Transferrin Receptor Binding Proteins and Conjugates

Abstract

Provided herein are proteins comprising one monovalent human TfR-binding domain ("human TfR-binding proteins"), proteins comprising one monovalent mouse TfR-binding domain ("mouse TfR-binding proteins"), conjugates comprising such human or mouse TfR-binding proteins, e.g., human TfR-binding protein-dsRNA conjugates, pharmaceutical compositions comprising the human TfR-binding proteins or conjugates, and methods of treating CNS diseases (e.g., neurodegenerative diseases such as neurodegenerative synucleinopathies or tauopathies) using the human TfR-binding proteins or conjugates.

Description

[Technical Field]

[0001] Sequence Listing This application has been submitted with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled "30369_WO," created on July 18, 2023, and is 667 kilobytes in size. The Sequence Listing information in ST.26 XML format is incorporated herein by reference in its entirety. [Background technology]

[0002] The blood-brain barrier (BBB) ​​is a selective, semipermeable boundary of capillary endothelial cells that prevents the passage of solutes, including pathogens, into the central nervous system (CNS). The BBB allows the passage of some small molecules by passive diffusion, and cells of the BBB actively transport metabolites important for neuronal function, such as glucose and amino acids, across the barrier using specific transport proteins. The BBB has a neuroprotective function by tightly controlling access to the brain, but it can also prevent therapeutic agents from accessing the CNS.

[0003] BBB shuttles have been described to improve the ability of therapeutic agents to cross the blood-brain barrier and enter the CNS. For example, International Publication No. 2003 / 009815 describes the use of antibodies against transferrin receptors ("transferrin receptors, TfR") to regulate blood-brain barrier transport. However, attempts to shuttle therapeutic agents across the BBB using anti-TfR antibodies have proven difficult. To date, no TfR shuttles or conjugates have been approved for the treatment of CNS diseases.

[0004] Thus, there remains a need for TfR-binding proteins and conjugates that can deliver therapeutic agents across the BBB to the CNS for the treatment of various CNS disorders. Summary of the Invention

[0005] Provided herein are proteins comprising one monovalent human TfR-binding domain ("human TfR-binding proteins"), proteins comprising one monovalent mouse TfR-binding domain ("mouse TfR-binding proteins"), conjugates comprising such human or mouse TfR-binding proteins, e.g., human TfR-binding protein-dsRNA conjugates, pharmaceutical compositions comprising the human TfR-binding proteins or conjugates, and methods of treating CNS diseases (e.g., neurodegenerative diseases such as neurodegenerative synucleinopathies or tauopathies) using the human TfR-binding proteins or conjugates.

[0006] In one aspect, provided herein is a protein comprising a unique monovalent human TfR-binding domain ("human TfR-binding protein"). In some embodiments, the monovalent human TfR-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3. In some embodiments, the monovalent human TfR-binding domain comprises a VH comprising HCDR1, HCDR2, and HCDR3 selected from Table 1, and / or a VL comprising LCDR1, LCDR2, and LCDR3 selected from Table 2. In some embodiments, the monovalent human TfR-binding domain comprises a VH and / or VL selected from Table 3.

[0007] In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 21, HCDR3 comprises SEQ ID NO: 22, LCDR1 comprises SEQ ID NO: 23, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24, or (b) A protein in which HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18.

[0008] In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (b) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 7, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (c) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 8, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (d) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; or (e) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 14, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; or (f) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18, or (g) A protein wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18.

[0009] In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH and VL comprise the following sequences: (a) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 27 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (b) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 29 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (c) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 30 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 31; or (d) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 32 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 33; or (e) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 34 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 35; or (f) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 36 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37; or (g) A protein, wherein the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 38 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37.

[0010] In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH and VL comprise the following sequences: (a) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; or (b) the VH comprises SEQ ID NO: 29 and the VL comprises SEQ ID NO: 28; or (c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; or (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 35; or (f) the VH comprises SEQ ID NO: 36 and the VL comprises SEQ ID NO: 37; or (g) a protein, wherein the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO: 37.

[0011] In some embodiments, the monovalent human TfR-binding domain is an antibody fragment, such as a Fab, scFv, Fv, or scFab (single-chain Fab). In some embodiments, the monovalent human TfR-binding domain is a Fab. In some embodiments, the human TfR-binding domain further comprises a heavy chain variable region and / or a light chain variable region.

[0012] In some embodiments, the human TfR binding proteins described herein further comprise a half-life extender, for example, a VHH that binds to an immunoglobulin Fc region or human serum albumin (HSA).

[0013] In some embodiments, the human TfR binding proteins described herein comprise one or more engineered cysteine ​​residues for conjugation. In some embodiments, the human TfR binding proteins described herein comprise one or more native cysteine ​​residues for conjugation.

[0014] In some embodiments, the human TfR binding protein described herein is any one of the human TfR binding proteins in Tables 6a and 6b. In some embodiments, the human TfR binding protein described herein has one heavy chain (HC) and one light chain (LC), e.g., TBP1, TBP2, TBP3, TBP4, TBP5, TBP6, TBP7, TBP8, or TBP9. In some embodiments, the human TfR binding protein has two heavy chains (HC1 and HC2) and two light chains (LC1 and LC2). In some embodiments, the human TfR binding protein described herein has a heterodimeric antibody format, e.g., TBP10, TBP11, TBP12, or TBP13.

[0015] In some embodiments, provided herein are proteins comprising one monovalent human transferrin receptor (TfR) binding domain, wherein the human TfR binding domain binds to an epitope comprising one or more residues in (a) residues 346-364 FGNMEGDCPSDWKTDSTCR (SEQ ID NO: 119), (b) residues 243-247 FEDLY (SEQ ID NO: 162), and residues 345-364 LFGNMEEGDCPSDWKTDSTCR) (SEQ ID NO: 163) of human TfR, or (c) residues 243-247 FEDLY (SEQ ID NO: 162), residues 259-263 AGKIT (SEQ ID NO: 164), and residues 532-538 (VEKLTLD) (SEQ ID NO: 165) of human TfR.

[0016] In another aspect, provided herein are proteins comprising one monovalent mouse TfR-binding domain ("mouse TfR-binding proteins"). These mouse TfR-binding proteins can function as surrogate molecules for human TfR-binding proteins in mouse models. In some embodiments, provided herein are proteins comprising one monovalent mouse TfR-binding domain, wherein the mouse TfR-binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 71, HCDR2 comprises SEQ ID NO: 72, HCDR3 comprises SEQ ID NO: 73, LCDR1 comprises SEQ ID NO: 74, LCDR2 comprises SEQ ID NO: 75, and LCDR3 comprises SEQ ID NO: 76. In some embodiments, provided herein is a protein comprising one monovalent mouse TfR binding domain, wherein the mouse TfR binding domain comprises a VH comprising SEQ ID NO: 77 and a VL comprising SEQ ID NO: 78.

[0017] Also provided herein are antibodies comprising a VH comprising an HCDR1, HCDR2, and HCDR3 selected from Table 1, and / or a VL comprising an LCDR1, LCDR2, and LCDR3 selected from Table 2. In some embodiments, such antibodies comprise a VH and / or VL selected from Table 3.

[0018] In another aspect, provided herein is a conjugate comprising a human or mouse TfR binding protein and a therapeutic agent. In some embodiments, the therapeutic agent is selected from double-stranded RNA (e.g., siRNA, saRNA), an oligonucleotide (e.g., an antisense oligonucleotide), a peptide, a small molecule, a nanoparticle, a lipid nanoparticle, an exosome, an antibody or antigen-binding fragment thereof, or a combination thereof. In some embodiments, the therapeutic agent is double-stranded RNA (dsRNA). In some embodiments, the dsRNA comprises a sense strand and an antisense strand, and the antisense strand is complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B mRNA. In some embodiments, the ratio of therapeutic agent to protein is about 1:1 to 3:1. In some embodiments, the ratio of therapeutic agent to protein is about 1:1. In some embodiments, the ratio of therapeutic agent to protein is about 2: 1. In some embodiments, the ratio of therapeutic agent to protein is about 3:1.

[0019] In some embodiments, the therapeutic agent is linked to the human or mouse TfR binding protein via a linker, which in some embodiments is a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker (the structures of these linkers are shown in Table 8).

[0020] In some embodiments, provided herein are conjugates of Formula (I):RLP, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human or mouse TfR binding domain; and L is a linker or, optionally, absent. In some embodiments, P is a human or mouse TfR binding protein described herein. In some embodiments, the ratio of R to P is about 1:1 to 3:1. In some embodiments, the ratio of R to P is about 1:1. In some embodiments, the ratio of R to P is about 2:1. In some embodiments, the ratio of R to P is about 3:1.

[0021] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, P is a protein comprising one monovalent human or mouse TfR binding domain, L is a linker or optionally absent, and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0022] In some embodiments, provided herein is a conjugate of Formula (I): RLP, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 21, HCDR3 comprises SEQ ID NO: 22, LCDR1 comprises SEQ ID NO: 23, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24, or (b) A conjugate wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18.

[0023] In some embodiments, provided herein is a conjugate of Formula (I): RLP, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (b) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 7, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (c) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 8, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (d) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; or (e) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 14, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; or (f) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18, or (g) A conjugate wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18.

[0024] In some embodiments, provided herein is a conjugate of Formula (I): RLP, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the following sequences: (a) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 27 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (b) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 29 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (c) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 30 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 31; or (d) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 32 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 33; or (e) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 34 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 35; or (f) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 36 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37; or (g) A conjugate, wherein the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 38 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37.

[0025] In some embodiments, provided herein is a conjugate of Formula (I): RLP, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the following sequences: (a) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; or (b) the VH comprises SEQ ID NO: 29 and the VL comprises SEQ ID NO: 28; or (c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; or (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 35; or (f) the VH comprises SEQ ID NO: 36 and the VL comprises SEQ ID NO: 37; or (g) A conjugate, wherein the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO: 37.

[0026] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3; and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 21, HCDR3 comprises SEQ ID NO: 22, LCDR1 comprises SEQ ID NO: 23, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24, or (b) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; n is 1 to 3, and is a conjugate.

[0027] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0028] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (b) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 7, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (c) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 8, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (d) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; or (e) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 14, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; or (f) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18, or (g) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; n is 1 to 3, and is a conjugate.

[0029] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0030] In some embodiments, provided herein is a conjugate of formula (II): (RL)nP, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the following sequences: (a) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 27 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (b) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 29 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (c) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 30 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 31; or (d) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 32 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 33; or (e) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 34 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 35; or (f) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 36 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37; or (g) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 38 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37; n is 1 to 3, and is a conjugate.

[0031] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0032] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, P is a protein comprising one monovalent human TfR binding domain, L is a linker or optionally absent, and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the following sequences: (a) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; or (b) the VH comprises SEQ ID NO: 29 and the VL comprises SEQ ID NO: 28; or (c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; or (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 35; or (f) the VH comprises SEQ ID NO: 36 and the VL comprises SEQ ID NO: 37; or (g) the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO: 37; n is 1 to 3, and is a conjugate.

[0033] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0034] In some embodiments, the linker (L) is a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker (see Table 8).

[0035] In some embodiments, the dsRNA comprises an antisense strand complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B mRNA. In some embodiments, the dsRNA comprises an antisense strand complementary to SNCA mRNA. In some embodiments, the dsRNA comprises an antisense strand complementary to MAPT mRNA.

[0036] Exemplary unmodified sense and antisense strand sequences of dsRNA targeting human SNCA mRNA are provided in Table 9a. In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 81 and the antisense strand comprises SEQ ID NO: 82; (b) the sense strand comprises SEQ ID NO: 83 and the antisense strand comprises SEQ ID NO: 84; (c) the sense strand comprises SEQ ID NO: 85 and the antisense strand comprises SEQ ID NO: 86; (d) the sense strand comprises SEQ ID NO: 87 and the antisense strand comprises SEQ ID NO: 88; (e) the sense strand comprises SEQ ID NO: 89 and the antisense strand comprises SEQ ID NO: 90; and (f) the sense strand comprises SEQ ID NO: 91 and the antisense strand comprises SEQ ID NO: 92; (g) the sense strand comprises SEQ ID NO: 116 and the antisense strand comprises SEQ ID NO: 82; Optionally, one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and optionally, one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages. In some embodiments, the sense strand comprises SEQ ID NO: 81 and the antisense strand comprises SEQ ID NO: 82.

[0037] Exemplary unmodified sense and antisense strand sequences of dsRNA targeting human MAPT mRNA are provided in Table 9b. In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 120 and the antisense strand comprises SEQ ID NO: 121; (b) the sense strand comprises SEQ ID NO: 122 and the antisense strand comprises SEQ ID NO: 123, and (c) the sense strand comprises SEQ ID NO: 124 and the antisense strand comprises SEQ ID NO: 125; Optionally, one or more nucleotides in the sense strand and the antisense strand are independently modified nucleotides, and optionally, one or more internucleotide linkages in the sense strand and the antisense strand are modified internucleotide linkages.

[0038] dsRNA may contain modifications. Modifications may be made to one or more nucleotides of the sense strand and / or antisense strand, or to the internucleotide linkage. In some embodiments, one or more nucleotides of the sense strand and / or antisense strand are independently modified nucleotides, meaning that the sense strand and the antisense strand may have different modified nucleotides. In some embodiments, each nucleotide of the sense strand is a modified nucleotide. In some embodiments, each nucleotide of the antisense strand is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-alkyl-modified nucleotide. In some embodiments, each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, for example, a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-O-alkyl-modified nucleotide.

[0039] In some embodiments, the sense strand has four 2'-fluoro modified nucleotides, e.g., at positions 7, 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, the other nucleotides in the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has four 2'-fluoro modified nucleotides, e.g., at positions 2, 6, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides in the antisense strand are 2'-O-methyl modified nucleotides.

[0040] In some embodiments, the sense strand has three 2'-fluoro modified nucleotides, e.g., at positions 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, the other nucleotides in the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 3, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides in the antisense strand are 2'-O-methyl modified nucleotides.

[0041] In some embodiments, the 5'-end of the antisense strand has a phosphate analog, for example, 5'-vinylphosphonate (5'-VP).

[0042] In some embodiments, the sense strand or the antisense strand comprises an abasic portion or an inverted abasic portion.

[0043] In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate linkages. In some embodiments, the sense strand has four or five phosphorothioate linkages. In some embodiments, the antisense strand has four or five phosphorothioate linkages. In some embodiments, the sense strand and the antisense strand each have four or five phosphorothioate linkages. In some embodiments, the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.

[0044] Exemplary modified sense and antisense strand sequences of dsRNAs targeting human SNCA mRNA are provided in Table 11a. Exemplary modified sense and antisense strand sequences of dsRNAs targeting human MAPT mRNA are provided in Table 11b.

[0045] In another aspect, provided herein is a method of treating a CNS disease, e.g., a neurodegenerative disease, in a patient in need thereof, comprising administering to the patient an effective amount of a human TfR binding protein, or conjugate, or pharmaceutical composition described herein.

[0046] In a further aspect, provided herein are methods of treating a neurodegenerative synucleinopathy in a patient in need thereof, comprising administering to the patient an effective amount of a human TfR binding protein, or conjugate, or pharmaceutical composition described herein (e.g., a TBP-SNCA siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-SNCA siRNA conjugate). In some embodiments, the neurodegenerative synucleinopathy is selected from Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies. The human TfR binding protein, conjugate, or pharmaceutical composition may be administered intravenously or subcutaneously to the patient.

[0047] In a further aspect, provided herein is a method of treating a tauopathy in a patient in need thereof, such method comprising administering to the patient an effective amount of a human TfR binding protein, or conjugate, or pharmaceutical composition described herein (e.g., a TBP-MAPT siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-MAPT siRNA conjugate). In some embodiments, the tauopathy is Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary progressive aphasia-semantic (PPA-S), primary progressive aphasia-logopenic (PPA-L), multiple system tauopathy with presenile dementia (MSD). Tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementiadementia (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C C, NP-C), non-guamanian motor neuron disease with neurofibrillary tangles, post-encephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle-type senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathies (GGT). The human TfR binding protein or conjugate or pharmaceutical composition may be administered intravenously or subcutaneously to the patient.

[0048] In another aspect, provided herein are human TfR binding proteins or conjugates described herein, or pharmaceutical compositions comprising such human TfR binding proteins or conjugates, for use in therapy. Also provided herein are human TfR binding proteins or conjugates described herein, or pharmaceutical compositions comprising such human TfR binding proteins or conjugates (e.g., TBP-SNCA siRNA conjugates described herein, or pharmaceutical compositions comprising such TBP-SNCA siRNA conjugates), for use in treating neurodegenerative synucleinopathies, such as Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies.Also provided herein are tauopathies, such as, for example, Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PPA-S), primary logopenic progressive aphasia (PPA-L), and the like. , multisystem tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, calcification Diffuse neurofibrillary tangles with atresia, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, cerebral amyloidosis A human TfR binding protein or conjugate described herein, or a pharmaceutical composition comprising such a human TfR binding protein or conjugate (e.g., a TBP-MAPT siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-MAPT siRNA conjugate), for use in treating transfibrillary tangle senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

[0049] In another aspect, provided herein is the use of a human TfR binding protein or conjugate described herein in the manufacture of a medicament for treating a CNS disease, e.g., a neurodegenerative disease. In some embodiments, the neurodegenerative disease is a neurodegenerative synucleinopathy, e.g., Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies. In some embodiments, the neurodegenerative disease is a tauopathy, e.g., Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PP) A-S), primary logopenic progressive aphasia (PPA-L), multisystem tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic Corticobasal degeneration (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C ( NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle-type senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or glioglobular tauopathy (GGT). [Brief explanation of the drawings]

[0050] [Figure 1A]1 shows an exemplary analytical anion exchange (aAEX) chromatogram of the DAR profile for a TBP11-dsRNA conjugate before purification. [Figure 1B] 1 shows an exemplary aAEX chromatogram of the DAR profile for a purified TBP14-dsRNA conjugate. [Figure 1C] 1 shows an exemplary aAEX chromatogram of the DAR profile for a TBP15-dsRNA conjugate before purification. [Figure 1D] 1 shows an exemplary aAEX chromatogram of the DAR profile for a purified TBP15-dsRNA conjugate. [Figure 1E] Illustrated are exemplary diagrams of TBP-dsRNA conjugates of DAR2 (top) or DAR1 (bottom). [Figure 2] 1 shows the in vitro binding, internalization, and degradation of the indicated molecules in mouse cortical neurons. [Figure 3] FIG. 1 shows the in vitro efficacy of the indicated molecules for knocking down mouse SNCA in primary mouse cortical neurons. [Figure 4] 1 shows in vitro binding, internalization, and degradation assessment of the indicated molecules in SHSY5Y cells. [Figure 5] FIG. 1 shows the in vitro efficacy of the indicated molecules for knocking down human SNCA in SH-SY5Y cells. [Figure 6A] Proof-of-concept data in mice are shown demonstrating the pharmacodynamic efficacy of the mTBP2-SNCA siRNA conjugate with multiple intravenous (IV) dosing at a single time point (28 days), showing a reduction in SNCA mRNA and protein in the mouse brain (Figure 6A), as well as a reduction in SNCA mRNA in the spinal cord (Figure 6B), and a reduction in SNCA mRNA in the lumbar dorsal root ganglion (Figure 6C). [Figure 6B]Proof-of-concept data in mice are shown demonstrating the pharmacodynamic efficacy of the mTBP2-SNCA siRNA conjugate with multiple intravenous (IV) dosing at a single time point (28 days), showing a reduction in SNCA mRNA and protein in the mouse brain (Figure 6A), as well as a reduction in SNCA mRNA in the spinal cord (Figure 6B), and a reduction in SNCA mRNA in the lumbar dorsal root ganglion (Figure 6C). [Figure 6C] Proof-of-concept data in mice are shown demonstrating the pharmacodynamic efficacy of the mTBP2-SNCA siRNA conjugate with multiple intravenous (IV) dosing at a single time point (28 days), showing a reduction in SNCA mRNA and protein in the mouse brain (Figure 6A), as well as a reduction in SNCA mRNA in the spinal cord (Figure 6B), and a reduction in SNCA mRNA in the lumbar dorsal root ganglion (Figure 6C). [Figure 7A] Figure 7 shows the pharmacodynamic efficacy time course data for the mTBP2-SNCA siRNA conjugate after a single IV dose, with mice sacrificed at multiple time points (7, 28, 70, and 120 days) after administration. The data show the pharmacodynamic time course of SNCA mRNA and protein reduction in the mouse brain (Figure 7A) and spinal cord (Figure 7B). Error bars in Figures 7A and 7B represent standard deviations. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison test relative to the PBS control group. Annotations indicate P values ​​>0.0001 = ****, >0.001 = ***, >0.01 = **, and >0.05 = *. [Figure 7B]Figure 7 shows the pharmacodynamic efficacy time course data for the mTBP2-SNCA siRNA conjugate after a single IV dose, with mice sacrificed at multiple time points (7, 28, 70, and 120 days) after administration. The data show the pharmacodynamic time course of SNCA mRNA and protein reduction in the mouse brain (Figure 7A) and spinal cord (Figure 7B). Error bars in Figures 7A and 7B represent standard deviations. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison test relative to the PBS control group. Annotations indicate P values ​​>0.0001 = ****, >0.001 = ***, >0.01 = **, and >0.05 = *. [Figure 8A] 1 shows SNCA mRNA reduction in cynomolgus monkey tissues 29 days after two consecutive single IV peripheral doses (given 2 hours apart) of 4.4 mg / kg siRNA of TBP10-SNCA siRNA (dsRNA #8 in Table 11a) conjugate. [Figure 8B] Figure 8A and 8B show SNCA mRNA reduction in cynomolgus monkey tissues 29 days after two consecutive single IV peripheral doses (given 2 hours apart) of 1.3 mg / kg siRNA of the TBP11-SNCA siRNA (dsRNA No. 8 in Table 11a) conjugate. Error bars in Figures 8A and 8B represent standard error of the mean. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison test relative to the PBS control group. Annotations indicate P values ​​>0.0001 to 0.05 = *. [Figure 8C] Figure 8C shows mouse brain efficacy comparison of mouse TfR-binding protein conjugates at NHP-equivalent siRNA doses adjusted for body weight. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test relative to the PBS control group. Annotations indicate P values ​​>0.0001 = ****, >0.001 = ***, >0.01 = **, >0.05 = *. [Figure 9A] 1 shows SNCA mRNA reduction in cynomolgus monkey tissues following three monthly peripheral intravenous (IV) administrations of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate at 10 mg / kg siRNA. [Figure 9B] 1 shows the reduction of α-synuclein protein in cynomolgus monkey tissues following three monthly peripheral IV administrations of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate at 10 mg / kg siRNA. [Figure 9C] 1 shows SNCA mRNA reduction in cynomolgus monkey tissues 85 days after a single peripheral IV administration of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate at 10 mg / kg siRNA. [Figure 9D] 1 shows the reduction of α-synuclein protein in cynomolgus monkey tissues 85 days after a single peripheral IV administration of 10 mg / kg siRNA of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate. [Figure 9E] 1 shows SNCA mRNA reduction in gastrocnemius muscle following single or three-monthly peripheral IV administration of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate at 10 mg / kg siRNA. [Figure 10A] 1 shows MAPT mRNA reduction in cynomolgus monkey tissues after three monthly peripheral IV administrations of TBP14-MAPT siRNA (dsRNA No. 38 in Table 11b) conjugate at 10 mg / kg siRNA. [Figure 10B] 1 shows the reduction of tau protein in cynomolgus monkey tissues following three monthly peripheral IV administrations of TBP14-MAPT siRNA (dsRNA No. 38 in Table 11b) conjugate at 10 mg / kg siRNA. [Figure 11A] 1 shows MAPT mRNA reduction in cynomolgus monkey tissues after three monthly peripheral IV administrations of 10 mg / kg of TBP14-MAPT siRNA (dsRNA No. 39 in Table 11b) conjugate. [Figure 11B] 1 shows the reduction of tau protein in cynomolgus monkey tissues following three monthly peripheral IV administrations of TBP14-MAPT siRNA (dsRNA No. 39 in Table 11b) conjugate at 10 mg / kg siRNA. [Figure 12A]1 shows MAPT mRNA reduction in cynomolgus monkey tissues after three monthly peripheral IV administrations of TBP14-MAPT siRNA (dsRNA No. 40 in Table 11b) conjugate at 10 mg / kg siRNA. [Figure 12B] 1 shows the reduction of tau protein in cynomolgus monkey tissues following three monthly peripheral IV administrations of TBP14-MAPT siRNA (dsRNA No. 40 in Table 11b) conjugate at 10 mg / kg siRNA. [Figure 13A] 1 shows SNCA mRNA reduction in cynomolgus monkey tissues one month after a single peripheral IV administration of TBP16-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) at 1 mg / kg siRNA. [Figure 13B] 13B shows SNCA mRNA reduction in selected cynomolgus monkey brain tissues one month after a single peripheral IV administration of TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) at 1 mg / kg (13B) and 10 mg / kg (13C) of siRNA. [Figure 13C] 13B shows SNCA mRNA reduction in selected cynomolgus monkey brain tissues one month after a single peripheral IV administration of TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) at 1 mg / kg (13B) and 10 mg / kg (13C) of siRNA. [Figure 13D] Figure 1 shows the plasma PK of conjugate-associated siRNA following a single peripheral IV administration of either 10 mg / kg of siRNA TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR2) or 10 mg / kg or 1 mg / kg of siRNA TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1). [Figure 13E] Figure 1 shows total siRNA concentrations in selected cynomolgus monkey brain tissues 29 days after a single peripheral IV administration of TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) at either 1 or 10 mg / kg siRNA. [Figure 14A] Figure 11 shows plasma PK of conjugate-associated siRNA in human TfR transgenic mice after a single peripheral IV administration of either TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR2) or TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) at 10 mg / kg siRNA. [Figure 14B] FIG. 11 shows brain tissue concentrations of total antisense siRNA in human TfR transgenic mice at 24 hours after a single peripheral IV administration of either TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR2) or TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) across a range of doses. [Figure 14C] Figure 11 shows brain tissue concentrations of total siRNA in human TfR transgenic mice 24 hours after a single peripheral IV administration of either a TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR2) or a TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) across various siRNA doses. [Figure 14D] Figure 1 shows the reduction of SNCA mRNA levels in whole brain homogenates on day 28 in human TfR transgenic mice after a single peripheral IV administration of either a TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR2) or a TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) across various siRNA doses. Error bars represent standard deviations. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test relative to the PBS control group. Annotations indicate P values ​​>0.0001 = ****, >0.001 = ***, >0.01 = **, and >0.05 = *. [Figure 14E]Figure 1 shows the reduction of SNCA mRNA levels in whole brain homogenates at day 28 in human TfR transgenic mice after a single subcutaneous administration of TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) across various siRNA doses. Error bars represent standard deviations. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test relative to the PBS control group. Annotations indicate P values ​​>0.0001 = ****, >0.001 = ***, >0.01 = **, and >0.05 = *. DETAILED DESCRIPTION OF THE INVENTION

[0051] Provided herein are proteins comprising one monovalent human TfR-binding domain ("human TfR-binding proteins"), proteins comprising one monovalent mouse TfR-binding domain ("mouse TfR-binding proteins"), conjugates comprising such human or mouse TfR-binding proteins, e.g., human TfR-binding protein-dsRNA conjugates, pharmaceutical compositions comprising the human TfR-binding proteins or conjugates, and methods of treating CNS diseases (e.g., neurodegenerative diseases such as neurodegenerative synucleinopathies or tauopathies) using the human TfR-binding proteins or conjugates.

[0052] Human TfR-binding protein In one aspect, provided herein is a protein comprising one monovalent human TfR-binding domain (a "human TfR-binding protein"). In some embodiments, the monovalent human TfR-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3. In some embodiments, the monovalent human TfR-binding domain comprises a VH comprising an HCDR1, HCDR2, and HCDR3 selected from Table 1. In some embodiments, the monovalent human TfR-binding domain comprises a VL comprising an LCDR1, LCDR2, and LCDR3 selected from Table 2. In some embodiments, the monovalent human TfR-binding domain comprises a VH comprising an HCDR1, HCDR2, and HCDR3 selected from Table 1, and / or a VL comprising an LCDR1, LCDR2, and LCDR3 selected from Table 2. In some embodiments, the monovalent human TfR binding domain comprises a VH and / or VL selected from Table 3. In some embodiments, the monovalent human TfR binding domain ("TfR binding domain, TBD") is TBD1, TBD2, TBD3, TBD4, TBD5, TBD6, TBD6, TBD7, TBD8, or TBD9. In some embodiments, the monovalent human TfR binding domain is TBD1, TBD2, TBD3, TBD4, TBD5, TBD6, TBD6, or TBD7. In some embodiments, the human TfR binding proteins described herein also bind to cynomolgus monkey TfR.

[0053] Table 1. Exemplary sequences of human TfR binding domain heavy chain CDRs [Table 1]

[0054] Table 2. Exemplary sequences of human TfR binding domain light chain CDRs [Table 2]

[0055] Table 3. Exemplary sequences of human TfR binding domains VH and VL [Table 3]

[0056] In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 21, HCDR3 comprises SEQ ID NO: 22, LCDR1 comprises SEQ ID NO: 23, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24, or (b) A protein in which HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18.

