t T*T n m C L n T L n m C L ; or

[0053] (d) m C L n T L n G L *T*T*A t G t A*C*A*T*T*C n A n T*T* m C L n T L n m C L

[0054] Here m C L , T L and G L are 5-methylcytosine lock nucleoside, thymine lock nucleoside, and guanosine lock nucleoside, respectively; A, C, T, and G are deoxyadenosine, deoxycytidine, deoxythymidine, and deoxyguanosine, respectively; and * is a PS nucleoside interlink, and n is a PN internucleoside bond, and t is a PS2 nucleoside bond.

[0055] In some embodiments, the oligonucleotide contains a 5' end group, and the 5' end group is a phosphodiester (PO) group, a phosphorothioate (PS) group, a phosphorodithioate (PS2) group, a mesyl-phosphoramidate (MsPA) group, a cyclic phosphoryl guanidine (PN) group, or O - It may be an isopropyl phosphorothioate (OiPS) group, but is not limited thereto. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' end group selected from the group consisting of PO, PS, and PN. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PO 5' end group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS 5' end group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PN 5' end group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS2 5' end group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains an MsPA 5' end group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains an OiPS 5' end group.

[0056] A MAPT ASO conjugated to a transferrin receptor (TfR)-targeted Fc polypeptide dimer is described. Any of the described MAPT ASOs may be conjugated to a TfR-targeted Fc polypeptide dimer. In some embodiments, the TfR-targeted 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 the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide. In some embodiments, the TfR-targeted Fc polypeptide dimer comprises a first Fc polypeptide that does not contain a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) and a second Fc polypeptide that contains a modified constant domain that specifically binds to TfR, and the first and second Fc polypeptides form an Fc dimer. In some embodiments, the first Fc polypeptide, the second Fc polypeptide, or both the first and second Fc polypeptides are modified to reduce effector function. In some embodiments, the first Fc polypeptide and the second Fc polypeptide are modified to reduce effector function. In some embodiments, the first Fc polypeptide comprises cysteine ​​at position 239 according to EU numbering, A at position 234, A at position 235, and serine at position 329, respectively. In some embodiments, the second Fc polypeptide comprises an A at position 234, an A at position 235, and a serine at position 329 according to EU numbering, and comprises a sequence having at least 90% sequence identity with SEQ ID NO. 12.

[0057] In some embodiments, the TfR-targeted Fc polypeptide dimer further comprises a non-targeted Fab (NTF) fused to the first Fc polypeptide through a hinge region to form a first Fab-Fc fusion polypeptide. In some embodiments, the TfR-targeted Fc polypeptide dimer further comprises an NTF fused to the second Fc polypeptide through a hinge region to form a second Fab-Fc fusion polypeptide. In some embodiments, the TfR-targeted Fc polypeptide dimer comprises a non-targeted Fab (NTF) fused to the first Fc polypeptide through a hinge region to form a first Fab-Fc fusion polypeptide and a non-targeted Fab (NTF) fused to the second Fc polypeptide through a hinge region to form a second Fab-Fc fusion polypeptide, wherein the first and second Fab-Fc fusion polypeptides form a Fab-Fc dimer. In some embodiments, the first and second NTFs each comprise a heavy chain segment comprising SEQ ID NO. 109 or 110 and a light chain comprising SEQ ID NO. 108. In some embodiments, the first and second NTFs each comprise a heavy chain segment comprising SEQ ID NO. 130 or 131 and a light chain comprising SEQ ID NO. 129. In some embodiments, the hinge region each comprises SEQ ID NO. 121.

[0058] In some embodiments, the TfR-targeted Fc polypeptide dimer further comprises a non-binding variable region (NBVR) fused to the first Fc polypeptide through a hinge region to form an NVBR-Fc fusion polypeptide. In some embodiments, the TfR-targeted Fc polypeptide dimer further comprises an NVBR fused to the second Fc polypeptide through a hinge region to form an NBVR-Fc fusion polypeptide. In some embodiments, the NVBR comprises the variable region of SEQ ID NO. 109 or 110, respectively. In some embodiments, the hinge region comprises SEQ ID NO. 121, respectively.

[0059] MAPT ASO is linked to the TfR-targeted Fc polypeptide dimer via a linker. The linker may be any linker available in the art suitable for linking an oligonucleotide to a polypeptide. MAPT ASO may be 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 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 the cysteine ​​at position 239. In some embodiments, the first Fc polypeptide comprises a CH1 domain, and MAPT ASO is linked to the first Fc polypeptide at a cysteine ​​at position 114 (according to Kabat numbering) or position 124 (according to EU numbering) (i.e., the CH1 domain comprises an A114C substitution or an S124C substitution). In some embodiments, MAPT ASO is 5 of MAPT ASO It is connected to the first Fc polypeptide through the terminal. In some embodiments, the first Fc polypeptide is cysteine ​​at position 239 and 5 of MAPT ASO. Terminal (or MAPT ASO's 5) It is connected to MAPT ASO through a connector attached to the terminal, and the connector includes the following,

[0060]

[0061] Among the formulas, bonds and intersect in the chemical structure ' ' indicates the point in the chemical structure where a wavy bond intersects the rest of the molecule.

[0062] In some embodiments, the modified constant domain of the second Fc polypeptide is, as numbered with respect to SEQ ID NO. 1, glutamate (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; alanine (A), I, V, serine (S), or T at position 162; N, S, arginine (R), or T at position 163; T, histidine (H), or S at position 186; It includes E, S, aspartate (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.

[0063] In some embodiments, the modified constant domain of the second Fc polypeptide comprises, according to EU numbering, E, L, S, V, W, or Y at position 380; 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.

[0064] In some embodiments, the modified constant domain of the second Fc polypeptide comprises, as numbered with reference to SEQ ID NO. 1, 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.

[0065] In some embodiments, the modified constant domain of the second Fc polypeptide further comprises, according to EU numbering, 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.

[0066] In some embodiments, the modified constant domain of the second Fc polypeptide comprises, according to EU numbering, 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.

[0067] In some embodiments, the second Fc polypeptide further comprises W at position 366 according to EU numbering, and the first Fc polypeptide further comprises serine at position 366 according to EU numbering, A at position 368, and valine at position 407. In some embodiments, the second Fc polypeptide further comprises serine at position 366 according to EU numbering, A at position 368, and valine at position 407, and the first Fc polypeptide further comprises W at position 366 according to EU numbering.

[0068] 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.

[0069] In some embodiments, the first and second NTFs each include a heavy chain variable region comprising SEQ ID NO. 109 or 110 and a light chain variable region comprising SEQ ID NO. 108.

[0070] 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.

[0071] Certain embodiments provide a pharmaceutical composition comprising the MAPT ASO conjugate described herein and a pharmaceutically acceptable carrier or diluent.

[0072] A specific embodiment provides a method for generating a neuron with reduced tau expression, the method comprising delivering the MAPT ASO conjugate described herein to a neuron, wherein the MAPT ASO reduces the expression level of an endogenous MAPT gene. The neuron may be a brain cell, a deep brain cell, or a spinal cord cell, but is not limited thereto.

[0073] Specific embodiments provide a method for modifying a neuron to reduce tau expression, the method comprising delivering the MAPT ASO conjugate described herein to a neuron, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene. The neuron may be, but is not limited to, brain cells, deep brain cells, or spinal cord cells.

[0074] A specific embodiment provides a method for delivering MAPT ASO to the CNS or cells of a human subject in need thereof, comprising administering the MAPT ASO conjugate described herein to the subject.

[0075] Delivery of MAPT ASO to neurons using the described MAPT ASO conjugate may be used to treat neurodegenerative disorders. Delivery of the described MAPT ASO to neurons may be used to treat tau-related neurodegenerative disorders. Neurodegenerative disorders may be, but are not limited to, Alzheimer's disease.

[0076] A specific embodiment provides a method for reducing MAPT messenger ribonucleic acid (mRNA) expression or levels in a human subject requiring this, and the method comprises administering the MAPT ASO conjugate described herein to a human subject. Brief explanation of the drawing

[0077] Figure 1 shows the plasma pharmacokinetic profile of modified MAPT ASO OTV. Figure 2 shows the single-dose brain ASO concentration of the modified MAPT ASO OTV. Figure 3 shows the multiple dose brain ASO concentrations of the modified MAPT ASO OTV. Figure 4 illustrates target MAPT knockdown after 4 administrations of modified MAPT ASO OTV. Figure 5 illustrates target MAPT knockdown after 8 administrations of modified MAPT ASO OTV. Figure 6 shows the brain ASO concentration of the unmodified MAPT ASO OTV. Figure 7 illustrates the target MAPT knockdown of the non-deformed MAPT ASO OTV. Figure 8 illustrates the efficacy of MAPT ASO. Figure 9 illustrates ASO stability in brain tissue. FIG. 10. Chemical structure diagram showing one embodiment of MAPT ASO. FIG. 11. Chemical structure diagram showing one embodiment of MAPT ASO. FIG. 12. Chemical structure diagram showing one embodiment of MAPT ASO. FIG. 13. Chemical structure diagram showing one embodiment of MAPT ASO. FIG. 14. Chemical structure diagram showing one embodiment of MAPT ASO. Specific details for implementing the invention

[0078] I. Definition

[0079] Before describing this teaching in detail, it should be understood that this disclosure is not limited to specific compositions or process steps, and that they may vary. It should be noted that, as used in this specification and the appended claims, singular nouns include plural objects unless otherwise indicated by the context. Thus, for example, references to 'oligonucleotides' include plural oligonucleotides, etc. The conjunction 'or' should be interpreted in an inclusive sense, that is, equivalent to 'and / or' unless the inclusive sense is unreasonable in the context.

[0080] Generally, the term “about” indicates a non-substantial variation in the amount of a component of a composition that does not have a significant effect on the activity or stability of the composition. Where the specification discloses a specific value for a parameter, it should be understood that the specification alternatively discloses the parameter at that value “about”. When the terms “about” and “approximately” are used to modify a quantity specified as a numerical value or range, they indicate that not only the numerical value but also a reasonable deviation from a value known to a person skilled in the art (e.g., the standard error limit of measurement (SEM)), such as ± 20%, ± 10%, or ± 5%, is within the intended meaning of the mentioned value.

[0081] A composition or method that 'comprising' or 'comprising' one or more cited elements may include other elements that are not specifically cited. For example, a composition that 'comprising' or 'comprising' an Fc polypeptide dimer may contain the Fc polypeptide dimer alone or in combination with other components.

[0082] The specification of a value range includes all integers within or defining the range, and all sub-ranges defined by the integers within the range.

[0083] The use of 'include,' 'comprising,' 'containing,' 'containing,' 'comprising,' 'inclusive,' and 'comprising' is not intended to be limiting and may include other elements not specifically mentioned. For example, a composition described as 'comprising' or 'comprising' an Fc polypeptide dimer may contain the Fc polypeptide dimer alone or in combination with other components. It should be understood that the foregoing general description and detailed description are merely illustrative and descriptive and are not intended to limit the teachings. To the extent that any material incorporated by reference is inconsistent with the express content of this disclosure, the express content shall prevail.

[0084] The specification of a value range includes all integers within or defining the range, and all sub-ranges defined by the integers within the range.

[0085] As used herein, the term “antibody” refers to a protein having an immunoglobulin fold that specifically binds to an antigen through its variable region. This term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-strand antibodies, multispecific antibodies such as 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 divalent scFv. Antibodies may contain a light chain classified as kappa or lambda. Antibodies may contain a heavy chain classified as gamma, mu, alpha, delta, or epsilon, which in turn define immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0086] The 'EU numbering system' is commonly used in the relevant technical field to refer to residues within the constant region of the antibody heavy chain. The EU numbering system is shown below for sequence number 5 (clone CH3C.35.23.2 knop).

[0087]

[0088] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two pairs of polypeptide chains, each pair having one 'light' chain (about 25 kD) and one 'heavy' chain (about 50 to 70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The term 'variable light chain' (V L ) and 'variable heavy chain'(V H ) refers to these light chains and heavy chains, respectively.

[0089] The term 'variable region' or 'variable domain' refers to a domain within the heavy or light chain of an antibody that is derived from germline variable (V) genes, diversity (D) genes, or binding (J) genes (not derived from constant (Cμ and Cδ) gene segments) and provides the antibody with specificity for binding to an antigen. Typically, the antibody variable region comprises four conserved 'framework' regions interspersed with three hypervariable 'complementarity determining regions'.

[0090] The term 'Complementary Determining Region' or 'CDR' refers to three hypervariable regions within each chain that interrupt the four framework regions established by the light and heavy chain variable regions. CDRs are primarily responsible for antibody binding to antigen epitopes. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 when numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which a specific CDR is located. Therefore, V H While CDR3 or CDR-H3 is located in the variable region of the heavy chain of the antibody in which it appears, V L CDR1 or CDR-L1 is CDR1 from the variable region of the light chain of the antibody in which it appears.

[0091] The 'framework regions' or 'FRs' of different light or heavy chains are relatively conserved within a species. The framework regions of antibodies, namely the combined framework regions of the constituent light and heavy chains, serve to localize and align CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from published references. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the 'VBASE2' germline variable gene sequence database for human and mouse sequences.

[0092] The amino acid sequences of the CDR and framework regions are defined by various definitions widely known in the art, e.g., Kabat, Chothia , It can be determined using the International ImMunoGeneTics Database (IMGT), AbM, and observed antigen contact ('Contact'). In some embodiments, the CDR is determined according to the definition of Contact. Literature [MacCallum et al., J. Mol. Biol. [262:732-745, 1996] Note. In some embodiments, the CDR is determined by a combination of the Kabat, Chothia, and / or Contact CDR definitions.

[0093] The term 'Fd portion' refers to the N-terminal portion of an immunoglobulin heavy chain. Typically, the Fd portion includes a heavy chain variable (VH) region and a heavy chain constant (CH1) region.

[0094] The term 'Fab' refers to an antigen-binding fragment composed of a light chain variable region, a light chain constant region, a heavy chain variable region, and a heavy chain CH1 constant region.

[0095] The term 'single-chain variable fragment' or 'scFv' refers to an antigen-binding fragment composed of a heavy-chain variable region and a light-chain variable region linked together via a peptide linker. The linker is V H The N-terminus of V L Connect to the C-terminus or V L The N-terminus of V H It can be connected to the C-terminus. scFv lacks an invariant region.

[0096] The term 'epitope' refers to a region or area of ​​an antigen to which a molecule, for example, the antibody's CDR, specifically binds, and may include several amino acids or portions of several amino acids, for example, five or six or more, for example, twenty or more amino acids or portions of such amino acids. In some cases, the epitope includes non-protein components, for example, components from carbohydrates, nucleic acids, or lipids. In some cases, the epitope is a three-dimensional moiety. Thus, for example, if the target is a protein, the epitope may consist of a continuous sequence of amino acids (e.g., a linear epitope) or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g., a discontinuous or stereoscopic epitope).

[0097] As used herein, the phrase 'recognizes an epitope' used in relation to antibodies means that the antibody CDR interacts with or specifically binds to the corresponding epitope or a part of the antigen containing the corresponding epitope.

[0098] The term ‘specifically binds’ refers to a molecule (e.g., Fab or scFv) that binds to an epitope or target with greater affinity, greater binding strength, and / or longer duration than that which binds to another epitope or non-target compound (e.g., a structurally different antigen) in a sample. In some embodiments, the Fab or scFv that specifically binds to the epitope or target is a Fab or scFv that binds to the epitope or target with at least 5 times greater affinity than the other epitope or non-target compound, e.g., at least 6 times, 7 times, 8 times, 9 times, 10 times, 25 times, 50 times, 100 times, 1000 times, or 10,000 times or more affinity. As used herein, the terms 'specific binding,' 'specifically binds,' or 'specific to' a specific epitope or target refer, for example, to the epitope or target to which it binds, for example 10 -4 M or less, e.g., 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Equilibrium dissociation constant K of M D It can be represented by molecules having. A person skilled in the art will recognize that Fab or scFv that specifically binds to a target from one species can also specifically bind to the ortolog of that target.

[0099] The term 'binding affinity' is used herein to refer to the strength of a non-covalent interaction between two molecules, for example, between Fab or scFv and an antigen. Thus, unless otherwise indicated or evident from the context, this term may refer to a 1:1 interaction between Fab or scFv and an antigen. Binding affinity is equal to the equilibrium dissociation constant (K DIt can be quantified by measuring ), which is the dissociation rate constant (k d , hour -1 ) is the coupling rate constant (k a , hour -1 M -1 It refers to what is divided by ). K D This can be determined by measuring the kinetics of complex formation and dissociation, for example, by the surface plasmon resonance (SPR) method, e.g., Biacore TM System; KinExA ® Dynamic exclusion tests such as; and biolayer interferometers (e.g., ForteBio ® It can be determined using the Octet platform. As used herein, 'binding affinity' refers not only to formal binding affinities, such as those reflecting the 1:1 interaction between Fab or scFv and the antigen, but also to K D It also includes apparent affinity, which is calculated and can reflect strong bonding forces.

[0100] Monoclonal antibodies and fragments thereof (including Fabs and conjugates), or other biological entities are typically provided in an isolated form. The antibody (or its fragment or conjugate) is typically at least 50% w / w pure from interfering proteins and other contaminants arising from its production or purification, but does not exclude the possibility that the antibody or Fab may be combined with an excess of pharmaceutically acceptable carrier(s) or other vehicle to facilitate its use. The antibody (or its fragment or conjugate) may be at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% w / w pure from interfering proteins and contaminants from its production or purification. The isolated antibody (or its fragment or conjugate) may be the major macromolecular species remaining after its purification.

[0101] 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 structural domain. The Fc polypeptide typically contains an invariant region sequence including at least a CH2 domain and / or a CH3 domain, and may contain at least a portion of a hinge region. Two Fc peptides dimerize to form an Fc region or an Fc fragment.

[0102] The terms 'polypeptide' and 'peptide' refer to polymers of amino acid residues within a single chain. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. 'Protein' may refer to polypeptides, polypeptide dimers, or polypeptide polymers. Single-chain polypeptides of protein dimers or polymers may be connected by covalent bonds (e.g., disulfide bonds) or non-covalent interactions.

[0103] For the purpose of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chain): met, ala, val, leu, ile; Group II (neutral hydrophilic side chain): cys, ser, thr; Group III (acidic side chain): asp, glu; Group IV (basic side chain): asn, gln, his, lys, arg; Group V (residues affecting chain orientation): gly, pro; and Group VI (aromatic side chain): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids within the same class. Non-conservative substitutions consist of exchanging a member of one class for a member of another class.

[0104] In the context of two or more polypeptide sequences, the terms 'identical' or percent 'identity' refer to two or more sequences or subsequences having a specific percentage of identical amino acid residues across a specified region, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as measured by using a sequence comparison algorithm or by manual alignment and visual inspection when compared and aligned for maximum correspondence across a comparison window or a specified region. For the sequence comparison of polypeptides, typically one amino acid sequence serves as the reference sequence, and candidate sequences are compared against it. Alignment can be performed using various methods available to a person skilled in the art, for example, by using publicly available software that uses visual alignment or known algorithms to achieve maximum alignment. These programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment can be determined by a person skilled in the art. For the purposes of this application, the standard protein BLAST algorithm is used to align two protein sequences using basic parameters for sequence comparison of polypeptide sequences.

[0105] Percent sequence identity is determined by the antibody or Fc polypeptide sequences aligned as much as possible according to Kabat numbering conventions. After alignment, when a target antibody region (e.g., a heavy chain or light chain or the entire mature variable region of the Fc polypeptide) is compared with the same region of a reference antibody, the percentage sequence identity between the target antibody region and the reference antibody region is the value obtained by dividing the number of positions occupied by the same amino acid in both the target antibody region and the reference antibody region by the total number of aligned positions in both regions, excluding gaps, and multiplying by 100 to convert to a percentage.