[0057] In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 21, HCDR3 comprises SEQ ID NO: 22, LCDR1 comprises SEQ ID NO: 23, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24. In some embodiments, provided herein is a protein comprising one monovalent human transferrin receptor (TfR) binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18.

[0058] In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (b) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 7, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (c) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 8, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (d) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; or (e) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 14, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; or (f) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18, or (g) A protein wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18.

[0059] In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 7, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 8, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12.In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 14, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18. In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18. In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18.

[0060] In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH and VL comprise the following sequences: (a) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 27 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (b) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 29 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (c) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 30 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 31; or (d) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 32 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 33; or (e) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 34 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 35; or (f) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 36 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37; or (g) A protein, wherein the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 38 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37.

[0061] In some embodiments, provided herein is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a VH and a VL, wherein the VH and VL comprise the following sequences: (a) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; or (b) the VH comprises SEQ ID NO: 29 and the VL comprises SEQ ID NO: 28; or (c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; or (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 35; or (f) the VH comprises SEQ ID NO: 36 and the VL comprises SEQ ID NO: 37; or (g) a protein, wherein the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO: 37.

[0062] In some embodiments, provided herein is a protein comprising one monovalent human TfR-binding domain, wherein the human TfR-binding domain comprises a VH and a VL, wherein the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28. In some embodiments, provided herein is a protein comprising one monovalent human TfR-binding domain, wherein the human TfR-binding domain comprises a VH and a VL, wherein the VH comprises SEQ ID NO: 29 and the VL comprises SEQ ID NO: 28. In some embodiments, provided herein is a protein comprising one monovalent human TfR-binding domain, wherein the human TfR-binding domain comprises a VH and a VL, wherein the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31. In some embodiments, provided herein is a protein comprising one monovalent human TfR-binding domain, wherein the human TfR-binding domain comprises a VH and a VL, wherein the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33. In some embodiments, provided herein is a protein comprising one monovalent human TfR-binding domain, wherein the human TfR-binding domain comprises a VH and a VL, wherein the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 35. In some embodiments, provided herein is a protein comprising one monovalent human TfR-binding domain, wherein the human TfR-binding domain comprises a VH and a VL, wherein the VH comprises SEQ ID NO: 36 and the VL comprises SEQ ID NO: 37. In some embodiments, provided herein is a protein comprising one monovalent human TfR-binding domain, wherein the human TfR-binding domain comprises a VH and a VL, wherein the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO: 37.

[0063] In some embodiments, the monovalent human TfR-binding domain is an antibody fragment, such as a Fab, scFv, Fv, or scFab (single-chain Fab). In some embodiments, the monovalent human TfR-binding domain is a Fab. In some embodiments, the human TfR-binding domain further comprises a heavy chain variable region and / or a light chain variable region.

[0064] In some embodiments, the human TfR binding proteins described herein further comprise a half-life extender, for example, a VHH that binds to an immunoglobulin Fc region or human serum albumin (HSA).

[0065] In some embodiments, the human TfR binding proteins described herein further comprise an immunoglobulin Fc region, such as a modified human IgG4 Fc region or a modified human IgG1 Fc region. In some embodiments, the human TfR binding proteins described herein further comprise a modified human IgG4 Fc region comprising a proline at residue 228 and an alanine at residues 234 and 235 (all residues are numbered according to the EU index numbering system, also referred to as the hIgG4PAA Fc region). In some embodiments, the human TfR binding proteins described herein further comprise a modified human IgG1 Fc region comprising an alanine at residues 234, 235, and 329, a serine at position 265, and an aspartic acid at position 436 (all residues are numbered according to the EU index numbering system, also referred to as the hIgG1 effector null or hIgG1EN Fc region). In some embodiments, a human TfR binding protein described herein comprises a modified human IgG1 or IgG4 Fc region, the Fc region comprising a first Fc CH3 domain comprising a serine at position 349, a methionine at position 366, a tyrosine at position 370, and a valine at position 409, and a second Fc CH3 domain comprising a glycine at position 356, an aspartic acid at position 357, a glutamine at position 364, and an alanine at position 407 (all residues are numbered according to EU index numbering). In some embodiments, a human TfR binding protein described herein comprises a modified human IgG1 or IgG4 Fc region comprising a first Fc CH3 domain comprising a leucine at residue 405, and a second Fc CH3 domain comprising an arginine at residue 409 (all residues are numbered according to EU index numbering).

[0066] In some embodiments, the human TfR-binding proteins described herein further comprise a VHH that binds to human HSA. In some embodiments, the VHH also binds to mouse, rat, and / or cynomolgus monkey albumin. Exemplary VHHs that bind to human HSA are shown in Table 4. In some embodiments, such a VHH comprises a CDR1 comprising SEQ ID NO: 39, a CDR2 comprising SEQ ID NO: 40, and a CDR3 comprising SEQ ID NO: 41. In some embodiments, such a VHH comprises SEQ ID NO: 42. In some embodiments, the VHH is linked to the TfR-binding domain via a peptide linker, e.g., (GGGGQ)4 (SEQ ID NO: 70).

[0067] Table 4. Exemplary sequences of VHHs that bind to human serum albumin (HSA) [Table 4]

[0068] In some embodiments, the human TfR binding proteins described herein are heterodimeric antibodies comprising a first arm comprising one monovalent human TfR binding domain and a second arm that is a null arm (e.g., an isotype arm) that does not bind to any known human target. Heterodimeric antibodies, such as heteromabs, orthomabs, or duobodies, are described in International Publication Nos. 2014150973, 2016118742, 2018118616, and 2011131746. In some embodiments, the first arm comprises any one of the monovalent human TfR binding domains described herein. In some embodiments, the second arm is a null arm (e.g., an isotype arm) that does not bind to any known human target and comprises a sequence in Table 5. In some embodiments, the second arm comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 43, HCDR2 comprises SEQ ID NO: 44, HCDR3 comprises SEQ ID NO: 45, LCDR1 comprises SEQ ID NO: 46, LCDR2 comprises SEQ ID NO: 47, and LCDR3 comprises SEQ ID NO: 48. In some embodiments, the second arm comprises a VH and a VL, wherein the VH comprises SEQ ID NO: 49 and the VL comprises SEQ ID NO: 50. In some embodiments, the second arm comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 51 and the LC comprises SEQ ID NO: 52.

[0069] In some embodiments, the human TfR binding proteins described herein comprise a heterodimeric mutation. In some embodiments, the human TfR binding proteins described herein comprise a modified Fc region comprising a first Fc CH3 domain comprising a serine at residue 349, a methionine at residue 366, a tyrosine at residue 370, and a valine at residue 409, and a second Fc CH3 domain comprising a glycine at residue 356, an aspartic acid at residue 357, a glutamine at residue 364, and an alanine at residue 407 (all residues are numbered according to EU index numbering). In some embodiments, the human TfR binding proteins described herein comprise a modified Fc region comprising a first Fc CH3 domain comprising a leucine at residue 405, and a second Fc CH3 domain comprising an arginine at residue 409 (all residues are numbered according to EU index numbering).

[0070] Table 5. Exemplary sequences of isotype or null arms that do not bind to any known target (isotype Ab) [Table 5]

[0071] In some embodiments, the human TfR binding proteins described herein comprise one or more native cysteine ​​residues that can be used for conjugation. For example, in some embodiments, the human TfR binding proteins described herein comprise a native cysteine ​​at position 220 of the light chain and / or a native cysteine ​​at position 226 of the heavy chain, which can be used for conjugation (all residues according to EU index numbering).

[0072] In some embodiments, the human TfR binding proteins described herein comprise engineered cysteine ​​residues for conjugation. Approaches involving engineered cysteines as a means for conjugation are described in WO 2018 / 232088. In some embodiments, the human TfR binding proteins described herein comprise a heavy chain comprising one or more cysteines at the following residues: 124, 157, 162, 262, 373, 375, 378, 397, 415 (all residues according to EU index numbering). In some embodiments, the human TfR binding proteins described herein comprise a light chain (e.g., a kappa light chain) comprising one or more cysteines at the following residues: 156, 171, 191, 193, 202, 208 (all residues according to EU index numbering). In some embodiments, the human TfR binding proteins described herein comprise a heavy chain constant region comprising a cysteine ​​at residue 124 (according to EU index numbering). In some embodiments, a human TfR binding protein described herein comprises a light chain constant region comprising a cysteine ​​at residue 156 (according to EU index numbering). In some embodiments, a human TfR binding protein described herein comprises an immunoglobulin Fc region comprising a cysteine ​​at residue 378 (according to EU index numbering).

[0073] In some embodiments, the human TfR binding protein described herein is any one of the human TfR binding proteins in Tables 6a and 6b. In some embodiments, the human TfR binding protein described herein has one heavy chain (HC) and one light chain (LC), e.g., TBP1, TBP2, TBP3, TBP4, TBP5, TBP6, TBP7, TBP8, or TBP9 (see Table 6a).

[0074] In some embodiments, the human TfR binding proteins described herein have a Fab-Fc format, e.g., TBP1, TBP2, TBP3, TBP4, TBP5, TBP6, or TBP7. In some embodiments, provided herein are human TfR binding proteins comprising one HC and one LC, wherein the HC comprises SEQ ID NO: 53 and the LC comprises SEQ ID NO: 54. In some embodiments, provided herein are human TfR binding proteins comprising one HC and one LC, wherein the HC comprises SEQ ID NO: 55 and the LC comprises SEQ ID NO: 54. In some embodiments, provided herein are human TfR binding proteins comprising one HC and one LC, wherein the HC comprises SEQ ID NO: 56 and the LC comprises SEQ ID NO: 57. In some embodiments, provided herein are human TfR binding proteins comprising one HC and one LC, wherein the HC comprises SEQ ID NO: 58 and the LC comprises SEQ ID NO: 59. In some embodiments, provided herein is a human TfR binding protein comprising one HC and one LC, wherein the HC comprises SEQ ID NO: 60 and the LC comprises SEQ ID NO: 61. In some embodiments, provided herein is a human TfR binding protein comprising one HC and one LC, wherein the HC comprises SEQ ID NO: 62 and the LC comprises SEQ ID NO: 63. In some embodiments, provided herein is a human TfR binding protein comprising one HC and one LC, wherein the HC comprises SEQ ID NO: 64 and the LC comprises SEQ ID NO: 63.

[0075] In some embodiments, the human TfR binding proteins described herein have a Fab format, e.g., TBP 8. In some embodiments, provided herein are human TfR binding proteins comprising one HC and one LC, wherein the HC comprises SEQ ID NO: 65 and the LC comprises SEQ ID NO: 59.

[0076] In some embodiments, the human TfR binding proteins described herein have a Fab-VHH format, e.g., TBP9. In some embodiments, provided herein are human TfR binding proteins comprising one HC and one LC, wherein the HC comprises SEQ ID NO: 66 and the LC comprises SEQ ID NO: 67.

[0077] Table 6a. Exemplary sequences of human TfR binding proteins (one HC and one LC) [Table 6-1]

[0078] (Continued from Table 6a) [Table 6-2]

[0079] (Continued from Table 6a) [Table 6-3]

[0080] In some embodiments, the human TfR binding proteins described herein have two or more heavy chains (HC) and / or two or more light chains (see Table 6b). In some embodiments, the human TfR binding proteins have two heavy chains (HC1 and HC2) and two light chains (LC1 and LC2). In some embodiments, the human TfR binding proteins described herein have a heterodimeric antibody format, e.g., TBP10, TBP11, TBP12, or TBP13.

[0081] In some embodiments, provided herein is a human TfR binding protein comprising two heavy chains, HC1 and HC2, and two light chains, LC1 and LC2, wherein HC1 comprises SEQ ID NO: 64, LC1 comprises SEQ ID NO: 63, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52. In some embodiments, provided herein is a human TfR binding protein comprising two heavy chains, HC1 and HC2, and two light chains, LC1 and LC2, wherein HC1 comprises SEQ ID NO: 55, LC1 comprises SEQ ID NO: 54, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52. In some embodiments, provided herein is a human TfR binding protein comprising two heavy chains, HC1 and HC2, and two light chains, LC1 and LC2, wherein HC1 comprises SEQ ID NO: 56, LC1 comprises SEQ ID NO: 57, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52. In some embodiments, provided herein is a human TfR binding protein comprising two heavy chains, HC1 and HC2, and two light chains, LC1 and LC2, wherein HC1 comprises SEQ ID NO: 58, LC1 comprises SEQ ID NO: 59, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52.

[0082] In some embodiments, the human TfR binding protein has two heavy chains (HC1 and HC2) and one light chain (LC1) (e.g., TBP14, TBP15, TBP16). In some embodiments, provided herein is a human TfR binding protein comprising two heavy chains, HC1 and HC2, and one light chain, LC1, wherein HC1 comprises SEQ ID NO: 68, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 69. In some embodiments, provided herein is a human TfR binding protein comprising two heavy chains, HC1 and HC2, and one light chain, LC1, wherein HC1 comprises SEQ ID NO: 138, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 139. In some embodiments, provided herein is a human TfR binding protein comprising two heavy chains, HC1 and HC2, and one light chain, LC1, wherein HC1 comprises SEQ ID NO: 166, LC1 comprises SEQ ID NO: 54, and HC2 comprises SEQ ID NO: 167.

[0083] Table 6b. Exemplary sequences of human TfR binding proteins (HCs and / or LCs) [Table 7-1]

[0084] (Continued from Table 6b) [Table 7-2]

[0085] In some embodiments, provided herein are proteins comprising one monovalent human transferrin receptor (TfR) binding domain, wherein the human TfR binding domain binds to an epitope comprising one or more residues in (a) residues 346-364 FGNMEGDCPSDWKTDSTCR (SEQ ID NO: 119), (b) residues 243-247 FEDLY (SEQ ID NO: 162) and residues 345-364 LFGNMEEGDCPSDWKTDSTCR) (SEQ ID NO: 163) of human TfR, or (c) residues 243-247 FEDLY (SEQ ID NO: 162), residues 259-263 AGKIT (SEQ ID NO: 164), and residues 532-538 (VEKLTLD) (SEQ ID NO: 165) of human TfR.

[0086] Also provided herein are antibodies comprising a VH comprising an HCDR1, HCDR2, and HCDR3 selected from Table 1, and / or a VL comprising an LCDR1, LCDR2, and LCDR3 selected from Table 2. In some embodiments, such antibodies comprise a VH and / or VL selected from Table 3.

[0087] The TfR-binding proteins or antibodies described herein can be recombinantly produced in host cells using, for example, an expression vector. For example, the expression vector may include a sequence encoding one or more signal peptides that facilitate secretion of the polypeptide from the host cell. An expression vector containing a polynucleotide of interest (e.g., a polynucleotide encoding the heavy or light chain of a TfR-binding protein or antibody) can be transferred into host cells by well-known methods. Additionally, the expression vector may include one or more selectable markers, such as, for example, tetracycline, neomycin, and dihydrofolate reductase, to facilitate detection of host cells transformed with the desired polynucleotide sequence.

[0088] Host cells include cells stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or a portion of the TfR binding proteins or antibodies described herein. According to some embodiments, host cells may be stably or transiently transfected, transformed, transduced, or infected with an expression vector expressing the HC polypeptide and an expression vector expressing the LC polypeptide of a TfR binding protein or antibody described herein. In some embodiments, host cells may be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing the HC and LC polypeptides of a TfR binding protein or antibody described herein. TfR binding proteins or antibodies may be produced in mammalian cells, such as CHO, NS0, HEK293, or COS cells, according to techniques well known in the art.

[0089] The medium into which the TfR-binding protein or antibody is secreted can be purified by conventional techniques, such as mixed-mode methods of ion exchange and hydrophobic interaction chromatography. For example, the medium can be applied to and eluted from a Protein A or Protein G column using conventional methods; mixed-mode methods of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. Various methods of protein purification can be used, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0090] Mouse TfR-binding protein In another aspect, provided herein are proteins comprising one monovalent mouse TfR-binding domain ("mouse TfR-binding protein" or mTBP). These mouse TfR-binding proteins can function in mouse models as surrogate molecules for the above-described human TfR-binding proteins. In some embodiments, the monovalent mouse TfR-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3. In some embodiments, the monovalent mouse TfR-binding domain comprises a VH comprising HCDR1, HCDR2, and HCDR3 selected from Table 7a, and / or a VL comprising LCDR1, LCDR2, and LCDR3 selected from Table 7a. In some embodiments, the monovalent human TfR-binding domain comprises a VH and / or VL selected from Table 7a.

[0091] In some embodiments, provided herein is a protein comprising one monovalent mouse TfR-binding domain, wherein the mouse TfR-binding domain comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 71, HCDR2 comprises SEQ ID NO: 72, HCDR3 comprises SEQ ID NO: 73, LCDR1 comprises SEQ ID NO: 74, LCDR2 comprises SEQ ID NO: 75, and LCDR3 comprises SEQ ID NO: 76. In some embodiments, provided herein is a protein comprising one monovalent mouse TfR-binding domain, wherein the mouse TfR-binding domain comprises a VH comprising SEQ ID NO: 77 and a VL comprising SEQ ID NO: 78.

[0092] In some embodiments, a mouse TfR binding protein described herein has one heavy chain (HC) and one light chain, e.g., mTBP1 in Table 7b. In some embodiments, a human TfR binding protein has two heavy chains (HC1 and HC2) and two light chains (LC1 and LC2), e.g., mTBP2 in Table 7b.

[0093] In some embodiments, provided herein is a protein comprising one monovalent mouse TfR binding domain, wherein the mouse TfR binding domain comprises a heavy chain (HC) comprising SEQ ID NO: 79 and a light chain (LC) comprising SEQ ID NO: 80.

[0094] In some embodiments, the mouse TfR binding protein described herein is a heterodimeric antibody comprising a first arm comprising one monovalent mouse TfR binding domain and a second arm that is a null arm (e.g., an isotype arm) that does not bind to any known human target. In some embodiments, provided herein is a mouse TfR binding protein comprising two heavy chains, HC1 and HC2, and two light chains, LC1 and LC2, wherein HC1 comprises SEQ ID NO:79, LC1 comprises SEQ ID NO:80, HC2 comprises SEQ ID NO:51, and LC2 comprises SEQ ID NO:52.

[0095] Also provided herein are antibodies comprising a VH comprising an HCDR1, HCDR2, and HCDR3 selected from Table 7a, and / or a VL comprising an LCDR1, LCDR2, and LCDR3 selected from Table 7a. In some embodiments, such antibodies comprise a VH and / or VL selected from Table 7a.

[0096] Table 7a. Exemplary sequences of mouse TfR binding domains [Table 8]

[0097] Table 7b. Exemplary sequences of mouse TfR binding proteins [Table 9]

[0098] Conjugates containing human or mouse TfR binding proteins In another aspect, provided herein is a conjugate comprising a human or mouse TfR binding protein or antigen described herein and a therapeutic agent. In some embodiments, the therapeutic agent is selected from double-stranded RNA (e.g., siRNA, saRNA), an oligonucleotide (e.g., an antisense oligonucleotide), a peptide, a small molecule, a nanoparticle, a lipid nanoparticle, an exosome, an antibody or antigen-binding fragment thereof, or a combination thereof. In some embodiments, the therapeutic agent is double-stranded RNA (dsRNA). In some embodiments, the dsRNA comprises a sense strand and an antisense strand, and the antisense strand is complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B mRNA. In some embodiments, the dsRNA comprises a sense strand and an antisense strand, and the antisense strand is complementary to SNCA mRNA. In some embodiments, the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to MAPT mRNA.

[0099] In some embodiments, the ratio of therapeutic agent to protein is about 1 to 3. In some embodiments, the ratio of therapeutic agent to protein is about 1. In some embodiments, the ratio of therapeutic agent to protein is about 2. In some embodiments, the ratio of therapeutic agent to protein is about 3.

[0100] In some embodiments, the human TfR binding proteins described herein comprise one or more native cysteine ​​residues that can be used for conjugation. For example, in some embodiments, the human TfR binding proteins described herein comprise a native cysteine ​​at position 220 of the light chain and / or a native cysteine ​​at position 226 of the heavy chain that can be used for conjugation (all residues according to EU index numbering).

[0101] In some embodiments, the human TfR binding proteins described herein comprise one or more engineered cysteine ​​residues for conjugation. Approaches involving engineered cysteines as a means for conjugation are described in WO 2018 / 232088. In some embodiments, the human TfR binding proteins described herein comprise a heavy chain comprising one or more cysteines at the following residues: 124, 157, 162, 262, 373, 375, 378, 397, 415 (all residues according to EU index numbering). In some embodiments, the human TfR binding proteins described herein comprise a light chain (e.g., a kappa light chain) comprising one or more cysteines at the following residues: 156, 171, 191, 193, 202, 208 (all residues according to EU index numbering). In some embodiments, the human TfR binding proteins described herein comprise a heavy chain constant region comprising a cysteine ​​at residue 124 (according to EU index numbering). In some embodiments, a human TfR binding protein described herein comprises a light chain constant region comprising a cysteine ​​at residue 156 (according to EU index numbering). In some embodiments, a human TfR binding protein described herein comprises an immunoglobulin Fc region comprising a cysteine ​​at residue 378 (according to EU index numbering).

[0102] In some embodiments, the therapeutic agent is linked to the human or mouse TfR binding protein via a linker, which in some embodiments is a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker (the structures of these linkers are shown in Table 8).

[0103] Table 8. Exemplary linker structures [Table 10-1]

[0104] (Continued from Table 8) [Table 10-2]

[0105] The conjugates described herein can be made by a variety of procedures known to those skilled in the art, some of which are illustrated in the preparations and examples below (e.g., Example 3). Those skilled in the art will recognize that the specific synthetic steps for each of the routes described can be combined in different ways or combined with steps from different schemes to prepare the conjugates. The products of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. The reagents and starting materials are readily available to those skilled in the art.

[0106] In some embodiments, a TfR binding protein having a native or engineered cysteine ​​described herein can be first treated with a reducing agent, such as DTT, and then reoxidized with an oxidizing agent, such as DHAA. The resulting oxidized TfR binding protein is then incubated with a linker-functionalized therapeutic agent, such as a linker-dsRNA, to produce a conjugate.

[0107] Human TfR-binding protein-dsRNA conjugate In some embodiments, provided herein are conjugates of Formula (I): RLP, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human or mouse TfR binding domain; and L is a linker or, optionally, absent. In some embodiments, P is a human or mouse TfR binding protein described herein. In some embodiments, the ratio of R to P is about 1-3. In some embodiments, the ratio of R to P is about 1. In some embodiments, the ratio of R to P is about 2. In some embodiments, the ratio of R to P is about 3.

[0108] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, P is a protein comprising one monovalent human or mouse TfR binding domain, L is a linker or optionally absent, and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0109] In some embodiments, provided herein is a conjugate of Formula (I): RLP, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 21, HCDR3 comprises SEQ ID NO: 22, LCDR1 comprises SEQ ID NO: 23, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24, or (b) A conjugate wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18.

[0110] In some embodiments, provided herein is a conjugate of Formula (I): RLP, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (b) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 7, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (c) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 8, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (d) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; or (e) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 14, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; or (f) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18, or (g) A conjugate wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18.

[0111] In some embodiments, provided herein is a conjugate of Formula (I): RLP, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the following sequences: (a) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 27 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (b) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 29 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (c) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 30 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 31; or (d) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 32 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 33; or (e) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 34 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 35; or (f) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 36 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37; or (g) A conjugate, wherein the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 38 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37.

[0112] In some embodiments, provided herein is a conjugate of Formula (I): RLP, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the following sequences: (a) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; or (b) the VH comprises SEQ ID NO: 29 and the VL comprises SEQ ID NO: 28; or (c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; or (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 35; or (f) the VH comprises SEQ ID NO: 36 and the VL comprises SEQ ID NO: 37; or (g) A conjugate, wherein the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO: 37.

[0113] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3; and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 21, HCDR3 comprises SEQ ID NO: 22, LCDR1 comprises SEQ ID NO: 23, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24, or (b) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; n is 1 to 3, and is a conjugate.

[0114] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0115] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR binding domain; L is a linker or, optionally, absent; the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (b) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 7, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (c) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 8, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (d) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; or (e) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 14, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; or (f) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18, or (g) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; n is 1 to 3, and is a conjugate.

[0116] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0117] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, P is a protein comprising one monovalent human TfR binding domain, L is a linker or optionally absent, and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the following sequences: (a) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 27 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (b) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 29 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 28; or (c) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 30 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 31; or (d) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 32 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 33; or (e) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 34 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 35; or (f) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 36 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37; or (g) the VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 38 and the VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 37; n is 1 to 3, and is a conjugate.

[0118] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0119] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, P is a protein comprising one monovalent human TfR binding domain, L is a linker or optionally absent, and the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the following sequences: (a) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; or (b) the VH comprises SEQ ID NO: 29 and the VL comprises SEQ ID NO: 28; or (c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; or (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 35; or (f) the VH comprises SEQ ID NO: 36 and the VL comprises SEQ ID NO: 37; or (g) the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO: 37; n is 1 to 3, and is a conjugate.

[0120] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0121] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR-binding domain; L is a linker or, optionally, absent; the human TfR-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3; and VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0122] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein comprising one monovalent human TfR-binding domain; L is a linker or optionally absent; the human TfR-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises SEQ ID NO: 32 and VL comprises SEQ ID NO: 33; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0123] In some embodiments, protein (P) also binds to cynomolgus monkey TfR. In some embodiments, the human TfR binding domain of protein (P) is a Fab, scFv, Fv, or scFab. In some embodiments, the human TfR binding domain of protein (P) is a Fab. In some embodiments, the human TfR binding domain protein (P) further comprises a heavy chain constant region comprising a cysteine ​​at residue 124 (according to EU index numbering). In some embodiments, the human TfR binding domain protein (P) further comprises a light chain constant region comprising a cysteine ​​at residue 156 (according to EU index numbering).

[0124] In some embodiments, the protein (P) further comprises a half-life extender, e.g., an immunoglobulin Fc region or a VHH that binds to human serum albumin (HSA). In some forms, the protein (P) comprises an immunoglobulin Fc region, e.g., a modified human IgG4 Fc region or a modified human IgG1 Fc region. In some embodiments, the protein (P) comprises a modified human IgG4 Fc region comprising a proline at residue 228 and an alanine at residues 234 and 235 (all residues are numbered according to the EU index numbering system, also referred to as a hIgG4PAA Fc region). In some embodiments, the protein (P) comprises a modified human IgG1 Fc region comprising an alanine at residues 234, 235, and 329, a serine at position 265, and an aspartic acid at position 436 (all residues are numbered according to the EU index numbering system, also referred to as a hIgG1 effector null or hIgG1EN Fc region). In some embodiments, protein (P) comprises a modified human IgG1 or IgG4 Fc region, the Fc region comprising a first Fc CH3 domain comprising serine at position 349, methionine at position 366, tyrosine at position 370, and valine at position 409, and a second Fc CH3 domain comprising glycine at position 356, aspartic acid at position 357, glutamine at position 364, and alanine at position 407 (all residues are numbered according to EU index numbering). In some embodiments, protein (P) comprises a modified human IgG1 or IgG4 Fc region comprising a first Fc CH3 domain comprising leucine at residue 405 and a second Fc CH3 domain comprising arginine at residue 409 (all residues are numbered according to EU index numbering).

[0125] In some embodiments, the protein (P) comprises a VHH that binds to human HSA. In some embodiments, the VHH also binds to mouse, rat, and / or cynomolgus monkey albumin. In some embodiments, such a VHH comprises a CDR1 comprising SEQ ID NO: 39, a CDR2 comprising SEQ ID NO: 40, and a CDR3 comprising SEQ ID NO: 41. In some embodiments, such a VHH comprises SEQ ID NO: 42. In some embodiments, the VHH is linked to the TfR-binding domain via a peptide linker, e.g., (GGGGQ)4 (SEQ ID NO: 70).

[0126] In some embodiments, the protein (P) comprises one heavy chain (HC) and one light chain (LC), wherein the HC and LC comprise the following sequences: (a) HC comprises SEQ ID NO: 53 and LC comprises SEQ ID NO: 54; or (b) HC comprises SEQ ID NO: 55 and LC comprises SEQ ID NO: 54; or (c) HC comprises SEQ ID NO: 56 and LC comprises SEQ ID NO: 57; or (d) HC comprises SEQ ID NO: 58 and LC comprises SEQ ID NO: 59; or (e) HC comprises SEQ ID NO: 60 and LC comprises SEQ ID NO: 61; or (f) HC comprises SEQ ID NO: 62 and LC comprises SEQ ID NO: 63; or (g) HC comprises SEQ ID NO: 64 and LC comprises SEQ ID NO: 63.

[0127] In some embodiments, the protein (P) comprises one HC and one LC, wherein the HC comprises SEQ ID NO:65 and the LC comprises SEQ ID NO:59.

[0128] In some embodiments, the protein (P) comprises one HC and one LC, wherein the HC comprises SEQ ID NO:66 and the LC comprises SEQ ID NO:67.

[0129] In some embodiments, the protein (P) comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 68, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 69.

[0130] In some embodiments, the protein (P) comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 138, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 139.

[0131] In some embodiments, the protein (P) comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 166, LC1 comprises SEQ ID NO: 54, and HC2 comprises SEQ ID NO: 167.