[0106] Nucleic acid sequence identity can be determined by aligning sequences using algorithms such as BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis., using basic gap parameters, by inspection, and by the best alignment (i.e., the alignment that results in the highest percentage of sequence similarity across the comparison window). The percentage of sequence identity is calculated by comparing two optimally aligned sequences across the comparison window, determining the number of locations where identical residues occur in both sequences to calculate the number of matching locations, dividing the number of matching and mismatching locations (i.e., the window size) for which gaps were not calculated in the comparison window, and multiplying the result by 100 to calculate the percentage of sequence identity. Unless otherwise indicated, the comparison window between two sequences is defined by the total length of the shorter of the two sequences.

[0107] Modified nucleoside bonds are nucleoside bonds other than naturally occurring phosphate bonds. Below is a modified nucleoside bond connecting the sugar groups (included in the structure) of the 5' and 3' nucleosides of the modified nucleoside bond.

[0108]

[0109] As used herein, PN refers to structure (b).

[0110] As used herein, 'stabilized nucleoside-binding' refers to modified nucleoside-bindings specifically introduced into the backbone of an oligonucleotide of an oligonucleotide polypeptide conjugate to remove a vulnerable site (e.g., a nuclease-sensitive site), increase the resistance of the oligonucleotide to cleavage, or otherwise modify one or more pharmacokinetic or pharmacodynamic properties of the oligonucleotide and / or oligonucleotide polypeptide conjugate into which it is introduced. Stabilized nucleoside-bindings do not contain phosphorothioates or phosphodiesters. An oligonucleotide and / or oligonucleotide polypeptide conjugate containing one or more stabilized nucleoside-bindings (stabilized oligonucleotides) has improved properties compared to an oligonucleotide or oligonucleotide polypeptide conjugate having the same oligonucleotide sequence but not containing one or more stabilized nucleoside-bindings. Stabilized oligonucleotides and oligonucleotide polypeptide conjugates have improved properties compared to those having only phosphorothioate nucleoside bonds or a combination of phosphodiester and phosphorothioate nucleoside bonds. Stabilized oligonucleotides may contain phosphorothioate nucleoside bond(s) and / or phosphodiester nucleoside bond(s) in addition to stabilizing nucleoside bonds. In some embodiments, one or more phosphorothioate and / or phosphodiester nucleoside bonds within the oligonucleotide (e.g., ASO; e.g., Gapmer) are replaced with stabilizing nucleoside bonds. In some embodiments, one or more phosphorothioate and / or phosphodiester nucleoside bonds may be replaced with one or more stabilizing nucleoside bonds at or near a site (e.g., a vulnerable site) identified in the oligonucleotide as sensitive to cleavage.In some embodiments, one or more phosphorothioate and / or phosphodiester nucleoside bonds within the wing segments of the gapmer are replaced with stabilized nucleoside bonds. In some embodiments, one or more phosphorothioate and / or phosphodiester nucleoside bonds within the gap segments of the gapmer are replaced with stabilized nucleoside bonds. Examples of modified nucleoside bonds that can be used as stabilized nucleoside bonds include, but are not limited to, phosphorodithioates, phosphoramidates, mesylphosphoramidates, phosphonates, phosphotriesters, and phosphoryl guanidines (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)] Note). Additional examples of modified nucleoside bonds that can be used as stabilized nucleoside bonds include, but are not limited to, modified nucleoside bonds s1, s2, s3, s4, s5, s6, s7, s8, s8, s10, s11, s12, s13, s14, s15, s16, s17 or s18 described in WO 2017210647, the disclosure thereof is incorporated by reference in its entirety.Further examples of modified nucleoside bonds that can be used as stabilized nucleoside bonds include, but are not limited to, modified nucleoside bonds n001, n002, n003, n004, n005, n006, n007, n008, n009, n010, n020, n025, or n026 described in WO 2022099159, the disclosure thereof is incorporated by reference in its entirety. In some embodiments, the stabilized nucleoside bond is selected from the group consisting of PS2, MsPA, OiPS, and PN nucleoside bonds.

[0111] 'LNA' refers to a noncyclic nucleoside analog (2' to 4' noncyclic nucleotide analog) containing a bridge between the 2' and 4' positions of a ribose ring, and is known as a 'lock nucleoside' or 'lock nucleoside'. As used herein, 'LNA oligonucleotide' refers to an oligonucleotide containing one or more of these noncyclic nucleoside analogs. Literature[ Biochemistry , 43(42):13233-13240 (2004)]. In some embodiments, the LNA provided herein has the following structure, where “Base” is a nucleobase.

[0112]

[0113] '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 may refer to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of proteins.

[0114] The term 'pharmaceutical acceptable' means that a carrier, diluent, excipient, or adjuvant is compatible with other components of the formulation and is not substantially harmful to the recipient.

[0115] 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 mammalian species. In some embodiments, the subject is a human. The term ‘subject’ includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0116] The terms 'disease' or 'pathological condition' refer to any abnormal state that impairs physiological function. This term is used broadly to encompass any disorder, disease, abnormality, pathology, illness, pathological condition, or syndrome in which physiological function is impaired, regardless of the nature of the etiology.

[0117] The terms 'treat' and 'treatment' refer to methods or steps taken to provide relief or alleviation from the number, severity, and / or frequency of one or more symptoms of a disease or condition in a subject. Treating generally refers to obtaining a desired pharmacological and / or physiological effect. An effect may be prophylactic in that it prevents or partially prevents the disease, symptom, or condition thereof, but is not necessarily so. An effect may be therapeutic in that it is the partial or complete cure of the disease, condition, symptom, or adverse effects attributable to the disease, disorder, or condition. The term treatment may include (a) preventing the occurrence of the disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease; (b) suppressing the disease, i.e., inhibiting its development; and (c) alleviating the disease, i.e., reducing or improving the disease and / or its symptoms or condition. Treating may refer to therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those requiring treatment (subjects requiring such treatment) may include those who already have a disease or condition, or those for whom a disease or condition must be prevented. Treatment may include suppressing the disease, disorder, or condition, e.g., inhibiting its progression; and alleviating the disease, disorder, or condition, e.g. causing the regression of the disease, disorder, and / or condition. Treating the disease, disorder, or condition may include improving at least one symptom of a specific disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the symptoms without affecting or eliminating the underlying cause of the symptoms. Treatment or improvement of symptoms may be based on objective or subjective parameters. The effectiveness of treatment may be compared with individuals or a pool of individuals not receiving treatment, or with the same patient at different points in time before or during treatment.

[0118] "Pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to an amount (dose) of the described active pharmaceutical ingredient or pharmaceutical composition to produce the intended pharmacological, therapeutic, or prophylactic result. "Effective amount" may also refer to an amount of, for example, an excipient in the pharmaceutical composition sufficient to achieve the desired characteristics of the composition. The effective amount may be administered as one or more doses, applications, or doses.

[0119] II. Overview

[0120] Oligonucleotide therapy for CNS disorders caused by genetic abnormalities or increased protein accumulation is becoming increasingly widely used as an approach to regulate gene expression in these neurological disorders. The blood-brain barrier (BBB) ​​presents difficulties in delivering systemically administered oligonucleotides to relevant sites of action within the CNS. Intrathecal (IT) delivery, in which the drug is administered directly into the cerebrospinal fluid (CSF) space, enables the bypassing of the BBB. However, one limitation of this approach is that delivering these oligonucleotide therapies directly into the CSF via the IT approach does not achieve uniform distribution throughout the CNS.

[0121] A MAPT ASO conjugate is described that can be administered intravenously to a subject and facilitates the transport of MAPT ASO across the BBB and the delivery of MAPT ASO to CNS cells, wherein MAPT ASO provides knockdown of MAPT expression in the brain and spinal cord. The MAPT ASO conjugate comprises a transferrin-targeted Fc polypeptide dimer linked to MAPT ASO. Conjugating MAPT ASO to the Fc polypeptide dimer can be used to increase the targeting efficacy of the molecule after administration into a subject. The Fc polypeptide dimer-cargo molecule conjugate has increased targeting efficacy compared to the unconjugated molecule. MAPT ASO includes a modification that reduces in vivo plasma clearance and / or degradation / cleavage when MAPT ASO is linked to the transferrin-targeted Fc polypeptide dimer.

[0122] In some embodiments, the described MAPT ASO conjugate provides knockdown of MAPT expression across CNS regions including the frontal lobe, parietal lobe, temporal lobe, occipital lobe, and cerebellum, as well as brain regions including deep brain regions. In some embodiments, the described MAPT ASO conjugate provides knockdown of MAPT expression across a number of CNS cell types including endothelial cells, neurons, astrocytes, oligodendrocytes, and microglia. In some embodiments, the described MAPT ASO conjugate provides knockdown of MAPT expression in the spinal cord.

[0123] In some embodiments, the described MAPT ASO conjugate provides knockdown of MAPT expression in skeletal muscle and cardiac muscle. Knockdown of MAPT expression in skeletal muscle, cardiac muscle and diaphragmatic muscle can be used to treat neuromuscular disorders.

[0124] The described MAPT ASO conjugate provides therapeutic-level targeted delivery of MAPT ASO to various tissues, including the CNS, following systemic administration. This targeted delivery enables the use of lower doses of MAPT ASO compared to the administration of MAPT ASO alone, i.e., non-targeted delivery.

[0125] A MAPT ASO conjugate comprising a transferrin receptor (TfR)-targeting Fc polypeptide dimer and a MAPT antisense oligonucleotide (ASO) is described, wherein

[0126] (a) The TfR-targeted Fc polypeptide dimer is

[0127] (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;

[0128] (ii) a second Fc polypeptide comprising a modified constant domain that specifically binds to serine and human transferrin receptor 1 (TfR) at position 234 A, position 235 A, and position 329, respectively, according to EU numbering (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

[0129] (iii) comprising a first non-targeted Fab (NTF) fused to a first Fc polypeptide through a first hinge region to form a first Fab-Fc fusion polypeptide and a second NTF fused to a second Fc polypeptide through a second hinge region to form a second Fab-Fc fusion polypeptide (the first and second NTFs each include SEQ ID NO. 109 or 110, wherein the hinge region each includes SEQ ID NO. 121), and

[0130] (b) MAPT ASO is

[0131] 5'-AL x d m C L x e m C L x f TTA p a A p b G p c TATTACT x g T L x h G L x i m C L Includes -3' (sequence number 111), but,

[0132] A L , m C L , G L and T L are adenine, 5-methylcytosine, guanosine, and thymine lock nucleosides, respectively, and

[0133] A, C, G, and T are deoxyadenosine, deoxycytidine, deoxyguanidine, and deoxythymidine nucleosides, respectively, and

[0134] Each p It independently includes phosphorothioate (PS) nucleoside inter-binding, phosphorodithioate (PS2) nucleoside inter-binding, and mesylphosphoramidate (MsPA) nucleoside inter-binding. O - It is an isopropyl phosphorothioate (OiPS) internucleoside bond or a phosphorylguanidine (PN) internucleoside bond, and

[0135] Each x is independently a PN nucleoside inter-bond, a PS nucleoside inter-bond, or a phosphodiester (PO) nucleoside inter-bond, and

[0136] Any internucleoside bond other than PO, PS2, MsPA, OiPS, or PN nucleosides is a PS nucleoside bond, but,

[0137] The first Fc polypeptide is linked to MAPT ASO through a linker.

[0138] In some embodiments, p a , p b and p c At least one of them is not a PS nucleoside bond. In some embodiments, p a , p b and p c At least two of them are not PS nucleoside bonds.

[0139] In some embodiments, x g , x h and x i At least two of them are PN nucleoside bonds.

[0140] In some embodiments, the modified constant domain of the second Fc polypeptide comprises, according to EU numbering, 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.

[0141] In some embodiments, MAPT ASO is linked to the cysteine ​​at position 239 (EU numbering) of the first Fc polypeptide. In some embodiments, the 5' end of MAPT ASO is linked to the cysteine ​​at position 239 of the first Fc polypeptide, wherein the linker is as follows,

[0142]

[0143] Among the formulas, bonds and intersect in the chemical structure ' ' indicates the point in the chemical structure where a wavy bond intersects the rest of the molecule.

[0144] In some embodiments, the NTF includes SEQ ID NO. 109 or 110 and SEQ ID NO. 108.

[0145] A MAPT ASO conjugate comprising a transferrin receptor (TfR)-targeting Fc polypeptide dimer and a MAPT antisense oligonucleotide (ASO) is described, wherein

[0146] (a) The TfR-targeted Fc polypeptide dimer is

[0147] (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;

[0148] (ii) a second Fc polypeptide comprising a modified constant domain that specifically binds to serine and human transferrin receptor 1 (TfR) at position 234 A, position 235 A, and position 329, respectively, according to EU numbering (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

[0149] (iii) comprising a first non-targeted Fab (NTF) fused to a first Fc polypeptide through a first hinge region to form a first Fab-Fc fusion polypeptide and a second NTF fused to a second Fc polypeptide through a second hinge region to form a second Fab-Fc fusion polypeptide (the first and second NTFs each include SEQ ID NO. 109 or 110, wherein the hinge region each includes SEQ ID NO. 121), and

[0150] (b) MAPT ASO is

[0151] 5'-C L x d T L x e G L x f T p j T p k A p l G p m ACATTC p n A p o TT x g C L x h T L x i C L Includes -3' (sequence number 112), but,

[0152] mCL, GL및 TL은 각각 5-메틸시토신, 구아노신 및 티민 잠금 뉴클레오시드이고,

[0153] A, C, G, and T are deoxyadenosine, deoxycytidine, deoxyguanidine, and deoxythymidine nucleosides, respectively, and

[0154] Each p It independently includes phosphorothioate (PS) nucleoside inter-binding, phosphorodithioate (PS2) nucleoside inter-binding, and mesylphosphoramidate (MsPA) nucleoside inter-binding. O - isopropyl phosphorothioate (OiPS) internucleoside bonding or phosphorylguanidine (PN) internucleoside bonding, and p a , p b and p c At least two of them are not PS nucleoside bonds, and

[0155] Each x Independently, it is a PN nucleoside inter-bond or a PS nucleoside inter-bond, and x g , xh and x i At least two of them are PN nucleoside interlinks, and

[0156] Any internucleoside bond other than PS2, MsPA, OiPS, or PN nucleoside bonds is a PS nucleoside bond, but,

[0157] The first Fc polypeptide is linked to MAPT ASO through a linker.

[0158] In some embodiments, the modified constant domain of the second Fc polypeptide comprises, according to EU numbering, 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. In some embodiments, MAPT ASO is linked to the cysteine ​​at position 239 (EU numbering) of the first Fc polypeptide. In some embodiments, the 5' end of MAPT ASO is linked to the cysteine ​​at position 239 of the first Fc polypeptide, wherein the linker is as follows,

[0159]

[0160] Among the formulas, bonds and intersect in the chemical structure ' ' indicates the point in the chemical structure where a wavy bond intersects the rest of the molecule.

[0161] In some embodiments, the NTF includes SEQ ID NO. 109 or 110 and SEQ ID NO. 108.

[0162] A. Transferrin receptor (TfR)-targeted Fc polypeptide dimer

[0163] In some embodiments, the first Fc polypeptide or the first Fab-Fc fusion polypeptide 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 polypeptide comprises cysteine ​​at positions 239, 442, 330, and 289 according to EU numbering (i.e., the Fc domain comprises an S239C substitution, an S442C substitution, an A330C substitution, a K149C substitution, or a T289C substitution). In some embodiments, the first Fc polypeptide comprises cysteine ​​at position 239 according to EU numbering. In some embodiments, the first Fc polypeptide further comprises a CH1 domain (i.e., a Fab-Fc fusion polypeptide), wherein the CH1 domain comprises cysteine ​​at position 114 (according to Kabat numbering) or position 124 (according to EU numbering) (i.e., the first Fc polypeptide comprises an A114C substitution or an S124C substitution). In some embodiments, the first Fc polypeptide comprises an LALA-PS mutation (A at position 234, A at position 235, and serine at position 329, respectively, according to EU numbering). In some embodiments, the first Fc polypeptide comprises cysteine ​​at position 239, according to EU numbering, an LALA-PS mutation, and a Knop or Hall mutation. In some embodiments, the first Fc polypeptide comprises a LALA-PS mutation, a Knop or a Hall mutation, and further comprises a CH1 domain, wherein the CH1 domain comprises a cysteine ​​at position 114 (according to Kabat numbering) or position 124 (according to EU numbering). 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 NOs 47 to 48, 51 to 54, 61 to 63, 66, 73 to 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 cysteine ​​at position 239, A at position 234, A at position 235, and serine at position 329 (each according to EU numbering), 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.

[0164] In some embodiments, the modified constant domain of the second Fc polypeptide comprises, according to EU numbering, 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. In some embodiments, the second Fc polypeptide or Fab-Fc fusion 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 numbered based on SEQ ID NO. 5. In some embodiments, the modified constant domain of the second Fc polypeptide comprises 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; Includes S at position 424; and S at position 426 (according to EU numbering), and has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with sequence number 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 numbered based on 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 numbering). In some embodiments, the second Fc polypeptide comprises LALA-PS mutations and Knop or Hall mutations. In some embodiments, the modified constant domain of the second Fc polypeptide comprises 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 includes S at position 426 (according to EU numbering), and has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with any of sequence numbers 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 numbered based on SEQ ID NO. 5, and has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NOs 4-29, 36, and 43-44.

[0165] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide is a knob mutation (e.g., T139W as numbered based on SEQ ID NO. 5), a hole mutation (e.g., T139S, L141A, and Y180V as numbered based on SEQ ID NO. 5), one or more mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S as numbered based on SEQ ID NO. 5 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)) and / or one or more mutations that increase serum stability (e.g., in the presence or absence of (i) M25Y, S27T, and T29E as numbered based on SEQ ID NO. 5, or (ii) M201L as numbered based on SEQ ID NO. 5). It includes one or more mutations or sets of mutations selected from the group consisting of N207S). If the first Fc polypeptide contains a KNOB mutation, the second Fc polypeptide contains a HALL mutation. If the second Fc polypeptide contains a KNOB mutation, the first Fc polypeptide contains a HALL mutation.

[0166] In some embodiments, both the first and second Fc polypeptides contain one or more mutations that reduce effector function. In some embodiments, the first and second Fc polypeptides each contain an LALA mutation. In some embodiments, the first and second Fc polypeptides each contain an LALA-PG mutation. In some embodiments, the first and second Fc polypeptides each contain an LALA-PD mutation.