[0132] In some embodiments, protein (P) is a heterodimeric antibody comprising a first arm comprising one monovalent human TfR binding domain and a second arm that is a null arm, e.g., an arm that does not bind to any known human target, e.g., an isotype arm of Table 5.

[0133] In some embodiments, the protein (P) comprises two heavy chains HC1 and HC2 and two light chains LC1 and LC2, wherein HC1, LC1, HC2, and LC2 comprise the following sequences: (a) HC1 comprises SEQ ID NO: 64, LC1 comprises SEQ ID NO: 63, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52; or (b) HC1 comprises SEQ ID NO: 55, LC1 comprises SEQ ID NO: 54, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52; or (c) HC1 comprises SEQ ID NO: 56, LC1 comprises SEQ ID NO: 57, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52, or (d) HC1 comprises SEQ ID NO:58, LC1 comprises SEQ ID NO:59, HC2 comprises SEQ ID NO:51, and LC2 comprises SEQ ID NO:52.

[0134] In some embodiments, the linker (L) is present and is selected from a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker (see Table 8). In some embodiments, the linker (L) is absent.

[0135] In some embodiments, the protein (P) is linked to the 3' end of the sense strand of the dsRNA. In some embodiments, the protein (P) is linked to the 5' end of the sense strand of the dsRNA. In some embodiments, the protein (P) is linked to an internal position of the sense strand of the dsRNA. In some embodiments, the protein (P) is linked to the 3' end of the antisense strand of the dsRNA. In some embodiments, the protein (P) is linked to an internal position of the antisense strand of the dsRNA.

[0136] In some embodiments, the dsRNA comprises an antisense strand complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B mRNA. In some embodiments, the dsRNA comprises an antisense strand complementary to SNCA mRNA. In some embodiments, the dsRNA comprises an antisense strand complementary to MAPT mRNA.

[0137] In some embodiments, the sense strand and antisense strand of the dsRNA are each 15 to 30 nucleotides in length, for example, 20 to 25 nucleotides in length. In some embodiments, the dsRNA has a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides. In some embodiments, the sense strand and antisense strand of the dsRNA may have an overhang (i.e., a 5' overhang or a 3' overhang) at either the 5' or 3' end. For example, the sense strand and antisense strand may have a 5' or 3' overhang of 1 to 5 nucleotides or 1 to 3 nucleotides. In some embodiments, the antisense strand includes a 3' overhang of 2 nucleotides.

[0138] Exemplary unmodified sense and antisense strand sequences of dsRNA targeting human SNCA mRNA are provided in Table 9a. Exemplary unmodified sense and antisense strand sequences of dsRNA targeting human MAPT mRNA are provided in Table 9b.

[0139] Table 9a. Unmodified nucleic acid sequences of dsRNA targeting human SNCA mRNA (SNCA siRNA) [Table 11]

[0140] Table 9b. Unmodified nucleic acid sequences of dsRNA targeting human MAPT mRNA (MAPT siRNA) [Table 12]

[0141] In some embodiments, the dsRNA targets SNCA mRNA. In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 81, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 82; (b) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 83, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 84; (c) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 85, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 86; (d) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 87, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 88; (e) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 89, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 90; (f) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 91, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 92; and (g) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 116, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 82; Optionally, one or more nucleotides in the sense strand and the antisense strand are independently modified nucleotides, and optionally, one or more internucleotide linkages in the sense strand and the antisense strand are modified internucleotide linkages.

[0142] In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 81, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 82; (b) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 83, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 84; (c) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 85, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 86; (d) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 87, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 88; (e) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 89, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 90; (f) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 91, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 92; and (g) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 116, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 82; Optionally, one or more nucleotides in the sense strand and the antisense strand are independently modified nucleotides, and optionally, one or more internucleotide linkages in the sense strand and the antisense strand are modified internucleotide linkages.

[0143] In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 81 and the antisense strand comprises SEQ ID NO: 82; (b) the sense strand comprises SEQ ID NO: 83 and the antisense strand comprises SEQ ID NO: 84; (c) the sense strand comprises SEQ ID NO: 85 and the antisense strand comprises SEQ ID NO: 86; (d) the sense strand comprises SEQ ID NO: 87 and the antisense strand comprises SEQ ID NO: 88; (e) the sense strand comprises SEQ ID NO: 89 and the antisense strand comprises SEQ ID NO: 90; (f) the sense strand comprises SEQ ID NO: 91 and the antisense strand comprises SEQ ID NO: 92; and (g) the sense strand comprises SEQ ID NO: 116 and the antisense strand comprises SEQ ID NO: 82; Optionally, one or more nucleotides in the sense strand and the antisense strand are independently modified nucleotides, and optionally, one or more internucleotide linkages in the sense strand and the antisense strand are modified internucleotide linkages.

[0144] In some embodiments, the dsRNA targets MAPT mRNA. In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 120, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 121; (b) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 122, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 123; and (c) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 124, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 125; Optionally, one or more nucleotides in the sense strand and the antisense strand are independently modified nucleotides, and optionally, one or more internucleotide linkages in the sense strand and the antisense strand are modified internucleotide linkages.

[0145] In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 120, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 121; (b) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 122, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 123; and (c) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 124, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 125; Optionally, one or more nucleotides in the sense strand and the antisense strand are independently modified nucleotides, and optionally, one or more internucleotide linkages in the sense strand and the antisense strand are modified internucleotide linkages.

[0146] In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 120 and the antisense strand comprises SEQ ID NO: 121; (b) the sense strand comprises SEQ ID NO: 122 and the antisense strand comprises SEQ ID NO: 123, and (c) the sense strand comprises SEQ ID NO: 124 and the antisense strand comprises SEQ ID NO: 125; Optionally, one or more nucleotides in the sense strand and the antisense strand are independently modified nucleotides, and optionally, one or more internucleotide linkages in the sense strand and the antisense strand are modified internucleotide linkages.

[0147] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, the sense strand comprises SEQ ID NO: 81 and the antisense strand comprises SEQ ID NO: 82; P is a protein comprising one monovalent human TfR-binding domain; L is a linker or is absent; the human TfR-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0148] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 81 and the antisense strand comprises SEQ ID NO: 82; P is a protein comprising one monovalent human TfR binding domain; L is a linker or is absent; the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0149] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 81 and the antisense strand comprises SEQ ID NO: 82; P is a protein comprising one monovalent human TfR binding domain; P comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 68, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 69; L is a linker or is absent; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0150] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 81 and the antisense strand comprises SEQ ID NO: 82; P is a protein comprising one monovalent human TfR binding domain; P comprises two heavy chains, HC1 and HC2, and one light chain, LC1, wherein HC1 comprises SEQ ID NO: 138, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 139; L is a linker or is absent; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0151] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, the sense strand comprises SEQ ID NO: 120 and the antisense strand comprises SEQ ID NO: 121; P is a protein comprising one monovalent human TfR-binding domain; L is a linker or is absent; the human TfR-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0152] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 120 and the antisense strand comprises SEQ ID NO: 121; P is a protein comprising one monovalent human TfR binding domain; L is a linker or is absent; the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0153] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 120 and the antisense strand comprises SEQ ID NO: 121; P is a protein comprising one monovalent human TfR binding domain; P comprises two heavy chains, HC1 and HC2, and one light chain, LC1, wherein HC1 comprises SEQ ID NO: 68, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 69; L is a linker or is absent; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0154] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 120 and the antisense strand comprises SEQ ID NO: 121; P is a protein comprising one monovalent human TfR binding domain; P comprises two heavy chains, HC1 and HC2, and one light chain, LC1, wherein HC1 comprises SEQ ID NO: 138, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 139; L is a linker or is absent; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0155] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, the sense strand comprises SEQ ID NO: 122 and the antisense strand comprises SEQ ID NO: 123; P is a protein comprising one monovalent human TfR-binding domain; L is a linker or is absent; the human TfR-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0156] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is a double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 122 and the antisense strand comprises SEQ ID NO: 123; P is a protein comprising one monovalent human TfR binding domain; L is a linker or is absent; the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0157] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is a double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 122 and the antisense strand comprises SEQ ID NO: 123; P is a protein comprising one monovalent human TfR binding domain; P comprises two heavy chains, HC1 and HC2, and one light chain, LC1, wherein HC1 comprises SEQ ID NO: 68, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 69; L is a linker or is absent; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0158] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is a double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 122 and the antisense strand comprises SEQ ID NO: 123; P is a protein comprising one monovalent human TfR binding domain; P comprises two heavy chains, HC1 and HC2, and one light chain, LC1, wherein HC1 comprises SEQ ID NO: 138, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 139; L is a linker or is absent; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0159] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, the sense strand comprises SEQ ID NO: 124 and the antisense strand comprises SEQ ID NO: 125; P is a protein comprising one monovalent human TfR-binding domain; L is a linker or is absent; the human TfR-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0160] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 124 and the antisense strand comprises SEQ ID NO: 125; P is a protein comprising one monovalent human TfR binding domain; L is a linker or is absent; the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0161] In some embodiments, provided herein are compounds of formula (II): (RL) n-P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 124 and the antisense strand comprises SEQ ID NO: 125; P is a protein comprising one monovalent human TfR binding domain; P comprises two heavy chains, HC1 and HC2, and one light chain, LC1, wherein HC1 comprises SEQ ID NO: 68, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 69; L is a linker or is absent; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0162] In some embodiments, provided herein are compounds of formula (II): (RL) n -P, wherein R is double-stranded RNA (dsRNA) having a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 124 and the antisense strand comprises SEQ ID NO: 125; P is a protein comprising one monovalent human TfR binding domain; P comprises two heavy chains, HC1 and HC2, and one light chain, LC1, wherein HC1 comprises SEQ ID NO: 138, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO: 139; L is a linker or is absent; and n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 8. In some embodiments, L is an SMCC linker in Table 8.

[0163] dsRNA can contain modifications. Modifications can be made to one or more nucleotides of the sense strand and / or antisense strand, or to the internucleotide linkage, which is the bond between two nucleotides in the sense strand or antisense strand. For example, some 2'-modifications of ribose or deoxyribose can increase RNA stability or DNA stability and half-life. Such 2'-modifications can be 2'-fluoro, 2'-O-methyl (i.e., 2'-methoxy), or 2'-O-alkyl.

[0164] In some embodiments, one or more nucleotides in the sense strand and / or antisense strand are independently modified nucleotides, meaning that the sense strand and the antisense strand can have different modified nucleotides. In some embodiments, each nucleotide in the sense strand is a modified nucleotide. In some embodiments, each nucleotide in the antisense strand is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-alkyl-modified nucleotide. In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a modified nucleotide, for example, a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-O-alkyl-modified nucleotide.

[0165] In some embodiments, the sense strand has four 2'-fluoro modified nucleotides, e.g., at positions 7, 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, the other nucleotides in the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has four 2'-fluoro modified nucleotides, e.g., at positions 2, 6, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides in the antisense strand are 2'-O-methyl modified nucleotides.

[0166] In some embodiments, the sense strand has three 2'-fluoro modified nucleotides, e.g., at positions 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, the other nucleotides in the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 3, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides in the antisense strand are 2'-O-methyl modified nucleotides.

[0167] In some embodiments, the 5' end of the antisense strand has a phosphate analog, for example, 5'-vinylphosphonate (5'-VP).

[0168] In some embodiments, the sense strand or antisense strand comprises an abasic portion or an inverted abasic portion, such as those shown in Table 10. In some embodiments, the sense strand comprises an abasic portion at the 10 position.

[0169] Table 10. Abasic or inverted abasic (iAb) moieties [Table 13] "5'" and "3'" indicate the 5' to 3' direction of the sequence.

[0170] In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate linkages. In some embodiments, the sense strand has four or five phosphorothioate linkages. In some embodiments, the antisense strand has four or five phosphorothioate linkages. In some embodiments, the sense strand and the antisense strand each have four or five phosphorothioate linkages. In some embodiments, the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.

[0171] Exemplary modified sense and antisense strand sequences of dsRNAs targeting human SNCA mRNA are provided in Table 11a. Exemplary modified sense and antisense strand sequences of dsRNAs targeting human MAPT mRNA are provided in Table 11b.

[0172] In some embodiments, the dsRNA comprises a sense strand comprising a sequence having one, two, or three differences from the sense strand sequence in Table 9a or Table 11a. In some embodiments, the dsRNA comprises an antisense strand comprising a sequence having one, two, or three differences from the antisense strand sequence in Table 9a or Table 11a.

[0173] In some embodiments, the dsRNA comprises a sense strand that comprises a sequence that has one, two, or three differences from the sense strand sequence in Table 9b or Table 11b. In some embodiments, the dsRNA comprises an antisense strand that comprises a sequence that has one, two, or three differences from the antisense strand sequence in Table 9b or Table 11b.

[0174] Table 11a: Modified nucleic acid sequences of dsRNA targeting human SNCA mRNA (SNCA siRNA) [Table 14-1]

[0175] (Continued from Table 11a) [Table 14-2] Abbreviations - "m" indicates 2'-OMe, "f" indicates 2'-fluoro, * " indicates phosphorothioate linkage, "VP" indicates 5'-vinylphosphonate, "iAb" indicates the inverted abasic moiety in Table 10, "S" indicates the sense strand, and "AS" indicates the antisense strand.

[0176] Table 11b: Modified nucleic acid sequences of dsRNA targeting human MAPT mRNA (MAPT siRNA) [Table 15] Abbreviations - "m" indicates 2'-OMe, "f" indicates 2'-fluoro, * " indicates phosphorothioate linkage, "VP" indicates 5'-vinylphosphonate, "S" denotes the sense strand, and "AS" denotes the antisense strand.

[0177] In some embodiments, the dsRNA targets SNCA mRNA. In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 93 or 140 and the antisense strand comprises SEQ ID NO: 94; (b) the sense strand comprises SEQ ID NO: 95 or 141 and the antisense strand comprises SEQ ID NO: 96; (c) the sense strand comprises SEQ ID NO: 95 or 141 and the antisense strand comprises SEQ ID NO: 97; (d) the sense strand comprises SEQ ID NO: 95 or 141 and the antisense strand comprises SEQ ID NO: 98; (e) the sense strand comprises SEQ ID NO: 99 or 142 and the antisense strand comprises SEQ ID NO: 94; (f) the sense strand comprises SEQ ID NO: 100 or 143 and the antisense strand comprises SEQ ID NO: 101; (g) the sense strand comprises SEQ ID NO: 102 or 144 and the antisense strand comprises SEQ ID NO: 103; (h) the sense strand comprises SEQ ID NO: 104 or 145 and the antisense strand comprises SEQ ID NO: 105; (i) the sense strand comprises SEQ ID NO: 106 or 146 and the antisense strand comprises SEQ ID NO: 107; (j) the sense strand comprises SEQ ID NO: 108 or 147 and the antisense strand comprises SEQ ID NO: 107; (k) the sense strand comprises SEQ ID NO: 117 or 148 and the antisense strand comprises SEQ ID NO: 97; and (l) the sense strand comprises SEQ ID NO: 118 or 149 and the antisense strand comprises SEQ ID NO: 97.

[0178] In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand consists of SEQ ID NO: 93 or 140 and the antisense strand consists of SEQ ID NO: 94; (b) the sense strand consists of SEQ ID NO: 95 or 141 and the antisense strand consists of SEQ ID NO: 96; (c) the sense strand consists of SEQ ID NO: 95 or 141 and the antisense strand consists of SEQ ID NO: 97; (d) the sense strand consists of SEQ ID NO: 95 or 141 and the antisense strand consists of SEQ ID NO: 98; (e) the sense strand consists of SEQ ID NO: 99 or 142 and the antisense strand consists of SEQ ID NO: 94; (f) the sense strand consists of SEQ ID NO: 100 or 143 and the antisense strand consists of SEQ ID NO: 101; (f) the sense strand consists of SEQ ID NO: 102 or 144 and the antisense strand consists of SEQ ID NO: 103; (h) the sense strand consists of SEQ ID NO: 104 or 145 and the antisense strand consists of SEQ ID NO: 105 (i) the sense strand consists of SEQ ID NO: 106 or 146, and the antisense strand consists of SEQ ID NO: 107; (j) the sense strand consists of SEQ ID NO: 108 or 147, and the antisense strand consists of SEQ ID NO: 107 (k) the sense strand consists of SEQ ID NO: 117 or 148 and the antisense strand consists of SEQ ID NO: 97; and (l) the sense strand consists of SEQ ID NO: 118 or 149, and the antisense strand consists of SEQ ID NO: 97.

[0179] In some embodiments, the dsRNA targets MAPT mRNA. In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 126 or 150 and the antisense strand comprises SEQ ID NO: 127; (b) the sense strand comprises SEQ ID NO: 128 or 151 and the antisense strand comprises SEQ ID NO: 129; (c) the sense strand comprises SEQ ID NO: 130 or 152 and the antisense strand comprises SEQ ID NO: 131; (d) the sense strand comprises SEQ ID NO: 132 or 153 and the antisense strand comprises SEQ ID NO: 133; (e) the sense strand comprises SEQ ID NO: 134 or 154 and the antisense strand comprises SEQ ID NO: 135; and (f) the sense strand comprises SEQ ID NO: 136 or 155 and the antisense strand comprises SEQ ID NO: 137.

[0180] In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand consists of SEQ ID NO: 126 or 150 and the antisense strand consists of SEQ ID NO: 127; (b) the sense strand consists of SEQ ID NO: 128 or 151 and the antisense strand consists of SEQ ID NO: 129; (c) the sense strand consists of SEQ ID NO: 130 or 152 and the antisense strand consists of SEQ ID NO: 131; (d) the sense strand consists of SEQ ID NO: 132 or 153, and the antisense strand consists of SEQ ID NO: 133; (e) the sense strand consists of SEQ ID NO: 134 or 154 and the antisense strand consists of SEQ ID NO: 135; and (f) the sense strand consists of SEQ ID NO: 136 or 155, and the antisense strand consists of SEQ ID NO: 137.

[0181] The sense and antisense strands of dsRNA can be synthesized using any nucleic acid polymerization method known in the art, such as solid-phase synthesis using phosphoramidite chemistry (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphotriester chemistry, or enzymatic synthesis. Automated commercially available synthesizers, such as MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems, can be used. Phosphorothioate linkages can be introduced using sulfurizing reagents such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazoline-3-thione). The use of similar techniques and commercially available modified amidites and Controlled-Pore Glass (CPG) products to synthesize modified and conjugated oligonucleotides is well known.

[0182] Purification methods can be used to remove unwanted impurities from final oligonucleotide products.The purification techniques commonly used for single-stranded oligonucleotides include reverse-phase ion pair high performance liquid chromatography (RP-IP-HPLC), capillary gel electrophoresis (CGE), anion exchange HPLC (AX-HPLC) and size exclusion chromatography (SEC).After purification, oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm.Then, sense strand and antisense strand can be annealed to form dsRNA.

[0183] Pharmaceutical Composition In another aspect, provided herein is a pharmaceutical composition comprising any of the human TfR binding proteins or conjugates described herein and a pharmaceutically acceptable carrier. Such pharmaceutical compositions may also include one or more pharmaceutically acceptable excipients, diluents, or carriers. Pharmaceutical compositions may be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A. Loyd et al., Academic Press).

[0184] Methods of Treatment and Therapeutic Use In another aspect, provided herein is a method of treating a CNS disease, e.g., a neurodegenerative disease, in a patient in need thereof, comprising administering to the patient an effective amount of a human TfR binding protein, or conjugate, or pharmaceutical composition described herein.

[0185] In a further aspect, provided herein is a method of treating a neurodegenerative synucleinopathy in a patient in need thereof, comprising administering to the patient an effective amount of a human TfR binding protein, conjugate, or pharmaceutical composition described herein, such as a TBP-SNCA siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-SNCA siRNA conjugate. Exemplary neurodegenerative synucleinopathies include, but are not limited to, Parkinson's disease, multiple system atrophy, dementia with Lewy bodies or dementia with Lewy bodies, pure autonomic failure, Alzheimer's disease, dysphagia with Lewy bodies, and concomitant Lewy body disease. In some embodiments, the neurodegenerative synucleinopathy is selected from Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies. The human TfR binding protein, conjugate, or pharmaceutical composition may be administered intravenously or subcutaneously to the patient.

[0186] In a further aspect, provided herein are methods of treating a tauopathy in a patient in need thereof, such methods comprising administering to the patient an effective amount of a human TfR binding protein, or conjugate, or pharmaceutical composition described herein, e.g., a TBP-MAPT siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-MAPT siRNA conjugate. Exemplary tauopathies include, but are not limited to, Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PPA- S), primary logopenic progressive aphasia (PPA-L), multisystem tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, and chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (N P-C), non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle-type senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT). The human TfR binding protein or conjugate or pharmaceutical composition can be administered intravenously or subcutaneously to a patient.

[0187] Human TfR binding domain or conjugate dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0188] Dosage values ​​may vary depending on the type and severity of the condition to be alleviated. It will be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[0189] In another aspect, provided herein are human TfR binding proteins or conjugates described herein, or pharmaceutical compositions comprising such human TfR binding proteins or conjugates, for use in therapy. Also provided herein are human TfR binding proteins or conjugates described herein, or pharmaceutical compositions comprising such human TfR binding proteins or conjugates (e.g., TBP-SNCA siRNA conjugates described herein, or pharmaceutical compositions comprising such TBP-SNCA siRNA conjugates), for use in treating neurodegenerative synucleinopathies, such as Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies.

[0190] Also provided herein are tauopathies, such as Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PPA-S), primary logopenic progressive aphasia (PPA-L), primary progressive aphasia (PPA-R), primary progressive aphasia (PPA-S), primary progressive aphasia (PPA-R ... Multisystem tauopathy with dementia in old age (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, and calcification. Diffuse neurofibrillary tangles with neurofibromatosis, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurological disorders A human TfR binding protein or conjugate described herein, or a pharmaceutical composition comprising such a human TfR binding protein or conjugate (e.g., a TBP-MAPT siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-MAPT siRNA conjugate), for use in treating fibrillary tangle senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

[0191] In another aspect, provided herein is the use of a human TfR binding protein or conjugate described herein in the manufacture of a medicament for treating a CNS disease, e.g., a neurodegenerative disease. In some embodiments, the neurodegenerative disease is a neurodegenerative synucleinopathy, e.g., Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies. In some embodiments, the neurodegenerative disease is a tauopathy, e.g., Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PP) A-S), primary logopenic progressive aphasia (PPA-L), multisystem tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic Corticobasal degeneration (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C ( NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle-type senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or glioglobular tauopathy (GGT).

[0192] definition As used herein, the terms "a," "an," "the," and similar terms as used in the context of this disclosure (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context.

[0193] As used herein, the term "alkyl" means a saturated straight- or branched-chain monovalent hydrocarbon radical containing the indicated number of carbon atoms. For example, "C1-C 20 Alkyl (C1-C 20 "Alkyl" means a radical having from 1 to 20 carbon atoms in a linear or branched arrangement.

[0194] As used herein, the term "antibody" refers to a molecule that binds to an antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, heterodimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0195] Immunoglobulin G (IgG) antibodies are composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of approximately 100 to 125 amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0196] The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). The CDRs are exposed on the surface of the protein and are critical regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs can be performed using the methods of Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics This may be done according to well-known schemes, including those described in the Imgt database, available at www.imgt.org (see Lefranc et al., Nucleic Acids Res. 1999;27:209-212).

[0197] Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments, as used herein, that comprise at least a portion of an antibody that retains the ability to specifically interact with an antigen or an epitope of an antigen, such as Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, scFab, disulfide-linked Fv (sdFv), Fd fragment, etc.

[0198] As used herein, the term "antigen-binding domain" refers to a portion of an antibody or antibody fragment that binds to an antigen or an epitope of an antigen. For example, a "TfR-binding domain" refers to a portion of an antibody or antibody fragment that binds to TfR or an epitope of TfR.

[0199] As used herein, the term "heterodimeric antibody" refers to an antibody that comprises two different antigen-binding domains.

[0200] As used herein, "antisense strand" refers to a single-stranded oligonucleotide that is complementary to a region of a target sequence. Similarly, as used herein, "sense strand" refers to a single-stranded oligonucleotide that is complementary to a region of the antisense strand.

[0201] As used herein, unless otherwise specified, the terms "bind" and "binds" refer to the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, bringing the two proteins or molecules into proximity as determined by common methods known in the art.

[0202] As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand, e.g., a hairpin) that allows the two nucleotides to base pair with each other. For example, purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid may base pair with each other by forming hydrogen bonds. Complementary nucleotides may base pair in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. Similarly, two nucleic acids may have regions of multiple nucleotides that are complementary to each other and form a region of complementarity, as described herein.

[0203] As used herein, "duplex," with respect to a nucleic acid or oligonucleotide, means the structure formed through complementary base pairing of two antiparallel sequences of nucleotides, whether formed by two separate nucleic acid strands or by a single folded strand (e.g., via a hairpin).

[0204] An "effective amount" refers to the amount (duration and means of administration) necessary to achieve the desired therapeutic result. An effective amount of a protein or conjugate may vary depending on factors such as the individual's condition, age, sex, and weight, and the ability of the protein or conjugate to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the protein or conjugate are outweighed by the therapeutically beneficial effects.

[0205] As referred to herein, the term "epitope" refers to the amino acid residues of an antigen that are bound by an antibody. An epitope can be a linear epitope, a conformational epitope, or a hybrid epitope. The term "epitope" may be used in reference to a structural epitope, which, according to some embodiments, may be used to describe the region of an antigen that is covered by an antibody or antigen-binding protein. In some embodiments, a structural epitope may describe amino acid residues of an antigen or binding protein that are within a particular proximity (e.g., within a particular number of angstroms) of amino acid residues of an antibody. The term "epitope" may also be used in reference to a functional epitope, which, according to some embodiments, may be used to describe amino acid residues of an antibody or antigen-binding protein that interact with amino acid residues of an antibody in a manner that contributes to the binding energy between the antigen and the antibody or antigen-binding protein.

[0206] Epitopes can be determined according to different experimental techniques, also known as "epitope mapping techniques." It is understood that the determination of an epitope may vary based on the different epitope mapping techniques used, and may also vary depending on the different experimental conditions used, for example, due to conformational changes or cleavage of the antigen induced by certain experimental conditions. Epitope mapping techniques are known in the art, including, but not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species swap mutagenesis, alanine scanning mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd ed. 2018).

[0207] As used herein, the term "Fc region" refers to a polypeptide comprising the CH2 and CH3 domains of the constant region of an immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4. Optionally, the Fc region may comprise a portion of or the entire hinge region of an immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is a human IgG Fc region, e.g., a human IgG1 Fc region, a human IgG2 Fc region, a human IgG3 Fc region, or a human IgG4 Fc region. In some embodiments, the Fc region is a modified IgG Fc region that has reduced or eliminated effector function compared to the corresponding wild-type IgG Fc region. The numbering of residues in the Fc region is based on the EU index, as described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: US Department of Health and Human Services, Public Health Service, National Institutes of Health, 1991). The boundaries of the Fc region of an immunoglobulin heavy chain might vary, and the human IgG heavy chain Fc region is usually defined as stretching from the N-terminus of the CH2 domain (e.g., amino acid residue 231 according to the EU index numbering) to the C-terminus of the CH3 domain (or the C-terminus of the immunoglobulin).

[0208] The term "knockdown" or "expression knockdown" refers to a reduction in the mRNA or protein expression of a gene following treatment with a reagent.

[0209] As used herein, "modified internucleotide linkage" refers to an internucleotide linkage that has one or more chemical modifications when compared to a reference internucleotide linkage having a phosphodiester bond. The modified internucleotide linkage may be a non-naturally occurring linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage.

[0210] As used herein, a "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide selected from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymidine deoxyribonucleotides. A modified nucleotide may have one or more chemical modifications, for example, in its sugar, nucleobase, and / or phosphate group. Additionally or alternatively, a modified nucleotide may have one or more chemical moieties conjugated to the corresponding reference nucleotide. In some embodiments, a modified nucleotide is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-alkyl-modified nucleotide. In some embodiments, a modified nucleotide has a phosphate analog, e.g., 5'-vinyl phosphonate. In some embodiments, a modified nucleotide has an abasic moiety or an inverted abasic moiety, e.g., those shown in Table 10.

[0211] As used herein, the term "neurodegenerative synucleinopathy" refers to a neurodegenerative disorder characterized by fibrillar aggregates of alpha-synuclein protein in the cytoplasm of selective populations of neurons and glia in the central and / or peripheral nervous system.

[0212] As used herein, "nucleotide" refers to an organic compound having a nucleoside (a nucleic acid base such as adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar such as ribose or 2'-deoxyribose) linked to a phosphate group. A "nucleotide" can function as a monomer unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0213] As used herein, a "null arm" means an antibody arm that does not bind to any known human target.

[0214] As used herein, "oligonucleotide" means a polymer of linked nucleotides, each of which may be modified or unmodified. Oligonucleotides are typically less than about 100 nucleotides in length.

[0215] As used herein, "overhang" refers to an unpaired nucleotide(s) that protrudes from the duplex structure of a double-stranded oligonucleotide. An overhang may include one or more unpaired nucleotides extending from the duplex region at the 5'-end or 3'-end of a double-stranded oligonucleotide. An overhang may be a 3'-overhang or a 5'-overhang on the antisense strand or the sense strand of a double-stranded oligonucleotide.

[0216] As used herein, the term "patient" refers to a human patient.

[0217] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. The 5' phosphate analog may include a phosphatase-resistant linkage. Examples of phosphate analogs include 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). In some embodiments, the phosphate analog is 5'-VP.

[0218] The term "% sequence identity" or "percent sequence identity" with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical to the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software programs, including those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp. 30, Section 7.7.18, Table 7.7.1), BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Clustal W2.0, or Clustal X2.0 software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, although the nucleic acid sequence fragment within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleotide, nucleoside, or nucleic acid base is present in both sequences to determine the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity. The output is the percent identity of the subject sequence to the query sequence.

[0219] As used herein, the term "polypeptide" or "protein" refers to a polymer of amino acid residues. This term applies to polymers containing naturally occurring amino acids as well as to polymers containing one or more non-naturally occurring amino acids.

[0220] As used herein, a "strand" refers to a single, contiguous sequence of nucleotides linked together by internucleotide linkages (e.g., phosphodiester or phosphorothioate linkages). A strand may have two free ends (e.g., a 5' end and a 3' end).

[0221] As used herein, "SNCA" refers to alpha-synuclein (SNCA) mRNA, protein, or polypeptide. The nucleic acid sequence of the human SNCA mRNA transcript can be found in NM_000345.4: 1 GGCGACGACC AGAAGGGGCC CAAGAGAGGG GGCGAGCGAC CGAGCGCCGC GACGCGGAAG 61 TGAGGTGCGT GCGGGCTGCA GCGCAGACCC CGGCCCGGCC CCTCCGAGAG CGTCCTGGGC 121 GCTCCCTCAC GCCTTGCCTT CAAGCCTTCT GCCTTTCCAC CCTCGTGAGC GGAGAACTGG 181 GAGTGGCCAT TCGACGACAG TGTGGTGTAA AGGAATTCAT TAGCCATGGA TGTATTCATG 241 AAAGGACTTT CAAAGGCCAA GGAGGGAGTT GTGGCTGCTG CTGAGAAAAC CAAACAGGGT 301 GTGGCAGAAG CAGCAGGAAA GACAAAAGAG GGTGTTCTCT ATGTAGGCTC CAAAACCAAG 361 GAGGGAGTGG TGCATGGTGT GGCAACAGTG GCTGAGAAGA CCAAAGAGCA AGTGACAAAT 421 GTTGGAGGAG CAGTGGTGAC GGGTGTGACA GCAGTAGCCC AGAAGACAGT GGAGGGAGCA 481 GGGAGCATTG CAGCAGCCAC TGGCTTTGTC AAAAAGGACC AGTTGGGCAA GAATGAAGAA 541 GGAGCCCCAC AGGAAGGAAT TCTGGAAGAT ATGCCTGTGG ATCCTGACAA TGAGGCTTAT 601 GAAATGCCTT CTGAGGAAGG GTATCAAGAC TACGAACCTG AAGCCTAAGA AATATCTTTG 661 CTCCCAGTTT CTTGAGATCT GCTGACAGAT GTTCCATCCT GTACAAGTGC TCAGTTCCAA 721 TGTGCCCAGT CATGACATTT CTCAAAGTTT TTACAGTGTA TCTCGAAGTC TTCCATCAGC 781 AGTGATTGAA GTATCTGTAC CTGCCCCCAC TCAGCATTTC GGTGCTTCCC TTTCACTGAA 841 GTGAATACAT GGTAGCAGGG TCTTTGTGTG CTGTGGATTT TGTGGCTTCA ATCTACGATG 901 TTAAAACAAA TTAAAAACAC CTAAGTGACT ACCACTTATT TCTAAATCCT CACTATTTTT 961 TTGTTGCTGT TGTTCAGAAG TTGTTAGTGA TTTGCTATCA TATATTATAA GATTTTTAGG 1021 TGTCTTTTAA TGATACTGTC TAAGAATAAT GACGTATTGT GAAATTTGTT AATATATA 1081 ATACTTAAAA ATATGTGAGC ATGAAACTAT GCACCTATAA ATACTAAATA TGAAATTTTA 1141 CCATTTTGCG ATGTGTTTTA TTCACTTGTG TTTGTATATA AATGGTGAGA ATTAAAATAA 1201 AACGTTATCT CATTGCAAAA ATATTTTATT TTTATCCCAT CTCACTTTAA TAATAAAAAT 1261 ASSISTANCE ATTAGCTTATA CHANGE ATTAGCTTATA 1321 ATTACKTT FREQUENCY FREQUENCY AAAATGAC ATTACK 1381 CACTCGGAAT TCCCTGAAGC AACACTGCCA GAAGTGTGTT TTGGTATGCA CTGGTTCCTT 1441 AAGTGGCTGT FAMILY TGAAAGTGGG GTGTTGAGA CCCCAACTAC TATTGTAGAG 1501 TGGTCTATTT CTCCCTTCAA TCCTGTCAAT GTTTGCTTTA CGTATTTTGG GGAACTGTTG 1561 TTTGATGTGT ATGTGTTTAT AATTGTTATA CATTTTAT TGAGCCTTTT ATTAACATAT 1621 ATTGTTATTT TTGTCTCGAA ATAATTTTTT AGTTAAAATC TATTTTGTCT GATATTGGTG 1681 TGAATGCTGT ACCTTTCTGA CATAAAATAA TATTCGACCA TGAATAAAAAAAAAAAAAAAA 1741 GTGGGTTCCC GGGAACTAAG CAGTGTAGAA GATGATTTTG ACTACACCCT CCTTAGAGAG 1801 CCATAAGACA CATTAGCACA TATTAGCACA TTCAAGGCTC TGAGAGAATG TGGTTAACTT 1861 TGTTTAACTC AGCATTCCTC ACTTTTTTTT TTTAATCATC AGAAATTCTC TCTCTCTCTC 1921 TCTCTTTTTC TCTCGCTCTC TTTTTTTTTT TTTTTTTACA GGAAATGCCT TTAAACATCG 1981 TTGGAACTAC CAGAGTCACC TTAAAGGAGA TCAATTCTCT AGACTGATAA AAATTTCATG 2041 GCCTCCTTTA AATGTTGCCA AATATATGAA TTCTAGGATT TTTCCTTAGG AAAGGTTTTT 2101 CTCTTTCAGG GAAGATCTAT TAACTCCCCA TGGGTGCTGA AAATAAACTT GATGGTGAAA 2161 AACTCTGTAT AAATTAATTT AAAAATTATT TGGTTTCTCT TTTTAATTAT TCTGGGGCAT 2221 AGTCATTTCT AAAAGTCACT AGTAGAAAGT ATAATTTCAA GACAGAATAT TCTAGACATG 2281 CTAGCAGTTT ATATGTATTC ATGAGTAATG TGATATATAT TGGGCGCTGG TGAGGAAGGA 2341 AGGAGGAATG AGTGACTATA AGGATGGTTA CCATAGAAAC TTCCTTTTTT ACCTAATTGA 2401 AGAGAGACTA CTACAGAGTG CTAAGCTGCA TGTGTCATCT TACACTAGAG AGAAATGGTA 2461 AGTTTCTTGT TTTATTTAAG TTATGTTTAA GCAAGGAAAG GATTTGTTAT TGAACAGTAT 2521 ATTTCAGGAA GGTTAGAAAG TGGCGGTTAG GATATATTTT AAATCTACCT AAAGCAGCAT 2581 ATTTTAAAAAA TTTAAAAGTA TTGGTATTAA ATTAAGAAAT AGAGGACAGA ACTAGACTGA 2641 TAGCAGTGAC CTAGAACAAT TTGAGATTAG GAAAGTTGTG ACCATGAATT TAAGGATTTA 2701 TGTGGATACA AATTCTCCTT TAAAGTGTTT CTTCCCTTAA TATTTATCTG ACGGTAATTT 2761 TTGAGCAGTG AATTACTTTA TATATCTTAA TAGTTTATTT GGGACCAAAC ACTTAAACAA 2821 AAAGTTCTTT AAGTCATATA AGCCTTTTCA GGAAGCTTGT CTCATATTCA CTCCCGAGAC 2881 ATTCACCTGC CAAGTGGCCT GAGGATCAAT CCAGTCCTAG GTTTATTTTG CAGACTTACA 2941 TTCTCCCAAG TTATTCAGCC TCATATGACT CCACGGTCGG CTTTACCAAA ACAGTTCAGA 3001 GTGCACTTTG GCACACAATT GGGAACAGAA CAATCTAATG TGTGGTTTGG TATTCCAAGT 3061 GGGGTCTTTT TCAGAATCTC TGCACTAGTG TGAGATGCAA ACATGTTTCC TCATCTTTCT 3121 GGCTTATCCA GTATGTAGCT ATTTGTGACA TAATAAATAT ATACATATAT GAAAATA (SEQ ID NO: 109).

[0222] The amino acid sequence of the human SNCA protein can be found in NP_000336.1: 1 MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK 61 EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP 121 DNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 110).

[0223] The nucleic acid sequence of the mouse SNCA mRNA transcript can be found at NM_001042451.2, and the amino acid sequence of the mouse SNCA protein can be found at NP_001035916.1. The nucleic acid sequence of the rat SNCA mRNA transcript can be found at NM_019169.3, and the amino acid sequence of the rat SNCA protein can be found at NP_062042.1. The nucleic acid sequence of the monkey SNCA mRNA transcript can be found at XM_005555422.2, and the amino acid sequence of the monkey SNCA protein can be found at XP_005555479.1.

[0224] As used herein, "MAPT" refers to the human MAPT mRNA transcript, which encodes the microtubule-associated protein tau. The nucleotide sequences of human MAPT transcript variants and the amino acid sequences of human tau protein isoforms can be found below. i. MAPT transcript variant 1 → tau protein isoform 1: NM_016835.5 (nucleotide sequence) → NP_058519.3 (amino acid sequence), ii. MAPT transcript variant 2 → tau protein isoform 2: NM_005910.6 (nucleotide sequence) → NP_005901.2 (amino acid sequence), iii. MAPT transcript variant 3 → tau protein isoform 3: NM_016834.5 (nucleotide sequence) → NP_058518.1 (amino acid sequence), iv. MAPT transcript variant 4 → tau protein isoform 4: NM_016841.5 (nucleotide sequence) → NP_058525.1 (amino acid sequence), v. MAPT transcript variant 5 → tau protein isoform 5: NM_001123067.4 (nucleotide sequence) → NP_001116539.1 (amino acid sequence), vi. MAPT transcript variant 6 → tau protein isoform 6: NM_001123066.4 (nucleotide sequence) → NP_001116538.2 (amino acid sequence), vii. MAPT transcript variant 7 → tau protein isoform 7: NM_001203251.2 (nucleotide sequence) → NP_001190180.1 (amino acid sequence); viii. MAPT transcript variant 8 → tau protein isoform 8: NM_001203252.2 (nucleotide sequence) → NP_001190181.1 (amino acid sequence); ix. MAPT transcript variant 9 → tau protein isoform 9: NM_001377265.1 (nucleotide sequence) → NP_001364194.1 (amino acid sequence), x. MAPT transcript variant 10 → tau protein isoform 10: NM_001377266.1 (nucleotide sequence) → NP_001364195.1 (amino acid sequence), xi. MAPT transcript variant 11 → tau protein isoform 11: NM_001377267.1 (nucleotide sequence) → NP_001364196.1 (amino acid sequence); xii. MAPT transcript variant 12 → tau protein isoform 4: NM_001377268.1 (nucleotide sequence) → NP_001364197.1 (amino acid sequence).

[0225] The nucleotide sequence of human MAPT transcript variant 6 (encoding 2N4R tau) can be found in NM_001123066.4: 1 GCAGTCACCG CCACCCACCA GCTCCGGCAC CAACAGCAGC GCCGCTGCCA CCGCCCACCT 61 TCTGCCGCCG CCACCACAGC CACCTTCTCC TCCTCCGCTG TCCTCTCCCG TCCTCGCCTC 121 TGTCGACTAT CAGGTGAACT TTGAACCAGG ATGGCTGAGC CCCGCCAGGA GTTCGAAGTG 181 ATGGAAGATC ACGCTGGGAC GTACGGGTTG GGGGACAGGA AAGATCAGGG GGGCTACACC 241 ATGCACCAAG ACCAAGAGGG TGACACGGAC GCTGGCCTGA AAGAATCTCC CCTGCAGACC 301 CCCACTGAGG ACGGATCTGA GGAACCGGGC TCTGAAACCT CTGATGCTAA GAGCACTCCA 361 ACAGCGGAAG ATGTGACAGC ACCCTTAGTG GATGAGGGAG CTCCCGGCAA GCAGGCTGCC 421 GCGCAGCCCC ACACGGAGAT CCCAGAAGGA ACCACAGCTG AAGAAGCAGG CATTGGAGAC 481 ACCCCCAGCC TGGAAGACGA AGCTGCTGGT CACGTGACCC AAGAGCCTGA AAGTGGTAAG 541 GTGGTCCAGG AAGGCTTCCT CCGAGAGCCA GGCCCCCCAG GTCTGAGCCA CCAGCTCATG 601 TCCGGCATGC CTGGGGCTCC CCTCCTGCCT GAGGGCCCCA GAGAGGCCAC ACGCCAACCT 661 TCGGGGACAG GACCTGAGGA CACAGAGGGC GGCCGCCACG CCCCTGAGCT GCTCAAGCAC 721 CAGCTTCTAG GAGACCTGCA CCAGGAGGGG CCGCCGCTGA AGGGGGCAGG GGGCAAAGAG 781 AGGCCGGGGA GCAAGGAGGA GGTGGATGAA GACCGCGACG TCGATGAGTC CTCCCCCCAA 841 GACTCCCCTC CCTCCAAGGC CTCCCCAGCC CAAGATGGGC GGCCTCCCCA GACAGCCGCC 901 AGAGAAGCCA CCAGCATCCC AGGCTTCCCA GCGGAGGGTG CCATCCCCCT CCCTGTGGAT 961 TTCCTCTCCA AAGTTTCCAC AGAGATCCCA GCCTCAGAGC CCGACGGGCC CAGTGTAGGG 1021 CGGGCCAAAG GGCAGGATGC CCCCCTGGAG TTCACGTTTC ACGTGGAAAT CACACCCAAC 1081 GTGCAGAAGG AGCAGGCGCA CTCGGAGGAG CATTTGGGAA GGGCTGCATT TCCAGGGGCC 1141 CCTGGAGAGG GGCCAGAGGC CCGGGGCCCC TCTTTGGGAG AGGACACAAA AGAGGCTGAC 1201 CTTCCAGAGC CCTCTGAAAA GCAGCCTGCT GCTGCTCCGC GGGGGAAGCC CGTCAGCCGG 1261 GTCCCTCAAC TCAAAGCTCG CATGGTCAGT AAAAGCAAAG ACGGGACTGG AAGCGATGAC 1321 AAAAAAGCCA AGACATCCAC ACGTTCCTCT GCTAAAACCT TGAAAAATAG GCCTTGCCTT 1381 AGCCCCAAAC ACCCCACTCC TGGTAGCTCA GACCCTCTGA TCCAACCCTC CAGCCCTGCT 1441 GTGTGCCCAG AGCCACCTTC CTCTCCTAAA TACGTCTCTT CTGTCACTTC CCGAACTGGC 1501 AGTTCTGGAG CAAAGGAGAT GAAACTCAAG GGGGCTGATG GTAAAACGAA GATCGCCACA 1561 CCGCGGGGAG CAGCCCCTCC AGGCCAGAAG GGCCAGGCCA ACGCCACCAG GATTCCAGCA 1621 AAAACCCCGC CCGCTCCAAA GACACCACCC AGCTCTGCGA CTAAGCAAGT CCAGAGAAGA 1681 CCACCCCCTG CAGGGCCCAG ATCTGAGAGA GGTGAACCTC CAAAATCAGG GGATCGCAGC 1741 GGCTACAGCA GCCCCGGCTC CCCAGGCACT CCCGGCAGCC GCTCCCGCAC CCCGTCCCTT 1801 CCAACCCCAC CCACCCGGGA GCCCAAGAAG GTGGCAGTGG TCCGTACTCC ACCCAAGTCG 1861 CCGTCTTCCG CCAAGAGCCG CCTGCCAGACA GCCCCCGTGC CCATGCCAGA CCTGAAGAAT 1921 GTCAAGTCCA AGATCGGCTC CACTGAGAAC CTGAAGCACC AGCCGGGAGG CGGGAAGGTG 1981 SUMMER ATAAGAAGCT GGATCTTAGC AACGTCCAGT CCAAGTGTGG CTCAAAGGAT 2041 AATATCAAAC ACGTCCCGGG AGGCGGCAGT GTGCAAATAG TCTACAAACC AGTTGACCTG 2101 AGCAAGGTGA CCTCCAAGTG TGGCTCATTA GGCAACATCC ATCATAAACC AGGAGGTGGC 2161 CAGGTGGAAG TAAAATCTGA GAAGCTTGAC TTCAAGGACA GAGTCCAGTC TAAATTGGG 2221 TCCCTGGACA ATATCACCCA CGTCCCTGGC GGAGGAAATA AAAAGATTGA AACCCACAAG 2281 CTGACCTTCC GCGAGAACGC CAAAGCCAAG ACAGACCACG GGGCGGAGAT CGTGTACAAG 2341 TCGCCAGTGG TGTCTGGGGA CACGTCTCCA CGGCATCTCA GCAATGTCTC CTCCACCGGC 2401 AGCATCGACA TGGTAGACTC GCCCCAGCTC GCCACGCTAG CTGACGAGGT GTCTGCCTCC 2461 CTGGCCAAGC AGGGTTTGTG ATCAGGCCCC TGGGGCGGTC AATAATTGTG GAGAGGAGAG 2521 AATGAGAGAG TGTGGAAAAA AAAAGAATAA TGACCCGGCC CCCGCCCTCT GCCCCCAGCT 2581 GCTCCTCGCA GTTCGGTTAA TTGGTTAATC ACTTAACCTG CTTTTGTCAC TCGGCTTTGG 2641 CTCGGGACTT CAAAATCAGT GATGGGAGTA AGAGCAAATT TCATCTTTCC AAATTGATGG 2701 GTGGGCTAGT AATAAAATAT TTAAAAAAAA ACATTCAAAA ACATGGCCAC ATCCAACATT 2761 TCCTCAGGCA ATTCCTTTTG ATTCTTTTTT CTTCCCCCTC CATGTAGAAG AGGGAGAAGG 2821 AGAGGCTCTG AAAGCTGCTT CTGGGGGATT TCAAGGGACT GGGGGTGCCA ACCACCTCTG 2881 GCCCTGTTGT GGGGGTGTCA CAGAGGCAGT GGCAGCAACA AAGGATTTGA AACTTGGTGT 2941 GTTCGTGGAG CCACAGGCAG ACGATGTCAA CCTTGTGTGA GTGTGACGGG GGTTGGGGTG 3001 GGGCGGGAGG CCACGGGGGA GGCCGAGGCA GGGGCTGGGC AGAGGGGAGA GGAAGCACAA 3061 GAAGTGGGAG TGGGAGAGGA AGCCACGTGC TGGAGAGTAG ACATCCCCCT CCTTGCCGCT 3121 GGGAGAGCCA AGGCCTATGC CACCTGCAGC GTCTGAGCGG CCGCCTGTCC TTGGTGGCCG 3181 GGGGTGGGGG CCTGCTGTGG GTCAGTGTGC CACCCTCTGC AGGGCAGCCT GTGGGAGAAG 3241 GGACAGCGGG TAAAAAGAGA AGGCAAGCTG GCAGGAGGGT GGCACTTCGT GGATGACCTC 3301 CTTAGAAAAG ACTGACCTTG ATGTCTTGAG AGCGCTGGCC TCTTCCTCCC TCCCTGCAGG 3361 GTAGGGGGCC TGAGTTGAGG GGCTTCCCTC TGCTCCACAG AAACCCTGTT TTATTGAGTT 3421 CTGAAGGTTG GAACTGCTGC CATGATTTTG GCCACTTTGC AGACCTGGGA CTTTAGGGCT 3481 AACCAGTTCT CTTTGTAAGG ACTTGTGCCT CTTGGGAGAC GTCCACCCGT TTCCAAGCCT 3541 GGGCCACTGG CATCTCTGGA GTGTGTGGGG GTCTGGGAGG CAGGTCCCGA GCCCCCTGTC 3601 CTTCCCACGG CCACTGCAGT CACCCCGTCT GCGCCGCTGT GCTGTTGTCT GCCGTGAGAG 3661 CCCAATCACT GCCTATACCC CTCATCACAC GTCACAATGT CCCGAATTCC CAGCCTCACC 3721 ACCCCTTCTC AGTAATGACC CTGGTTGGTT GCAGGAGGTA CCTACTCCAT ACTGAGGGTG 3781 AAATTAAGGG AAGGCAAAGT CCAGGCACAA GAGTGGGACC CCAGCCTCTC ACTCTCAGTT 3841 CCACTCATCC AACTGGGACC CTCACCACGA ATCTCATGAT CTGATTCGGT TCCCTGTCTC 3901 CTCCTCCCGT CACAGATGTG AGCCAGGGCA CTGCTCAGCT GTGACCCTAG GTGTTTCTGC 3961 CTTGTTGACA TGGAGAGAGC CCTTTCCCCT GAGAAGGCCT GGCCCCTTCC TGTGCTGAGC 4021 CCACAGCAGC AGGCTGGGTG TCTTGGTTGT CAGTGGTGGC ACCAGGATGG AAGGGCAAGG 4081 CACCCAGGGC AGGCCCACAG TCCCGCTGTC CCCCACTTGC ACCCTAGCTT GTAGCTGCCA 4141 ACCTCCCAGA CAGCCCAGCC CGCTGCTCAG CTCCACATGC ATAGTATCAG CCCTCCACAC 4201 CCGACAAAGG GGAACACACC CCCTTGGAAA TGGTTCTTTT CCCCCAGTCC CAGCTGGAAG 4261 CCATGCTGTC TGTTCTGCTG GAGCAGCTGA ACATATACAT AGATGTTGCC CTGCCCTCCC 4321 CATCTGCACC CTGTTGAGTT GTAGTTGGAT TTGTCTGTTT ATGCTTGGAT TCACCAGAGT 4381 GACTATGATA GTGAAAAGAA AAAAAAAAAAA AAAAAAGGAC GCATGTATCT TGAAATGCTT 4441 GTAAAGAGGT TTCTAACCCA CCCTCACGAG GTGTCTCTCA CCCCCACACT GGGACTCGTG 4501 TGGCCTGTGT GGTGCCACCC TGCTGGGGCC TCCCAAGTTT TGAAAGGCTT TCCTCAGCAC 4561 CTGGGACCCA ACAGAGACCA GCTTCTAGCA GCTAAGGAGG CCGTTCAGCT GTGACGAAGG 4621 CCTGAAGCAC AGGATTAGGA CTGAAGCGAT GATGTCCCCT TCCCTACTTC CCCTTGGGGC 4681 TCCCTGTGTC AGGGCACAGA CTAGGTCTTG TGGCTGGTCT GGCTTGCGGC GCGAGGATGG 4741 TTCTCTCTGG TCATAGCCCG AAGTCTCATG GCAGTCCCAA AGGAGGCTTA CAACTCCTGC 4801 ATCACAAGAA AAAGGAAGCC ACTGCCAGCT GGGGGGATCT GCAGCTCCCA GAAGCTCCGT 4861 GAGCCTCAGC CACCCCTCAG ACTGGGTTCC TCTCCAAGCT CGCCCTCTGG AGGGGCAGCG 4921 CAGCCTCCCA CCAAGGGCCC TGCGACCACA GCAGGGATTG GGATGAATTG CCTGTCCTGG 4981 ATCTGCTCTA GAGGCCCAAG CTGCCTGCCT GAGGAAGGAT GACTTGACAA GTCAGGAGAC 5041 ACTGTTCCCA AAGCCTTGAC CAGAGCACCT CAGCCCGCTG ACCTTGCACA AACTCCATCT 5101 GCTGCCATGA GAAAAGGGAA GCCGCCTTTG CAAAACATTG CTGCCTAAAG AAACTCAGCA 5161 GCCTCAGGCC CAATTCTGCC ACTTCTGGTT TGGGTACAGT TAAAGGCAAC CCTGAGGGAC 5221 TTGGCAGTAG AAATCCAGGG CCTCCCCTGG GGCTGGCAGC TTCGTGTGCA GCTAGAGCTT 5281 TACCTGAAAG GAAGTCTCTG GGCCCAGAAC TCTCCACCAA GAGCCTCCCT GCCGTTCGCT 5341 GAGTCCCAGC AATTCTCCTA AGTTGAAGGG ATCTGAGAAG GAGAAGGAAA TGTGGGGTAG 5401 ATTTGGTGGT GGTTAGAGAT ATGCCCCCCT CATTACTGCC AACAGTTTCG GCTGCATTTC 5461 TTCACGCACC TCGGTTCCTC TTCCTGAAGT TCTTGTGCCC TGCTCTTCAG CACCATGGGC 5521 CTTCTTATAC GGAAGGCTCT GGGATCTCCC CCTTGTGGGG CAGGCTCTTG GGGCCAGCCT 5581 AAGATCATGG TTTAGGGTGA TCAGTGCTGG CAGATAAATT GAAAAGGCAC GCTGGCTTGT 5641 GATCTTAAAT GAGGACAATC CCCCCAGGGC TGGGCACTCC TCCCCTCCCC TCACTTCTCC 5701 CACCTGCAGA GCCAGTGTCC TTGGGTGGGC TAGATAGGAT ATACTGTATG CCGGCTCCTT 5761 CAAGCTGCTG ACTCACTTTA TCAATAGTTC CATTTAAATT GACTTCAGTG GTGAGACTGT 5821 ATCCTGTTTG CTATTGCTTG TTGTGCTATG GGGGGAGGGG GGAGGAATGT GTAAGATAGT 5881 TAACATGGGC AAAGGGAGAT CTTGGGGTGC AGCACTTAAA CTGCCTCGTA ACCCTTTTCA 5941 TGATTTCAAC CACATTTGCT AGAGGGAGGG AGCAGCCACG GAGTTAGAGG CCCTTGGGGT 6001 TTCTCTTTTC CACTGACAGG CTTTCCCAGG CAGCTGGCTA GTTCATTCCC TCCCCAGCCA 6061 GGTGCAGGCG TAGGAATATG GACATCTGGT TGCTTTGGCC TGCTGCCCTC TTTCAGGGGT 6121 CCTAAGCCCA CAATCATGCC TCCCTAAGAC CTTGGCATCC TTCCCTCTAA GCCGTTGGCA 6181 CCTCTGTGCC ACCTCTCCACA CTGGCTCCAG ACACACAGCC TGTGCTTTTG GAGCTGAGAT 6241 CACTCGCTTC ACCCTCCTCA TCTTTGTTCT CCAAGTAAAG CCACGAGGTC GGGGCGAGGG 6301 CAGAGGTGAT CACCTGCGTG TCCCATCTAC AGACCTGCAG CTTCATAAAA CTTCTGATTT 6361 CTCTTCAGCT TTGAAAAGGG TTACCCTGGG CACTGGCCTA GAGCCTCACC TCCTAATAGA 6421 CTTAGCCCCA TGAGTTTGCC ATGTTGAGCA GGACTATTTC TGGCACTTGC AAGTCCCATG 6481 ATTTCTTCGG TAATTCTGAG GGTGGGGGGA GGGACATGAA ATCATCTTAG CTTAGCTTTC 6541 TGTCTGTGAA TGTCTATATA GTGTATTGTG TGTTTTAACA AATGATTTAC ACTGACTGTT 6601 GCTGTAAAAG TGAATTTGGA AATAAAGTTA TTACTCTGAT TAAA (SEQ ID NO: 156).

[0226] The corresponding amino acid sequence of human tau protein isoform 6 can be found in NP_001116538.2: 1 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 61 SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 121 HVTQEPESGK VVQEGFLREP GPPGLSHQLM SGMPGAPLLP EGPREATRQP SGTGPEDTEG 181 GRHAPELLKH QLLGDLHQEG PPLKGAGGKE RPGSKEEVDE DRDVDESSPQ DSPPSKASPA 241 QDGRPPQTAA REATSIPGFP AEGAIPLPVD FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE 301 FTFHVEITPN VQKEQAHSEE HLGRAAFPGA PGEGPEARGP SLGEDTKEAD LPEPSEKQPA 361 AAPRGKPVSR VPQLKARMVS KSKDGTGSDD KKAKTSTRSS AKTLKNRPCL SPKHPTPGSS 421 DPLIQPSSPA VCPEPPSSPK YVSSVTSRTG SSGAKEMKLK GADGKTKIAT PRGAAPPGQK 481 GQANATRIPA KTPPAPKTPP SSATKQVQRR PPPAGPRSER GEPPKSGDRS GYSSPGSPGT 541 PGSRSRTPSL PTPPTREPKK VAVVRTPPKS PSSAKSRLQT APVPMPDLKN VKSKIGSTEN 601 LKHQPGGGKV QIINKKLDLS NVQSKCGSKD NIKHVPGGGS VQIVYKPVDL SKVTSKCGSL 661 GNIHHKPGGG QVEVKSEKLD FKDRVQSKIG SLDNITHVPG GGNKKIETHK LTFRENAKAK 721 TDHGAEIVYK SPVVSGDTSP RHLSNVSSTG SIDMVDSPQL ATLADEVSAS LAKQGL (SEQ ID NO: 157).