[0167] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a Knob mutation (e.g., T139W as numbered based on SEQ ID NO. 5). In some embodiments, the second Fc polypeptide contains a Knob mutation and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0168] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a nop mutation (e.g., T139W as numbered based on SEQ ID NO. 5) and one or more mutations that modulate effector function (e.g., L7A, L8A and / or P102G or P102S as numbered based on SEQ ID NO. 5 (e.g., L7A and L8A; L7A, L8A and P102G; or L7A, L8A and P102S)). In some embodiments, the second Fc polypeptide contains a NOP mutation and one or more mutations that modulate effector function, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0169] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a NOP mutation (e.g., T139W as numbered based on SEQ ID NO. 5) and one or more mutations that increase serum stability (e.g., M25Y, S27T, and T29E as numbered based on SEQ ID NO. 5). In some embodiments, the second Fc polypeptide contains a NOP mutation and one or more mutations that increase serum stability and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0170] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a NOP mutation (e.g., T139W as numbered based on SEQ ID NO. 5) and one or more mutations that increase serum stability (e.g., N207S in the presence or absence of M201L as numbered based on SEQ ID NO. 5). In some embodiments, the second Fc polypeptide contains a NOP mutation and one or more mutations that increase serum stability and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0171] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a nop mutation (e.g., T139W as numbered based on SEQ ID NO. 5), one or more mutations that modulate effector function (e.g., L7A, L8A and / or P102G or P102S as numbered based on SEQ ID NO. 5 (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., M25Y, S27T and T29E as numbered based on SEQ ID NO. 5). In some embodiments, the second Fc polypeptide contains a NOP mutation, one or more mutations that regulate effector function, and one or more mutations that increase serum stability, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0172] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a nop mutation (e.g., T139W as numbered based on SEQ ID NO. 5), one or more mutations that modulate effector function (e.g., L7A, L8A and / or P102G or P102S as numbered based on SEQ ID NO. 5 (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., N207S in the presence or absence of M201L as numbered based on SEQ ID NO. 5). In some embodiments, the second Fc polypeptide contains a NOP mutation, one or more mutations that modulate effector function, and one or more mutations that increase serum stability, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with the sequence of SEQ ID NO. 17 or 18. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO. 17 or 18.

[0173] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a hole mutation (e.g., T139S, L141A, and Y180V as numbered based on SEQ ID NO. 5). In some embodiments, the second Fc polypeptide contains a hole mutation and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0174] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a hole mutation (e.g., T139S, L141A, and Y180V as numbered based on SEQ ID NO. 5) and one or more mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S as numbered based on SEQ ID NO. 5 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, the second Fc polypeptide may contain a hole mutation and one or more mutations that modulate effector function and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0175] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a hole mutation (e.g., T139S, L141A, and Y180V as numbered based on SEQ ID NO. 5) and one or more mutations that increase serum stability (e.g., M25Y, S27T, and T29E as numbered based on SEQ ID NO. 5). In some embodiments, the second Fc polypeptide contains a hole mutation and one or more mutations that increase serum stability and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0176] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a hole mutation (e.g., T139S, L141A, and Y180V as numbered based on SEQ ID NO. 5) and one or more mutations that increase serum stability (e.g., N207S in the presence or absence of M201L as numbered based on SEQ ID NO. 5). In some embodiments, the second Fc polypeptide contains a hole mutation and one or more mutations that increase serum stability and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0177] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a hole mutation (e.g., T139S, L141A, and Y180V as numbered based on SEQ ID NO. 5), one or more mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S as numbered based on SEQ ID NO. 5 (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., M25Y, S27T, and T29E as numbered based on SEQ ID NO. 5). In some embodiments, the second Fc polypeptide contains a hole mutation, one or more mutations that modulate effector function, and one or more mutations that increase serum stability, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0178] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a hole mutation (e.g., T139S, L141A, and Y180V as numbered based on SEQ ID NO. 5), one or more mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S as numbered based on SEQ ID NO. 5 (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., N207S in the presence or absence of M201L as numbered based on SEQ ID NO. 5). In some embodiments, the second Fc polypeptide contains a hole mutation, one or more mutations that modulate effector function, and one or more mutations that increase serum stability, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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.

[0179] In some embodiments, the N-terminus of the second Fc polypeptide comprises a hinge sequence or a portion of a hinge sequence (e.g., SEQ ID NO. 49 or 50 for the second Fc polypeptide). In some embodiments, the N-terminus of the first Fc polypeptide is additionally connected to a CH1 region (e.g., SEQ ID NO. 86, 87 or 94) or an NTF heavy chain sequence.

[0180] 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 one of SEQ ID NOs 4, 5, 6, 7, 9, 11, 50, 86, and 87, 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 numbered based on SEQ ID NO. 5, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises SEQ ID NOs 54, 63, 66 and It includes any one of the sequences of 90. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide includes Q at position 192; Q at position 193; S at position 197; and S at position 199, as numbered based on SEQ ID NO. 5. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide includes the sequence of SEQ ID NO. 7, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide includes the sequence of SEQ ID NO. 54. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide includes the sequence of SEQ ID NO. 9, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide includes 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.

[0181] In some embodiments, the N-terminus of the first and / or second Fc polypeptide comprises all or part of a hinge region (e.g., TCPPCP (SEVENTEEN NO. 121), DKTHTCP (SEVENTEEN NO. 91), or DKTHTCPPCP (SEVENTEEN NO. 92)). Accordingly, in some embodiments, the second Fc polypeptide comprises the sequence of SEVENTEEN NO. 50, and the first Fc polypeptide comprises the sequence of SEVENTEEN NO. 90.

[0182] 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 one of SEQ ID NOs 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 numbered based on SEQ ID NO. 5, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises SEQ ID NOs 48, 77, 78, and 80 It includes any one of the sequences of and 101. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide includes Q at position 192; Q at position 193; S at position 197; and S at position 199, as numbered based on SEQ ID NO. 5. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide includes the sequence of SEQ ID NO. 8, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide includes the sequence of SEQ ID NO. 77. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide includes the sequence of SEQ ID NO. 10, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide includes 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 and / or second Fc polypeptide comprises a portion of a hinge region (e.g., DKTHTCP (SEQ ID NO. 91) or DKTHTCPPCP (SEQ ID NO. 92)).

[0183] 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 one of SEQ ID NOs 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, F at position 194 (as numbered based on SEQ ID NO. 5), and Ser at position 239 (according to EU numbering), and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises SEQ ID NOs 47, 48, 51 to It comprises any one of the sequences 54, 61 to 63, 66, 73 to 78, 80, and 90. In some embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises Q at position 192; Q at position 193; S at position 197; and S at position 199, as numbered based on SEQ ID NO. 5. In some embodiments, the second polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO. 29, and the first polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO. 48, 53, 54, 74, or 77. In some embodiments, the second polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO. 36, and the first polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO. 61, 66, 75, or 78. In some embodiments, the second polypeptide or the second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO. 44, and the first polypeptide or the first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO. 74 or 77.

[0184] 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 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 numbered based on SEQ ID NO. 5, and the first Fc polypeptide or the first Fab-Fc fusion polypeptide comprises any one of the sequences of SEQ ID NOs 48, 73 to 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.

[0185] 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 one of SEQ ID NOs 4, 5, 7, 9, 50, 86, and 87, 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 numbered based on SEQ ID NO. 5, and the first Fab-Fc fusion polypeptide comprises any one of SEQ ID NOs 52, 53, and 61.

[0186] 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 one of SEQ ID NOs 6, 8, 10, 43, 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 numbered based on SEQ ID NO. 5, and the first Fab-Fc fusion polypeptide comprises any one of SEQ ID NOs 73 to 75.

[0187] 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 one of SEQ ID NOs 6, 8, 10, 43, 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 numbered based on SEQ ID NO. 5, and the first Fab-Fc fusion polypeptide comprises any one of SEQ ID NOs 73 to 75.

[0188] 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 according to EU numbering (each according to EU numbering), 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.

[0189] B. Non-targeted Fab

[0190] The untargeted Fab (NTF) comprises a light chain and a heavy chain, where the light chain is V L It includes a region and a light chain invariant region (CL), and the heavy chain is V H It includes a region and a heavy chain CH1 constant region. In some embodiments, the NTF does not specifically bind to an epitope that occurs naturally in the subject. In some embodiments, the NTF does not specifically bind to an antigen 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 an antigen from an infectious organism such as a virus, bacteria, fungus, or parasite. The mammal may be, but is not limited to, non-human primates, humans, or rodents (e.g., mice).

[0191] The specific binding of antibodies to antigens is at least 10 6 M -1It refers to the affinity. Specific binding has a detectably higher magnitude than non-specific binding occurring to at least one unrelated target and is distinguishable from non-specific binding. Non-specific binding is often the result of van der Waals forces. Non-targeting does not mean that NTF does not bind to any antigen with any affinity. Rather, in some embodiments, NTF does not exhibit specific binding to (a) any protein or epitope in mammalian cells, mammalian tissues, or within 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 within mammals.

[0192] 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 each comprise SEQ ID NOs 105, 106, or 128 and 107, and the light chain CDRs CDR-L1, CDR-L2, and CDR-L1 each comprise SEQ ID NOs 102, 103, and 104. In some embodiments, the heavy chain CDRs CDR-H1, CDR-H2, and CDR-H1 each comprise SEQ ID NOs 125, 126, or 128 and 127, and the light chain CDRs CDR-L1, CDR-L2, and CDR-L1 each comprise SEQ ID NOs 122, 123, and 124.

[0193] In some embodiments, the NTF comprises a heavy chain having 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 having 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.

[0194] In some embodiments, the NTF comprises a heavy chain having 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 having 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.

[0195] In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO. 109 or 110 and a light chain sequence comprising SEQ ID NO. 108. In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO. 109 and a light chain sequence comprising SEQ ID NO. 108. In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO. 110 and a light chain sequence comprising SEQ ID NO. 108.

[0196] In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO. 130 or 131 and a light chain sequence comprising SEQ ID NO. 129. In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO. 130 and a light chain sequence comprising SEQ ID NO. 129. In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO. 131 and a light chain sequence comprising SEQ ID NO. 129.

[0197] In some embodiments, the NTF comprises a light chain having an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO. 108, and an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO. 109 or 110, and comprises the 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 comprises a light chain comprising SEQ ID NO. 108 and SEQ ID NO. 109 or It includes a heavy chain that maintains the non-targeting characteristics of an NTF containing a heavy chain containing 110.

[0198] In some embodiments, the NTF comprises a light chain having an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO. 129, and an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO. 130 or 131, and comprises the 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 comprises a light chain comprising SEQ ID NO. 129 and SEQ ID NO. 130 or It includes a heavy chain that maintains the non-targeting characteristics of an NTF containing a heavy chain containing 131.

[0199] The NTF light chain and / or heavy chain may contain one or more modifications that facilitate conjugation to one or more cargo molecules. The modifications may be amino acid substitutions or insertions. Amino acid substitutions may be, but are not limited to, a substitution from lysine to cysteine ​​at position 149 (EU numbering) of the light chain, a substitution from serine to cysteine ​​at position 156 (EU numbering) of the light chain, a substitution from A to cysteine ​​at position 118 (EU numbering (position 114 according to Kabat numbering)) of the heavy chain, or a substitution from serine to cysteine ​​at position 124 (UE numbering) of the heavy chain.

[0200] In some embodiments, the first Fab-Fc fusion polypeptide comprises cysteine ​​at position 239, A at position 234, A at position 235, and serine at position 329 (each according to EU numbering), 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 cysteine ​​at position 114 (according to Kabat numbering) or position 124 (according to EU numbering), and A at position 234, A at position 235, and serine at position 329 (according to EU numbering). 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 numbering (each according to EU numbering), 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.

[0201] C. MAPT ASO

[0202] 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.

[0203] 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.

[0204] In some embodiments, MAPT ASO is connected to a delivery vehicle (e.g., a TfR-targeted Fc polypeptide dimer) through the 5' end of an oligonucleotide (e.g., the delivery vehicle is connected to the 5' end of MAPT ASO).

[0205] A MAPT ASO comprising SEQ ID NO. 111 or 112 is described having a modification that reduces non-specific plasma clearance and / or degradation / cleavage of the MAPT ASO when the MAPT ASO is linked to a peptide delivery vehicle (e.g., a TfR-targeted Fc polypeptide dimer). In some embodiments, the MAPT ASO is a capmer. In some embodiments, the MAPT ASO sequence consists of the nucleobase sequence of SEQ ID NO. 111 or 112.

[0206] In some embodiments, the MAPT capmer comprises a 5' wing segment having 3, 4, or 5 nucleosides. In some embodiments, the MAPT capmer comprises a 3' wing segment having 3, 4, or 5 nucleosides. In some embodiments, the MAPT capmer comprises a 5' wing segment having 3, 4, or 5 nucleosides and a 3' wing segment having 3, 4, or 5 nucleosides.

[0207] In some embodiments, the MAPT capmer comprises a 5' wing segment having three nucleosides. In some embodiments, the MAPT capmer comprises a 3' wing segment having three nucleosides. In some embodiments, the MAPT capmer comprises a 5' wing segment having three nucleosides and a 3' wing segment having three nucleosides.

[0208] In some embodiments, the MAPT gapmer comprises a gap segment containing 8 to 16 nucleosides. In some embodiments, the MAPT gapmer comprises a gap segment containing 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleosides. In some embodiments, the MAPT gapmer comprises a gap segment containing 10 to 14 nucleosides. In some embodiments, the MAPT gapmer comprises a gap segment containing 12 nucleosides. In some embodiments, the MAPT gapmer comprises a gap segment containing 13 nucleosides. In some embodiments, the MAPT gapmer comprises a gap segment containing 14 nucleosides. In some embodiments, each nucleoside within the gap segment comprises a 2'-deoxyribose sugar.

[0209] In some embodiments, MAPT Gapmer is X a -Y b -Z c The formula comprises, wherein 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 nucleoside comprises a modified sugar, each Y nucleoside has a 2' deoxyribose, and each Z nucleoside comprises a modified sugar. In some embodiments, the MAPT capmer comprises a 3-12-3 capmer. In some embodiments, the MAPT capmer comprises a 3-13-3 capmer. In some embodiments, a and c are each 3, b is 12, and each X and Z nucleoside is an LNA.

[0210] In some embodiments, the wing nucleoside of the MAPT Gammer comprises at least one nucleoside having a modified sugar (modified ribose). In some embodiments, all nucleosides within the 5' wing segment, 3' wing segment, or both 5' and 3' wing segments of the MAPT Gammer comprise the modified sugar. The modified sugar may be, but is not limited to, a 2'-O-methoxyethyl (MOE) sugar moiety or an acyclic sugar moiety comprising a 2'-4' bridge. The 2'-4' bridge may be -O-CH2- (locked nucleic acid or locked nucleoside (LNA)) or -O-CH(CH)- (pharmaceutical ethyl (cEt)). In some embodiments, each wing nucleoside of the MAPT Gammer comprises an acyclic sugar moiety comprising an -O-CH2- 2'-4' bridge (LNA).

[0211] i. Internucleoside binding:

[0212] In some embodiments, all nucleoside-to-nucleoside bonds in MAPT ASO include modified bonds (i.e., bonds other than naturally occurring phosphate bonds).

[0213] In some embodiments, the MAPT ASO comprises at least two stabilizing nucleoside bonds at the MAPT ASO end opposite to the end connected to the delivery vehicle. When the delivery vehicle is connected to the 5' end of the MAPT ASO, at least two nucleoside bonds at the 3' end of the MAPT ASO comprise stabilizing nucleoside bonds. In some embodiments, the MAPT ASO further comprises a stabilizing nucleoside bond at the 5' end. In some embodiments, the stabilizing nucleoside bonds have increased stability or nuclease resistance compared to PS nucleoside bonds. In some embodiments, the stabilizing nucleoside bonds are PN nucleoside bonds.

[0214] In some embodiments, the MAPT ASO comprises at least two PN bonds at the 3' end (i.e., PN bonds between the nucleoside at position n-2 (n minus 2) and n-1, and between the nucleoside at position n-1 and n, where n is the 3' end nucleoside). In some embodiments, the MAPT ASO further comprises at least two PN bonds at the 5' end (i.e., PN bonds between the nucleosides at position 1 and 2, and between the nucleosides at position 2 and 3).

[0215] In some embodiments, the MAPT ASO comprises at least three PN bonds at the 3' end (i.e., PN bonds between nucleosides at position n-3 and n-2, between nucleosides at position n-2 and n-1, and between nucleosides at position n-1 and n, where n is a 3' end nucleoside). In some embodiments, the MAPT ASO further comprises at least three PN bonds at the 5' end (i.e., PN bonds between nucleosides at position 1 and 2, between nucleosides at position 2 and 3, and between nucleosides at position 3 and 4).

[0216] In some embodiments, the MAPT ASO comprises at least two stabilizing nucleoside inter-links between nucleosides at positions 6–9. In some embodiments, the stabilizing nucleoside inter-links have increased stability or nuclease resistance compared to the PS nucleoside inter-links. In some embodiments, the stabilizing nucleoside inter-links are located at and / or near sites identified as vulnerable or catabolic sites of the MAPT ASO. The stabilizing nucleoside inter-links may be, but are not limited to, PS2 bonds, MsPA bonds, OiPS bonds, or PN bonds.

[0217] In some embodiments, the MAPT ASO comprises at least two PS2 nucleoside interlinks between nucleosides at positions 6–9. In some embodiments, the MAPT ASO comprises PS2 nucleoside interlinks between nucleosides at positions 6–7 and between nucleosides at positions 7–8.

[0218] In some embodiments, the MAPT ASO comprises at least two MsPA nucleoside interbonds between nucleosides at positions 6–9. In some embodiments, the MAPT ASO comprises MsPA nucleoside interbonds between nucleosides at positions 6–7 and between nucleosides at positions 7–8. In some embodiments, the MAPT ASO comprises MsPA nucleoside interbonds between nucleosides at positions 6–7, between nucleosides at positions 7–8, and between nucleosides at positions 8–9.

[0219] In some embodiments, the MAPT ASO comprises at least two OiPS nucleoside interbonds between nucleosides at positions 6–9. In some embodiments, the MAPT ASO comprises OiPS nucleoside interbonds between nucleosides at positions 6–7 and between nucleosides at positions 7–8. In some embodiments, the MAPT ASO comprises OiPS nucleoside interbonds between nucleosides at positions 6–7, between nucleosides at positions 7–8, and between nucleosides at positions 8–9.

[0220] In some embodiments, the MAPT ASO comprises at least two PN nucleoside interbonds between nucleosides at positions 6–9. In some embodiments, the MAPT ASO comprises PN nucleoside interbonds between nucleosides at positions 7–8 and between nucleosides at positions 8–9. In some embodiments, the MAPT ASO comprises PN nucleoside interbonds between nucleosides at positions 6–7, between nucleosides at positions 7–8, and between nucleosides at positions 8–9.

[0221] In some embodiments, MAPT ASO includes the following formula, and

[0222] X a -Y b -Z c ,

[0223] During the meal,

[0224] (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, and

[0225] (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, and

[0226] (c) Each internucleoside bond between X nucleosides comprises a PS bond or a PN bond, and each internucleoside bond between Z nucleosides comprises a PN bond, and

[0227] (d) The internucleoside bonds between X and Y nucleosides and the internucleoside bonds between Y and Z nucleosides independently include PS bonds or PN bonds, and

[0228] (e) Each nucleoside bond between Y nucleosides comprises a modified bond, wherein Yb comprises 2 to 3 adjacent PS2 bonds, 2 to 3 adjacent MsPA bonds, 2 to 3 adjacent OiPS bonds or 2 to 3 adjacent PN bonds, wherein other nucleoside bonds between Y nucleosides comprise PS bonds.

[0229] 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.

[0230] In some embodiments, a and c are each 3 and b is 12 to 13, and each nucleoside bond between X nucleosides includes a PS bond.

[0231] In some embodiments, a and c are each 3 and b is 12 to 13, and each nucleoside bond between X nucleosides includes a PN bond.

[0232] In some embodiments, a and c are each 3 and b is 12 to 13, and each internucleoside bond between X nucleosides includes a PS bond, the internucleoside bond between X and Y nucleosides includes a PS bond, and the internucleoside bond between Y and Z nucleosides includes a PS bond.

[0233] In some embodiments, a and c are each 3 and b is 12 to 13, and each internucleoside bond between X nucleosides includes a PS bond, the internucleoside bond between X and Y nucleosides includes a PS bond, and the internucleoside bond between Y and Z nucleosides includes a PN bond.

[0234] In some embodiments, a and c are each 3 and b is 12 to 13, and each nucleoside bond between X nucleosides includes a PN bond, the nucleoside bond between X and Y nucleosides includes a PS bond, and the nucleoside bond between Y and Z nucleosides includes a PS bond.

[0235] In some embodiments, a and c are each 3 and b is 12 to 13, and each internucleoside bond between X nucleosides includes a PN bond, the internucleoside bond between X and Y nucleosides includes a PN bond, and the internucleoside bond between Y and Z nucleosides includes a PN bond.

[0236] Gapmers having PS2, MsPA, OiPS, or PN bonds within the gap

[0237] In some embodiments, a and c are each 3 and b is 12 to 13, and Yb comprises 2 to 3 adjacent PS2 bonds. In some embodiments, Yb comprises 2 adjacent PS2 bonds. In some embodiments, the 2 adjacent PS2 bonds are between the third and fourth nucleosides of Yb and between the fourth and fifth nucleosides. In some embodiments, Yb comprises 3 adjacent PS2 bonds.

[0238] In some embodiments, a and c are each 3 and b is 12 to 13, and Yb comprises 2 to 3 adjacent MsPA bonds. In some embodiments, Yb comprises 2 adjacent MsPA bonds. In some embodiments, 2 adjacent MsPA bonds are between the third and fourth nucleosides of Yb and between the fourth and fifth nucleosides. In some embodiments, Yb comprises 3 adjacent MsPA bonds. In some embodiments, 3 adjacent MsPA bonds are between the third and fourth nucleosides of Yb, between the fourth and fifth nucleosides, and between the fifth and sixth nucleosides.

[0239] In some embodiments, a and c are each 3 and b is 12 to 13, and Yb comprises 2 to 3 adjacent OiPS bonds. In some embodiments, Yb comprises 2 adjacent OiPS bonds. In some embodiments, 2 adjacent OiPS bonds are between the 3rd and 4th nucleosides of Yb and between the 4th and 5th nucleosides. In some embodiments, Yb comprises 3 adjacent OiPS bonds. In some embodiments, 3 adjacent OiPS bonds are between the 3rd and 4th nucleosides of Yb, between the 4th and 5th nucleosides, and between the 5th and 6th nucleosides.

[0240] In some embodiments, a and c are each 3 and b is 12 to 13, and Yb comprises 2 to 3 adjacent PN bonds. In some embodiments, Yb comprises 2 adjacent PN bonds. In some embodiments, the 2 adjacent PN bonds are between the 4th and 5th nucleosides of Yb and between the 5th and 6th nucleosides. In some embodiments, Yb comprises 3 adjacent PN bonds.

[0241] In some embodiments, a and c are each 3 and b is 12 to 13, and Yb comprises 2 to 4 inter-nucleoside stabilization bonds. In some embodiments, Yb comprises PN bonds 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 comprises two different inter-nucleoside stabilization bonds. In some embodiments, Yb comprises PN bonds between the first and second nucleosides of Yb and PS2 bonds between the third and fourth nucleosides of Yb and between the eleventh and twelfth nucleosides. In some embodiments, Yb comprises two adjacent PS2 bonds and two adjacent PN bonds. In some embodiments, two adjacent PS2 bonds are between the third and fourth nucleosides of Yb and between the fourth and fifth nucleosides, and two adjacent PN bonds are between the tenth and eleventh nucleosides of Yb and between the eleventh and twelfth nucleosides.

[0242] In some embodiments, X a or Z c If at least one C nucleoside is present, it comprises 5-methylcytosine. In some embodiments, X a or Z c If any of my C nucleosides are present, they contain 5-methylcytosine.

[0243] ii. Specific combination

[0244] In some embodiments, a and c are each 3 and b is 12 to 13, each internucleoside bond between X nucleosides comprises a PS bond, the internucleoside bond between X and Y nucleosides comprises a PS bond, the internucleoside bond between Y and Z nucleosides comprises a PS bond, the internucleoside bond connecting the 4th and 5th nucleosides of Yb and the 5th and 6th nucleosides comprises a PN bond, and X a or Z c If any of my C nucleosides are present, they contain 5-methylcytosine.

[0245] In some embodiments, MAPT ASO is

[0246] 5' A L * m C L * m C L *T*T*A*A n G n T*A*T*T*A*C*T*T L n G L n m C L Includes 3' (sequence number 111), but,

[0247] A L is an adenine-locked nucleic acid, and

[0248] mCL은 5-메틸시토신 잠금 핵산이고,

[0249] T L It is a thymine-locked nucleic acid, and

[0250] G L It is a guanine-locked nucleic acid, and

[0251] A is deoxyadenosine, and

[0252] C is deoxycytidine, and

[0253] T is deoxythymidine, and

[0254] G is deoxyguanosine, and

[0255] * is a bond between PS nucleosides, and

[0256] n is a PN nucleoside interlink (see Fig. 10).

[0257] In some embodiments, a and c are each 3 and b is 12, each internucleoside bond between X nucleosides comprises a PN bond, the internucleoside bond between X and Y nucleosides comprises a PN bond, the internucleoside bond between Y and Z nucleosides comprises a PN bond, the internucleoside bond connecting the third and fourth nucleosides of Yb and the fourth and fifth nucleosides comprises a PS2 bond, and X a or Z c If any of my C nucleosides are present, they contain 5-methylcytosine.

[0258] In some embodiments, MAPT ASO is

[0259] 5' A L n m C L n m C L n T*T*A s 2 A s 2 G*T*A*T*T*A*C*T n T L n G L n m C L Includes 3' (sequence number 111), but,

[0260] A L is an adenine-locked nucleic acid, and

[0261] mCL은 5-메틸시토신 잠금 핵산이고,

[0262] T L It is a thymine-locked nucleic acid, and

[0263] G L It is a guanine-locked nucleic acid, and

[0264] A is deoxyadenosine, and

[0265] C is deoxycytidine, and

[0266] T is deoxythymidine, and

[0267] G is deoxyguanosine, and

[0268] * is a bond between PS nucleosides, and

[0269] s 2 is a PS2 internucleoside bond, and

[0270] n is a PN nucleoside interlink (see Fig. 11).

[0271] In some embodiments, a and c are each 3 and b is 12, each internucleoside bond between X nucleosides comprises a PN bond, the internucleoside bond between X and Y nucleosides comprises a PS bond, the internucleoside bond between Y and Z nucleosides comprises a PS bond, and the internucleoside bonds connecting the third and fourth nucleosides of Yb and the fourth and fifth nucleosides comprise a PS2 bond, and X a or Z c If any of my C nucleosides are present, they contain 5-methylcytosine.

[0272] In some embodiments, MAPT ASO is

[0273] 5' A L n m C L n m C L *T*T*A s 2 A s 2 G*T*A*T*T*A*C*T*T L n G L n m C L Includes 3' (sequence number 111), but,

[0274] A L is an adenine-locked nucleic acid, and

[0275] mCL은 5-메틸시토신 잠금 핵산이고,

[0276] TL It is a thymine-locked nucleic acid, and

[0277] G L It is a guanine-locked nucleic acid, and

[0278] A is deoxyadenosine, and

[0279] C is deoxycytidine, and

[0280] T is deoxythymidine, and

[0281] G is deoxyguanosine, and

[0282] * is a bond between PS nucleosides, and

[0283] s 2 is a PS2 internucleoside bond, and

[0284] n is a PN nucleoside interlink (see Fig. 12).

[0285] In some embodiments, a and c are each 3 and b is 12, and each internucleoside bond between X nucleosides comprises a PS bond, the internucleoside bond between X and Y nucleosides comprises a PS bond, the internucleoside bond between Y and Z nucleosides comprises a PN bond, and the internucleoside bonds connecting the third and fourth nucleosides of Yb, the fourth and fifth nucleosides, and the fifth and sixth nucleosides comprise an MsPA bond, and X a or Z c If any of my C nucleosides are present, they contain 5-methylcytosine.

[0286] In some embodiments, MAPT ASO is

[0287] 5' A L s m C L s m C L *T*T*A u A u G u T*A*T*T*A*C*T n T L n G L n m C LIncludes 3' (sequence number 111), but,

[0288] A L is an adenine-locked nucleic acid, and

[0289] mCL은 5-메틸시토신 잠금 핵산이고,

[0290] T L It is a thymine-locked nucleic acid, and

[0291] G L It is a guanine-locked nucleic acid, and

[0292] A is deoxyadenosine, and

[0293] C is deoxycytidine, and

[0294] T is deoxythymidine, and

[0295] G is deoxyguanosine, and

[0296] * is a bond between PS nucleosides, and

[0297] u Is MsPA is an internucleoside bond, and

[0298] n is a PN nucleoside interlink (see Fig. 13).

[0299] In some embodiments, a and c are each 3 and b is 12, each internucleoside bond between X nucleosides comprises a PN bond, the internucleoside bond between X and Y nucleosides comprises a PS bond, the internucleoside bond between Y and Z nucleosides comprises a PS bond, and the internucleoside bonds connecting the third and fourth nucleosides of Yb and the fourth and fifth nucleosides comprise an MsPA bond, and X a or Z c If any of my C nucleosides are present, they contain 5-methylcytosine.

[0300] In some embodiments, MAPT ASO is

[0301] 5' A L n m C L n m C L *T*T*Au A u G*T*A*T*T*A*C*T*T L n G L n m C L Includes 3' (sequence number 111), but,

[0302] A L is an adenine-locked nucleic acid, and

[0303] mCL은 5-메틸시토신 잠금 핵산이고,

[0304] T L It is a thymine-locked nucleic acid, and

[0305] G L It is a guanine-locked nucleic acid, and

[0306] A is deoxyadenosine, and

[0307] C is deoxycytidine, and

[0308] T is deoxythymidine, and

[0309] G is deoxyguanosine, and

[0310] * is a bond between PS nucleosides, and

[0311] u is an MsPA internucleoside bond, and

[0312] n is a PN nucleoside interlink (see Fig. 14).

[0313] D. Connector

[0314] MAPT ASO may be linked to the first Fc polypeptide, the second Fc polypeptide, the first NTF and / or the second NTF. In some embodiments, MAPT ASO is covalently linked to the first Fc polypeptide, the second Fc polypeptide, the first NTF and / or the second NTF. The first Fc polypeptide, the second Fc polypeptide, the first NTF and / or the second NTF may contain amino acid substitutions (e.g., cysteine ​​substitutions) to facilitate the attachment of MAPT ASO.

[0315] In some embodiments, MAPT ASO is linked to the first Fc polypeptide. In some embodiments, MAPT ASO is linked to a cysteine ​​residue in the first Fc polypeptide. In some embodiments, the cysteine ​​residue in the first Fc polypeptide comprises a cysteine ​​substitution at position 239 (according to EU numbering). In some embodiments, the first Fc polypeptide comprises cysteine ​​at positions 239, 442, 330, and 289 (according to EU numbering). In some embodiments, the cysteine ​​residue in the first Fab-Fc fusion polypeptide comprises a cysteine ​​substitution at position 114 (according to Kabat numbering) or position 124 (according to EU numbering).

[0316] MAPT ASO may be connected to a first Fc polypeptide, a second Fc polypeptide, or a first NTF and / or a second NTF through any linker available in the art suitable for connecting an oligonucleotide (e.g., ASO, e.g., Gapmer) to a polypeptide (e.g., Fc polypeptide or Fab polypeptide). In some embodiments, the linker comprises any known linker which is a difunctional linker capable of covalently connecting an oligonucleotide to a polypeptide.

[0317] Oligonucleotide polypeptide conjugates can be produced using well-known chemical crosslinking agents and protocols that covalently link oligonucleotides and polypeptides through linkers. For example, there are numerous chemical crosslinking agents known to those skilled in the art that are useful for crosslinking proteins with agents of interest. For example, a crosslinking agent may be a heterofunctional crosslinking agent that can be used to link molecules in a stepwise manner. Heterofunctional crosslinking agents allow for the design of more specific coupling methods for protein conjugation, thereby reducing the occurrence of unwanted side reactions such as homoprotein polymers. N-hydroxysuccinimide (NHS) or its water-soluble analogs N-hydroxysulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); Various heterofunctional crosslinking agents are known in the art, including but not limited to N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Such crosslinkers having an N-hydroxysuccinimide moiety can generally be obtained as N-hydroxysulfosuccinimide analogs having greater water solubility. Additionally, such crosslinkers having a disulfide bridge within the linking chain can be synthesized as alkyl derivatives instead to reduce the amount of linker cleavage in vivo. In addition to heterofunctional crosslinkers, a number of other crosslinkers exist, including homofunctional and photoreactive crosslinkers. Disuccinimidyl subverate (DSS), bismaleimidohexane (BMH), and dimethylpimelimidate.2HCl (DMP) is an example of a useful homofunctional crosslinking agent, and bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenyl-amino)hexanoate (SANPAH) are examples of useful photoreactive crosslinking agents.

[0318] Non-limiting examples of conjugate linkers include those described in WO2023 / 279099 and WO2023 / 056388, each incorporated herein by reference in its entirety. In some embodiments, the linker includes a val-cit linker as described in US 6,214,345, which is incorporated herein by reference. In some embodiments, the linker includes those described in WO2020 / 028840 and WO2022 / 212886, each incorporated herein by reference in its entirety. Other linkers [Bioconjugate Chemistry 2023 34 It may include the items described in

[11] , 2096-2111].

[0319] A linker may be attached to any region of the polypeptide (e.g., an N-terminal region, a C-terminal region, or amino acids within the protein such as cysteine ​​residues or glutamine residues) as long as the oligonucleotide does not interfere with the binding of the oligonucleotide to the TfR of the second Fc polypeptide. Similarly, a linker may be attached to any region of the oligonucleotide (e.g., a 5' end, a 3' end, or an intramolecular nucleic acid residue) as long as the polypeptide does not interfere with the functionality of the oligonucleotide (e.g., complementary binding to a target nucleic acid). For example, a linker may be attached to the oligonucleotide through any number of synthetically possible points located throughout the oligonucleotide, such as 3' or 5' terminal residues of the oligo; sugar moiety; base moiety; or residues located within the backbone. In some embodiments, the linker is attached to the oligonucleotide at the 5' terminal residue of the oligonucleotide.

[0320] In some embodiments, the connector includes a spacer. The spacer may be a hydrophilic spacer, but is not limited thereto. The hydrophilic spacer may be polyethylene glycol (PEG), but is not limited thereto.

[0321] The linker may be a cleavable linker or a non-cleavable linker. In some embodiments, the linker is cleavable. The cleavable linker contains a cleavable linker. The cleavable linker includes, but is not limited to, nuclease-cleavable linkers, acid-unstable linkers, peptidase-sensitive linkers, photounstable linkers, dimethyl linkers, and disulfide-containing linkers (Reference [Chari et al., Cancer Res. 52:127-131 (1992)]; US Patent No. 5,208,020).

[0322] In some embodiments, the connector comprises maleimide. In some embodiments, the connector has the following structure,

[0323]

[0324] In the formula, L' is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, wherein one or more of the carbon atoms (e.g., 1, 2, 3, or 4) are optionally replaced by (-O-), (-NH-), (-S-), amino acids, hydrazones (-C(R')=N=N(R')-), nucleotides, or 3 to 12-membered divalent heterocycles, wherein the chain and any 3 to 12-membered divalent heterocycles are optionally (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, It is substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of oxo (=O), hydrazone (=N=N(R')-), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R' is independently H or (C1-C6)alkyl, where the valence indicated by * is attached to the polypeptide and the valence indicated by ** 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 contains 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 comprises one or more hydrazone groups added to the chain or to carbon atoms 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 comprises one or more amino acids within 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 within 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 within 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 another embodiment, L' comprises one or more nucleotides having an unmodified base, an unmodified sugar group, and / or an unmodified phosphate group.

[0325] In some embodiments, the connector includes the following structure,

[0326]

[0327] Among the formulas, bonds and intersect in the chemical structure ' ' indicates the point in the chemical structure where a wavy bond intersects the rest of the molecule. In some embodiments, the valence marked * is attached to the TfR-targeted Fc polypeptide dimer, and the valence marked ** is attached to MAPT ASO. In some embodiments, the valence marked ** is attached to MAPT ASO through the 5' end group of MAPT ASO. In some embodiments, the valence marked ** is attached to MAPT ASO through the 5' end PO group of MAPT ASO. In some embodiments, the valence marked ** is attached to MAPT ASO through the 5' end PS group of MAPT ASO. In some embodiments, the valence marked ** is attached to MAPT ASO through the 5' end PS2 group of MAPT ASO. In some embodiments, the valence marked ** is attached to MAPT ASO through the 5' end PN group of MAPT ASO. In some embodiments, the valence denoted by ** is attached to MAPT ASO through the 5' terminal MsPA group of MAPT ASO. In some embodiments, the valence denoted by ** is attached to MAPT ASO through the 5' terminal OiPS group of MAPT ASO.

[0328] In some embodiments, the connector includes the following,

[0329]

[0330] In the formula, wavy lines that intersect bonds in the chemical structure ' indicates the point in the chemical structure where a wavy bond intersects the rest of the molecule.

[0331] In some embodiments, the stabilized oligonucleotide polypeptide conjugate comprises the following,

[0332]

[0333] In the formula, P is a polypeptide, and the phosphodiester (PO) group is the 5' terminal group of an oligonucleotide.

[0334] In some embodiments, the stabilized oligonucleotide polypeptide conjugate comprises the following,

[0335]

[0336] In the formula, P is a polypeptide, and the phosphothioate (PS) group is the 5' terminal group of an oligonucleotide.

[0337] In some embodiments, the stabilized oligonucleotide polypeptide conjugate comprises the following,

[0338]

[0339] In the formula, P is a polypeptide, and the cyclic phosphorylguanidine (PN) group is the 5' terminal group of an oligonucleotide.

[0340] nucleic acids

[0341] The present invention describes nucleic acids encoding any heavy and light chains of any of the described Fc peptides, Fc polypeptide dimers, NTFs, and / or Fab-Fc fusion polypeptides. Optionally, such nucleic acids further encode signal peptides. The coding sequence of the nucleic acid may be operably linked to one or more regulatory sequences to facilitate the 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 an isolated form or may be cloned into one or more vectors. The nucleic acids may be synthesized, for example, by solid-phase synthesis or by PCR of nested oligonucleotides. The nucleic acids encoding the heavy and light chains may be linked as a single continuous nucleic acid, for example, within an expression vector, or may be separate, for example, each may be cloned into its own expression vector.

[0342] In some embodiments, the nucleic acid encoding the 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 peptides, Fc polypeptide dimers, NTFs, and / or Fab-Fc fusion polypeptides. The nucleic acid sequence may be provided within an expression vector to facilitate the expression of heavy chains and / or light chains in a host cell. The nucleic acid or the expression vector containing the nucleic acid is transformed into a host cell. The host cell may then be used to produce the heavy chains and / or light chains of the NTF.

[0343] In some embodiments, the nucleic acid encoding the TfR-targeted Fc polypeptide dimer 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 NOs 1, 47 to 48, 51 to 54, 61 to 63, 66, 73 to 78, 80, and 90, and the second nucleic acid sequence encodes any one of SEQ ID NOs 4 to 29, 36, 43 to 44, and 49 to 50.

[0344] In some embodiments, the nucleic acid encoding the TfR-targeted Fc polypeptide dimer 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 an NTF light chain. In some embodiments, the first nucleic acid sequence encodes any one of SEQ ID NOs 100 to 101 and 130 to 131, the second nucleic acid sequence encodes any one of SEQ ID NOs 98 to 99, and the third nucleic acid sequence encodes any one of SEQ ID NOs 108 and 129.

[0345] 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.