[0227] The nucleotide sequence of human MAPT transcript variant 5 (encoding 1N4R tau) can be found in NM_001123067.4: 1 GCAGTCACCG CCACCCACCA GCTCCGGCAC CAACAGCAGC GCCGCTGCCA CCGCCCACCT 61 TCTGCCGCCG CCACCACAGC CACCTTCTCC TCCTCCGCTG TCCTCTCCCG TCCTCGCCTC 121 TGTCGACTAT CAGGTGAACT TTGAACCAGG ATGGCTGAGC CCCGCCAGGA GTTCGAAGTG 181 ATGGAAGATC ACGCTGGGAC GTACGGGTTG GGGGACAGGA AAGATCAGGG GGGCTACACC 241 ATGCACCAAG ACCAAGAGGG TGACACGGAC GCTGGCCTGA AAGAATCTCC CCTGCAGACC 301 CCCACTGAGG ACGGATCTGA GGAACCGGGC TCTGAAACCT CTGATGCTAA GAGCACTCCA 361 ACAGCGGAAG CTGAAGAAGC AGGCATTGGA GACACCCCCA GCCTGGAAGA CGAAGCTGCT 421 GGTCACGTGA CCCAAGCTCG CATGGTCAGT AAAAGCAAAG ACGGGACTGG AAGCGATGAC 481 AAAAAAGCCA AGGGGGCTGA TGGTAAAACG AAGATCGCCA CACCCGGGGG AGCAGCCCCT 541 CCAGGCCAGA AGGGCCAGGC CAACGCCACC AGGATTCCAG CAAAAACCCC GCCCGCTCCA 601 AAGACACCAC CCAGCTCTGG TGAACTCCA AAATCAGGGG ATCGCAGCGG CTACAGCAGC 661 CCCGGCTCCC CAGGCACTCC CGGCAGCCGC TCCCGCACCC CGTCCCTTCC AACCCCACCC 721 ACCCGGGAGC CCAAGAAGGT GGCAGTGGTC CGTACTCCAC CCAAGTCGCC GTCTTCCGCC 781 AAGAGCCGCC TGCAGACAGC CCCCGTGCCC ATGCCAGACC TGAAGAATGT CAAGTCCAAG 841 ATCGGCTCCA CTGAGAACCT GAAGCACCAG CCGGGAGGCG GGAAGGTGCCA GATAATTAAT 901 AAGAAGCTGG ATCTTAGCAA CGTCCAGTCC AAGTGTGGCT CAAAGTAA TATCAAACAC 961 GTCCCGGGAG GCGGCAGTGT GCAAATAGTC TACAAACCAG TTGACCTGAG CAAGGTGACC 1021 TCCAAGTGTG GCTCATTAGG CAACATCCAT CATAAACCAG GAGGTGGCCA GGTGGAAGTA 1081 AAATCTGAGA AGCTTGACTT CAAGGACAGA GTCCAGTCGA AGATTGGGTC CCTGGACAAT 1141 ATCACCCACG TCCCTGGCGG AGGAAATAAA AAGATTGAAA CCCACAAGCT GACCTTCCGC 1201 GAGAACGCCA AAGCCAAGAC AGACCACGGG GCGGAGATCG TGTACAAGTC GCCAGTGGTG 1261 TCTGGGGACA CGTCTCCACG GCATCTCAGC AATGTCTCCT CCACCGGCAG CATCGACATG 1321 GTAGACTCGC CCCAGCTCGC CACGCTAGCT GACGAGGTGT CTGCCTCCCT GGCCAAGCAG 1381 GGTTTGTGAT CAGGCCCCTG GGGCGGTCAA TAATTGTGGA GAGGAGAGAA TGAGAGAGTG 1441 TGGAAAAAAA AAGAATAATG ACCCGGCCCC CGCCCTCTGC CCCCAGCTGC TCCTCGCAGT 1501 TCGGTTAATT GGTTAATCAC TTAACCTGCT TTTGTCACTC GGCTTTGGCT CGGGACTTCA 1561 AAATCAGTGA TGGGAGTAAG AGCAAATTTC ATCTTTCCAA ATTGATGGGT GGGCTAGTAA 1621 TAAAATATTT AAAAAAAAAAC ATTCAAAAAC ATGGCCACAT CCAACATTTC CTCAGGCAAT 1681 TCCTTTTGAT TCTTTTTTCT TCCCCCTCCA TGTAGAAGAG GGAGAAGGAG AGGCTCTGAA 1741 AGCTGCTTCT GGGGGATTTC AAGGGACTGG GGGTGCCAAC CACCTCTGGC CCTGTTGTGG 1801 GGGTGTCACA GAGGCAGTGG CAGCAACAAA GGATTTGAAA CTTGGTGTGT TCGTGGAGCC 1861 ACAGGCAGAC GATGTCAACC TTGTGTGAGT GTGACGGGGG TTGGGGTGGG GCGGGAGGCC 1921 ACGGGGGAGG CCGAGGCAGG GGCTGGGCAG AGGGAGAGG AGCACAAGA AGTGGGAGTG 1981 GGAGGAAG CCACGTGCTG GGAGGAAG ATCCCCCTCC TTGCCGCTGG GGAGCCAAG 2041 GCCTATGCCA CCTGCAGCGT CTGAGCGGCC GCCTGTCCTT GGTGGCCGGG GGTGGGGGCC 2101 TGCTGTGGGT CAGTGTGCCA CCCTCTGCAG GGCAGCCTGT GGGAGAAGGG ACAGCGGGTA 2161 AAAAGAGAAG GCAAGCTGGC AGGAGGTGG CACTTCGTGG ATGACCTCCT TAGAAAAGAC 2221 TGACCTTGAT GTCTTGAGAG CGCTGGCCTC TTCCTCCCTC CCTGCAGGGT AGGGGGCCTG 2281 AGTTGAGGGG CTTCCCTCTG CTCCACAGAA ACCCTGTTTT ATTGAGTTCT GAAGGTTGGA 2341 ACTGCTGCCA TGATTTTGGC CACTTTGCAG ACCTGGGACT TTAGGGCTAA CCAGTTCTCT 2401 TTGTAAGGAC TTGTGCCTCT TGGGAGACGT CCACCCGTTT CCAAGCCTGG GCCACTGGCA 2461 TCTCTGGAGT GTGTGGGGGT CTGGGAGGCA GGTCCCGAGC CCCCTGTCCT TCCCACGGCC 2521 ACTGCAGTCA CCCCGTCTGC GCCGCTGTGC TGTTGTCTGC CGTGAGAGCC CAATCACTGC 2581 CTATACCCCT CATCACACGT CACAATGTCC CGAATTCCCA GCCTCACCAC CCCTTCTCAG 2641 TAATGACCCT GGTTGGTTGC AGGAGTACC TACTCCATAC TGAGGGTGAA ATTAAGGGAA 2701 GGCAAAGTCC AGGCACAAGA GTGGGACCCC AGCCTCTCAC TCTCAGTTCC ACTCATCCAA 2761 CTGGGACCCT CACCACGAAT CTCATGATCT GATTCGGTTC CCTGTCTCCT CCTCCCGTCA 2821 CAGATGTGAG CCAGGGCACT GCTCAGCTGT GACCCTAGGT GTTTCTGCCT TGTTGACATG 2881 GAGAGAGCCC TTTCCCCTGA GAAGGCCTGG CCCCTTCCTG TGCTGAGCCC ACAGCAGCAG 2941 GCTGGGTGTC TTGGTTGTCA GTGGTGGCAC GGGCAAGGCA CCCAGGGCAG 3001 GCCCACAGTC CCGCTGTCCC CCACTTGCAC CCTAGCTTGT AGCTGCCAAC CTCCCAGACA 3061 GCCCAGCCCG CTGCTCAGCT CCACATGCAT AGTATCAGCC CTCCACACCC GACAAAGGGG 3121 AACACACCCC CTTGGAAATG GTTCTTTTCC CCCAGTCCCA GCTGGAAGCC ATGCTGTCTG 3181 TTCTGCTGGA GCAGCTGAAC ATTACK ATGTTGCCCT GCCCTCCCCA TCTGCACCCT 3241 GTTGAGTTGT AGTTGGATTT GTCTGTTTAT GCTTGGATTC ACCAGAGTGA CTATGATAGT 3301 GAAAAGAAAA AAAAAAAAAA AAAAGGACGC ATGTATCTTG AAATGCTTGT AAAGAGGTTT 3361 CTAACCCACC CTCACGAGGT GTCTCTCACC CCCACACTGG GACTCGTGTG GCCTGTGTGG 3421 TGCCACCCTG CTGGGGCCTC CCAAGTTTTG AAAGGCTTTC CTCAGCACCT GGGACCCAAC 3481 AGAGACCAGC TTCTAGCAGC TAAGGAGGCC GTTCAGCTGT GACGAAGGCC TGAAGCACAG 3541 GATTAGGACT GAAGCGATGA TGTCCCCTTC CCTACTTCCC CTTGGGGCTC CCTGTGTCAG 3601 GGCACAGACT AGGTCTTGTG GCTGGTCTGG CTTGCGGCGC GAGGATGGTT CTCTCTGGTC 3661 ATAGCCCGAA GTCTCATGGC AGTCCCAAAG GAGGCTTACA ACTCCTGCAT CACAAGAAAA 3721 AGGAAGCCAC TGCCAGCTGG GGGGATCTGC AGCTCCCAGA AGCTCCGTGA GCCTCAGCCA 3781 CCCCTCAGAC TGGGTTCCTC TCCAAGCTCG CCCTCTGGAG GGGCAGCGCA GCCTCCCACC 3841 AAGGGCCCTG CGACCACAGC AGGGATTGGG ATGAATTGCC TGTCCTGGAT CTGCTCTAGA 3901 GGCCCAAGCT GCCTGCCTGA GGAAGGATGA CTTGACAAGT CAGGAGACAC TGTTCCCAAA 3961 GCCTTGACCA GAGCACCTCA GCCCGCTGAC CTTGCACAAA CTCCATCTGC TGCCATGAGA 4021 AAAGGGAAGC CGCCTTTGCA AAACATTGCT GCCTAAAGAA ACTCAGCAGC CTCAGGCCCA 4081 ATTCTGCCAC TTCTGGTTTG GGTACAGTTA AAGGCAACCC TGAGGGACTT GGCAGTAGAA 4141 ATCCAGGGCC TCCCCTGGGG CTGGCAGCTT CGTGTGCAGC TAGAGCTTTA CCTGAAAGGA 4201 AGTCTCTGGG CCCAGAACTC TCCACCAAGA GCCTCCCTGC CGTTCGCTGA GTCCCAGCAA 4261 TTCTCCTAAG TTGAAGGGAT CTGAGAAGGA GAAGGAAATG TGGGGTAGAT TTGGTGGTGG 4321 TTAGAGATAT GCCCCCCTCA TTACTGCCAA CAGTTTCGGC TGCATTTCTT CACGCACCTC 4381 GGTTCCTCTT CCTGAAGTTC TTGTGCCCTG CTCTTCAGCA CCATGGGCCT TCTTATACGG 4441 AAGGCTCTGG GATCTCCCCC TTGTGGGGCA GGCTCTTGGG GCCAGCCTAA GATCATGGTT 4501 TAGGGTGATC AGTGCTGGCA GATAAATTGA AAAGGCACGC TGGCTTGTGA TCTTAAATGA 4561 GGACAATCCC CCCAGGGCTG GGCACTCCTC CCCTCCCCTC ACTTCTCCCA CCTGCAGAGC 4621 CAGTGTCCTT GGGTGGGCTA GATAGGATAT ACTGTATGCC GGCTCCTTCA AGCTGCTGAC 4681 TCACTTTATC AATAGTTCCA TTTAAATTGA CTTCAGTGGT GAGACTGTAT CCTGTTTGCT 4741 ATTGCTTGTT GTGCTATGGG GGGAGGGGGG AGGAATGTGT AAGATAGTTA ACATGGGCAA 4801 AGGGAGATCT TGGGGTGCAG CACTTAAACT GCCTCGTAAC CCTTTTCATG ATTTCAACCA 4861 CATTTGCTAG AGGGAGGGAG CAGCCACGGA GTTAGAGGCC CTTGGGGTTT CTCTTTTCCA 4921 CTGACAGGCT TTCCCAGGCA GCTGGCTAGT TCATTCCCTC CCCAGCCAGG TGCAGGCGTA 4981 GGAATATGGA CATCTGGTTG CTTTGGCCTG CTGCCCTCTT TCAGGGGTCC TAAGCCCACA 5041 ATCATGCCTC CCTAAGACCT TGGCATCCTT CCCTCTAAGC CGTTGGCACC TCTGTGCCAC 5101 CTCTCACACT GGCTCCAGAC ACACAGCCTG TGCTTTTGGA GCTGAGATCA CTCGCTTCAC 5161 CCTCCTCATC TTTGTTCTCC AAGTAAAGCC ACGAGGTCGG GGCGAGGGCA GAGGTGATCA 5221 CCTGCGTGTC CCATCTACAG ACCTGCAGCT TCATAAAACT TCTGATTTCT CTTCAGCTTT 5281 GAAAAGGGTT ACCCTGGGCA CTGGCCTAGA GCCTCACCTC CTAATAGACT TAGCCCCATG 5341 AGTTTGCCAT GTTGAGCAGG ACTATTTCTG GCACTTGCAA GTCCCATGAT TTCTTCGGTA 5401 ATTCTGAGGG TGGGGGGAGG GACATGAAAT CATCTTAGCT TAGCTTTCTG TCTGTGAATG 5461 TCTATATAGT GTATTGTGTG TTTTAACAAA TGATTTACAC TGACTGTTGC TGTAAAAGTG 5521 AATTTGGAAA TAAAGTTATT ACTCTGATTA AA (SEQ ID NO: 158).

[0228] The corresponding amino acid sequence of human tau protein isoform 5 can be found in NP_001116539.1: 1 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 61 SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKT 121 KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 181 SRTPSLPTPP TREPKKVAVV RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 241 PGGGKVQIIN KKLDLSNVQS KCGSKDNIKH VPGGGSVQIV YKPVDLSKVT SKCGSLGNIH 301 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG 361 AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL (SEQ ID NO: 159).

[0229] The nucleotide sequence of human MAPT transcript variant 4 (encoding 0N3R tau) can be found in NM_016841.5: 1 GCAGTCACCG CCACCCACCA GCTCCGGCAC CAACAGCAGC GCCGCTGCCA CCGCCCACCT 61 TCTGCCGCCG CCACCACAGC CACCTTCTCC TCCTCCGCTG TCCTCTCCCG TCCTCGCCTC 121 TGTCGACTAT CAGGTGAACT TTGAACCAGG ATGGCTGAGC CCCGCCAGGA GTTCGAAGTG 181 ATGGAAGATC ACGCTGGGAC GTACGGGTTG GGGGACAGGA AAGATCAGGG GGGCTACACC 241 ATGCACCAAG ACCAAGAGGG TGACACGGAC GCTGGCCTGA AAGCTGAAGA AGCAGGCATT 301 GGAGACACCC CCAGCCTGGA AGACGAAGCT GCTGGTCACG TGACCCAAGC TCGCATGGTC 361 AGTAAAAGCA AAGACGGGAC TGGAAGCGAT GACAAAAAAG CCAAGGGGGC TGATGGTAAA 421 ACGAAGATCG CCACACCGCG GGGAGCAGCC CCTCCAGGCC AGAAGGGCCA GGCCAACGCC 481 ACCAGGATTC CAGCAAAAAC CCCGCCCGCT CCAAAGACAC CACCCAGCTC TGGTGAACCT 541 CCAAAATCAG GGGATCGCAG CGGCTACAGC AGCCCCGGCT CCCCAGGCAC TCCCGGCAGC 601 CGCTCCCGCA CCCCGTCCCT TCCAACCCCA CCCACCCGGG AGCCCAAGAA GGTGGCAGTG 661 GTCCGTACTC CACCCAAGTC GCCGTCTTCC GCCAAGAGCC GCCTGCAGAC AGCCCCCGTG 721 CCCATGCCAG ACCTGAAGAA TGTCAAGTCC AAGATCGGCT CCACTGAGAA CCTGAAGCAC 781 CAGCCGGGAG GCGGGAAGGT GCAAATAGTC TACAAACCAG TTGACCTGAG CAAGGTGACC 841 TCCAAGTGTG GCTCATTAGG CAACATCCAT CATAAACCAG GAGGTGGCCA GGTGGAAGTA 901 AAATCTGGA AGCTTGACTT CAAGGACAGA GTCCAGTCGA AGATTGGGTC CCTGGACAAT 961 ATCACCCACG TCCCTGGCGG AGGAAATAAA AAGATTGAAA CCCACAAGCT GACCTTCCGC 1021 GAGAACGCCA AAGCCAAGAC AGACCACGGG GCGGAGATCG TGTACAAGTC GCCAGTGGTG 1081 TCTGGGGACA CGTCTCCACG GCATCTCAGC AATGTCTCCT CCACCGGCAG CATCGACATG 1141 GTAGACTCGC CCCAGCTCGC CACGCTAGCT GACGAGGTGT CTGCCTCCCT GGCCAAGCAG 1201 GGTTTGTGAT CAGGCCCCTG GGGCGGTCAA TAATTGTGGA GAGGAGAGAA TGAGAGAGTG 1261 TGGAAAAAAA AAGAATAATG ACCCGGCCCC CGCCCTCTGC CCCCAGCTGC TCCTCGCAGT 1321 TCGGTTAATT GGTTAATCAC TTAACCTGCT TTTGTCACTC GGCTTTGGCT CGGGACTTCA 1381 AAATCAGTGA TGGGAGTAAG AGCAAATTTC ATCTTTCCAA ATTGATGGGT GGGCTAGTAA 1441 TAAAATATTT AAAAAAAAAAC ATTCAAAAAAC ATGGCCACAT CCAACATTTC CTCAGGCAAT 1501 TCCTTTTGAT TCTTTTTTCT TCCCCCTCCA TGTAGAAGAG GGAGAAGGAG AGGCTCTGAA 1561 AGCTGCTTCT GGGGGATTTC AAGGGACTGG GGGTGCCAAC CACCTCTGGC CCTGTTGTGG 1621 GGGTGTCACA GAGGCAGTGG CAGCAACAAA GGATTTGAAA CTTGGTGTGT TCGTGGAGCC 1681 ACAGGCAGAC GATGTCAACC TTGTGTGAGT GTGACGGGGG TTGGGGTGGG GCGGGAGGCC 1741 ACGGGGGAGG CCGAGGCAGG GGCTGGGCAG AGGGAGAGG AGCACAAGA AGTGGGAGTG 1801 GGAGGAAG CCACGTGCTG GGAGGAAG ATCCCCCTCC TTGCCGCTGG GGAGCCAAG 1861 GCCTATGCCA CCTGCAGCGT CTGAGCGGCC GCCTGTCCTT GGTGGCCGGG GGTGGGGGCC 1921 TGCTGTGGGT CAGTGTGCCA CCCTCTGCAG GGCAGCCTGT GGGAGAAGGG ACAGCGGGTA 1981 AAAAGAGAAG GCAAGCTGGC AGGAGGTGG CACTTCGTGG ATGACCTCCT TAGAAAAGAC 2041 TGACCTTGAT GTCTTGAGAG CGCTGGCCTC TTCCTCCCTC CCTGCAGGGT AGGGGGCCTG 2101 AGTTGAGGGG CTTCCCTCTG CTCCACAGAA ACCCTGTTTT ATTGAGTTCT GAAGGTTGGA 2161 ACTGCTGCCA TGATTTTGGC CACTTTGCAG ACCTGGGACT TTAGGGCTAA CCAGTTCTCT 2221 TTGTAAGGAC TTGTGCCTCT TGGGAGACGT CCACCCGTTT CCAAGCCTGG GCCACTGGCA 2281 TCTCTGGAGT GTGTGGGGGT CTGGGAGGCA GGTCCCGAGC CCCCTGTCCT TCCCACGGCC 2341 ACTGCAGTCA CCCCGTCTGC GCCGCTGTGC TGTTGTCTGC CGTGAGAGCC CAATCACTGC 2401 CTATACCCCT CATCACACGT CACAATGTCC CGAATTCCCA GCCTCACCAC CCCTTCTCAG 2461 TAATGACCCT GGTTGGTTGC AGGAGTACC TACTCCATAC TGAGGGTGAA ATTAAGGGAA 2521 GGCAAAGTCC AGGCACAAGA GTGGGACCCC AGCCTCTCAC TCTCAGTTCC ACTCATCCAA 2581 CTGGGACCCT CACCACGAAT CTCATGATCT GATTCGGTTC CCTGTCTCCT CCTCCCGTCA 2641 CAGATGTGAG CCAGGGCACT GCTCAGCTGT GACCCTAGGT GTTTCTGCCT TGTTGACATG 2701 GAGAGAGCCC TTTCCCCTGA GAAGGCCTGG CCCCTTCCTG TGCTGAGCCC ACAGCAGCAG 2761 GCTGGGTGTC TTGGTTGTCA GTGGTGGCAC GGGCAAGGCA CCCAGGGCAG 2821 GCCCACAGTC CCGCTGTCCC CCACTTGCAC CCTAGCTTGT AGCTGCCAAC CTCCCAGACA 2881 GCCCAGCCCG CTGCTCAGCT CCACATGCAT AGTATCAGCC CTCCACACCC GACAAAGGGG 2941 AACACACCCC CTTGGAAATG GTTCTTTTCC CCCAGTCCCA GCTGGAAGCC ATGCTGTCTG 3001 TTCTGCTGGA GCAGCTGAAC ATATACATAG ATGTTGCCCT GCCCTCCCCA TCTGCACCCT 3061 GTTGAGTTGT AGTTGGATTT GTCTGTTTAT GCTTGGATTC ACCAGAGTGA CTATGATAGT 3121 GAAAAGAAAA AAAAAAAAAA AAAAGGACGC ATGTATCTTG AAATGCTTGT AAAGAGGTTT 3181 CTAACCCACC CTCACGAGGT GTCTCTCACC CCCACACTGG GACTCGTGTG GCCTGTGTGG 3241 TGCCACCCTG CTGGGGCCTC CCAAGTTTTG AAAGGCTTTC CTCAGCACCT GGGACCCAAC 3301 AGAGACCAGC TTCTAGCAGC TAAGGAGGCC GTTCAGCTGT GACGAAGGCC TGAAGCACAG 3361 GATTAGGACT GAAGCGATGA TGTCCCCTTC CCTACTTCCC CTTGGGGCTC CCTGTGTCAG 3421 GGCACAGACT AGGTCTTGTG GCTGGTCTGG CTTGCGGCGC GAGGATGGTT CTCTCTGGTC 3481 ATAGCCCGAA GTCTCATGGC AGTCCCAAAG GAGGCTTACA ACTCCTGCAT CACAAGAAAA 3541 AGGAAGCCAC TGCCAGCTGG GGGGATCTGC AGCTCCCAGA AGCTCCGTGA GCCTCAGCCA 3601 CCCCTCAGAC TGGGTTCCTC TCCAAGCTCG CCCTCTGGAG GGGCAGCGCA GCCTCCCACC 3661 AAGGGCCCTG CGACCACAGC AGGGATTGGG ATGAATTGCC TGTCCTGGAT CTGCTCTAGA 3721 GGCCCAAGCT GCCTGCCTGA GGAAGGATGA CTTGACAAGT CAGGAGACAC TGTTCCCAAA 3781 GCCTTGACCA GAGCACCTCA GCCCGCTGAC CTTGCACAAA CTCCATCTGC TGCCATGAGA 3841 AAAGGGAAGC CGCCTTTGCA AAACATTGCT GCCTAAAGAA ACTCAGCAGC CTCAGGCCCA 3901 ATTCTGCCAC TTCTGGTTTG GGTACAGTTA AAGGCAACCC TGAGGGACTT GGCAGTAGAA 3961 ATCCAGGGCC TCCCCTGGGG CTGGCAGCTT CGTGTGCAGC TAGAGCTTTA CCTGAAAGGA 4021 AGTCTCTGGG CCCAGAACTC TCCACCAAGA GCCTCCCTGC CGTTCGCTGA GTCCCAGCAA 4081 TTCTCCTAAG TTGAAGGGAT CTGAGAAGGA GAAGGAAATG TGGGGTAGAT TTGGTGGTGG 4141 TTAGAGATAT GCCCCCCTCA TTACTGCCAA CAGTTTCGGC TGCATTTCTT CACGCACCTC 4201 GGTTCCTCTT CCTGAAGTTC TTGTGCCCTG CTCTTCAGCA CCATGGGCCT TCTTATACGG 4261 AAGGCTCTGG GATCTCCCCC TTGTGGGGCA GGCTCTTGGG GCCAGCCTAA GATCATGGTT 4321 TAGGGTGATC AGTGCTGGCA GATAAATTGA AAAGGCACGC TGGCTTGTGA TCTTAAATGA 4381 GGACAATCCC CCCAGGGCTG GGCACTCCTC CCCTCCCCTC ACTTCTCCCA CCTGCAGAGC 4441 CAGTGTCCTT GGGTGGGCTA GATAGGATAT ACTGTATGCC GGCTCCTTCA AGCTGCTGAC 4501 TCACTTTATC AATAGTTCCA TTTAAATTGA CTTCAGTGGT GAGACTGTAT CCTGTTTGCT 4561 ATTGCTTGTT GTGCTATGGG GGGAGGGGGG AGGAATGTGT AAGATAGTTA ACATGGGCAA 4621 AGGGAGATCT TGGGGTGCAG CACTTAAACT GCCTCGTAAC CCTTTTCATG ATTTCAACCA 4681 CATTTGCTAG AGGGAGGGAG CAGCCACGGA GTTAGAGGCC CTTGGGGTTT CTCTTTTCCA 4741 CTGACAGGCT TTCCCAGGCA GCTGGCTAGT TCATTCCCTC CCCAGCCAGG TGCAGGCGTA 4801 GGAATATGGA CATCTGGTTG CTTTGGCCTG CTGCCCTCTT TCAGGGGTCC TAAGCCCACA 4861 ATCATGCCTC CCTAAGACCT TGGCATCCTT CCCTCTAAGC CGTTGGCACC TCTGTGCCAC 4921 CTCTCACACT GGCTCCAGAC ACACAGCCTG TGCTTTTGGA GCTGAGATCA CTCGCTTCAC 4981 CCTCCTCATC TTTGTTCTCC AAGTAAAGCC ACGAGGTCGG GGCGAGGGCA GAGGTGATCA 5041 CCTGCGTGTC CCATCTACAG ACCTGCAGCT TCATAAAACT TCTGATTTCT CTTCAGCTTT 5101 GAAAAGGGTT ACCCTGGGCA CTGGCCTAGA GCCTCACCTC CTAATAGACT TAGCCCCATG 5161 AGTTTGCCAT GTTGAGCAGG ACTATTTCTG GCACTTGCAA GTCCCATGAT TTCTTCGGTA 5221 ATTCTGAGGG TGGGGGGAGG GACATGAAAT CATCTTAGCT TAGCTTTCTG TCTGTGAATG 5281 TCTATATAGT GTATTGTGTG TTTTAACAAA TGATTTACAC TGACTGTTGC TGTAAAAGTG 5341 AATTTGGAAA TAAAGTTATT ACTCTGATTA AA (SEQ ID NO: 160).

[0230] The corresponding amino acid sequence of human tau protein isoform 4 can be found in NP_058525.1: 1 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 61 AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA 121 PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS 181 AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH 241 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG 301 AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL (SEQ ID NO: 161).

[0231] As used herein, the term "tauopathy" refers to a disease associated with abnormal tau protein expression, secretion, phosphorylation, cleavage, and / or aggregation.

[0232] As used herein, "TfR" refers to a transferrin receptor protein or polypeptide, e.g., a human or mouse transferrin receptor protein or polypeptide. The amino acid sequence of the human transferrin receptor protein (hTFR) can be found in NP_001121620.1: 1 MMDQARSAFS NLFGGEPLSY TRFSLARQVD GDNSHVEMKL AVDEEENADN NTKANVTKPK 61 RCSGSICYGT IAVIVFFLIG FMIGYLGYCK GVEPKTECER LAGTESPVRE EPGEDFPAAR 121 RLYWDDLKRK LSEKLDSTDF TGTIKLLNEN SYVPREAGSQ KDENLALYVE NQFREFKLSK 181 VWRDQHFVKI QVKDSAQNSV IIVDKNGRLV YLVENPGGYV AYSKAATVTG KLVHANFGTK 241 KDFEDLYTPV NGSIVIVRAG KITFAEKVAN AESLNAIGVL IYMDQTKFPI VNAELSFFGH 301 AHLGTGDPYT PGFPSFNHTQ FPPSRSSGLP NIPVQTISRA AAEKLFGNME GDCPSDWKTD 361 STCRMVTSES KNVKLTVSNV LKEIKILNIF GVIKGFVEPD HYVVVGAQRD AWGPGAAKSG 421 VGTALLLKLA QMFSDMVLKD GFQPSRSIIF ASWSAGDFGS VGATEWLEGY LSSLHLKAFT 481 YINLDKAVLG TSNFKVSASP LLYTLIEKTM QNVKHPVTGQ FLYQDSNWAS KVEKLTLDNA 541 AFPFLAYSGI PAVSFCFCED TDYPYLGTTM DTYKELIERI PELNKVARAA AEVAGQFVIK 601 LTHDVELNLD YERYNSQLLS FVRDLNQYRA DIKEMGLSLQ WLYSARGDFF RATSRLTTDF 661 GNAEKTDRFV MKKLNDRVMR VEYHFLSPYV SPKESPFRHV FWGSGSHTLP ALLENLKLRK 721 QNNGAFNETL FRNQLALATW TIQGAANALS GDVWDIDNEF (SEQ ID NO: 111).