[0346] A method for preparing the described Fc polypeptide, Fc polypeptide dimer, NTF, Fab-Fc fusion polypeptide, or antibody comprises, but is not limited to, the steps of: expressing one or more nucleic acids encoding the heavy and light chains of the Fc polypeptide, Fc polypeptide dimer, NTF, Fab-Fc fusion polypeptide, or NTF in a plurality of host cells; proliferating the cells under conditions suitable for the expression of the heavy and light chains of the Fc polypeptide, Fc polypeptide dimer, NTF, Fab-Fc fusion polypeptide, or NTF within the cells; and purifying the heavy and light chains of the Fc polypeptide, Fc polypeptide dimer, NTF, Fab-Fc fusion polypeptide, or NTF. The Fc polypeptide, Fc polypeptide dimer, Fab-Fc fusion polypeptide, or purified NTF may be conjugated to one or more of the described MAPT ASOs.

[0347] Cells containing nucleic acids encoding the described Fc polypeptide, Fc polypeptide dimer, NTF, Fab-Fc fusion polypeptide, or the heavy and light chains of Fab are described. The cells may be bacterial cells, yeast cells, insect cells, or mammalian cells. The cells may be used to express the Fc polypeptide, Fc polypeptide dimer, NTF, or Fc polypeptide-NTF heavy chain fusion polypeptide. The expressed polypeptides may then be isolated from the cells and selectively purified.

[0348] How to use

[0349] The MAPT ASO conjugates described herein may be used for various purposes, including therapeutic indications.

[0350] 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 may be used to transport MAPT ASO across the endothelium, e.g., the blood-brain barrier, so that MAPT ASO is absorbed into the brain.

[0351] In some embodiments, a method for reducing the expression of the MAPT gene in a subject is described, said method comprising the step of administering an effective amount of the MAPT ASO conjugate or a composition thereof as described herein to the subject. In some embodiments, the MAPT ASO conjugate or a composition thereof is provided for use in reducing the expression of the MAPT gene in the cells of the subject. In some embodiments, the MAPT ASO binds to the MAPT transcript and mobilizes RNase H, which degrades the transcript. In some embodiments, administering the described MAPT ASO conjugate or a composition thereof to a cell or subject reduces the expression of the MAPT gene in the cell or subject. Expression may be reduced to 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% compared to expression in a control group (e.g., cells or subjects not administered with the MAPT ASO, MAPT ASO conjugate, or composition described herein) or compared to the level of MAPT expression in cells or subjects prior to administration of MAPT ASO.

[0352] For example, certain embodiments provide a method for transcytosis of MAPT ASO across the endothelium, said method comprising the step of bringing the endothelium (e.g., blood-brain barrier (BBB)) into contact with a MAPT ASO conjugate as described herein. Accordingly, certain embodiments provide a method for transporting MAPT ASO across the BBB of a subject requiring this, which comprises the step of administering a MAPT ASO conjugate as described herein to the subject. In some embodiments, a MAPT ASO conjugate as described herein is provided for use in transporting MAPT ASO across the BBB of a subject requiring this. In some embodiments, the present invention provides a method for delivering MAPT ASO to the CNS. In some embodiments, the present invention provides a method for delivering MAPT ASO to deep brain regions (e.g., cortex, brainstem, hippocampus, striatum, cerebellum, thalamus, caudate horn, and substantia nigra). In some embodiments, the present invention provides a method for delivering MAPT ASO to deep brain regions and the spinal cord (e.g., cervical, lumbar). In some embodiments, the present invention provides a method for delivering MAPT ASO to the CNS and muscles (e.g., cardiac muscle and skeletal muscle). In some embodiments, the present invention provides a method for delivering MAPT ASO to the CNS, peripheral nerves (e.g., retina, sciatic nerve), muscles (e.g., quadriceps femoris), and other peripheral organs (e.g., heart, diaphragm, spleen, intestine, lung, liver, and kidney).

[0353] Certain embodiments also provide a method for controlling the expression of MAPT in a subject requiring this, comprising the step of administering an effective amount of a MAPT ASO conjugate as described herein to the subject. In some embodiments, the MAPT ASO conjugate as described herein is intended for use in controlling the expression of MAPT in the subject. In some embodiments, MAPT is expressed in cells within the subject's brain.

[0354] MAPT ASO or MAPT ASO conjugates as described herein may be administered to subjects at a therapeutically effective amount or dose. However, the dose may vary depending on several factors, including the selected route of administration, the formulation of the composition, patient response, severity of the condition, the subject's body weight, and the prescribing physician's judgment. The dose may be increased or decreased over time as required by the individual patient.

[0355] In various embodiments, the MAPT ASO or MAPT ASO conjugate as described herein is administered parenterally. In some embodiments, the MAPT ASO or MAPT ASO conjugate is administered intravenously. Intravenous administration may be by infusion and may be carried out, for example, over a period of about 10 minutes to about 30 minutes, or over a period of at least 1 hour, 2 hours, or 3 hours. In some embodiments, the MAPT ASO or MAPT ASO conjugate is administered as an intravenous bolus. A combination of infusion and bolus administration may also be used.

[0356] In some parenteral embodiments, the MAPT ASO or MAPT ASO conjugate is administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, the MAPT ASO or MAPT ASO conjugate is administered intradermally or intramuscularly. In some embodiments, the MAPT ASO or MAPT ASO conjugate is administered intrathecally or intraventricularly, as by epidural administration.

[0357] In other embodiments, the MAPT ASO or MAPT ASO conjugate as described herein may be administered orally, by pulmonary administration, by nasal administration, by ocular administration, or by topical administration. Pulmonary administration may also be utilized, for example, by the use of an inhaler or nebulizer and by formulation with an aerosolizing agent.

[0358] The MAPT ASO and MAPT ASO conjugates described herein may also be used to treat, prevent, or improve tau-related diseases, disorders, and pathological conditions. Accordingly, in some embodiments, the present invention provides a method for treating, preventing, or improving tau-related diseases, disorders, and pathological conditions in subjects requiring such methods. In some embodiments, tau-related diseases are tau-related neurodegenerative disorders. In some embodiments, tau-related diseases include, but are not limited to, tauopathy, Alzheimer's disease, frontotemporal dementia (FTD), FTDP-17, progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), epilepsy, and Dravet syndrome.

[0359] Accordingly, a specific embodiment provides a method for treating a tau-related neurodegenerative disorder in a subject requiring such treatment, said method comprising the step of administering to the subject a MAPT ASO, a MAPT ASO conjugate, or a composition as described herein.

[0360] In some embodiments, the tau-related neurodegenerative syndrome is Alzheimer's disease. Accordingly, certain embodiments provide a method for treating Alzheimer's disease, said method comprising the step of administering a MAPT ASO, a MAPT ASO conjugate, or a composition as described herein to a subject in need thereof. The subject may be diagnosed with Alzheimer's disease, diagnosed with one or more symptoms of Alzheimer's disease, or at risk of developing Alzheimer's disease or one or more symptoms associated with Alzheimer's disease.

[0361] A method for targeting and delivering MAPT ASO to a patient's CNS tissue is described, comprising the step of administering any described MAPT ASO conjugate or pharmaceutical composition to a subject. In some embodiments, MAPT ASO is distributed throughout the entire CNS. In some embodiments, MAPT ASO is distributed throughout 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 regulates the expression of a target gene. In some embodiments, the regulation of target gene expression is the repression of gene expression (i.e., gene knockdown).

[0362] In a specific embodiment, the object is a human object.

[0363] Pharmaceutical composition and kit

[0364] In some embodiments, pharmaceutical compositions and kits comprising a MAPT ASO conjugate as described herein are provided.

[0365] A. Pharmaceutical composition

[0366] Guidelines for preparing formulations for use as described herein can be found in any number of handbooks for pharmaceutical manufacturing and formulation known to a person skilled in the art.

[0367] In some embodiments, the pharmaceutical composition comprises a MAPT ASO conjugate as described herein and further comprises one or more pharmaceutically acceptable carriers and / or excipients.

[0368] In certain embodiments, the composition comprises a plurality of MAPT ASO conjugates as described herein (e.g., a mixture of different conjugates) that may be identical or different.

[0369] In some embodiments, the MAPT ASO conjugate as described herein comprises 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 1 (1) MAPT ASO linked to an amino acid of the MAPT ASO conjugate. In some embodiments, the MAPT ASO conjugate comprises 2 (2) MAPT ASOs linked to 2 amino acids of the MAPT ASO conjugate. In some embodiments, the MAPT ASO conjugate comprises 2 (2) MAPT ASOs linked to 1 amino acid of the MAPT ASO conjugate. In some embodiments, the MAPT ASO conjugate comprises 4 (4) MAPT ASOs linked to 4 amino acids of the MAPT ASO conjugate. In some embodiments, the MAPT ASO conjugate comprises four (four) MAPT ASOs (two MAPT ASOs linked to each of the two amino acids) linked to two amino acids of the MAPT ASO conjugate. In some embodiments, the MAPT ASO conjugate comprises four (four) MAPT ASOs linked to one amino acid of the MAPT ASO conjugate.

[0370] In some embodiments, a single MAPT ASO is attached to a TfR-targeted Fc dimer or a Fab-Fc dimer. In some embodiments, two or more MAPT ASOs are attached to a TfR-targeted Fc dimer or a Fab-Fc dimer. Two or more MAPT ASOs may be connected to a single linker, or two or more MAPT ASOs may also be connected to a TfR-targeted Fc dimer or a Fab-Fc dimer through two or more linkers. Two or more linkers may be the same or different. In some embodiments, two MAPT ASOs are attached to a TfR-targeted Fc dimer or a Fab-Fc dimer. In some embodiments, four MAPT ASOs are attached to a TfR-targeted Fc dimer or a Fab-Fc dimer.

[0371] 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 tandem to a single linker (L). In the case of tandem 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, in the case of tandem 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 a nucleic acid linker or a non-nucleic acid cleavage linker.

[0372] In some embodiments, two or more MAPT ASOs are connected to a single branched linker (L). The linker may be a branched linker in which two or more oligonucleotides are separately attached to a single linker (L) (i.e., y is two or more).

[0373] In some embodiments, the ratio of oligonucleotides to proteins in the composition is about 1:1 to about 4:1. In some embodiments, the ratio of oligonucleotides to proteins in the composition is 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 about 2:1 to about 3:1. In some embodiments, the ratio of oligonucleotides to proteins in the composition is about 2.5.

[0374] As used herein, the term "pharmaceuticalally acceptable carrier" comprises any solvent, dispersion medium, or coating that is physiologically suitable and preferably does not interfere with or otherwise inhibit the activity of the activator. 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. The pharmaceutically acceptable carrier may contain one or more physiologically acceptable compounds that act to stabilize the composition or to increase or decrease the absorption of the MAPT ASO conjugate, for example. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, and dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of the activator; or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and formulations thereof are also available in the art.

[0375] The pharmaceutical compositions described herein may be prepared in a manner known to a person skilled in the art, for example by conventional mixing, dissolving, granulating, making coated tablets, emulsifying, encapsulating, capturing, or freeze-drying processes. The following methods and excipients are merely exemplary and are not limiting in any way.

[0376] For administration to a subject, the MAPT ASO conjugate as described herein may be formulated by combining it with a pharmaceutically acceptable carrier well known in the art. Such a carrier enables the compound to be formulated into tablets, pills, coated tablets, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc. A pharmaceutical formulation for administration to a subject may be obtained by mixing the MAPT ASO conjugate with a solid excipient, optionally grinding the resulting mixture, adding a suitable adjuvant if desired, and then processing the mixture of granules to obtain a tablet or coated tablet core. Suitable excipients include, for example, fillers such as sugars containing lactose, sucrose, mannitol, or sorbitol; For example, it includes cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone. If desired, a disintegrant such as cross-linked polyvinylpyrrolidone, agar, or salts thereof such as alginic acid or sodium alginate may be added.

[0377] As disclosed above, the MAPT ASO conjugate as described herein may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. For injection, the MAPT ASO conjugate may be formulated by dissolving, suspending, or emulsifying it in an aqueous or non-aqueous solvent such as vegetable oil or other similar oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the MAPT ASO conjugate may be formulated in an aqueous solution, preferably in a physiologically suitable buffer such as Hanks solution, Ringer solution, or physiological saline buffer. The injectable formulation may be provided as a single-dose form, for example, in an ampoule or in a multi-dose container, with added preservatives. The composition may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulation agents such as suspending agents, stabilizers, and / or dispersing agents.

[0378] Typically, pharmaceutical compositions intended for in vivo administration are in a sterile state. Sterilization can be achieved by methods known in the art, e.g., heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0379] The dosage and target drug concentration of the pharmaceutical composition as described herein may vary depending on the specific intended use. The determination of the appropriate dosage or route of administration is fully within the scope of the skill of a person skilled in the art. Suitable dosages are also described above.

[0380] B. Kit

[0381] In some embodiments, a kit comprising a MAPT ASO conjugate as described herein is provided. In some embodiments, the kit is intended for use in controlling 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 intended for use in controlling the expression of a target gene.

[0382] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises a MAPT ASO conjugate as described herein and further comprises one or more additional therapeutic agents. In some embodiments, the kit further comprises explanatory material containing instructions (i.e., protocols) for carrying out the method described herein (e.g., instructions regarding using the kit to administer a composition across the blood-brain barrier). The explanatory material typically comprises, but is not limited to, written or printed material. Any medium capable of storing such instructions and delivering them to an end user is considered herein. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips) and optical media (e.g., CD-ROMs). Such media may include the address of an internet site providing such explanatory material.

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391] Examples

[0392] Example 1. OTV:MAPT ASO Conjugate Construction

[0393] i. Design and cloning of the Fab-Fc dimer fusion. A Fab-Fc dimer fusion was designed comprising a first Fc polypeptide containing S239C cysteine ​​substitution and a hole and LALAPS mutation (SEQ No. 63), a second Fc polypeptide containing a modified constant domain that forms an Fc dimer with the first Fc polypeptide and specifically binds to a transferrin receptor (35.23.2), a kop and a LALAPS mutation (SEQ No. 11), wherein the first and second Fc polypeptides are fused to a non-targeted Fab (SEQ Nos. 108 and 109), respectively. Thus, the first Fab-Fc polypeptide fusion has the sequence of SEQ No. 100, and the second Fab-Fc polypeptide fusion has the sequence of SEQ No. 98.

[0394] Additionally, as described herein, the C-terminal lysine residue of the Fc polypeptide may be completely or partially removed by the cellular machinery during protein synthesis. Thus, the first Fc polypeptide may comprise SEQ ID NO. 80, and the second Fc polypeptide may comprise SEQ ID NO. 12. Similarly, the first Fab-Fc polypeptide fusion may have the sequence of SEQ ID NO. 101, and the second Fab-Fc polypeptide fusion may have the sequence of SEQ ID NO. 99.

[0395] The construct was cloned into the mammalian expression vector pRK5 by gene synthesis and Gibson assembly.

[0396] ii. Expression and purification of Fab-Fc dimer fusion protein.The vector was co-transfected into Expi293 cells with the corresponding light chain vector to achieve a knop:hole:light chain ratio of 1:1:2. The expressed protein was purified from the 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 containing 150 mM NaCl, pH 3.0, and immediately neutralized with 200 mM arginine, 137 mM succinic acid, pH 5.0. The protein was further purified by size exclusion chromatography (SEC) (GE Superdex200) using 200 mM arginine, 137 mM succinic acid, pH 5.0 as the running buffer. The purified protein was identified by Intact Mass LC / MS, and a purity greater than 95% was confirmed by SDS-PAGE and analytical HPLC-SEC.

[0397] iii. Synthesis of 5'-Maleimide Modified ASOs

[0398] Preparation of 5'-amino-modified ASOs

[0399] Solid-phase oligonucleotide synthesisOligonucleotide synthesis was performed on a MerMade 12 (LGC) DNA / RNA synthesizer at a 100 μmol scale according to a standard solid-phase oligonucleotide synthesis protocol. All lock nucleic acid (LNA) and deoxyribonucleic acid (DNA) phosphoramidites, including LNA-A (Bz), LNA-5MeC (Bz), LNA-T, LNA-G (dmf), and dA (Bz), dC (Ac), dT, and dG (dmf), were purchased from Hongene Biotech Corporation. LNA-5MeC (Bz) was dissolved in a mixed solvent of DCM / acetonitrile (1:1, v / v), while all other phosphoramidites were dissolved in acetonitrile and a molecular sieve (3 Å) was added. The parent antisense oligonucleotide (ASO) sequence was first assembled on a UnyLinker CPG solid support, and then an amino linker was attached to the 5'-terminus using 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (CAS: 133975-85-6; Glen Research Cat. # 10-1916). The synthesis cycle for adding a single nucleotide (or non-nucleic acid) unit consists of four separate steps: detritylation, coupling, oxidation (or sulfidation), and capping. 5-ethylthio-1H-tetrazole (ETT, 0.25 M in acetonitrile) was used as the activator solution. Phosphorothioate bonds were introduced using a 0.2 M solution of PADS (phenylacetyl disulfide) in 50% pyridine / 50% acetonitrile. The detailed protocol for 100 μmol scale ASO synthesis is Tables 2A to 2D below It is summarized in.

[0400]

[0401]

[0402]

[0403]

[0404]

[0405] Manipulation after synthesis When the synthesis of the solid-phase oligonucleotide was complete, the phosphate protecting group (2-cyanoethyl group) was removed in a 20% solution of diethylamine (DEA) in acetonitrile for 1 hour. Cleavage and nucleobase deprotection (C&D) from the solid support were performed in NH4OH / EtOH (3:1) at 45°C for 20 hours. The crude oligonucleotide solution was concentrated by centrifugal evaporation under reduced pressure, and the solid residue was reconstituted in water for prep-HPLC purification.

[0406] Purification of 5'-amino ASO by prep-HPLC. The crude 5'-amino ASO was purified by reverse-phase ion-pair HPLC. The detailed parameters are Table 3 It is summarized in ). Appropriate fractions were combined and freeze-dried to obtain purified 5'-amino ASO.

[0407]

[0408] Synthesis of 5'-amino-modified ASOs . under Table 4 and Table 5 The 5'-amino modified ASO shown in was synthesized according to the general method mentioned above.

[0409] Preparation of 5'-Maleimide Modified ASOGeneral procedure: A solution of 3-maleimidopropionic acid N-hydroxysuccinimide ester (MCOSu, 10 equivalents) in DMF (2.5 mL) was added to a solution of ASO amine TEA salt (30 mg, based on OD) in PBS buffer (TEKNOVA 10X PBS stock solution, pH 6.0, 2.5 mL) at room temperature. The resulting solution was shaken at room temperature for 18 hours. Upon completion, the solution was desalted by passing it through a sephadex G25 column (2.5X40 cm) in AKTA pure 25 M, eluted with Milli Q water at 3 mL / min, and monitored by UV 260 / 280 and conductivity. Appropriate fractions were combined and freeze-dried to obtain the desired 5'-maleimide ASO.

[0410] iv. BioconjugationThe Fab-Fc dimer fusion product generated above, containing the S239C cysteine ​​modification for conjugation, was first reduced with 30 molar equivalents of TCEP and 2 mM EDTA at 37°C for 1 hour. Reduction was confirmed by LC / MS. After reduction, the remaining TCEP was removed by dialysis (e.g., purification by dialysis) using 1X PBS containing 2 mM EDTA, pH 6.8, and the Fab-Fc dimer fusion product 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 product and reacted at room temperature for 1 hour. The generated 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 unconjugated products. The purity of the MAPT ASO conjugate was determined by LC / MS and SEC. The generated conjugate is referred to as OTV or OTV:MAPT.

[0411] Example 2. In vitro screening of ASO for the MAPT gene

[0412] The selection of an ASO sequence specific to a given target sequence is based on the analysis of the selected target sequence and the determination of multiple factors, including in vitro and in vivo efficacy and hepatotoxicity profiles.

[0413] Initial MAPT ASO screening. The initial identification of 393 MAPT ASO sequences 100% complementary to MAPT pre-mRNA (Ensembl ENST0000034429) and / or MAPT mRNA (refseq NM_001123066) sequence number 115 was based on computational prediction of target binding, specificity, efficacy, and safety.

[0414] The knockdown efficacy of MAPT-specific ASOs was tested in human MiaPaCa-2 and HDLM2 cells. Cells were treated with MAPT-specific ASOs or control oligonucleotides at a concentration of 5 μM without the use of transfection reagents. After 3 days of treatment, the cells were lysed. MAPT and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher). MAPT expression values ​​were normalized to HPRT1 values, and the degree of knockdown was determined by comparison with mock-treated cells.

[0415] 2 concentration screenings. Fifty-five MAPT-specific ASOs with knockdown efficacy exceeding 99.9% (at 5 μM) in both tested cell lines were screened in MiaPaCa-2 cells at two concentrations (100 nM, 1000 nM). Cells were lysed after 3 days of treatment. MAPT and HPRT1 mRNA expression were analyzed using the QuantiGene Singleplex assay (ThermoFisher). MAPT expression values ​​were normalized to HPRT1 values, and the degree of knockdown was determined by comparison to mock-treated cells. TLR9 activating ability and concentration-response relationship (IC10) were analyzed. 50 22 ASOs were selected for the investigation of the decision.

[0416] Investigation of TLR9 activation ability. Twenty-two ASOs were tested for TLR9-dependent pro-inflammatory potential. Reporter cell lines (HEK-Blue-hTLR9 cells, Invivogen) were treated with MAPT-specific ASOs, positive controls, and negative controls at a concentration of 5 μM without the use of transfection reagents. After 20 hours, QUANTI-Blue TM A solution (Invivogen) was added to the cells. Optical density was measured after 2 hours to determine the TLR9 activating ability of the tested ASO.

[0417] Example 3. In vivo knockdown screening

[0418] An ASO containing SEQ ID NO. 111 was synthesized for in vivo administration according to the procedure described below. The ASO was diluted in sterile saline and administered to hTau mice (mTau- / -) by intraventricular (ICV) injection as a single dose of 50 μg or 100 μg at a total volume of 10 μL per mouse. The 50 μg or 100 μg dose was determined based on in vivo tolerability.

[0419] Two weeks after administration, tissues (brain and spinal cord) were harvested and terminal blood was collected. MAPT expression was measured in the brain and spinal cord as follows. Sections of the frontal lobe and spinal cord were homogenized in Trizol using a bead homogenizer for bulk RNA isolation. Briefly, the homogenized tissues were incubated with chloroform for 3 to 5 minutes to induce phase separation after centrifugation. Subsequently, the aqueous phase was incubated with isopropanol for 10 minutes to induce RNA precipitation, followed by washing with 75% ethanol and resuspension in nuclease-free water. MAPT expression was then measured by qPCR using the Express One-Step Superscript Kit and normalized to the expression of the housekeeping gene Gapdh. To determine whether ASO induced a chronic neuroinflammatory response, a panel of glial cell activation markers (Aif1, Gfap, Tlr9, Itgax) was also quantified by qPCR using the Express One-Step Superscript kit.

[0420] ASO6 knocked down human tau expression by 76% at a dose of 50 μg.

[0421] Example 4. In vivo hepatotoxicity screening

[0422] The hepatic safety of ASO6 was evaluated. Alanine transaminase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) are enzymes found in large quantities in the liver. When liver cells are damaged, they release these enzymes into the bloodstream. Therefore, serum ALT, AST, and LDH levels were measured to determine whether ASO induced liver damage.

[0423] ASO was diluted in sterile saline and administered subcutaneously to wild-type mice at a dose of 20 mpk daily for 5 consecutive days. Mouse body weight was measured daily for 12 days, and blood was collected on days 5, 8, and 12. Serum was prepared by coagulating the blood samples and centrifuging them. Subsequently, ALT, AST, and LDH levels were measured in the serum samples. After administration of ASO6, ALT, AST, and LDH levels were low. Additionally, there was almost no change in body weight.

[0424] To determine whether internal cytosine methylation—that is, the methylation of cytosine within the gap region of ASO—affected the hepatic safety profile of ASO, the internal cytosine in ASO 6 was replaced with 5-methylcytosine. Hepatic safety was analyzed using the same protocol described above. The addition of 5-methylcytosine had only a mild beneficial effect on the hepatic safety profile of ASO 6; that is, a slight increase in liver enzyme levels was improved by cytosine methylation.

[0425] Example 5. In vivo ED50 screening

[0426] ASO6 was diluted in sterile saline and administered via ICV injection in a final volume of 10 μL in various dose ranges including 5 and 15 μg (0 and 50 μg data were used from previous studies) as hTau (mTau - / - ) was administered to mice.

[0427] Two weeks after administration, tissues were harvested, and MAPT expression in the brain and spinal cord was measured by qPCR of MAPT and Gapdh after bulk RNA isolation according to the protocol described above. In vivo ED50 was observed to be 17.7 μg and 13.1 μg, respectively.

[0428] Example 6. Determination of In Vivo ASO Half-life

[0429] Dilute ASO6 in sterile saline and administer hTau (mTau) via ICV injection at an intermediate dose of 25 μg in a total volume of 10 μL. - / - ) was administered to mice. Tissues were harvested at 5 and 9 weeks after administration to determine the duration of tau knockdown. MAPT expression was measured in the brain and spinal cord by qPCR of MAPT and Gapdh after bulk RNA isolation according to the protocol described above. MAPT knockdown persisted in the brain and spinal cord for at least 9 weeks.

[0430] Example 7. Determination of in vivo rat nephrotoxicity

[0431] Rats are considered the most sensitive species for detecting ASO-mediated nephrotoxicity and are a preferred species for risk assessment because they can overestimate nephrotoxicity susceptibility in humans. The purpose of this study was to determine if there were any effects on renal function and morphology (gross and histological evaluation) after five repeated doses of ASO6 in a 2-week rat study.

[0432] Five male Wistar Hannover rats (7 to 8 weeks old) were administered two subcutaneous injections of 40 mg / kg of the control ASO A, ASO6, A, or saline (vehicle control) on days 1 and 8, and the animals were euthanized on day 15. Body weight was measured before administration on days 1 and 8, and on days 5, 10, and 15. Clinical observations were performed after administration on days 1 and 8. 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 damage.

[0433] ASO6 was well tolerated in Wistar Hannover rats throughout the treatment period without clinical signs or changes in body weight. Serum BUN and creatinine levels were within the normal historical range in all treatment groups and were similar to the vehicle control group. On day 15, the change in the urinary KIM-1:creatinine ratio was below the critical limit and was considered to be of the magnitude of change typically observed in rats under similar study conditions.

[0434] Microscopic minimal to mild proximal tubular epithelial degeneration associated with minimal regeneration and minimal to mild interstitial, perivascular, and / or periglomerular mononuclear cell infiltration was observed in the kidney. These microscopic changes were considered non-harmful based on the minimal to mild severity and clinical signs, as well as the absence of corresponding functional changes in body weight and serum and urine renal biomarkers (BUN, creatinine, and KIM-1). In conclusion, no evidence of adverse effects on renal function or histopathology was detected in Wistar Han rats.

[0435]

[0436] Example 8: Evaluation of the in vivo efficacy and safety of MAPT ASO

[0437] ASO sequence number 111 is as follows Table 5 It was further modified as shown in [figure].

[0438]

[0439] Dilute ASO in sterile saline and add 10 or 25 μg at a total volume of 10 μL per mouse (below Table 6 As a single dose of (according to) via intraventricular (ICV) injection in hTau mice (mTau - / - It was administered to ) 10 to 14 days after administration (according to Table 6 below), brain tissue was harvested to measure relative MAPT RNA levels (according to the tissue homogenization and qPCR procedures below) and total ASO concentration (according to the tissue homogenization and total ASO assay procedures below).

[0440]

[0441] The results are shown in Figure 8 (relative MAPT RNA levels) and Figure 9 (total ASO). These results suggest enhanced efficacy of the ASO base sequence compared to the existing control MAPT ASO and enhanced tissue stability of the modified ASO (e.g., ASOM4 and ASOM5) compared to the unmodified version (ASOM_unmod), which will extend the duration of action of the modified ASO in vivo.

[0442] Example 9: Modified OTV: In vivo OTV brain uptake and MAPT knockdown after MAPT administration

[0443] Peripheral medication. The above five modified ASOs selected for efficacy were synthesized according to the procedure described above and bioconjugated to TV proteins to produce five MAPT OTV molecules (OTVM1, OTVM2, OTVM3, OTVM4, OTVM5) to evaluate tau knockdown in the brain after peripheral intravenous administration.

[0444] TfR ms / huEach thawed mouse (see US Patent No. 10,143,187 incorporated herein by reference) was administered a single dose of 25 mpk of OTV. MAPT OTV was diluted in sterile saline and administered intravenously. To determine the appropriate dose, the body weight of the mice was measured before each dose administration. Plasma was collected at 4, 24, and 72 hours after administration, while terminal plasma and tissue samples (i.e., brain and liver) were collected 1 week after administration to determine the total full-length ASO levels deposited in the brain according to the procedure below (Figs. 1 and 2).

[0445] Additionally, TfR ms / hu A melted mouse (see US Patent No. 10,143,187 incorporated herein by reference) hTau + / - Transformed for (human Tau) and mTau - / - Mice with mouse tau knockout were bred, and the resulting mice were administered 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 MAPT OTV. MAPT OTV was diluted in sterile saline and administered intravenously to the mice. Anti-CD4 was administered before the initial administration and before the Week 3 administration to prevent anti-drug antibody reactions in the mice. To determine the appropriate dose, the body weight of the mice was measured before each dose administration. Brain samples were collected 1 week after the final administration, and brain ASO concentrations (Fig. 3) were determined using the total ASO assay described below, and human tau knockdown levels relative to Gapdh and saline-treated mice were determined according to the procedure below (Figs. 4 and 5).

[0446] Subsequently, these values ​​were compared with a separate study using the same mouse strain to evaluate the brain uptake and targeted knockdown of OTV molecules conjugated to unmodified ASOs. In this study, mice were administered a single dose (Day 0) or four doses (Day 0, Week 1, Week 2, and Week 3) of unmodified MAPT OTV. MAPT OTV was diluted in sterile saline and administered intravenously to the mice. Anti-CD4 was administered before the initial administration and before the Week 3 administration to prevent anti-drug antibody responses in the mice. To determine the appropriate dose, the body weight of the mice was measured before each dose administration. Brain samples were collected one week after the final administration, and brain ASO concentrations (Fig. 6) and human tau knockdown levels relative to Gapdh and saline-treated mice were determined using the total ASO assay (Fig. 7).

[0447] In summary, these results demonstrate superior ASO brain uptake and targeted knockdown in modified MAPT OTV compared to non-modified MAPT OTV. We observed 20 to 27 nM of ASO brain uptake (modified sequence) (Figs. 1 and 2) 1 week after a single 25 mpk IV dose administration, compared to 18 nM (unmodified) (Fig. 6); 33 to 53 nM of ASO brain uptake (modified sequence) 1 week after four 25 mpk IV dose administrations (Fig. 3), compared to 18 nM (unmodified) (Fig. 6); 53 to 75% of MAPT knockdown (modified sequence) in the brain 1 week after four 25 mpk IV dose administrations (Fig. 4), compared to 21% (unmodified) (Fig. 7); and 84% of MAPT knockdown in the brain (Figs. 3 and 2) along with 137 nM of ASO brain uptake 1 week after eight 25 mpk IV dose administrations (Fig. 2). 5) was observed.

[0448] In vivo method

[0449] method

[0450] Mouse handling and tissue collection. The therapeutic treatment was administered peripherally to mice via intravenous (IV) tail vein injection (total volume approximately 200 μL). For live plasma collection, blood was collected via submandibular puncture and transferred to an EDTA-coated tube; the blood was then centrifuged at 12,700 rpm for 7 minutes at 4°C before collecting the upper plasma layer. For tissue collection, animals were anesthetized with tribromoethanol, and whole blood was collected into an EDTA-coated tube via cardiac puncture to evaluate plasma drug concentrations. After transferring to an EDTA-coated tube, the whole blood was centrifuged at 12,700 rpm for 7 minutes at 4°C before collecting the upper plasma layer. Subsequently, mice were transcardially perfused with ice-cold PBS at a rate of 5 mL / min for 5 minutes. For biochemical analysis, tissues were collected, weighed, rapidly frozen over dry ice, and stored at -80°C.

[0451] Intraventricular bolus (ICV) surgeryThe 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; incorporated herein by reference) and is briefly summarized as follows. For surgical preparation, the surgical site was sterilized with 70% ethanol. Mice were anesthetized with 4% isoflurane. Before placing the mice on a stereoscopic surface, the head was shaved from the shoulder area to between the eyes. At a maintenance level of 2% isoflurane, an incision was made from the base of the neck to between the eyes. After rinsing with hydrogen peroxide, a needle was slowly inserted through the skull at a speed of 1 mm / s. After allowing a period of 2 to 3 minutes for brain sealing around the needle to occur, a 10 μL ASO dose was administered at a rate of 1 μL / s. With a cotton swab placed against the skull at the base of the needle, the needle was lifted at a speed of 1 mm / s. To limit drug leakage, a cotton swab was held at the injection site for 1 minute. After the ICV bolus, the incision was sutured and treated with antibiotic ointment. The mice were transferred to a warm recovery pad and observed until full recovery. The mice were monitored daily to check for postoperative pain, discomfort, or infection.

[0452] Drug concentration measurement and protein test Tissue homogenization for.Weighed frozen tissue samples were treated for biochemical assays by adding 10 times the volume of cooled 1% NP40 + PBS homogenization buffer containing the cOmplete protease inhibitor (Roche #04693132001) and PhosStop (Roche 04906837001) phosphatase inhibitor. The samples were homogenized in 1.5 mL Eppendorf tubes using 3 mm tungsten carbide beads and shaken using a Qiagen TissueLyzer II (Cat No. / ID: 85300) (twice at 27 Hz for 3 minutes each). For protein assays, the samples were then centrifuged at 17,000 Hz for 15 minutes, and the supernatant was removed for use in the assay.

[0453] Tissue homogenization for RNA measurement. Weighed frozen tissue samples were treated for RNA assay by adding 10 times the volume of Qiazol reagent. The samples were homogenized in 2 mL Eppendorf tubes using 5 mm tungsten carbide beads and shaken using a Qiagen TissueLyzer II (Cat No. / ID: 85300) (twice at 27 Hz for 3 minutes). After lysis, the samples were incubated at room temperature for 5 minutes, followed by the addition of chloroform. The samples were vortexed, incubated at room temperature for 3 minutes, and then centrifuged at 12,000 µg at 4°C for 15 minutes. The aqueous phase was then isolated. Subsequently, RNA was isolated by adding isopropanol, vortexing, incubating at room temperature for 10 minutes, and then centrifuging at 12,000 µg at 4°C for 10 minutes. The resulting pellet was resuspended in 75% ethanol, vortexed, and centrifuged at 7500 lb for 5 minutes at 4°C. The final pellet was resuspended in water.

[0454] huIgG test.Quantification of humanized antibodies in mouse plasma and tissue lysates was measured using general electrochemiluminescence immunoassay (ECLIA). Briefly, the working concentration of the prepared biotinylated goat anti-human IgG polyclonal primary antibody (Southern Biotech, Birmingham, AL) in the assay dilution was added to the wells of MSD GOLD 96-well streptavidin-coated microtiter plates (Meso Scale Discovery, Rockville, MD) and incubated for approximately 1 hour. After this incubation and plate washing step, the prepared test samples (including sample pre-dilutions where appropriate) and related standards were added to the assay plates and incubated for approximately 1 hour. After the test sample incubation and plate washing step, the working concentration of the secondary ruthenylated (SULFO-TAG) goat anti-human IgG antibody (Meso Scale Discovery, Rockville, MD) in the assay dilution was added to the assay plates and incubated for approximately 1 hour. After washing the plates, 1× MSD lead buffer T (Meso Scale Discovery, Rockville, MD) was added to generate electrochemiluminescence (ECL) calibration signals, which were subsequently expressed in ECL units (ECLU). All calibration reaction steps were performed at ambient temperature with shaking on a plate shaker (where appropriate); all test samples were pre-diluted to calibration MRD 1:20 before analysis on the calibration plate. The sample ECLU signals subsequently generated from the calibration were treated as concentrations by inverse calculation from the calibration calibration (CS) curve. The calibration CS curve was fitted with weighted 4-parameter nonlinear logistic regression to be used for calculating concentrations for unknown / test samples.

[0455] Total ASO test.Quantification of total ASO (conjugated and free forms) in mouse plasma and tissue homogenates was measured using hybridization-based electrochemiluminescence immunoassay (ECLIA). Briefly, custom biotinylated and digoxigenin-conjugated antisense probes (synthesized by Integrated DNA Technologies, Coralville, IA) at working concentrations were combined with test samples (including sample pre-dilution where appropriate) and associated standards prepared in TE buffer (10 mM Tris-HCl containing 1 mM EDTA). The samples prepared in TE buffer were added as a 1:1 mixture to 1X SSC buffer (Sigma-Aldrich, St. Louis, MO) containing the working concentration of recombinant protease K enzyme (ThermoFisher, Waltham, MA). The hybridization / enzyme mixture was then digested, denatured, annealed, and cooled in a thermal cycler. After hybridization product incubation, samples were added to the wells of MSD GOLD 96-well streptavidin-coated microtiter plates (Meso Scale Discovery, Rockville, MD) and incubated for approximately 30 minutes. Following the incubation and plate washing steps, secondary ruthenylated (SULFO-TAG) anti-digoxigenin antibody (Novus Biologicals, Littleton, CO) at the working concentration in the assay dilution was added to the plates and incubated for approximately 30 minutes. After plate washing, 1X MSD Reed Buffer T (Meso Scale Discovery, Rockville, MD) was added to generate an electrochemiluminescence (ECL) assay signal, which was subsequently expressed in ECL units (ECLU). All assay reaction steps were performed at ambient temperature with shaking on a plate shaker (where appropriate); all test samples were pre-diluted to the assay MRD 1:20 prior to analysis on the assay plates. The sample ECLU signal subsequently generated in the calibration was processed into concentration by inverse calculation from the calibration correction (CS) curve.The test CS curve was fitted with weighted 4-parameter nonlinear logistic regression to be used to calculate the concentration for the unknown / test sample.

[0456] qPCR analysis. To evaluate target mRNA levels, qRT-PCR was performed on RNA extracted from tissue lysates. Target mRNA levels were assessed using Taqman probes (hMAPT, mGapdh) and the Express One-Step Kit. For each sample, hMAPT mRNA levels were normalized to the housekeeping gene Gapdh. qRT-PCR was performed using the QuantStudio 6 Flex system (Applied Biosystems), and mean CT values ​​were measured for each probe using technical double replicates. Next, delta-delta CT (ΔΔCT) values ​​were calculated for the non-ASO treated groups and plotted as relative expression levels.

[0457] Example 10. Vulnerable area identification verification

[0458] In order to determine the catabolism and biotransformation of oligonucleotides (ASOs) and to further characterize the oligonucleotides and oligonucleotide polypeptide conjugates described herein, it was necessary to develop stability assays to identify highly catabolistic sites (clipping sites) of ASOs (referred to herein as "vulnerable sites"). The catabolism assays described in detail below were used to identify vulnerable sites for potential modifications to design and provide more stable molecules. Previous analytical methods for the in vitro assay of oligonucleotides are described, for example, in the literature [Basiri et al. in Molecular Therapy: Nucleic Acids; 21: 725-736 (2020)].