[0233] The amino acid sequence of the mouse transferrin receptor protein (mTFR) can be found in NP_001344227.1: 1 MMDQARSAFS NLFGGEPLSY TRFSLARQVD GDNSHVEMKL AADEEENADN NMKASVRKPK 61 RFNGRLCFAA IALVIFFLIG FMSGYLGYCK RVEQKEECVK LAETEETDKS ETMETEDVPT 121 SSRLYWADLK TLLSEKLNSI EFADTIKQLS QNTYTPREAG SQKDESLAYY IENQFHEFKF 181 SKVWRDEHYV KIQVKSSIGQ NMVTIVQSNG NLDPVESPEG YVAFSKPTEV SGKLVHANFG 241 TKKDFEELSY SVNGSLVIVR AGEITFAEKV ANAQSFNAIG VLIYMDKNKF PVVEADLALF 301 GHAHLGTGDP YTPGFPSFNH TQFPPSQSSG LPNIPVQTIS RAAAEKLFGK MEGSCPARWN 361 IDSSCKLELS QNQNVKLIVK NVLKERRILN IFGVIKGYEE PDRYVVVGAQ RDALGAGVAA 421 KSSVGTGLLL KLAQVFSDMI SKDGFRPSRS IIFASWTAGD FGAVGATEWL EGYLSSLHLK 481 AFTYINLDKV VLGTSNFKVS ASPLLYTLMG KIMQDVKHPV DGKSLYRDSN WISKVEKLSF 541 DNAAYPFLAY SGIPAVSFCF CEDADYPYLG TRLDTYEALT QKVPQLNQMV RTAAEVAGQL 601 IIKLTHDVEL NLDYEMYNSK LLSFMKDLNQ FKTDIRDMGL SLQWLYSARG DYFRATSRLT 661 TDFHNAEKTN RFVMREINDR IMKVEYHFLS PYVSPRESPF RHIFWGSGSH TLSALVENLK 721 LRQKNITAFN ETLFRNQLAL ATWTIQGVAN ALSGDIWNID NEF (SEQ ID NO: 112).

[0234] As used herein, "treatment" or "treating" refers to any process that may slow, control, retard, or stop the progression of a disorder or disease disclosed herein, or ameliorate the symptoms of the disorder or disease, but does not necessarily indicate the complete elimination of all disorder or disease symptoms. Treatment includes the administration of a protein or nucleic acid or vector or composition for the treatment of a disease or condition in a patient, particularly a human.

[0235] The following examples are offered to illustrate, but not to limit, the claimed invention. [Example]

[0236] Example 1: Generation and characterization of TfR-binding proteins Generation of human or mouse TfR-binding proteins Antibodies against mouse TfR were generated by immunizing New Zealand White rabbits with the extracellular domain (ECD) of the mouse transferrin receptor 1 protein with a His-tag (mTfR-ECD-6His, SEQ ID NO: 113, see Table 12). mTfR antigen-positive B cells were sorted from peripheral blood, and the binding of individual antibodies cloned from those B cells was verified on the His-tagged mTfR.

[0237] Antibodies against human TfR were generated by immunizing AlivaMab® transgenic mice with the extracellular domain of His-tagged human transferrin receptor 1 protein (hTfR-ECD-6His, SEQ ID NO: 114, see Table 12) and mouse transferrin receptor protein (mTfR, SEQ ID NO: 110). Antigen-positive B cells were sorted from pooled spleens. Individual antibodies cloned from these B cells were tested for binding to the His-tagged hTfR-ECD.

[0238] Additional antibodies against the human TfR were generated by immunizing AlivaMab® transgenic mice with the apical domain of the human transferrin receptor 1 protein with a His-tagged tag (hTfR-ApD-6His, SEQ ID NO: 115, see Table 12). Antigen-positive B cells were sorted from pooled spleens. Individual antibodies cloned from these B cells were tested for binding to the His-tagged hTfR-ECD.

[0239] Table 12. Sequences of immunogens used to generate human or mouse TfR antibodies. [Table 16]

[0240] Affinity variants of the resulting human or mouse TfR antibodies were generated by systematically introducing mutations into individual CDRs of each antibody. The resulting variants were subjected to multiple rounds of selection with decreasing antigen concentration and / or increasing dissociation time to isolate clones with improved affinity. The sequences of individual variants were used to construct combinatorial libraries, which were subjected to additional rounds of selection with increasing stringency to identify additive or synergistic mutation pairings between individual CDR regions. Individual combination clones were sequenced. The heavy and light chain CDR and VH / VL sequences of human TfR binding domains TBD1-7 are provided in Tables 1-3. The heavy and light chain CDR and VH / VL sequences of the mouse TfR binding protein (mTBP1) are provided in Table 7.

[0241] Human or mouse TfR-binding proteins were produced by recombinant DNA technology. Such TfR-binding proteins can be expressed in mammalian cell lines, such as HEK293 or CHO, either transiently or stably transfected with an expression system using an optimal, predetermined HC:LC vector ratio, or with a single vector system encoding both the HC and LC. The clarified medium into which the protein is secreted can be purified using commonly used techniques.

[0242] Binding affinity at 25°C The binding affinity and stoichiometry of an exemplary mouse TfR-binding protein for mouse TFR were determined using a surface plasmon resonance assay on a Biacore T200 instrument primed with HBS-EP+ (10 mM Hepes pH 7.4+150 mM NaCl+3 mM EDTA+0.05% (w / v) surfactant P20) running buffer and the analysis temperature set at 25°C. A human Fab capture kit (Cytiva P / N 28958325) was immobilized on a CM5 chip (Cytiva P / N 29104988) using standard NHS-EDC amine coupling on all four flow cells (Fc). The mouse TfR-binding protein was prepared at 10 μg / mL by dilution into running buffer. The target (mouse TfR-mIgG1-Fc) was prepared by dilution in running buffer at final concentrations of 100.0, 25.0, 6.25, 1.56, 0.39, 0.097, 0.024, and 0 (blank) nM.

[0243] Each analysis cycle consisted of (1) capturing the capture antibody samples onto separate flow cells (Fc2, Fc3, and Fc4), (2) injecting each concentration of TfR across the entire Fc at 100 μL / min for 60 seconds, followed by a return to buffer flow for 1800 seconds to monitor the dissociation phase, (3) regenerating the chip surface by injecting 10 mM glycine, pH 1.5, across the entire cell at 10 μL / min for 30 seconds, and (4) equilibrating the chip surface with a 10 μL (60 seconds) injection of HBS-EP+. Data were processed using standard double referencing and fitted to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0.3, to determine the association rate (k on , M -1 s -1 units), dissociation rate (k off , s -1 units), and R max (unit: RU) was determined. D =k off / k on From the relationship, the equilibrium dissociation constant (K D) was calculated and is in units of moles. The results are provided in Table 13.

[0244] Table 13: Binding affinity of exemplary mTfR binding proteins to mouse TFR at 25°C. [Table 17]

[0245] These results demonstrate that the exemplified mouse TfR binding proteins and conjugates bind to mouse TfR with high affinity at 25°C.

[0246] The binding affinity and stoichiometry of exemplary human TfR-binding proteins for human and cynomolgus monkey TfR were determined using surface plasmon resonance assays on a Biacore 8K instrument primed with HBS-EP+ (10 mM Hepes pH 7.4+150 mM NaCl+3 mM EDTA+0.05% (w / v) surfactant P20) running buffer and the analysis temperature set at 25°C. Anti-His antibodies were immobilized on a CM5 chip (Cytiva P / N 29104988) using standard NHS-EDC amine coupling on all four flow cells (Fc). Targets (human or cynomolgus monkey TfR ECD) were prepared in running buffer at a final concentration of 500 μg / mL. The TfR-binding proteins were prepared at final concentrations of 1, 0.2, 0.04, 0.008, and 0.0016 μM, respectively, by diluting the stock solutions into running buffer.

[0247] Binding analyses were performed in single-cycle kinetic mode. Each analysis cycle consisted of: (1) capture of target (His-tagged human or cynomolgus TfR ECD) samples onto separate flow cells (Fc2, Fc3, and Fc4); (2) injection of the lowest to highest concentration of antibody or protein across the entire Fc at 30 μL / min for 900 seconds, followed by a return to buffer flow for 1800 seconds to monitor the dissociation phase; (3) regeneration of the chip surface by injecting 10 mM glycine, pH 1.5, across the entire cell at 10 μL / min for 30 seconds; and (4) equilibration of the chip surface with a 10 μL (60 seconds) injection of HBS-EP+. Data were processed using standard double referencing and fitted to a 1:1 binding model using Biacore 8K Evaluation software to determine the association rate (k on , M -1 s -1 units), dissociation rate (k off , s -1 units), and R max (unit: RU) was determined. D =k off / k on From the relationship, the equilibrium dissociation constant (K D ) was calculated and is in units of moles. The results are provided in Table 14A.

[0248] Antibody / protein binding to cell-bound TfR was assessed using the human endothelial line hCMEC-D3 (EMD Millipore SC066), which endogenously expresses the human TfR, and an MDCK cell line (ATCC CCL-34) engineered to express the cynomolgus monkey TfR. Cells were grown and maintained at submaximal confluence, detached from the culture vessel using Accutase cell detachment solution, washed, and plated at 50,000 cells / well for binding assessment. Cells were treated with a viability stain and then incubated on ice with titrated concentrations of TfR-binding proteins. Cells were washed, and binding of the test antibody or protein was detected using a PE-labeled secondary reagent. Cells were then washed and read the same day using a BioRad ZE5 cytometer. Post-acquisition analysis was performed in FlowJo to analyze the fluorescence of single, viable, non-debris events. EC50 values ​​were derived by plotting the geometric median PE intensity values ​​across a given sample titration and fitting a sigmoidal (4PL) response curve in GraphPad Prism 8.3.0.

[0249] Table 14A: Binding affinity of exemplary human TfR binding proteins to human or cynomolgus monkey TfR at 25°C or 0°C. [Table 18]

[0250] Binding affinity at 37°C The binding affinity and stoichiometry of exemplary human TfR-binding proteins for human and cynomolgus monkey TfR were further characterized using surface plasmon resonance assays on a Biacore 8K instrument primed with HBS-EP+ (10 mM Hepes pH 7.4+150 mM NaCl+3 mM EDTA+0.05% (w / v) surfactant P20) running buffer and the analysis temperature set at 37°C. Target human and cynomolgus monkey TfR ECDs were immobilized on a CM4 chip (Cytiva P / N 29104989) using standard NHS-EDC amine coupling. TfR-binding proteins were prepared at final concentrations of 0.3, 0.1, 0.033, 0.01, 0.0033, 0.001, 0.00033, and 0.0001 μM, respectively, by diluting stock solutions into running buffer.

[0251] Binding analysis was performed in multi-cycle kinetic mode. Each analysis cycle consisted of (1) a 140-second injection of the lowest to highest concentration protein across the entire Fc at 50 μL / min, followed by a 400-second return to buffer flow to monitor the dissociation phase; (2) regenerating the chip surface by injecting 3 M magnesium chloride across the entire cell at 100 μL / min for 30 seconds; and (3) equilibrating the chip surface with a 50 μL (30-second) injection of HBS-EP+. Data were processed using standard double referencing and fitted to a 1:1 binding model using Biacore 8K Evaluation software to determine the association rate (k on , M -1 s -1 units), dissociation rate (k off , s -1 units), and R max (unit: RU) was determined. D =k off / k on From the relationship, the equilibrium dissociation constant (K D ) was calculated and is in units of moles. The results are provided in Table 14B.

[0252] Table 14B. Binding Affinities of Exemplary Human TfR Binding Proteins to Human or Cynomolgus TfR at 37°C [Table 19]

[0253] Epitope mapping by hydrogen-deuterium exchange mass spectrometry (HDX-MS) Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) was performed to determine where the exemplified TfR-binding proteins bind to the human TfR extracellular domain (TfR-ECD).

[0254] Peptide identification of human TfR-ECD was performed on a Waters Synapt G2Si (Waters Corporation) instrument using 5 μg of human TfR-ECD protein in zero exchange (1:10 dilution in 0.1X phosphate-buffered saline in HO) using nepenthesin II (Nep II) digestion followed by in-line treatment with PNGaseDj. The mass spectrometer was set to HDMSe (mobility ESI+ mode) using a mass acquisition range of m / z 255.00–1950.00 with a scan time of 0.4 s. Data were processed using PLGS 2.3.02 (Waters Corporation). For exchange experiments, complexes of human TfR-ECD protein and individual TfR-binding proteins were prepared at a molar ratio of 1:1.2 in 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl (1X PBS buffer). Using a custom TECAN sample preparation system, experiments were initiated by adding 25 μL of DO buffer containing 0.1× PBS to 2.5 μL of TfR-ECD (0.9 mg / mL) or TfR-ECD + protein complexes for various time periods (0 s, 10 s, 2 min, 10 min, and 60 min) at 15 °C (Espada et al. 2019, J Am Soc Mass Spectrom. 2019 Dec;30(12):2580-2583). The reaction was stopped with an equal volume of 0.32 M TCEP, 3 M guanidine HCl, 0.1 M phosphate pH 2.5 for 2 min at 4 °C and immediately frozen at -70 °C. The sample injection system consisted of a UR3 robot, a LEAP PAL3 HDX autosampler, and an HPLC system interfaced with a Waters Synapt G2Si (Waters Corporation), modified as described (Espada et al., 2019, J Am Soc Mass Spectrom. 2019 Dec;30(12):2580-2583). The LC mobile phase consisted of water (A) and acetonitrile (B) containing 0.2% formic acid, respectively.Each sample was thawed for 1 min using 50 μL of 1.5 M guanidine HCl, 0.1 M phosphate pH 2.5, and then injected onto a Nep II column and aged for 2.5 min at 4 °C using mobile phase A at a flow rate of 250 μL / min. The resulting peptides were captured on a Waters BEH Vanguard Pre-column at 4 °C and chromatographically separated using a Waters Acquity UPLC BEH C18 analytical column at 4 °C using a gradient of 3% to 85% mobile phase B over 7 min at a flow rate of 200 μL / min. The resulting peptides were then transferred to a mass spectrometer for mass analysis. The Synapt G2Si was calibrated with Glu-fibrinopeptide (Waters Corporation) prior to use. Mass spectra were acquired in HDMS mode over an m / z range of 255–1950, with a lock mass m / z of 556.2771 (Leucine Enkephalin, Waters Corporation). The relative deuterium uptake of each peptide was determined by processing the MS data of the deuterated samples and undeuterated controls using the identified peptide list in DynamX 3.0 (Waters Corporation). The free and bound states of TfR-ECD were compared for differences in deuterium uptake to identify conserved regions representing binding epitopes. The overall sequence coverage of the human TfR ECD was 90.4%.

[0255] For human TfR binding protein 1 (TBP1), decreased deuterium uptake upon binding to the human TfR-ECD was observed at residues 346-364 FGNMEGDCPSDWKTDSTCR (SEQ ID NO: 119), indicating a putative epitope region. For human TfR binding protein 13 (TBP13), decreased deuterium uptake upon binding to the human TfR was observed at residues 243-247 (FEDLY) (SEQ ID NO: 162) and 345-364 (LFGNMEEGDCPSDWKTDSTCR) (SEQ ID NO: 163), indicating a putative epitope region. For human TfR binding protein 10 (TBP10), reduced deuterium incorporation upon binding to human TfR was observed at residues 243-247 (FEDLY) (SEQ ID NO: 162), 259-263 (AGKIT) (SEQ ID NO: 164), and 532-538 (VEKLTLD) (SEQ ID NO: 165), indicating putative epitope regions.

[0256] Example 2: Synthesis and characterization of dsRNA (e.g., siRNA) targeting SNCA The single strands (sense and antisense) of the dsRNA duplex were synthesized on a solid support via MerMade™ 12 (LGC Biosearch Technologies). The sequences of the sense and antisense strands are shown in Table 11. The sense strand was synthesized using phthalamidoamino C6 lcaa CPG 500Å (Chemgenes), while the antisense strand used a standard support (LGC Biosearch Technologies). Oligonucleotides were synthesized via phosphoramidite chemistry at either a 5, 10, or 50 μmol scale.

[0257] Standard reagents were used for oligo synthesis (Table 16), where 0.1 M hydrogenated xanthan gum in pyridine was used as the sulfurizing reagent and 20% DEA in ACN was used as a post-synthesis auxiliary wash. All monomers (Table 17) were made up at 0.1 M in ACN and contained a molecular sieve trap bag.

[0258] The oligonucleotides were cleaved and deprotected (C / D) at 45°C for 20 hours. The sense strand was C / Ded from the CPG using cold 50% (methylamine / ammonia hydroxide) for 3 hours at room temperature, while 3% DEA in ammonia hydroxide (28-30%, cold) was used for the antisense strand. C / D was determined to be complete by IP-RP LCMS when the resulting mass data confirmed sequence identity. Depending on scale, the CPG was filtered through a 0.45 μm PVDF syringeless filter, a 0.22 μm PVDF Steriflip® vacuum filter, or a 0.22 μm PVDF Stericup® Quick Release filter. The CPG was backwashed / rinsed with 30% EtOH / RNAse-free water or 30% EtOH, then filtered through the same filtration device and combined with the first filtrate. This was repeated twice. The material was then divided equally between 50 mL Falcon tubes and filtered through a Genevac™ to remove organics. After concentration, the crude oligonucleotide was returned to synthesis scale by dilution with RNase-free water and filtered through either a 0.45 μm PVDF syringeless filter, a 0.22 μm PVDF Steriflip® vacuum filtration, or a 0.22 μm PVDF Stericup® Quick release.

[0259] The crude oligonucleotides were purified via an AKTA™ Pure purification system using anion exchange (AEX). For AEX, an ES Industry Source™ 15Q column was used, with MPA: 20 mM NaH2PO4, 15% ACN, pH 7.4, and MPB: 20 ​​mM NaH2PO4, 1 M NaBr, 15% ACN, pH 7.4, with the column temperature maintained at 65°C. Fractions containing greater than 85% mass purity and containing no impurities >5% were combined.

[0260] The purified oligonucleotides were desalted using a 15 mL 3K MWCO centrifuge spin tube at 3500 x g for approximately 30 minutes. The oligonucleotides were rinsed with RNase-free water until the eluate reached a conductivity of <100 μg / cm. After desalting was complete, 2-3 mL of RNase-free water was added, followed by 10 cycles of aspiration and transfer of the retention to a 50 mL Falcon tube. This was repeated until complete transfer of the oligos by measuring the concentration of the compound on the filter via nanodrop. The final oligonucleotides were then nanofiltered twice at 3500 x g for 2 minutes through a 15 mL 100K MWCO centrifuge spin tube. The final desalted oligonucleotides were analyzed for concentration (nanodrop at A260) and characterized by IP-RP LC / MS for mass purity (Table 15) and UPLC for UV purity.

[0261] Table 15: Exemplary LC / MS data [Table 20]

[0262] Table 16 - Oligonucleotide synthesis reagents [Table 21]

[0263] Table 17 - Phosphoramidites [Table 22]

[0264] Example 3: Generation of TfR binding protein-dsRNA conjugates Certain abbreviations are defined as follows: "acetonitrile (ACN)" refers to acetonitrile; "aAEX" refers to analytical anion exchange; "AS" refers to antisense strand; "drug / siRNA to antibody / protein ratio (DAR)" refers to drug / siRNA to antibody / protein ratio; "dichloromethane (DCM)" refers to dichloromethane; "dehydroascorbic acid (DHAA)" refers to dehydroascorbic acid; "N,N-diisopropylethylamine (DIEA)" refers to N,N-diisopropylethylamine; "dimethylformamide (DMF)" refers to dimethylformamide; "double stranded ribonucleic acid (dsRNA)" refers to double-stranded ribonucleic acid; "dithiothreitol (DTT)" refers to dithiothreitol; "ethyl acetate (EtOAc)" refers to ethyl acetate; "fluorinated ethylene propylene (FEP)" refers to fluorinated ethylene propylene; and "FMI" refers to Fluid Metering. "Hour, h" refers to time; "hexafluorophosphate azabenzotriazole tetramethyl uranium, HATU" refers to hexafluorophosphate azabenzotriazole tetramethyl uranium; "high-performance liquid chromatography, HPLC" refers to high-performance liquid chromatography; "Liquid Chromatography-Mass Spectrometry, LC / MS" refers to liquid chromatography-mass spectrometry; "linear ion trap mass spectrometer, LTQ / MS" refers to linear ion trap mass spectrometer; "minute, min" refers to minutes; "methyl tert-butyl ether, MTBE" refers to methyl tert-butyl ether; "molecular weight, MW" refers to molecular weight; "N-hydroxysuccinimide, NHS" refers to N-hydroxysuccinimide; and "optical"density, OD" refers to optical density, "phosphate-buffered saline, PBS" refers to phosphate-buffered saline, "polyethylene glycol, PEG" refers to polyethylene glycol, "revolutions per minute, rpm" refers to revolutions per minute, "SEC" refers to size exclusion chromatography, "small interfering RNA, siRNA" refers to small interfering RNA, "succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxamide, SMCC" refers to succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, "SS" refers to the sense strand, "trans-cyclo-octene, TCO" refers to trans-cyclo-octene, "triethylamine, TEA" refers to triethylamine, and "trifluoroacetic acid" refers to trifluoroacetic acid. "TFA" refers to trifluoroacetic acid, "TfR" refers to transferrin receptor, "tetrahydrofuran" refers to tetrahydrofuran, "tris(hydroxymethyl)aminomethane" refers to tris(hydroxymethyl)aminomethane, and "ultraviolet" refers to ultraviolet light.

[0265] Scheme 1

[0266] [ka] Scheme 1, Step A, depicts the coupling of compound (1) with furan-2,5-dione in a solvent such as acetic acid, followed by treatment with acetic anhydride and sodium acetate in a solvent such as toluene to give compound (2). Step B depicts acidic deprotection of compound (2) with an acid such as TFA in a suitable solvent such as DCM, followed by amide coupling with methyltetrazine-PEG4-acid using an amide coupling reagent such as HATU with a suitable base such as N,N-diisopropylamine in a solvent system such as DMF and THF to give compound (3). Those skilled in the art will recognize that a variety of coupling reagents, bases, and solvents can be used to carry out the amide coupling.

[0267] Scheme 2

[0268] [ka] Scheme 2, Step A, shows the transformation of cis-olefin compound 4 to trans-olefin compounds 5 and 6 using a closed-loop flow apparatus employing irradiation and capture on a column of silver nitrate absorbed on silica gel. Step B shows the reaction of compound 5 with N,N'-disuccinimidyl carbonate using a suitable base such as TEA in a solvent system such as ACN to give compound 7.

[0269] Scheme 3

[0270] [ka] Scheme 3, Step A depicts the one-pot reaction of compound (8) with glutaric anhydride using a suitable base such as DIEA in a solvent such as THF, followed by amide coupling with N-hydroxysuccinimide using a suitable coupling reagent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with a suitable base such as 4-dimethylaminopyridine to give compound (9). Those skilled in the art will recognize that a variety of coupling reagents, bases, and solvents can be used to carry out the amide coupling.

[0271] Scheme 4

[0272] [ka] Scheme 4, Step A depicts the coupling of compound 10 with furan-2,5-dione in a solvent such as acetic acid, followed by treatment with TEA in a solvent such as toluene to give compound 11. Step B depicts the conversion of compound 11 to compound 12 in essentially the same manner as Scheme 1, Step B.

[0273] Preparation 1 tert-Butyl 4-[(2-(2,5-dioxopyrrol-1-yl)ethyl]piperazine-1-carboxylate

[0274] [ka] tert-Butyl 4-(2-aminoethyl)piperazine-1-carboxylate (3.00 g, 13.1 mmol) was dissolved in acetic acid (6 mL). Furan-2,5-dione (1.28 g, 13.1 mmol) was added and stirred at ambient temperature for 7 hours. The mixture was then stored in the refrigerator for 18 hours. Most of the acetic acid was removed under vacuum at 50°C. Acetic anhydride (10 mL, 106 mmol) and sodium acetate (1.6 g, 20 mmol) were added, followed by heating to 80°C for 2 hours. Toluene was added, and most of the acetic anhydride was removed under vacuum. The mixture was poured into saturated aqueous ammonium chloride solution (60 mL) and extracted with DCM (3 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give the crude product as a dark oil. Purification via silica gel flash chromatography eluting with EtOAc / hexanes afforded the title compound (2.1 g, 52%). LC / MS m / z 310.3(M+H).

[0275] Preparation 2 tert-Butyl 4-[(3-(2,5-dioxopyrrol-1-yl)propyl]piperazine-1-carboxylate

[0276] [ka] Furan-2,5-dione (789 mg, 7.97 mmol) was added to tert-butyl 4-(3-aminopropyl)piperazine-1-carboxylate (2.00 g, 7.97 mmol) in acetic acid (8 mL, 140 mmol). The mixture was stirred at ambient temperature for 12 hours and then concentrated in vacuo to give the crude intermediate (Z)-4-[3-(4-tert-butoxycarbonylpiperazin-1-yl)propylamino]-4-oxo-but-2-enoic acid (2.72 g, 7.97 mmol), which was then dissolved in toluene (80 mL). TEA (5.6 mL, 40 mmol) and 4 Å molecular sieves (8.8 g) were added. The flask was equipped with a Dean-Stark trap, and the mixture was heated at 120 °C for 48 hours. After cooling to ambient temperature, the solid was removed by filtration and washed with DCM (40 mL). Volatiles were removed under reduced pressure to give a residue, which was dried under vacuum. The thick residue was purified by normal phase chromatography eluting with (10% MeOH / MTBE) / DCM to give the title compound as a yellow flaky powder (353 mg, 13.7%). LC / MS m / z 324 (M+H).

[0277] Preparation 3 1-[2-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperazin-1-yl]ethyl]pyrrole-2,5-dione

[0278] [ka] tert-Butyl 4-[2-(2,5-dioxopyrrol-1-yl)ethyl]piperazine-1-carboxylate (150 mg, 0.485 mmol) was dissolved in DCM (2 mL). TFA (1 mL, 13 mmol) was added and stirred at ambient temperature for 1 hour. Concentration in vacuo and further drying under high vacuum for 18 hours gave the intermediate 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione trifluoroacetate. This material and methyltetrazine-PEG4-acid (130 mg, 0.283 mmol) were dissolved in DMF (2.0 mL) and THF (2 mL). HATU (380 mg, 0.969 mmol) was then added, followed by N,N-diisopropylamine (0.45 mL, 2.6 mmol). Stirring was continued at ambient temperature for 2 hours. Diluted with DCM (50 mL) and washed with saturated aqueous ammonium chloride (30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the crude product as a red solid. Purification via silica gel chromatography eluting with 0-20% MeOH / EtOAc afforded the title compound (150 mg, 49%) as a red solid. LC / MS m / z 628.6 (M+H).

[0279] Preparation 4 1-[3-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperazin-1-yl]propyl]pyrrole-2,5-dione

[0280] [ka] The title compound was prepared in a manner essentially similar to that found in Preparation 3 using tert-butyl 4-[3-(2,5-dioxopyrrol-1-yl)propyl]piperazine-1-carboxylate. LC / MS m / z 642 (M+H).

[0281] Preparation 5 (1R,4E)-Cyclooct-4-en-1-ol (axial) and (1R,4E)-Cyclooct-4-en-1-ol (equatorial)

[0282] [ka] A closed-loop flow apparatus was constructed that allowed irradiation of a solution of cis-olefins and circulation of the solution through silver nitrate absorbed on a silica gel cartridge. Since only the trans-olefins were retained in the silica gel, the cis-olefins were recycled to the irradiation step.

[0283] Equipment: (A) UV lamp (Pen-Ray 099912-1, 254 nM), power supply 99-0055-01, lamp current 18 mA / AC. According to the manufacturer's description, this lamp produces an intensity of 4400-4750 microwatts / cm² at 0.75 inches for 254 nM light. (B) FMI pump set at 10 mL / min, drawing the reaction mixture from a Pyrex® round-bottom flask (250 mL). This was connected to FEP 1 / 16 inch tubing wrapped around a cold finger (7 mL total loop, air-cooled). The UV lamp was placed in the center of the cold finger to irradiate the sample with air cooling. After irradiation, the sample tube was followed by an ISCO SLM containing 25 g of silver nitrate impregnated silica gel (see Fox, et. al., Angewandte Chemie, International Edition Engl 2009, 48(38), 7013-7016; Synthesis 2018, 50, 4875).