[0459] Tissue homogenization and incubation—Frozen mouse livers were homogenized to a tissue concentration of 200 mg / ml in PBS buffer (pH 7.4) containing 1% NP-40. 200 μL of the homogenate was spiked with ASO at a concentration of 1 to 2 mM and incubated at 37°C for 48 hours (300 g shaking).

[0460] Sample manufacturing ― 200 mL of 10% phosphoric acid was added to 200 mL of incubated tissue homogenate and vortexed for 5 minutes. 600 mL of Clarity OTX lysis-loading buffer (Phenomenex, PN AL0-8579) was added to each tube, vortexed for 5 minutes, and then centrifuged at 3200 rpm for 10 minutes at 4°C. Clarity OTX SPE plates were used. Table 5 shows the detailed procedure.

[0461]

[0462] Sample analysis was performed by liquid chromatography and mass spectrometry.

[0463] Liquid Chromatography (LC): ASO separation was performed by ion-pair chromatography at 70°C using a Waters BEH oligonucleotide 2.1 × 50 mm column. Mobile phase buffers A and B were water and methanol containing 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, and 10-5. The total run time was 10 minutes, and the LC output was converted to waste at 0 to 1 minute and 7 to 10 minutes.

[0464] Mass Spectrometry (MS): High-resolution MS equipped with the IDA method was used to identify catabolic products. Identified Vulnerabilities—The table below shows the vulnerabilities identified according to the methods provided herein. If one or more vulnerabilities are identified, the associated nucleoside bonds are replaced with PS2, MsPA, OiPS, or PN groups. One or more nucleoside bonds adjacent to the vulnerabilities may be replaced. This process may be repeated as needed after stability testing to improve the stability of the ASO.

[0465]

[0466] Example 11. In vivo plasma pharmacokinetics

[0467] ASO sequence numbers 111 and 112 are as follows Table 9 It was further modified as shown in [figure].

[0468]

[0469]

[0470] To evaluate the pharmacokinetics of additional oligonucleotide polypeptide conjugates (see Table 7B), additional conjugates were administered to Sprague Dawley rats by intravenous injection at a concentration of 10 mg / kg as described above. All oligonucleotide polypeptide conjugates were administered at a dosage volume of 2 mL / kg. Plasma was collected at 0.25, 4, 24, 48, and 72 hours after injection. huIgG and ASO concentrations were measured in plasma as described above.

[0471] The results are shown in Table 10. These results demonstrate that adding stabilizing deformations in various placements and forms consistently improves huIgG clearance, and that deforming the vulnerable area or the area near it with backbone deformation can adjust ASO clearance by protecting or moving the clipping area.

[0472]

[0473] ASO (non-conjugated) was administered as a single dose of 10 μL total volume per mouse via intraventricular (ICV) injection to hTau mice (mTau - / - It was administered to ). Brain tissue was harvested to measure relative MAPT RNA levels. Results demonstrating a significant knockdown of MAPT expression across the tested ASOs are shown in Figure 12.