[0284] The following procedure was performed. A 50 g silica gel cartridge was loaded with 25 g of silver nitrate adsorbed onto the silica gel top, covered with aluminum foil, and conditioned by pumping a 1:1 hexane / diethyl ether solvent mixture for 1 h. A solution of mixed (4Z)-cyclooct-4-en-1-ol, racemic at the hydroxyl position (2.00 g, 15.8 mmol) and methyl benzoate (2.0 mL, 16 mmol) in n-hexane (220 mL) and diethyl ether (220 mL) was circulated through the silica gel / silver nitrate cartridge at a flow rate of 10 mL / min for 96 h with the UV lamp on, by circulating the solution through a coil around the cold finger and back through the system. The silica cartridge was flushed with EtOAc (200 mL) and air-dried. The filtrate was discarded. The dried silica cartridge was rinsed with concentrated NH4OH (150 mL) followed by DCM (150 mL). Separate the layers and extract the aqueous solution with DCM (2 x 50 mL). Wash the combined organic layers with saturated aqueous sodium chloride, dry over MgSO4, filter, and concentrate under reduced pressure. Purification via silica gel chromatography eluting with 0-45% MTBE / hexanes afforded two products as a clear liquid. Axial-(1R,4E)-cyclooct-4-en-1-ol (569.8 mg, 28.5%). 1 H NMR (CDCl) 5.63-5.55 (m, 1H), 5.44-5.36 (m, 1H), 3.50-3.45 (m, 1H), 2.39-2.32 (m, 3H), 2.00-1.94 (m, 4H), 1.73-1.66 (m, 3H). Equatorial-(1R,4E)-cyclooct-4-en-1-ol (673.6 mg, 33.7%). 1 H NMR(CDCl3):5.60-5.57(m,2H),4.05(dd,J=5.3,10.2Hz,1H),2.44-2.37(m,1H) ),2.29-2.22(m,2H),2.18-2.13(m,2H),1.93-1.86(m,4H),1.32-1.25(m,1H).

[0285] Preparation 6 (1R,4E)-cyclooct-4-en-1-yl](2,5-dioxopyrrolidin-1-yl) carbonate

[0286] [ka] To a mixture of 1R,4E)-cyclooct-4-en-1-ol (axial) (569 mg, 4.50 mmol) and TEA (2.5 mL, 18 mmol) in ACN (25 mL) was added N,N'-disuccinimidyl carbonate (2.79 g, 10.3 mmol) in small portions (approximately 250-300 mg each addition, 5 min apart). The mixture was covered with aluminum foil and stirred at ambient temperature for 60 h. The solvent was removed under reduced pressure to give an oil, which was partitioned between water (20 mL) and diethyl ether (50 mL). The layers were separated, and the aqueous solution was extracted with diethyl ether (2 × 50 mL). The organic layer was washed with saturated aqueous ammonium chloride, then saturated aqueous sodium chloride, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification using silica gel chromatography, eluting with 0-60% MTBE / hexane, gave the title compound as a colorless residue which formed a white solid (732 mg, 61%). LC / MS m / z 324 (M+H).

[0287] Preparation 7 (2,5-dioxopyrrolidin-1-yl)4-[[2-methyl-2-(2-pyridyldisulfanyl)propyl]amino]-4-oxo-butanoate

[0288] [ka] 2-Methyl-2-(2-pyridyldisulfanyl)propan-1-amine hydrochloride (245 mg, 0.976 mmol), glutaric anhydride (112 mg, 0.972 mmol), and DIEA (360 μL, 2.16 mmol) were added together in THF (4 mL) and heated at 45° C. with vigorous stirring for 12 hours. After this time, the mixture was cooled to ambient temperature, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (224 mg, 1.17 mmol) and 4-dimethylaminopyridine (25 mg, 0.20 mmol) were added. After stirring the mixture at ambient temperature for 5 minutes, N-hydroxysuccinimide (126 mg, 1.07 mmol) was added in one portion, followed by stirring for 36 hours. The mixture was filtered, and the resulting filtrate was loaded directly onto silica gel (2 g). Purification using silica gel chromatography eluting with 75% EtOAc / hexanes gave the title compound as a light cloudy residue (100.2 mg, 24%). LC / MS m / z 426 (M+H) (hydrolyzed NHS ester).

[0289] TCO-functionalized SNCA In a set of four 50 mL Falcon™ tubes, 10 mL each of the sense strand of SNCA dsRNA (SNCA_SS-3C6A) with a hexylamine chain attached to the 3' end (measured SS concentration calculated to be 412.5 or 2 mM OD / mL, 120 mL, 0.24 mmol) and 6 mL of 20x borate buffer were aliquoted and treated with 7.5 mL of a solution of [(1R,4E)-cyclooct-4-en-1-yl](2,5-dioxopyrrolidin-1-yl)carbonate (1.65 g, 6.17 mmol) dissolved in 1,4-dioxane (100 mL). Mixing was performed at 25 °C and 600 rpm for 30 minutes. The remainder of the SS sample was divided and reacted in the same manner, yielding a total of 12 sample vessels, each containing approximately 150 mg of crude SS starting material. Dioxane was removed by placing the Falcon™ tube on a Genevac evaporator. The remaining aqueous solutions were combined and filtered to remove suspended solids. Purification was performed on an AKTA™ pure chromatography system using 13-45% ACN in 50 mM NaOAc (aq) at a flow rate of 40 mL / min. The appropriate fractions were combined and removed from the organics on a SpeedVac™ before desalting and concentrating to give 214 mL, which measured an OD / mL of 127.3, equivalent to 624 μM, and a total of 973 mg. LTQ / MS m / z 7292, UV purity 99+%.

[0290] TCO-SNCA duplex The Nanodrop concentrations (five replicate averages) of each strand in water were measured as SS = 624 μM and AS = 1094 μM. 210 mL of SS and 113.7 mL of AS were mixed and then shaken at ambient temperature for 30 minutes. The amount of remaining SS strand was measured to completion, requiring the addition of an additional 21.9 mL of AS. The resulting 345 mL solution was measured (Nanodrop™ Lite, six replicate averages, 20-fold dilution) to have an OD / mL of 159.5, which equates to 421 μM, for a total of 2.19 g. LTQ / MS m / z 7291, 7825, UV purity >99%.

[0291] SMCC-functionalization of SNCA dsRNA A freshly prepared solution of (2,5-dioxopyrrolidin-1-yl) 4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarboxylate (185 mg, 0.542 mmol) in THF (50 mL) was added to SNCA_SS-3C6A (44 mL, 0.0528 mmol, 250.4 OD / mL in 0.2 M phosphate buffer (44 mL), or approximately 1200 μM (approximately 8.8 mg / mL)). The mixture was vortexed vigorously for 2 min and then shaken at 900 rpm at ambient temperature for a total of 2 h. Analysis by LTQ indicated approximately 94-95% conversion. The mixture was acidified to pH 4 with 20-30 drops of 5 N HCl, followed by removal of organics in a Genevac concentrator. The mixture was desalted by centrifugal filtration in a 3K spin filter (4 × 4000 rpm, 30 min), and the retentate was pooled. The OD measurement of the solution (average of three measurements, 10-fold dilution) was 266, equivalent to 1.3 mM and a total of 316 mg. The extinction coefficient was 204.12. LTQ / MS m / z 7358.

[0292] SMCC-SNCA duplex The Nanodrop concentrations of each strand in water (averaged three times) were measured as SS = 1322 μM and AS = 1108 μM. 32 mL of SS and 36.2 mL of AS were mixed and shaken at 30°C for 30 minutes. The amount of residual SS strand was measured to completion, requiring the addition of an additional 360 μL of AS. Endotoxin was removed by filtration through a 0.45 μM filter. The resulting 75 mL solution was measured (Nanodrop™ Lite, average of five times, 10-fold dilution) to be 217 OD / mL, which is equivalent to 575 μM, for a total of 653 mg. LTQ / MS m / z 7358, 7825, UV purity 99+%.

[0293] GDM-functionalized SNCA In a 15 mL Falcon™ tube, SNCA_SS-3C6A (measured SS concentration calculated to be 247.6 or 1.21 mM OD / mL, 3 mL, 0.0036 mmol) was diluted with 20× borate buffer (0.3 mL) and water (3 mL, 166.530 mmol), followed by the addition of (2,5-dioxopyrrolidin-1-yl)5-[[2-methyl-2-(2-pyridyldisulfanyl)propyl]amino]-5-oxo-pentanoate (3.6 mL, 0.75 M in dioxane). The mixture was mixed at 200 rpm for 1 hour. The organics were removed with a SpeedVac™, desalted, and concentrated three times with water to give SNCA_SS-3C6A-GDM in a total yield of 13.2 mL (35.88 OD / mL, equivalent to 175.8 μM and a total of 17.3 mg). The extinction coefficient was 204.12. LTQ1 MS m / z m / z 7449 (UV purity 95+%).

[0294] 2 Tris(2-carboxyethyl)phosphine hydrochloride (75 μL of a 100 mM solution in water) was added to SNCA_SS-3C6A-GDM. Shaking was continued for 4 hours at 10°C, followed by 16 hours at ambient temperature. Additional tris(2-carboxyethyl)phosphine hydrochloride (75 μL of a 100 mM solution in water) was added and the mixture was shaken for an additional 16 hours. The mixture was desalted by centrifugal filtration through a 3K spin filter (3 × 40 minutes, 4000 rpm), and the retentate was pooled to obtain 10 mL. The OD measurement of the solution (average of four measurements, 10-fold dilution) was 63.6, equivalent to 311.4 μM and a total of 22.9 mg. The extinction coefficient was 204.12. LTQ / MS m / z 7340, UV purity 99+%.

[0295] GDM annealing process The Nanodrop concentrations of each strand in water (averaged four times) are SS = 311.4 μM and AS = 431.3 μM. 10 mL of SS and 6.7 mL of AS were mixed with 5 mL of water and shaken for 30 minutes. The amount of remaining SS strand was measured to completion, requiring the addition of an additional 560 μL of AS. Concentration was performed with a 3K MW cutoff filter (20 minutes), followed by 50K spin filtration and further concentration through a 3K filter. The resulting 6 mL solution was measured (Nanodrop™ Lite, average of five times, 20-fold dilution) to be 181.62 OD / mL, which is equivalent to 486 μM, for a total of 44.2 mg. LTQ / MS m / z 7340, 7825, UV purity 99+%.

[0296] MAPT dsRNA functionalization and annealing can be carried out in the same manner as for SNCA dsRNA described above.

[0297] Conjugation of dsRNA to TfR-binding proteins dsRNA was conjugated using site-specific native or engineered cysteine ​​amino acid residues in the TfR-binding protein. Cysteines can be engineered into the primary amino acid sequence of the TfR-binding protein. Approaches for introducing cysteines as a means for conjugation are described in International Publication No. 2018 / 232088, both of which are incorporated by reference in their entireties, and specifically incorporated with respect to conjugation via cysteine ​​residues. For engineered cysteine ​​conjugation, the TfR-binding protein was first reduced with 40 molar equivalents of the reducing agent dithiothreitol (DTT) at 37°C for 2 hours, followed by dialysis or desalting via a desalting column to remove the reducing agent. After this, the TfR-binding protein was reoxidized to reform the structural disulfide by incubating with 10 molar equivalents of dehydroascorbic acid (DHAA) at room temperature for 2 hours. The oxidizing agent was then removed by desalting.

[0298] Conjugation of dsRNA to TfR binding proteins was performed using the following method.

[0299] Conjugation Scheme 1 In the first method, a bifunctional maleimide-methyl-tetrazine linker was conjugated to an engineered cysteine ​​of the TfR-binding protein at neutral pH by adding the linker to the TfR-binding protein at 20 molar equivalents and incubating at ambient temperature for 1 hour. A desalting step was then performed to remove excess linker. Then, trans-cyclooctene (TCO)-functionalized dsRNA was added to the protein linker at 4 molar equivalents for overnight conjugation at 4°C. Step 1a: Conjugation of TfR-binding proteins using a maleimide-methyl-tetrazine linker

[0300] [ka] Step 1b: Conjugation of TfR-binding proteins via maleimide-methyl-tetrazine linker ring opening

[0301] [ka] Step 2a: dsRNA conjugation with a protein-linker intermediate

[0302] [ka] Step 2b: dsRNA conjugation with the protein-linker intermediate (open ring)

[0303] [ka]

[0304] Conjugation Scheme 2 The second conjugation method utilized SMCC-functionalized dsRNA to conjugate onto the engineered cysteine ​​of the TfR-binding protein. For this method, the TfR-binding protein was prepared as described above, and the engineered thiol was made available for conjugation by undergoing a reduction and oxidation process of the TfR-binding protein. Following this, the SMCC-dsRNA was incubated with 4 molar equivalents of the TfR-binding protein at 4°C overnight for conjugation.

[0305] Optionally, a maleimide hydrolysis step can be performed after conjugation to lock the linker-payload in place and prevent deconjugation in the human systemic circulation via retro-Michael addition. This succinimide ring hydrolysis process was carried out by increasing the conjugate pH to 9.0 using 50 mM arginine (a 0.7 M arginine stock solution, pH 9.0 was used) and incubating the solution at 37 °C for 20 hours. The hydrolysis state of the maleimide was confirmed by LCMS characterization of +18 Da resulting from water addition to the succinimide ring.

[0306] Step 1a: Conjugation of TfR-binding protein with SMCC linker [ka]

[0307] Step 1b: Conjugation of TfR-binding protein by SMCC linker opening [ka]

[0308] Conjugation Scheme 3 The third conjugation method utilized GDM-functionalized dsRNA to conjugate to an engineered cysteine ​​of the TfR-binding protein via a disulfide bond. For this method, the TfR-binding protein was prepared as described above, and the engineered thiol was made available for conjugation by undergoing a reduction and oxidation process of the TfR-binding protein. Dithiobis(5-nitropyridine) was then added to the protein at 20 molar equivalents to generate an intermediate prior to dsRNA conjugation. Excess dithiobis(5-nitropyridine) was removed by desalting. In the second step, GDM-functionalized dsRNA was added to the protein intermediate at 4 molar equivalents. Dithiobis(5-nitropyridine) acts as a leaving group in this reaction and is displaced by GDM-dsRNA.

[0309] Step 1: TfR-binding protein conjugation with dithiobis(5-nitropyridine) to generate intermediates [ka]

[0310] Step 2: dsRNA conjugation with GDM-functionalized dsRNA [ka]

[0311] Conjugation was monitored using analytical anion exchange chromatography using a ProPac™ SAX-10 HPLC column (10 μm particles, 4 mm diameter, 250 mm length) with the following procedure: flow rate 1 mL / min, Buffer A: 20 mM TRIS pH 7.0, Buffer B: 20 ​​mM TRIS pH 7.0 + 1.5 M NaCl, 30° C.

[0312] Table 18A: HPLC gradients used to assess dsRNA conjugation to TfR binding proteins TBP10 and TBP11 [Table 23]

[0313] Table 18B: HPLC gradients used to assess dsRNA conjugation to the TfR binding protein TBP14 [Table 24]

[0314] The drug / siRNA antibody / protein ratio (DAR) was calculated based on the peak area percentage from analytical anion exchange (aAEX) chromatogram. An illustrative example of a chromatogram of a TBP11-dsRNA conjugate before purification is shown in Figure 1A. Figure 1C shows an exemplary aAEX chromatogram of the DAR profile for a TBP15-dsRNA conjugate before purification.

[0315] After conjugation of dsRNA to the TfR-binding protein, excess dsRNA and unconjugated proteins were removed by further purification. Either preparative size-exclusion chromatography (SEC) or preparative analytical anion-exchange chromatography was used to purify the final conjugate. Preparative SEC was performed using Cytiva Superdex® 200 in 1×PBS pH 7.2 under isocratic conditions. Alternatively, anion-exchange chromatography, such as ThermoFisher POROS™ XQ, was used with a starting buffer of 20 mM TRIS pH 7.0 and eluted with a 20-column volume gradient using a buffer containing 20 mM TRIS pH 7.0 and 1 M NaCl. These results yielded purified TfR-binding protein-dsRNA conjugates lacking excess dsRNA and minimal unconjugated proteins. The resulting conjugate profiles were analyzed by analytical anion exchange for final DAR quantification (see Figures 1B and 1D and Table 19). An example chromatogram of the purified TBP14-dsRNA conjugate is shown in Figure 1B. Figure 1D shows an exemplary aAEX chromatogram of the DAR profile for the purified TBP15-dsRNA conjugate.

[0316] Table 19. siRNA / drug to TBP / antibody ratio (DAR) [Table 25]

[0317] Example 4: In vitro characterization of mouse TfR binding protein-dsRNA conjugates Assessment of in vitro binding, internalization, and degradation in mouse cortical neurons The fluorescent signals corresponding to the total levels and internalization of TfR-binding protein or TfR-binding protein-siRNA conjugate (ARC) were measured in primary mouse cortical neurons by performing a high-content live-cell imaging assay. Briefly, primary mouse cortical neurons were isolated from wild-type C57BL6 mouse embryos at E18. Cells were plated at a density of 40,000 cells / well in poly-D-lysine-coated 96-well plates and cultured in NbActiv1 (BrainBits, LLC) containing 1% antibiotic / antimycotic (Corning) at 37°C for 7 days in a tissue culture incubator in a humidified chamber with 5% CO2. On day 7, the medium was removed from each well and the wells were incubated with DyLight 650 (Thermo Biosciences) along with BHQ3 dye (BioSearch Tech BHQ-3000S-5) or pHAb dye (Promega #G9845) in culture medium containing 6.7 uM (1 mg / ml) goat gamma globulin (Jackson Immuno #005-000-002) in culture medium containing 5 ug / ml (33 nM) of either (i) isotype Ab (isotype control antibody), (ii) mTBP2 (heterodimeric antibody comprising a monovalent mouse TfR-binding arm and an isotype control arm), (iii) isotype Ab-SNCA siRNA (isotype control antibody with dsRNA number 8 linked to heavy chain constant region 1), or (iv) mTBP2-SNCA siRNA (mTBP2 with dsRNA number 8 linked to heavy chain constant region 1). The cells were substituted with 10 μg / ml (0.2 μM) of anti-human IgG Fcγ fragment-specific Fab fragment (Jackson Immuno #109-007-008) labeled with either DL650 (Fisher #62266) or DL650, and incubated overnight with live cells grown at 37°C in a 96-well plate.

[0318] The next day, cells were washed, incubated with NucBlue Hoechst dye (Thermo Fisher #R37605) for 20 minutes, washed again, and then imaged using a Cytation5 high-content imager (Biotek). The DyLight650 signal measures total TfR-bound protein levels, the DyLight650+BHQ3 signal measures the degradation signal resulting from increased DyLight 650 fluorescence when the BHQ3 dye is released and FRET quenching is lost, and the pHAb pH sensor dye signal measures internalized fluorescence only. Excess goat gamma globulin was added to reduce nonspecific binding and uptake of the antibody into the cells. The signal intensity per cell was determined by dividing the signal intensity in each well by the number of Hoechst-stained nuclei. Wells were analyzed in duplicate, and approximately 20,000 cells were analyzed for each well from images taken with a 4x objective. Background signal was determined from a human IgG isotype control and subtracted from the final value.

[0319] The results are shown in Figure 2. High-content imaging data demonstrate the cellular activity (binding, internalization, and degradation properties) of the exemplary mouse TfR-binding proteins and isotype control antibodies. While the isotype control antibody and isotype control antibody-dsRNA conjugate lacked activity, binding, internalization, and degradation activity was demonstrated for the exemplary mouse TfR-binding proteins in mouse primary cortical neurons. Additionally, conjugation to dsRNA does not substantially alter the activity of the exemplary mouse TfR-binding proteins.

[0320] In vitro potency assessment in mouse cortical neurons Mouse primary cortical neurons were isolated from wild-type C57BL6 mouse embryos at E18 and cultured as described above. On day 7, half of the medium was removed from each well, and a two-fold concentration of one of the following treatments was added in culture medium containing 2% FBS: (i) chol-teg-siSNCA (cholesterol-conjugated dsRNA #7), (ii) naked SNCA siRNA (unconjugated SNCA siRNA), (iii) isotype Ab-SNCA siRNA (isotype control antibody with dsRNA #7 linked to HC constant region 1), or (iv) mTBP2-SNCA siRNA (mTBP2-dsRNA #8 conjugate, dsRNA linked to HC constant region 1 of mTBP2). The cells were incubated for another 7 days. At the end of the treatment, RT-qPCR was performed to quantify targeted mRNA levels using the TaqMan Fast Advanced Cell-to-CT kit. Specifically, cells were lysed, cDNA was generated using a Mastercycler X50a (Eppendorf), and qPCR was performed using a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). SNCA gene expression levels were normalized to β-actin using the respective probes (ThermoFisher).

[0321] The results are provided in Figure 3 and Table 20. The results provided in Table 20 demonstrate that exemplified mouse TfR binding protein-siRNA conjugates (e.g., mTfR2-dsRNA No. 8 conjugates) successfully target mouse SNCA, providing potency several orders of magnitude greater than unconjugated siRNA (i.e., naked siRNA) and isotype Ab-SNCA siRNA, and comparable to or superior to the potency of cholesterol-conjugated siRNA.

[0322] Table 20: In vitro potency of the indicated molecules to reduce mouse SNCA mRNA in mouse cortical neurons [Table 26]

[0323] Example 5: In vitro characterization of human TfR binding protein-dsRNA conjugates Assessment of in vitro binding, internalization, and degradation in SHSY5Y cells SH-SY5Y cells (ATCC CRL-2266, passages 5–20) were maintained in a medium consisting of 225 ml of MEM / EBSS (Hyclone: ​​SH30024.02, Gibco 11095-072), 10% heat-inactivated fetal bovine serum (Hyclone: ​​SH30071.03), 1× sodium pyruvate (100×, Hyclone: ​​SH30239.01), 1× non-essential amino acids (100×, Hyclone: ​​SH30238.01), and 7.5% sodium bicarbonate (Hyclone: ​​SH30033.01), and 225 ml of HAM's F12 (Corning Cellgro 10-080CV). Cells were plated at 120,000 / well and grown in fibronectin-coated black 96-well plates (Falcon #353219) at 37°C and 90% humidity in a tissue culture incubator (Thermo Scientific Forma Series 3 Water Jacketed) for 4 days. On day 4, the medium was removed from each well and replaced with culture medium containing 5 μg / ml (33 nM) of either an isotype control antibody (isotype Ab), a conjugate linked to TBP10, TBP11, or SNCA siRNA (dsRNA #8), along with 10 μg / ml (0.2 μM) of an anti-human IgG Fcγ fragment-specific Fab fragment (Jackson Immuno #109-007-008) labeled with either DyLight 650 (Thermo Fisher #62266), DL650, along with BHQ3 dye (BioSearch Tech BHQ-3000S-5) or pHAb dye (Promega #G9845) in culture medium containing 6.7 μM (1 mg / ml) goat gamma globulin (Jackson Immuno #005-000-002), and incubated overnight at 37°C with live cells grown in a 96-well plate.

[0324] The next day, cells were washed, incubated with NucBlue Hoechst dye (Thermo Fisher #R37605) for 20 minutes, washed again, and then imaged using a Cytation5 high-content imager (Biotek). The DyLight650 signal measures total TfR-bound protein levels, the DyLight650+BHQ3 signal measures the degradation signal resulting from increased DyLight650 fluorescence when the BHQ3 dye is released and FRET quenching is lost, and the pHAb pH sensor dye signal measures internalized fluorescence only. Excess goat gamma globulin was added to reduce nonspecific binding and uptake of the antibody into the cells. The signal intensity per cell was determined by dividing the signal intensity in each well by the number of Hoechst-stained nuclei. Wells were analyzed in duplicate, and approximately 20,000 cells were analyzed for each well from images taken with a 4x objective. Background signal was determined from a human IgG isotype control and subtracted from the final value.

[0325] The results are shown in Figure 4. High-content imaging data demonstrate the cellular activity (binding, internalization, and degradation characteristics) of the exemplary human TfR binding proteins and isotype control antibodies. Binding, internalization, and degradation activity were demonstrated for the exemplary human TfR binding proteins on SH-SY5Y cells, whereas the isotype control antibody lacked substantial activity. Additionally, conjugation to dsRNA does not reduce the activity of the exemplary human TfR binding proteins.

[0326] In vitro potency evaluation in SYSY5Y cells SH-SY5Y cells (ATCC CRL-2266, passages 5–20) were maintained as described above. On day 4, the medium was removed from each well and replaced with culture medium containing one of the following: an isotype control antibody siRNA conjugate (isotype Ab-SNCA siRNA), a TBP10-SNCA siRNA conjugate, or a TBP11-SNCA siRNA conjugate in culture medium with 2% FBS added for treatment. The cells were then incubated for another 7 days. At the end of the treatment, RT-qPCR was performed to quantify target mRNA levels using the TaqMan Fast Advanced Cell-to-CT kit. Specifically, cells were lysed, cDNA was generated using a Mastercycler X50a (Eppendorf), and qPCR was performed using a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). SNCA gene expression levels were normalized to β-actin using the respective probes (ThermoFisher).

[0327] The results are provided in Table 21 and Table 5. The results provided in Table 21 demonstrate that exemplary human TFR binding protein-siRNA conjugates provide potency in knocking down the human SNCA gene, while the isotype control antibody conjugates showed low activity.

[0328] Table 21. In vitro potency for reducing human SNCA mRNA in SH-SY5Y cells [Table 27]

[0329] Example 6: Demonstration of in vivo proof-of-concept of pharmacodynamic efficacy of mouse TfR binding protein-dsRNA conjugates in the CNS with peripheral delivery In vivo pharmacodynamic evaluation in mice using multiple IV dosing To demonstrate that mouse TfR-binding protein-siRNA conjugates cross the BBB and deliver siRNA cargo to the CNS to reduce SNCA mRNA gene expression, a series of proof-of-concept studies were performed to evaluate the pharmacodynamic efficacy of the constructs using peripheral delivery in mice. Eight-week-old FVB mice were administered PBS control, isotype Ab-SNCA siRNA, or mTBP2-SNCA siRNA (mTBP2 SNCA-dsRNA no. 8 conjugate) at an effective siRNA concentration of 10 mg / kg. Four weekly doses were administered, with sacrifice at 28 days after the first dose (see Figures 6A and 6B), or a single dose was administered, with sacrifice at 7, 28, 70, or 120 days (see Figures 6C and 6D). In addition, mouse anti-CD4 antibody (GK1.5) was administered at 10 mg / kg for 2–3 days prior to the study to deplete CD4+ T cells and mitigate undesirable pharmacokinetic consequences resulting from spurious anti-drug antibody responses to the injected compound. At the designated time points, mice were fully anesthetized and then cardiac-perfused with cold PBS (6 ml / min for 5 min) until blood was completely removed. Brains and spinal cords were collected and target mRNA levels in tissue homogenates were assessed by RT-qPCR, and target protein levels were assessed by ELISA. For RT-qPCR, RNA was isolated using the RNeasy Plus Universal Mini Kit (Qiagen 73404). Briefly, hemibrain, spinal cord, and DRG tissue homogenates were prepared with FastPrep-24 Lysing Matrix D beads. The tissues were homogenized in an MP Fastprep 24 (MP Biomedical) at 6 m / s for 40 s at 4°C. The vials were then centrifuged to collect the supernatant. RNA was then collected and the amount of RNA determined by the A260 / A280 ratio using a spectrophotometer. cDNA was then generated using a Mastercycler X50a (Eppendorf) and qPCR was performed using a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). SNCA gene expression levels were normalized to β-actin using the respective probes (ThermoFisher).

[0330] The results are shown in Figures 6A-6C. IV administration of multiple doses of mTBP2-SNCA-siRNA in mice resulted in a robust 91% reduction in SNCA mRNA and a 41% reduction in SNCA protein in the brain 28 days after the first dose compared with PBS-treated controls (Figure 6A). Importantly, isotype Ab-SNCA siRNA did not induce a significant reduction in SNCA mRNA, demonstrating the need for active TfR-mediated transport to deliver siRNA cargo to the CNS, demonstrating crossing of the BBB and delivery to the brain. Furthermore, evaluation of the spinal cord 28 days after the first dose also demonstrated robust reductions in SNCA mRNA in the cervical, thoracic, and lumbar regions, with 79%, 79%, and 73% reductions in SNCA mRNA, respectively, with mTBP2-SNCA siRNA compared with PBS-treated controls (Figure 6B). There was also a significant 61% reduction in SNCA mRNA in the lumbar dorsal root ganglia (DRG) (Figure 6C). Interestingly, with isocontrol Ab-SNCA siRNA, there was a low but significant reduction in SNCA mRNA in the cervical and thoracic spinal cord and lumbar DRG, suggesting that the level of spinal cord and DRG siRNA delivery may be limited without TfR-mediated delivery.

[0331] Example 7: Demonstration of in vivo proof-of-concept of time-course efficacy of mouse TfR binding protein-dsRNA conjugates in the CNS with peripheral delivery In vivo pharmacodynamic time course evaluation in mice using a single IV dose The high efficacy of mTBP2-SNCA siRNA in the brain and spinal cord with multiple IV doses suggested that significant efficacy from a single dose was likely. Therefore, a follow-up proof-of-concept study was conducted to determine single IV dose efficacy and the pharmacodynamic efficacy over time for SNCA mRNA and protein levels to inform subsequent study design in non-human primates. For each time point, five mice were sacrificed and tissues were collected for the above analyses.