Claims

Claim 1 A microtubule-associated protein tau (MAPT) antisense oligonucleotide (ASO) conjugate comprising a transferrin receptor (TfR)-targeted Fc polypeptide dimer and a MAPT ASO, wherein (a) the TfR-targeted Fc polypeptide dimer comprises (i) a first Fc polypeptide; (ii) a second Fc polypeptide, wherein the second Fc polypeptide comprises a modified constant domain comprising a sequence that specifically binds to human transferrin receptor 1 (TfR) and has at least 90% sequence identity with SEQ ID NO. 12, and the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide; and (b) the MAPT ASO is 5'-A L x d m C L x e m C L x f TTA p a A p b G p c TATTACT x g T L x h G L x i m C L -3'(sequence number 111) or 5'-C L x d T L x e G x f T p j T p k A p l G p m A*C*A*T*T*C p n A p o T*T x g C L x h T L x i C L Includes -3' (sequence number 112), but A L , m C L , G L and T L are adenine, 5-methylcytosine, guanine, and thymine lock nucleosides, respectively, and A, C, G, and T are deoxyadenosine, deoxycytidine or 5-methyldeoxycytidine, deoxyguanidine, and deoxythymidine nucleosides, respectively, and each p is independently a phosphorothioate (PS) nucleoside inter-nucleoside bond or a stabilized nucleoside inter-nucleoside bond, and each x A MAPT ASO conjugate, wherein the first Fc polypeptide is independently a PS nucleoside bond, a phosphodiester (PO) nucleoside bond, or a stabilized nucleoside bond, and any nucleoside bond that is not a stabilized nucleoside bond is a PS nucleoside bond or a phosphodiester nucleoside bond, and the first Fc polypeptide is connected to the MAPT ASO. Claim 2 A MAPT ASO conjugate according to claim 1, wherein the stabilizing nucleoside inter-bonding is independently selected from the group consisting of phosphorodithioate (PS2) nucleoside inter-bonding, phosphorylguanidine (PN) nucleoside inter-bonding, mesylphosphoramidate (MsPA) nucleoside inter-bonding and O-isopropylphosphorothioate (OiPS) nucleoside inter-bonding. Claim 3 In paragraph 1, each p and x is a MAPT ASO conjugate, which is a PS nucleoside bond. Claim 4 In paragraph 1 or 2, (a) x g , x h and x i are each PS nucleoside bonds, or (b) x g is a PS internucleoside bond and x h and x i are respectively PN nucleoside interlinks, or (c) x g , x h and x i are respectively PN nucleoside interlinks, or (d) x g and x h are respectively PN nucleoside interlinks and x i is a PO nucleoside bond, or (e) x g and x i are respectively PN nucleoside interlinks and x h is a PO nucleoside bond, or (f) x h and x i are respectively PN nucleoside interlinks and x g is a MAPT ASO conjugate, which is a PO nucleoside bond. Claim 5 In any one of paragraphs 1, 2 and 4, (a) x d , x e and x f are each PS nucleoside bonds, or (b) x d and x e is a PN internucleoside bond and x f is a PS nucleoside bond, or (c) x d is a PS internucleoside bond and x e and x f are respectively PN nucleoside interlinks, or (d) x d is a PN internucleoside bond and x e and x f are respectively PS nucleoside bonds or, (e) x d , x e and x f are respectively PN nucleoside interlinks, or (d) x d and x e are respectively PN nucleoside interlinks and x f is a PO nucleoside bond, or (e) x d and x f are respectively PN nucleoside interlinks and x e is a PO nucleoside bond, or (f) x e and x f are respectively PN nucleoside interlinks and x d is a MAPT ASO conjugate, which is a PO nucleoside bond. Claim 6 In paragraph 4 or 5, (a) x d , x e , x f , x g , x h and x i are each PS nucleoside bonds, or (b) x d , x e and x f are respectively PS nucleoside bonds and x g is a PS internucleoside bond, and x h and x i are respectively PN nucleoside interlinks, or (c) x d , x e and x f are respectively PS nucleoside bonds and x g , x h and x i are respectively PN nucleoside interlinks, or (d) x d and x e is a PN internucleoside bond and x f is a PS internucleoside bond, and x g , x h and x i are respectively PS nucleoside bonds or, (e) x d and x e is a PN internucleoside bond and x f is a PS internucleoside bond and x g is a PS internucleoside bond, and x h and x i are respectively PN nucleoside interlinks, or (f) x d is a PS internucleoside bond and x e and x f are respectively PN nucleoside interlinks and x g is a PS internucleoside bond, and x h and x i are respectively PN nucleoside interlinks, or (g) x d is a PN internucleoside bond, and x e and x f are respectively PS nucleoside bonds and x g is a PS internucleoside bond and x h and x i are respectively PN nucleoside interlinks, or (h) x d , x e , x f , x g , x h and x i are each PN nucleoside bonds, or (i) x d , x e and x f are respectively PN nucleoside interlinks and x g and x h are respectively PN nucleoside interlinks and x i is a PO nucleoside bond, or (j) x d , x e and x f are respectively PN nucleoside interlinks and x g and x i are respectively PN nucleoside interlinks and x h is a PO nucleoside bond, or (k) x d , x e and x f are respectively PN nucleoside interlinks and x h and x i are respectively PN nucleoside interlinks and x g is a PO nucleoside bond, or (l) x d and x e are respectively PN nucleoside interlinks and x f is a PO internucleoside bond and x g , x h and x i are respectively PN nucleoside interlinks, or (m) x d and x f are respectively PN nucleoside interlinks and x e is a PO internucleoside bond and x g , x h and x i are respectively PN nucleoside interlinks, or (n) x e and x f are respectively PN nucleoside interlinks and x d is a PO internucleoside bond and x g , x h and x i MAPT ASO conjugates, each consisting of PN nucleosides. Claim 7 In any one of claims 1, 2 and 4 through 6, the MAPT ASO comprises SEQ ID NO. 111, and (a) p a , p b and p c is a PS nucleoside bond, or (b) p a is a stabilized internucleoside bond and p b and p c is a PS nucleoside bond, or (c) p b is a stabilized internucleoside bond and p a and p c is a PS nucleoside bond, or (d) p a and p b is a stabilized internucleoside bond and p c is a PS nucleoside bond, or (e) p b and p c is a stabilized internucleoside bond and p a is a PS nucleoside bond, or (f) p a , p b and p c MAPT ASO conjugate, which is a stabilized nucleoside bond. Claim 8 In paragraph 7, (a) p a is a PS2 combination p b and p c is a PS nucleoside bond, or (b) p b is a PS2 combination p a and p c is a PS nucleoside bond, or (c) p b is a PN bond and p a and p c is a PS nucleoside bond, or (d) p a and p b is a PN internucleoside bond and p c is a PS nucleoside bond, or (e) p a and p b is a PS2 internucleoside bond and p c is a PS nucleoside bond, or (f) p a and p b is an MsPA internucleoside bond and p c is a PS nucleoside bond, or (g) p a and p b is an OiPS internucleoside bond and p c is a PS nucleoside bond, or (h) p b and p c is a PN internucleoside bond and p a is a PS nucleoside bond, or (i) p a , p b and p c is a PN internucleoside bond, or (j) p a , p b and p c is a MAPT ASO conjugate, which is an MsPA nucleoside bond. Claim 9 In claim 1, the MAPT ASO comprises the following oligonucleotide, (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*A n A n G n T*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*A n G n T*A*T*T*A*C*T*T L n G L n m C L ;(e) A L * m C L * m C L *T*T*A n A n G n T*A*T*T*A*C*T*T L n G L n m C L ;(f) A L * m C L * m C L *T*T*A u A u G u T*A*T*T*A*C*T n T L n G L n m C L ;(g) A L n m C L * m C L *T*T*A n A n G*T*A*T*T*A*C*T*T L n G L n m C L ;(h) A L * m C L n m C L n T*T*A n A n G*T*A*T*T*A*C*T*T L n G L n m C L ;(i) A L n m C L n m C L *T*T*A n A n G*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*A n G n T*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*A n G*T*A*T*T*A*C*T*T L n G L n m C L ;(l) A L n m C L n m C L *T*T*A u A u G*T*A*T*T*A*C*T*T L n G L n m C L ;(m) A L n m C L n m C L *T*T*A t A t G*T*A*T*T*A*C*T*T L n G L n m C L ;(n) A L n m C L n m C L *T*T*A*A u G u T*A*T*T*A*C*T*T L n G L n m C L ;(o) A L n m C L n m C L n T*T*A*A*G*T*A*T*T*A*C*T n T L n G L n m C L ;(p) A L n m C L n m C L n T*T*A t A*G*T*A*T*T*A*C*T n T L n G L n m C L ;(q) A L n m C L n m C L n T*T*A*A t G*T*A*T*T*A*C*T n T L n G L n m C L ;(r) A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L ;(s) A L n m C L n m C L n T*T*A o A o G*T*A*T*T*A*C*T n T L n G L n m C L ;(t) A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L P m C L ;(u) A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T n T L P G L n m C L ;(v) A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T P T L n G L n m C L ;(w) A L n m C L n m C P T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L ;(x) A L n m C L P m C n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L ; or(y) A L P m C L n m C n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L ;Here A L is an adenine lock nucleoside, and mCL은 5-메틸시토신 잠금 뉴클레오시드이고, TL은 티민 잠금 뉴클레오시드이고, GL은 구아닌 잠금 뉴클레오시드이고, A는 데옥시아데노신이고, C는 데옥시시티딘이고,mC는 5-메틸데옥시시티딘이고, T는 데옥시티미딘이고, G는 데옥시구아노신이고, *는 PS 뉴클레오시드간 결합이고,n은 PN 뉴클레오시드간 결합이고,t는 PS2 뉴클레오시드간 결합이고,u는MsPA 뉴클레오시드간 결합이고,o는 OiPS 뉴클레오시드간 결합이며,P는 PO 뉴클레오시드간 결합인, MAPT ASO 접합체. Claim 10 In any one of claims 1 to 2 and 4 to 6, the MAPT ASO comprises SEQ ID NO. 112, and (a) p k , p l , p n and p o is a stabilized nucleoside bond, or (b) p k , p l , p n and p o is a stabilized internucleoside bond and p j and p m is a PS nucleoside bond, or (c) p j , p l and p o is a stabilized nucleoside bond, or (d) p j , p l and p o is a stabilized internucleoside bond and p k , p m and p n is a PS nucleoside bond, or (e) p l p m , p n and p o is a stabilized nucleoside bond, or (d) p l p m , p n and p o is a stabilized internucleoside bond and p j and p k is a MAPT ASO conjugate, which is a PS nucleoside bond. Claim 11 In paragraph 10, (a) p k , p l , p n and p o is a PN nucleoside interlink, or (b) p k , p l , p n and p o is a PN internucleoside bond and p j and p m is a PS nucleoside bond, or (c) p j is a PN bond and p l and p o is a PS2 nucleoside interlink, or (d) p j is a PN bond and p l and p o is a PS2 internucleoside bond and p k , p m and p n is a PS nucleoside bond, or (e) p l and p m is a PS2 internucleoside bond and p n and p o is a PN nucleoside interlink, or (d) p l and p m is a PS2 internucleoside bond and p n and p o is a PN internucleoside bond and p j and p k is a MAPT ASO conjugate, which is a PS nucleoside bond. Claim 12 In claim 1, the MAPT ASO comprises the following oligonucleotides, (a) m C L n T L n G L *T*T n A n G*A*C*A*T*T*C n A n T*T* m C L *T L * m C L ;(b) m C L *T L *G L *T*T n A n G*A*C*A*T*T*C n A n T*T* m C L n T L n m C L ;(c) m C L n T L n G L n T n T*A t G*A*C*A*T*T*C*A t T*T n m C L n T L n m C L ; or(d) m C L n T L n G L *T*T*A t G t A*C*A*T*T*C n A n T*T* m C L n T L n m C L Here mCL은 5-메틸시토신 잠금 뉴클레오시드이고, TL은 티민 잠금 뉴클레오시드이고, GL은 구아노신 잠금 뉴클레오시드이고, A는 데옥시아데노신이고, C는 데옥시시티딘이고, T는 데옥시티미딘이고, G는 데옥시구아노신이고, *는 PS 뉴클레오시드간 결합이고,n은 PN 뉴클레오시드간 결합이고,t는 PS2 뉴클레오시드간 결합인, MAPT ASO 접합체. Claim 13 A MAPT ASO conjugate according to any one of claims 1 to 12, wherein the modified constant domain specifically binding to TfR comprises glutamate at position 380, Y at position 384, T at position 386, glutamate at position 387, tryptophan at position 388, alanine at position 389, N at position 390, T at position 413, glutamate at position 415, glutamate at position 416, and phenylalanine at position 421 according to EU numbering. Claim 14 A MAPT ASO conjugate according to any one of claims 1 to 13, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises at least one cysteine ​​substitution. Claim 15 In claim 14, the MAPT ASO conjugate wherein at least one cysteine ​​substitution is selected from the group consisting of S239C substitution, S442C substitution, A330C substitution, K149C substitution, or T289C substitution. Claim 16 In claim 15, the above-mentioned first Fc polypeptide is a MAPT ASO conjugate comprising at least one cysteine ​​substitution. Claim 17 In any one of claims 1 to 16, the MAPT ASO comprises a phosphodiester group, a phosphorothioate group, a phosphorodithioate group, a mesylphosphoramidate group, a cyclophosphoryl guanine group and O - A MAPT ASO conjugate comprising a 5' end group selected from the group consisting of isopropyl phosphorothioate groups. Claim 18 In any one of claims 14 to 17, the Fc dimer is connected to the MAPT ASO through the at least one cysteine ​​substitution and a linker attached to the 5' end of the MAPT ASO, wherein the linker is as follows, A MAPT ASO conjugate, wherein the valence marked * is attached to the TfR-targeted Fc polypeptide dimer and the valence marked ** is attached to the MAPT ASO. Claim 19 A MAPT ASO conjugate according to any one of claims 1 to 18, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprise one or more mutations that regulate effector function. Claim 20 In claim 19, the MAPT ASO conjugate wherein the first Fc polypeptide and / or the second Fc polypeptide each comprise alanine at position 234 according to EU numbering, alanine at position 235, and serine or glycine at position 329. Claim 21 A MAPT ASO conjugate according to any one of claims 1 to 20, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprise one or more mutations that increase serum stability. Claim 22 A MAPT ASO conjugate according to any one of claims 1 to 21, wherein the second Fc polypeptide further comprises tryptophan at position 366 according to EU numbering. Claim 23 In claim 22, the above-mentioned first Fc polypeptide is a MAPT ASO conjugate further comprising serine at position 366 according to EU numbering, alanine at position 368, and valine at position 407. Claim 24 A MAPT ASO conjugate according to any one of claims 1 to 21, wherein the 2 Fc polypeptide comprises serine at position 366 according to EU numbering, alanine at position 368, and valine at position 407. Claim 25 In paragraph 24, the above-mentioned first Fc polypeptide is a MAPT ASO conjugate comprising tryptophan at position 366 according to EU numbering. Claim 26 A MAPT ASO conjugate according to any one of claims 1 to 12, wherein the first Fc polypeptide comprises any one of the amino acid sequences of SEQ ID NOs 1, 47 to 48, 51 to 54, 61 to 63, 66, 73 to 78, 80 and 90, and the second Fc polypeptide comprises any one of the amino acid sequences of SEQ ID NOs 4 to 29, 36, 43 to 44, 49 and 50. Claim 27 A MAPT ASO conjugate according to 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. Claim 28 A MAPT ASO conjugate according to any one of claims 1 to 27, wherein the first Fc polypeptide is fused to the first non-targeting Fab (NTF) through the first hinge region to form the first Fab-Fc fused polypeptide. Claim 29 A MAPT ASO conjugate according to any one of claims 1 to 27, wherein the second Fc polypeptide is fused to the second NTF through the second hinge region to form a second Fab-Fc fused polypeptide. Claim 30 A MAPT ASO conjugate according to any one of claims 1 to 27, wherein the first Fc polypeptide is fused to an NTF through a first hinge region to form a first Fab-Fc fusion polypeptide, and the second Fc polypeptide is fused to a second NTF through a second hinge region to form a second Fab-Fc fusion polypeptide. Claim 31 A MAPT ASO conjugate according to any one of claims 28 to 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 heavy chain position 114 (according to Kabat numbering), heavy chain position 124 (according to EU numbering), light chain position 149 (according to EU numbering), or light chain position 156 (according to EU numbering). Claim 32 A MAPT ASO conjugate according to claim 30 or 31, wherein the first and second NTFs each comprise a heavy chain comprising SEQ ID NO. 109 or 130 and a light chain comprising SEQ ID NO. 108 or 129, optionally wherein the heavy chain further comprises a hinge region comprising SEQ ID NO. 91, 92, 93 or 121. Claim 33 A MAPT ASO conjugate according to 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. Claim 34 A MAPT ASO conjugate comprising a TfR-targeted Fc polypeptide dimer and at least one MAPT ASO, wherein (a) the TfR-targeted Fc polypeptide dimer comprises (i) a first Fc polypeptide, (ii) a second Fc polypeptide, wherein the second Fc polypeptide comprises a modified constant domain comprising a sequence having alanine at position 234 according to EU numbering, alanine at position 235, and serine at position 329, specifically binding to human transferrin receptor 1 (TfR), and having at least 90% sequence identity with SEQ ID NO. 12, and the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide, (iii) a first non-targeted Fab (NTF) fused to the first Fc polypeptide through a first hinge region to form a first Fab-Fc fusion polypeptide, and a second (b) comprising a second NTF fused to the second Fc polypeptide through a second hinge region to form a Fab-Fc fusion polypeptide, and at least one MAPT ASO is 5'-A L x d m C L x e m C L x f TTA p a A p b G p c TATTACT x g T L x h G L x i m C L -3'(sequence number 111) or 5'-C L x d T L x e G x f T p j T p k A p l G p m A*C*A*T*T*C p n A p o T*T x g C L x h T L x i C L Includes -3' (sequence number 112), but A L , m C L , G L and T L are adenine, 5-methylcytosine, guanine, and thymine lock nucleosides, respectively, and A, C, G, and T are deoxyadenosine, deoxycytidine or 5-methyldeoxycytidine, deoxyguanidine, and deoxythymidine nucleosides, respectively, and each p is independently a phosphorothioate (PS) nucleoside inter-nucleoside bond or a stabilized nucleoside inter-nucleoside bond, and each x A MAPT ASO conjugate, wherein the bonds are independently PS nucleoside bonds, phosphodiester (PO) nucleoside bonds, or stabilized nucleoside bonds, and any nucleoside bond that is not a stabilized nucleoside bond is a PS nucleoside bond or a phosphodiester nucleoside bond, wherein the first Fab-Fc fusion polypeptide and / or the second Fab-Fc fusion polypeptide comprises at least one cysteine ​​substitution, and the at least one MAPT ASO is conjugated to the first Fab-Fc fusion polypeptide and / or the second Fab-Fc fusion polypeptide at the at least one cysteine ​​substitution via a linker. Claim 35 As MAPT ASO, 5'-A L x d m C L x e m C L x f TTA p a A p b G p c TATTACT x g T L x h G L x i m C L -3'(sequence number 111) or 5'-C L x d T L x e G x f T p j T p k A p l G p m A*C*A*T*T*C p n A p o T*T x g C L x h T L x i C L Includes -3' (sequence number 112), but A L , m C L , G L and T L are adenine, 5-methylcytosine, guanine, and thymine lock nucleosides, respectively, and A, C, G, and T are deoxyadenosine, deoxycytidine or 5-methyldeoxycytidine, deoxyguanidine, and deoxythymidine nucleosides, respectively, and each p is independently a phosphorothioate (PS) nucleoside inter-nucleoside bond or a stabilized nucleoside inter-nucleoside bond, and each x is independently a PS nucleoside bond, a phosphodiester (PO) nucleoside bond, or a stabilized nucleoside bond, and any nucleoside bond that is not a stabilized nucleoside bond is a PS nucleoside bond or a phosphodiester nucleoside bond, and the first Fc polypeptide is connected to the MAPT ASO, MAPT ASO. Claim 36 MAPT ASO according to claim 29, wherein the stabilizing nucleoside inter-bonding is independently selected from the group consisting of phosphorodithioate (PS2) nucleoside inter-bonding, phosphorylguanidine (PN) nucleoside inter-bonding, mesylphosphoramidate (MsPA) nucleoside inter-bonding, and O-isopropylphosphorothioate (OiPS) nucleoside inter-bonding. Claim 37 In Paragraph 35, each p and x MAPT ASO, which is a PS nucleoside bond. Claim 38 In paragraph 35 or 36, (a) x g , x h and x i are each PS nucleoside bonds, or (b) x g is a PS internucleoside bond and x h and x i are respectively PN nucleoside interlinks, or (c) x g , x h and x i are respectively PN nucleoside interlinks, or (d) x g and x h are respectively PN nucleoside interlinks and x i is a PO nucleoside interlink, or (d) x g and x i are respectively PN nucleoside interlinks and x h is a PO nucleoside bond, or (f) x h and x i are respectively PN nucleoside interlinks and x g MAPT ASO, which is a PO nucleoside bond. Claim 39 In any one of paragraphs 35, 36 and 38, (a) x d , x e and x f are each PS nucleoside bonds, or (b) x d and x e is a PN internucleoside bond and x f is a PS nucleoside bond, or (c) x d is a PS internucleoside bond and x e and x f are respectively PN nucleoside interlinks, or (d) x d is a PN internucleoside bond and x e and x f are respectively PS nucleoside bonds or, (e) x d , x e and x f are respectively PN nucleoside interlinks, or (d) x d and x e are respectively PN nucleoside interlinks and x f is a PO nucleoside bond, or (e) x d and x f are respectively PN nucleoside interlinks and x e is a PO nucleoside bond, or (f) x e and x f are respectively PN nucleoside interlinks and x d MAPT ASO, which is a PO nucleoside bond. Claim 40 In paragraph 38 or 39, (a) x d , x e , x f , x g , x h and x i are each PS nucleoside bonds, or (b) x d , x e and x f are respectively PS nucleoside bonds and x g is a PS internucleoside bond, and x h and x i are respectively PN nucleoside interlinks, or (c) x d , x e and x f are respectively PS nucleoside bonds and x g , x h and x i are respectively PN nucleoside interlinks, or (d) x d and x e is a PN internucleoside bond and x f is a PS internucleoside bond, and x g , x h and x i are respectively PS nucleoside bonds or, (e) x d and x e is a PN internucleoside bond and x f is a PS internucleoside bond and x g is a PS internucleoside bond, and x h and x i are respectively PN nucleoside interlinks, or (f) x d is a PS internucleoside bond and x e and x f are respectively PN nucleoside interlinks and x g is a PS internucleoside bond, and x h and x i are respectively PN nucleoside interlinks, or (g) x d is a PN internucleoside bond, and x e and x f are respectively PS nucleoside bonds and x g is a PS internucleoside bond, and x h and x i are respectively PN nucleoside interlinks, or (h) x d , x e , x f , x g , x h and x i are each PN nucleoside bonds, or (i) x d , x e and x f are respectively PN nucleoside interlinks and x g and x h are respectively PN nucleoside interlinks and x i is a PO nucleoside bond, or (j) x d , x e and x f are respectively PN nucleoside interlinks and x g and x i are respectively PN nucleoside interlinks and x h is a PO nucleoside bond, or (k) x d , x e and x f are respectively PN nucleoside interlinks and x h and x i are respectively PN nucleoside interlinks and x g is a PO nucleoside bond, or (l) x d and x e are respectively PN nucleoside interlinks and x f is a PO internucleoside bond and x g , x h and x i are respectively PN nucleoside interlinks, or (m) x d and x f are respectively PN nucleoside interlinks and x e is a PO internucleoside bond and x g , x h and x i are respectively PN nucleoside interlinks, or (n) x e and x f are respectively PN nucleoside interlinks and x d is a PO internucleoside bond and x g , x h and x i MAPT ASO, which are PN nucleoside interlinks, respectively. Claim 41 In any one of paragraphs 35 to 36 and 38 to 40, the MAPT ASO comprises SEQ ID NO. 111, (a) p a , p b and p c is a PS nucleoside bond, or (b) p a is a stabilized internucleoside bond and p b and p c is a PS nucleoside bond, or (c) p b is a stabilized internucleoside bond and p a and p c is a PS nucleoside bond, or (d) p a and p b is a stabilized internucleoside bond and p c is a PS nucleoside bond, or (e) p b and p c is a stabilized internucleoside bond and p a is a PS nucleoside bond, or (f) p a , p b and p c MAPT ASO, which is a stabilized nucleoside-nucleoside bond. Claim 42 In paragraph 41, (a) p a is a PS2 combination p b and p c is a PS nucleoside bond, or (b) p b is a PS2 combination p a and p c is a PS nucleoside bond, or (c) p b is a PN bond and p a and p c is a PS nucleoside bond, or (d) p a and p b is a PN internucleoside bond and p c is a PS nucleoside bond, or (e) p a and p b is a PS2 internucleoside bond and p c is a PS nucleoside bond, or (f) p a and p b is an MsPA internucleoside bond and p c is a PS nucleoside bond, or (g) p a and p b is an OiPS internucleoside bond and p c is a PS nucleoside bond, or (h) p b and p c is a PN internucleoside bond and p a is a PS nucleoside bond, or (i) p a , p b and p c is a PN internucleoside bond, or (j) p a , p b and p c MAPT ASO, which is an MsPA nucleoside bond. Claim 43 In paragraph 35, the above MAPT ASO comprises the following oligonucleotide, (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*A n A n G n T*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*A n G n T*A*T*T*A*C*T*T L n G L n m C L ;(e) A L * m C L * m C L *T*T*A n A n G n T*A*T*T*A*C*T*T L n G L n m C L ;(f) A L * m C L * m C L *T*T*A u A u G u T*A*T*T*A*C*T n T L n G L n m C L ;(g) A L n m C L * m C L *T*T*A n A n G*T*A*T*T*A*C*T*T L n G L n m C L ;(h) A L * m C L n m C L n T*T*A n A n G*T*A*T*T*A*C*T*T L n G L n m C L ;(i) A L n m C L n m C L *T*T*A n A n G*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*A n G n T*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*A n G*T*A*T*T*A*C*T*T L n G L n m C L ;(l) A L n m C L n m C L *T*T*A u A u G*T*A*T*T*A*C*T*T L n G L n m C L ;(m) A L n m C L n m C L *T*T*A t A t G*T*A*T*T*A*C*T*T L n G L n m C L ;(n) A L n m C L n m C L *T*T*A*A n G n T*A*T*T*A*C*T*T L n G L n m C L ;(o) A L n m C L n m C L n T*T*A*A*G*T*A*T*T*A*C*T n T L n G L n m C L ;(p) A L n m C L n m C L n T*T*A t A*G*T*A*T*T*A*C*T n T L n G L n m C L ;(q) A L n m C L n m C L n T*T*A*A t G*T*A*T*T*A*C*T n T L n G L n m C L ;(r) A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L ;(s) A L n m C L n m C L n T*T*A o A o G*T*A*T*T*A*C*T n T L n G L n m C L ;(t) A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L P m C L ;(u) A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T n T L P G L n m C L ;(v) A L n m C L n m C L n T*T*A t A t G*T*A*T*T*A*C*T P T L n G L n m C L ;(w) A L n m C L n m C P T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L ;(x) A L n m C L P m C n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L ; or(y) A L P m C L n m C n T*T*A t A t G*T*A*T*T*A*C*T n T L n G L n m C L ;Here A L is an adenosine-locked nucleic acid, and mCL은 5-메틸시토신 잠금 핵산이고, TL은 티미딘 잠금 핵산이고, GL은 구아노신 잠금 핵산이고, A는 데옥시아데노신이고, C는 데옥시시티딘이고,mC는 5-메틸데옥시시티딘이고, T는 데옥시티미딘이고, G는 데옥시구아노신이고, *는 PS 뉴클레오시드간 결합이고,n은 PN 뉴클레오시드간 결합이고,t는 PS2 뉴클레오시드간 결합이고,u는MsPA 뉴클레오시드간 결합이고,o는 OiPS 뉴클레오시드간 결합이며,P는 PO 뉴클레오시드간 결합인, MAPT ASO. Claim 44 In any one of paragraphs 35 to 36 and 38 to 40, the MAPT ASO comprises SEQ ID NO. 112, and (a) p k , p l , p n and p o is a stabilized nucleoside bond, or (b) p k , p l , p n and p o is a stabilized internucleoside bond and p j and p m is a PS nucleoside bond, or (c) p j , p l and p o is a stabilized nucleoside bond, or (d) p j , p l and p o is a stabilized internucleoside bond and p k , p m and p n is a PS nucleoside bond, or (e) p l p m , p n and p o is a stabilized nucleoside bond, or (d) p l p m , p n and p o is a stabilized internucleoside bond and p j and p k is a MAPT ASO conjugate, which is a PS nucleoside bond. Claim 45 In paragraph 44, (a) p k , p l , p n and p o is a PN nucleoside interlink, or (b) p k , p l , p n and p o is a PN internucleoside bond and p j and p m is a PS nucleoside bond, or (c) p j is a PN bond and p l and p o is a PS2 nucleoside interlink, or (d) p j is a PN bond and p l and p o is a PS2 internucleoside bond and p k , p m and p n is a PS nucleoside bond, or (e) p l and p m is a PS2 internucleoside bond and p n and p o is a PN nucleoside interlink, or (d) p l and p m is a PS2 internucleoside bond and p n and p o is a PN internucleoside bond and p j and p k is a MAPT ASO conjugate, which is a PS nucleoside bond. Claim 46 In paragraph 35, the above MAPT ASO comprises the following oligonucleotides, (a) m C L n T L n G L *T*T n A n G*A*C*A*T*T*C n A n T*T* m C L *T L m *C L ;(b) m C L *T L *G L *T*T n A n G*A*C*A*T*T*C n A n T*T* m C L n T L n m C L ;(c) m C L n T L n G L n T n T*A t G*A*C*A*T*T*C*A t T*T n m C L n T L n m C L ; or(d) m C L n T L n G L *T*T*A t G t A*C*A*T*T*C n A n T*T* m C L n T L n m C L Here mCL은 5-메틸시토신 잠금 뉴클레오시드이고, TL은 티민 잠금 뉴클레오시드이고, GL은 구아노신 잠금 뉴클레오시드이고, A는 데옥시아데노신이고, C는 데옥시시티딘이고, T는 데옥시티미딘이고, G는 데옥시구아노신이고, *는 PS 뉴클레오시드간 결합이고,n은 PN 뉴클레오시드간 결합이고,t는 PS2 뉴클레오시드간 결합인, MAPT ASO 접합체. Claim 47 A pharmaceutical composition comprising a MAPT ASO conjugate of any one of claims 1 to 34 or a MAPT ASO of any one of claims 35 to 46, and a pharmaceutically acceptable carrier or excipient. Claim 48 A method for generating neuronal cells with reduced tau expression, the method comprising the step of delivering to a neuronal cell a MAPT ASO conjugate of any one of claims 1 to 34, a MAPT ASO of any one of claims 35 to 46, or a pharmaceutical composition of claim 47, wherein the MAPT ASO reduces the expression level of an endogenous MAPT gene in the neuronal cell. Claim 49 A method for modifying neuronal cells to reduce tau expression, the method comprising the step of delivering to neuronal cells a MAPT ASO conjugate of any one of claims 1 to 34, a MAPT ASO of any one of claims 35 to 46, or a pharmaceutical composition of claim 47, wherein the MAPT ASO reduces the expression level of an endogenous MAPT gene. Claim 50 A method for modifying neuronal cells to reduce tau expression, the method comprising the step of delivering to neuronal cells a MAPT ASO conjugate of any one of claims 1 to 34, a MAPT ASO of any one of claims 35 to 46, or a pharmaceutical composition of claim 47, wherein the MAPT ASO specifically reduces the expression level of a MAPT transcript in the cells. Claim 51 In claim 50, the method of reducing the expression level of endogenous MAPT comprises specifically reducing the expression level of MAPT transcripts in cells. Claim 52 A method for reducing the expression of tau in spinal cord cells of a subject, comprising the step of administering the MAPT ASO conjugate of any one of claims 1 to 34, the MAPT ASO of any one of claims 35 to 46, or the pharmaceutical composition of claim 47 by intrathecal administration. Claim 53 A method for reducing tau expression in a subject, comprising the step of administering to the subject a MAPT ASO conjugate of any one of claims 1 to 34, a MAPT ASO of any one of claims 35 to 46, or a pharmaceutical composition of claim 47. Claim 54 In paragraph 53, a method in which tau expression is reduced in the subject's CNS. Claim 55 In paragraph 53, the method wherein the MAPT ASO conjugate, the MAPT ASO, or the pharmaceutical composition is administered to a subject by intrathecal administration or intravenous injection or intravenous infusion. Claim 56 A method according to any one of claims 48 to 55, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene or reduces the level of the 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 a level where the pharmaceutical composition is not administered. Claim 57 In paragraph 56, a method in which the expression of the endogenous MAPT gene or the level of the MAPT transcript is reduced by at least about 50%. Claim 58 In paragraph 56, a method in which the expression of the endogenous MAPT gene or the level of the MAPT transcript is reduced by at least about 70%. Claim 59 A method for treating a tau-related neurodegenerative disorder in a human subject requiring this, the method comprising the step of administering to a human subject a MAPT ASO conjugate of any one of claims 1 to 34, a MAPT ASO of any one of claims 35 to 46, or a pharmaceutical composition of claim 47. Claim 60 In paragraph 59, the above tau-related neurodegenerative syndrome is Alzheimer's disease, method. Claim 61 A method for treating Alzheimer's disease, comprising the step of administering a MAPT ASO conjugate of any one of claims 1 to 34, a MAPT ASO of any one of claims 35 to 46, or a pharmaceutical composition of claim 47 to a human subject in need thereof. Claim 62 A method for reducing MAPT messenger ribonucleic acid (mRNA) expression in a human subject requiring reduction of MAPT messenger ribonucleic acid (mRNA) expression, the method comprising the step of administering to a human subject a MAPT ASO conjugate of any one of claims 1 to 34, a MAPT ASO of any one of claims 35 to 46, or a pharmaceutical composition of claim 47. Claim 63 A pharmaceutical composition according to claim 47, wherein the MAPT ASO is to be used to deliver MAPT ASO to the CNS of a human subject requiring delivery of MAPT ASO to the CNS of a human subject, and said MAPT ASO reduces the expression level of the endogenous MAPT gene. Claim 64 A pharmaceutical composition according to claim 47 for use in treating a tau-related neurodegenerative disorder in a human subject requiring treatment for the tau-related neurodegenerative disorder. Claim 65 In paragraph 64, the above tau-related neurodegenerative disorder is Alzheimer's disease, a pharmaceutical composition. Claim 66 A pharmaceutical composition according to claim 47 for use in reducing MAPT mRNA expression in human subjects requiring reduction of MAPT mRNA expression. Claim 67 A nucleic acid sequence encoding an Fc polypeptide dimer comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes any one of SEQ ID NOs 1, 47 to 48, 51 to 54, 61 to 63, 66, 73 to 78, 80, and 90, and the second nucleic acid sequence encodes any one of SEQ ID NOs 4 to 29, 36, 43 to 44, and 49 to 50. Claim 68 In claim 67, the nucleic acid sequence 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. Claim 69 A nucleic acid sequence according to claim 68, wherein the first nucleic acid sequence encodes any one of SEQ ID NOs 100 to 101 and 130 to 131, the second nucleic acid sequence encodes any one of SEQ ID NOs 98 to 99, and the nucleic acid sequence further comprises a third nucleic acid sequence encoding SEQ ID NO. 108 or 129. Claim 70 A nucleic acid sequence according to claim 69, wherein the first nucleic acid sequence comprises a sequence having at least 75% identity with SEQ ID NO. 133 or SEQ ID NO. 133, the second nucleic acid sequence comprises a sequence having at least 75% identity with SEQ ID NO. 132 or SEQ ID NO. 132, and the third nucleic acid sequence comprises a sequence having at least 75% identity with SEQ ID NO. 134 or SEQ ID NO. 134.

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