[0332] As shown in Figure 7A, a single IV administration of mTBP2 SNCA-siRNA in mice resulted in a robust reduction of SNCA in the brain compared with PBS-treated controls, beginning at day 7 post-dose (60% mRNA reduction, 22% protein reduction), with a maximum reduction at day 28 (73% mRNA reduction, 41% protein reduction), followed by a sustained reduction at day 70 (34% mRNA reduction, 45% protein reduction), and returning to the PBS baseline group at day 120 (6% mRNA reduction and 19% protein reduction).

[0333] Furthermore, as shown in Figure 7B, a single IV administration of mTBP2 SNCA also resulted in a robust reduction of SNCA in the spinal cord compared to the PBS-treated control group, beginning at day 7 after mRNA-only dosing (48% mRNA reduction, 3% protein reduction), followed by a reduction in both mRNA and protein at day 28 (48% mRNA reduction, 27% protein reduction), followed by a sustained reduction at day 70 (32% mRNA reduction, 48% protein reduction), and returning to the PBS baseline group at day 120 (23% mRNA reduction and 14% protein reduction).

[0334] Example 8: In vivo characterization of human TfR binding protein-dsRNA conjugates 8A. In vivo pharmacodynamic evaluation of a single dose of human TfR binding protein-SNCA siRNA conjugate in non-human primates (NHPs) after 29 days.

[0335] Following the robust proof-of-concept demonstration of peripheral siRNA delivery across the BBB to the CNS in mice, the pharmacodynamic properties of the human TfR binding protein-SNCA siRNA conjugate were evaluated in NHPs as follows. Cynomolgus monkeys weighing 2-3 kg were intravenously administered i) PBS (n = 8), ii) TBP10-SNCA siRNA (TB10-dsRNA No. 8 conjugate) at an effective siRNA concentration of 8.8 mg / kg (n = 6), or iii) TBP11-SNCA siRNA (TBP11-dsRNA No. 8 conjugate) at an effective siRNA concentration of 2.6 mg / kg (n = 6) into the femoral saphenous vein and sacrificed 29 days after the first dose. Deeply anesthetized animals underwent cardiac perfusion, and brain, spinal cord, and peripheral tissues were then collected. Brains were sectioned coronally, and 3 mm punches were collected from the indicated subregions and frozen. Tissues were collected from the spinal cord, liver, and muscle, and target mRNA levels in tissue homogenates were assessed by RT-qPCR. Total RNA from NHP tissues was isolated manually using the RNadvance Tissue Kit (Beckman Coulter, Indianapolis, IN) or on a Biomek i7 liquid handler (Beckman Coulter) according to the manufacturer's protocol with some modifications. Briefly, frozen tissue sections were mixed with one 5 mm stainless steel bowl, lysis buffer, and proteinase K and homogenized in a 2010 GenoGrinder (SPEX SamplePrep, Metuchen, NJ) at 1200 rpm for five 30-second cycles with a 20-second interval between cycles. Tissues from several regions were shaved on dry ice before homogenization. The homogenates were incubated at 37°C for 1 hour and then extracted with an equal volume of phenol-chloroform. RNA in the supernatant was purified using the RNadvance tissue kit, which included a 30-minute digestion with DNase. The concentration and purity (A260 / A280) of the RNA eluate were determined spectrophotometrically. RNA was normalized to 15 ng / 10 μL PCR and re-digested with ezDNase (dsDNA-specific) before reverse transcription using the SSIV VILO kit (Thermo Fisher Scientific, Waltham, MA).The expression of each gene target in the cDNA was determined using TaqMan qPCR assays on the QuantStudio 7 Pro platform (ThermoFisher Scientific). SNCA gene expression was normalized by β-actin using the respective probes (ThermoFisher). The analyzed tissues and their acronyms are as follows: liver; gastrocnemius; AN, arcuate nucleus; MedEm, median eminence; LSC, lumbar spinal cord; medulla, pons; CB, cerebellum, midbrain; SN, substantia nigra; caudate nucleus; PUT, putamen; HT, hypothalamus; H, hippocampus; PFC, prefrontal cortical gray matter; PFC, prefrontal cortical white matter.

[0336] Peripheral IV administration of 8.8 mg / kg TBP10-SNCA siRNA in NHPs resulted in a significant reduction in SNCA mRNA in key brain regions and the lumbar spinal cord compared with the PBS-treated group at 29 days post-dosing. As shown in Figure 8A, significant SNCA mRNA reductions were demonstrated in the liver (48%), arcuate nucleus (58%), lumbar spinal cord (82%), medulla (71%), pons (77%), midbrain (56%), substantia nigra (76%), caudate nucleus (81%), putamen (76%), hypothalamus (64%), hippocampus (83%), prefrontal cortical gray matter (74%), and prefrontal cortical white matter (76%). Other brain regions and tissues evaluated did not demonstrate significant reductions in SNCA mRNA, as shown (Figure 8A).

[0337] Peripheral IV administration of TBP11-SNCA siRNA at a lower dose of 2.6 mg / kg in NHPs also resulted in a significant reduction in SNCA mRNA in key brain regions and the lumbar spinal cord compared with the PBS-treated group 29 days after dosing. As shown in Figure 8B, significant SNCA mRNA reductions were demonstrated in the lumbar spinal cord (62%), medulla (63%), pons (48%), substantia nigra (66%), caudate nucleus (59%), hippocampus (72%), and prefrontal cortical gray matter (39%). Other brain regions and tissues evaluated did not demonstrate significant reductions in SNCA mRNA, as shown (Figure 8B).

[0338] To determine the expected level of brain SNCA mRNA reduction at NHP-equivalent doses, cohorts of mice received a single IV dose of mTBP2-SNCA siRNA at equivalent 8.8 mg / kg and 2.6 mg / kg concentrations, treated as above, and assessed the translatability of mRNA KD efficacy by RT-qPCR.

[0339] Mice administered intravenously at a dose equivalent to an effective siRNA concentration of 8.8 mg / kg demonstrated a 69% reduction in SNCA mRNA in the brain, while dosing at an effective siRNA concentration of 2.6 mg / kg demonstrated a 53% reduction in SNCA mRNA in the brain, demonstrating that similar efficacy translates from rodents to NHPs ( Figure 8C ).

[0340] 8B. In vivo pharmacodynamic evaluation of human TfR binding protein-SNCA siRNA conjugate in NHPs after 85 days of a single dose or three monthly doses. To determine the efficacy of the human TfR binding protein-SNCA siRNA conjugate, a pharmacodynamic study was conducted after a single dose or three monthly doses. Cynomolgus monkeys weighing 2-3 kg were administered either a single intravenous dose of 10 mg / kg TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (n=5) into the femoral saphenous vein, or three monthly intravenous doses of 10 mg / kg i) PBS (n=5) or ii) TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (n=5) into the femoral saphenous vein. To mitigate anti-drug antibody responses, all groups received 30 mg / kg of anti-CD4 antibody immediately after the test substance dose. Eighty-five days after a single dose or after the first dose in a three-times-monthly dosing regimen, deeply anesthetized animals underwent cardiac perfusion, followed by collection of brain, spinal cord, and peripheral tissues.

[0341] Brains were sectioned coronally, and 4 mm punches were collected and frozen from the indicated subregions. Tissues were collected from the spinal cord, liver, and muscle. Target mRNA and protein levels in tissue homogenates were assessed by RT-qPCR and ELISA, respectively. To determine mRNA levels, total RNA from NHP tissues was isolated manually using the RNadvance Tissue Kit (Beckman Coulter, Indianapolis, IN) or on a Biomek i7 liquid handler (Beckman Coulter) according to the manufacturer's protocol with some modifications. Briefly, frozen tissue sections were mixed with one 5 mm stainless steel bowl, lysis buffer, and proteinase K and homogenized in a 2010 GenoGrinder (SPEX SamplePrep, Metuchen, NJ) at 1200 rpm for five 30-second cycles with a 20-second interval between cycles. Tissues from several regions were shaved on dry ice prior to homogenization. The homogenate was incubated at 37°C for 1 hour and then extracted with an equal volume of phenol-chloroform. RNA in the supernatant was purified using the Rnadvance Tissue Kit, which included a 30-minute digestion with DNase. The concentration and purity (A260 / A280) of the RNA eluate were determined spectrophotometrically. RNA was normalized to 15 ng / 10 μL PCR and re-digested with ezDNase (dsDNA-specific) before reverse transcription using the SSIV VILO kit (Thermo Fisher Scientific, Waltham, MA). Expression of each gene target in the cDNA was determined using TaqMan qPCR assays on the QuantStudio 7 Pro platform (Thermo Fisher Scientific). Gene expression levels of SNCA were normalized to β-actin using the respective probes for CNS regions (ThermoFisher) and GAPDH (ThermoFisher) for gastrocnemius muscle.

[0342] To determine α-synuclein protein levels, frozen 4 mm punches of neural tissue biopsies were mixed with cold RIPA buffer (Pierce #89901, Thermo Scientific, Waltham, MA) containing protease and phosphatase inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail, Thermo Scientific) at a ratio of 20 mL of buffer to 1 gram of tissue. The tissue-RIPA mixture was homogenized using 5 mm stainless steel beads on a 2010 GenoGrinder (Spex SamplePrep, Metuchen, NJ). The homogenate was then centrifuged in a refrigerated centrifuge (Eppendorf, Hamburg, Germany), and the supernatant was decanted, aliquoted, and stored at -80°C for further analysis.

[0343] The protein concentration in the protein lysate was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's instructions. Specifically, serially diluted bovine serum albumin (BSA) standards were analyzed in duplicate, while each protein lysate sample was diluted 10-fold or 20-fold in water and then analyzed in singlets. The protein concentration in the undiluted sample was then obtained by averaging the protein concentrations from the 10-fold dilution and the 20-fold dilution.

[0344] The levels of α-synuclein protein in the protein lysates were measured using a sandwich ELISA developed in-house. Briefly, half-area 96-well flat-bottom UV-transparent microplates (Corning, Corning, NY) were coated with a capture antibody (α-synuclein: anti-synuclein antibody, Syn42, Eli Lilly, Indianapolis, IN) overnight at 4°C with agitation. The wells were blocked with 2% bovine serum albumin (BSA) (Thermo Scientific) in phosphate-buffered saline Tween 20™ solution (PBST) (Thermo Scientific) for 60 minutes at room temperature (RT). After washing, the protein lysates or recombinant human α-synuclein protein (α-synuclein: rPeptide, Watkinsville, GA) diluted in PBST containing 2% BSA were added to the wells of each plate for ELISA. The plates were incubated overnight at 4°C with agitation.

[0345] Plates for α-Syn ELISA were washed and then incubated with detection antibody (rabbit pAb anti-α-synuclein, US Biological, Salem, MA) in PBST containing 2% BSA for 3 hours at room temperature. Plates were washed again and then incubated with anti-rabbit HRP-linked antibody in PBST containing 2% BSA for 1 hour at room temperature.

[0346] To minimize variability, all biopsies from the same brain region and a set of serially diluted recombinant human α-synuclein protein standards were analyzed on the same ELISA plate. All samples, including recombinant protein standards, were analyzed in duplicate. The arithmetic mean OD450 values ​​from the duplicates after subtracting the plate blank was used for further calculations. A standard curve was generated on each ELISA plate by fitting the OD450 (Y-axis) and protein concentration (X-axis) of each serially diluted protein standard to a logistic 4P nonlinear regression model using JMP software (SAS Institute, Cary, NY). The concentration of each protein in each diluted sample was then back-calculated from its OD450 based on the standard curve. The α-synuclein level in each sample was normalized to the level of total protein, and α-synuclein protein expression in the treatment group was calculated as a percentage of the residual α-synuclein protein expression in the treatment group relative to the average expression of that protein in the aCSF or PBS control group.

[0347] The tissues and their acronyms analyzed for mRNA or protein levels are gastrocnemius muscle; LSC, lumbar spinal cord; SN, substantia nigra; caudate nucleus; PUT, putamen; H, hippocampus; PFC, prefrontal cortical grey matter; and LDRG, lumbar DRG.

[0348] Peripheral IV administration of a 10 mg / kg dose of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate three times a month in NHPs resulted in a significant reduction in SNCA mRNA in key brain regions and the lumbar spinal cord compared to the PBS-treated group 85 days after the first dose. As shown in Figure 9A, significant reductions in SNCA mRNA were demonstrated in the lumbar spinal cord (72%), substantia nigra (76%), caudate nucleus (81%), putamen (66%), hippocampus (76%), and prefrontal cortex gray matter (73%). Figure 9B demonstrates a significant reduction in α-synuclein protein in key brain regions and the lumbar spinal cord compared to the PBS-treated control group 85 days after the first dose. As shown in Figure 9B, significant reductions in α-synuclein protein were observed in the lumbar spinal cord (50%), substantia nigra (45%), caudate nucleus (43%), putamen (54%), hippocampus (48%), and prefrontal cortex (54%).

[0349] A single peripheral IV administration of a 10 mg / kg dose of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate in NHPs resulted in a significant reduction in SNCA mRNA in key brain regions and the lumbar spinal cord compared to the PBS-treated group 85 days after dosing. As shown in Figure 9C, significant SNCA mRNA reduction was demonstrated in the lumbar caudate nucleus (54%) and putamen (45%). Other brain regions and tissues evaluated did not demonstrate a significant reduction in SNCA mRNA, as shown (Figure 9C). Figure 9D demonstrates a significant reduction in α-synuclein protein in key brain regions and the lumbar spinal cord compared to the PBS-treated control group 85 days after the first dose. As shown in Figure 9D, significant reductions in α-synuclein protein were observed in the lumbar spinal cord (52%), caudate nucleus (36%), putamen (39%), hippocampus (43%), and prefrontal cortex (33%). Other brain regions and tissues evaluated did not demonstrate a significant decrease in α-synuclein protein, as shown (FIG. 9D).

[0350] The reduction of SNCA mRNA in gastrocnemius muscle after single or three monthly doses is shown in Figure 9E.

[0351] 8C. In vivo pharmacodynamic evaluation in NHPs after three monthly doses of human TfR binding protein-MAPT siRNA conjugate. A pharmacodynamic study was conducted to determine the efficacy of the human TfR binding protein-MAPT siRNA conjugate after three monthly doses. Groups of cynomolgus monkeys weighing 2-3 kg were intravenously administered monthly into the femoral saphenous vein with either i) PBS (n=5) or ii) TBP14-MAPT siRNA (dsRNA No. 38 in Table 11b) (n=5) at an effective siRNA concentration of 10 mg / kg. Another group of cynomolgus monkeys weighing 2-3 kg were intravenously administered monthly into the femoral saphenous vein with either i) PBS (n=5), ii) TBP14-MAPT siRNA (dsRNA No. 39 in Table 11b) at an effective siRNA concentration of 10 mg / kg (n=5), or iii) TBP14-MAPT siRNA (dsRNA No. 40 in Table 11b) at an effective siRNA concentration of 10 mg / kg (n=5). To mitigate anti-drug antibody responses, all groups received 30 mg / kg of anti-CD4 antibody immediately after the test substance dose. Approximately 85 days after the first dose, deeply anesthetized animals underwent cardiac perfusion, and then brain, spinal cord, and peripheral tissues were collected.

[0352] Brains were sectioned coronally, and 4 mm punches were collected and frozen from the indicated subregions. Tissues were collected from the spinal cord, liver, and muscle. Target mRNA and protein levels in tissue homogenates were assessed by RT-qPCR and ELISA, respectively. To determine mRNA levels, total RNA from NHP tissues was isolated manually using the RNadvance Tissue Kit (Beckman Coulter, Indianapolis, IN) or on a Biomek i7 liquid handler (Beckman Coulter) according to the manufacturer's protocol with some modifications. Briefly, frozen tissue sections were mixed with one 5 mm stainless steel bowl, lysis buffer, and proteinase K and homogenized in a 2010 GenoGrinder (SPEX SamplePrep, Metuchen, NJ) at 1200 rpm for five 30-second cycles with a 20-second interval between cycles. Tissues from several regions were shaved on dry ice prior to homogenization. The homogenate was incubated at 37°C for 1 hour and then extracted with an equal volume of phenol-chloroform. RNA in the supernatant was purified using the RNadvance Tissue Kit, which included a 30-minute digestion with DNase. The concentration and purity (A260 / A280) of the RNA eluate were determined spectrophotometrically. RNA was normalized to 15 ng / 10 μL PCR and re-digested with ezDNase (dsDNA-specific) before reverse transcription using the SSIV VILO kit (Thermo Fisher Scientific, Waltham, MA). Expression of each gene target in the cDNA was determined using TaqMan qPCR assays on the QuantStudio 7 Pro platform (Thermo Fisher Scientific). Gene expression levels of MAPT were normalized to β-actin using the respective probes for CNS regions (ThermoFisher) and GAPDH (ThermoFisher) for gastrocnemius muscle.

[0353] To determine tau protein levels, frozen 4 mm punches of neural tissue biopsies were mixed with cold RIPA buffer (Pierce #89901, Thermo Scientific, Waltham, MA) containing protease and phosphatase inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail, Thermo Scientific) at a ratio of 20 mL of buffer to 1 gram of tissue. The tissue-RIPA mixture was homogenized using 5 mm stainless steel beads on a 2010 GenoGrinder (Spex SamplePrep, Metuchen, NJ). The homogenate was then centrifuged in a refrigerated centrifuge (Eppendorf, Hamburg, Germany), and the supernatant was decanted, aliquoted, and stored at -80°C for further analysis.

[0354] The protein concentration in the protein lysate was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's instructions. Specifically, serially diluted bovine serum albumin (BSA) standards were analyzed in duplicate, while each protein lysate sample was diluted 10-fold or 20-fold in water and then analyzed in singlets. The protein concentration in the undiluted sample was then obtained by averaging the protein concentration from the 10-fold dilution and the protein concentration from the 20-fold dilution.

[0355] Tau protein levels in the protein lysates were measured using a sandwich ELISA developed in-house. Briefly, half-area 96-well flat-bottom UV-transparent microplates (Corning, Corning, NY) were coated with a capture antibody (tau: anti-human tau antibody, Tau5, Eli Lilly, Indianapolis, IN) overnight at 4°C with agitation. The wells were blocked with 2% bovine serum albumin (BSA) (Thermo Scientific) in phosphate-buffered saline Tween 20™ solution (PBST) (Thermo Scientific) for 60 minutes at room temperature (RT). After washing, protein lysates or recombinant human tau protein (tau: Tau441, Eli Lilly) and detection antibody (tau: anti-human tau antibody, Biotinylated DA9, Eli Lilly) diluted in PBST containing 2% BSA were added to each well of the plate. The plate was incubated overnight at 4°C with agitation.

[0356] The next day, plates were washed and then incubated with Pierce™ High Sensitivity Streptavidin-conjugated horseradish peroxidase (HRP) (Thermo Scientific) in PBST containing 2% BSA for 30 minutes at room temperature. The HRP enzyme reaction was visualized by adding TMB substrate solution (T0440, Sigma Aldrich, St. Louis, MO) and stopped by adding sulfuric acid (ELISA Stop solution, Thermo Scientific). The optical density (OD) of the samples was measured at 450 nm (OD450) using an Envision plate reader (PerkinElmer, Waltham, MA).

[0357] To minimize variability, all biopsies from the same brain region and a set of serially diluted recombinant human tau protein standards were analyzed on the same ELISA plate. All samples, including recombinant protein standards, were analyzed in duplicate. The arithmetic mean OD450 from the duplicates after subtracting the plate blank was used for further calculations. A standard curve was generated on each ELISA plate by fitting the OD450 (Y-axis) and protein concentration (X-axis) of each serially diluted protein standard to a logistic 4P nonlinear regression model using JMP software (SAS Institute, Cary, NY). The concentration of each protein in each diluted sample was then back-calculated from its OD450 based on the standard curve. The tau protein level in each sample was normalized to the level of total protein, and residual tau protein expression in the treatment group was calculated as a percentage of the mean expression of that protein in the aCSF or PBS control group.

[0358] The tissues and their acronyms analyzed for mRNA or protein levels are: LSC, lumbar spinal cord; SN, substantia nigra; caudate nucleus; PUT, putamen; H, hippocampus; and PFC, prefrontal cortical gray matter.

[0359] Peripheral IV administration of 10 mg / kg of TBP14-MAPT siRNA (dsRNA No. 38 in Table 11b) conjugate three times a month in NHPs resulted in a significant reduction in MAPT mRNA in key brain regions and the lumbar spinal cord compared to the PBS-treated group 85 days after the first dose. As shown in Figure 10A, significant reductions in MAPT mRNA were demonstrated in the lumbar spinal cord (24%), caudate nucleus (31%), putamen (38%), hippocampus (41%), and prefrontal cortical gray matter (40%). Other brain regions and tissues evaluated did not demonstrate a significant reduction in MAPT mRNA, as shown (Figure 10A). Figure 10B demonstrates a significant reduction in tau protein in key brain regions and the lumbar spinal cord compared to the PBS-treated control group 85 days after the first dose. As shown in Figure 10B, significant reductions in tau protein were observed in the lumbar spinal cord (29%), caudate nucleus (26%), putamen (28%), hippocampus (27%), and prefrontal cortex (34%). Other brain regions and tissues evaluated did not demonstrate significant reductions in tau protein, as shown (Figure 10B).

[0360] Peripheral IV administration of 10 mg / kg of TBP14-MAPT siRNA (dsRNA No. 39 in Table 11b) conjugate three times a month in NHPs resulted in a significant reduction in MAPT mRNA in key brain regions and the lumbar spinal cord compared to the PBS-treated group 85 days after the first dose. As shown in Figure 11A, significant reductions in MAPT mRNA were demonstrated in the lumbar spinal cord (41%), substantia nigra (41%), caudate nucleus (67%), putamen (67%), hippocampus (57%), and prefrontal cortex gray matter (65%). Figure 11B demonstrates a significant reduction in tau protein in key brain regions and the lumbar spinal cord compared to the PBS-treated control group 85 days after the first dose. As shown in Figure 11B, significant reductions in tau protein were observed in the lumbar spinal cord (38%), substantia nigra (56%), caudate nucleus (63%), putamen (77%), hippocampus (59%), and prefrontal cortex (76%).

[0361] Peripheral IV administration of a 10 mg / kg dose of TBP14-MAPT siRNA (dsRNA No. 40 in Table 11b) conjugate three times a month in NHPs resulted in a significant reduction in MAPT mRNA in key brain regions and the lumbar spinal cord compared to the PBS-treated group 85 days after the first dose. As shown in Figure 12A, significant reductions in MAPT mRNA were demonstrated in the lumbar spinal cord (37%), substantia nigra (35%), caudate nucleus (61%), putamen (54%), hippocampus (36%), and prefrontal cortex gray matter (61%). Figure 12B demonstrates a significant reduction in tau protein in key brain regions and the lumbar spinal cord compared to the PBS-treated control group 85 days after the first dose. As shown in Figure 12B, significant reductions in tau protein were observed in the lumbar spinal cord (31%), substantia nigra (47%), caudate nucleus (57%), putamen (72%), hippocampus (45%), and prefrontal cortex (70%).

[0362] 8D. In vivo pharmacodynamic evaluation in NHPs after 1 month of a single dose of a BBB-penetrating antibody targeting SNCA human TfR-binding protein-SNCA siRNA conjugate (DAR1) Following the demonstration of central efficacy with peripheral siRNA delivery in cynomolgus monkeys (Macaca fascicularis) using a DAR2-average human TfR binding protein-SNCA siRNA conjugate, a 1-month efficacy study with a DAR1-average human TfR binding protein-SNCA siRNA conjugate was performed to determine efficacy differences. The pharmacodynamic properties of the human TfR binding protein-siRNA conjugate were evaluated in NHPs as follows. Cynomolgus monkeys weighing 2-3 kg were intravenously administered i) PBS (n=4), ii) TBP16-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) at an effective siRNA concentration of 1 mg / kg (n=4), or iii) TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) at an effective siRNA concentration of 1 mg / kg or 10 mg / kg (n=4 each) via the femoral saphenous vein and sacrificed 29 days after the first dose. For takedown, deeply anesthetized animals underwent cardiac perfusion, and brain tissue was collected and processed for RT-qPCR in tissue homogenates.

[0363] RT-qPCR data demonstrated the high efficacy of the DAR1 conjugate, showing a robust reduction of SNCA mRNA in all key brain regions, ranging from 60 to 80% at a 1 mg / kg siRNA dose (Figures 13A and 13B). A 10-fold increase in dose to 10 mg / kg only induced a further 5 to 10% reduction in mRNA from 1 mg / kg, suggesting that TfR-mediated drug delivery was already saturated (Figure 13C).

[0364] 8E. Exposure-Response Relationship To understand the exposure-response relationship, plasma pharmacokinetics (PK), and biodistribution of siRNA after a single IV dose for the studies described in Examples 8B and 8D above, plasma samples from the studies were collected, and the exposure of conjugate-associated siRNA in plasma or total siRNA in tissues was quantified by HR-LC / MS (Figures 13D and 13E). Briefly, liquid chromatography / mass spectrometry (LC / MS) was used to measure conjugate-associated or total siRNA levels in cynomolgus monkey plasma and tissue samples. Plasma standards were prepared by spiking control monkey plasma. Tissue standards were prepared in control tissue homogenates. An internal standard was added to all standards and samples to control assay variability.

[0365] For conjugate-associated siRNA, plasma standards and samples were incubated with biotinylated polyclonal goat anti-human IgG antibody (Southern Biotech, Birmingham, AL), followed by a second incubation with streptavidin beads (Promega, Madison, WI). The IgG-siRNA-streptavidin bead complexes were isolated using a magnetic separator, and the supernatant was discarded. The samples and standards were washed with phosphate-buffered saline, and then the conjugate-associated siRNA was eluted from the beads with triethylamine. The standards and samples were injected into the LC / MS system.

[0366] Tissue samples were homogenized in cell lysis buffer. For total siRNA measurement, tissue standards and samples were digested with proteinase K and then loaded onto an Oasis Wax microelution solid phase extraction (SPE) plate (Waters Inc, Milford, MA) for isolation. The SPE plate was washed with wash buffer, and then the analytes were eluted with elution buffer. The eluate from the SPE plate was dried, reconstituted, and injected into the LC / MS system.

[0367] Conjugate-associated siRNA or total siRNA was measured using a Thermo Orbitrap Exploris 240 (Thermo Scientific, San Jose, CA) mass spectrometer using the antisense strand peak for quantification. The mass spectrometer was operated in negative ion detection mode. All data were processed using Xcalibur version 4.4 (Thermo Scientific, San Jose, CA).

[0368] For the TBP15-SNCA conjugate (DAR1), based on AUC (0-168 hours), plasma PK appears to be greater than dose proportional between the 1 mg / kg siRNA dose and the 10 mg / kg ssiRNA dose (7.8 mM * Time vs. 111.6mM * The plasma PK is consistent with TMDD-mediated clearance. For the 10 mg / kg siRNA DAR2 conjugate, TBP14-SNCA siRNA (DAR2), the AUC (0-72 hours) was 78 mM. * time, an exposure approximately 1.8-fold lower than that observed for TBP15-SNCA siRNA(DAR1) at the same dose.

[0369] For TBP15-SNCA siRNA (DAR1), the dose-dependent plasma PK translated into brain distribution, albeit with a dose-proportional profile that was lower than the plasma exposure (Figure 13E). For a given dose, exposure across different brain regions was similar. Brain exposure for TBP14-SNCA (DAR2) at 3 months was undetectable, consistent with lower plasma exposure (data not shown).

[0370] Example 9. Further characterization of human TfR binding protein-dsRNA conjugates in human TfR (hTfR) transgenic mice To understand the impact of DAR on plasma PK and biodistribution of siRNA after a single IV dose of human TfR binding protein-dsRNA conjugates, TBP14-SNCA siRNA conjugate (DAR1) and TBP14-SNCA siRNA conjugate (DAR2) were administered to hTfR transgenic mice at 10 mg / kg, plasma samples were collected, and conjugate-associated siRNA exposure was quantified by HR-LC / MS at various time points up to 1 month post-dose (Figure 14A). Based on the AUC (0-168 hours), the plasma PK of DAR1 was 3.5-fold greater than that of DAR2 (323 mM). * Time vs. 92mM * (Figure 14B). This plasma PK is consistent with TMDD-mediated clearance. This dose-dependent plasma PK translates to the brain, with up to a 3.6-fold higher exposure observed for DAR1 versus DAR2 (Figure 14B).

[0371] Figure 14C shows brain tissue concentrations of total siRNA in human TfR transgenic mice 24 hours after a single peripheral IV administration of either a TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR2) or a TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) across various siRNA doses.

[0372] The pharmacodynamic efficacy relationship of the human TfR binding protein-dsRNA conjugates DAR1 and DAR2 was evaluated at various doses with matched antibody and siRNA concentrations to determine the effect of dose reduction. As shown in Figure 14D, the TBP15-SNCA siRNA conjugate (DAR1) and TBP14-SNCA siRNA conjugate (DAR2) were administered to hTfR transgenic mice via a single IV injection at 20, 10, 5, 2.5, and 0.5 mg / kg siRNA, and compared with a PBS-treated group (n=4 each). For takedown, deeply anesthetized animals underwent cardiac perfusion 28 days after IV dosing. Brain tissue was then collected and processed for RT-qPCR in tissue homogenates. As shown in Figure 14D, the TBP15-SNCA siRNA conjugate (DAR1) demonstrated higher efficacy of SNCA mRNA KD at all matched dose levels compared to the TBP14-SNCA siRNA conjugate (DAR2). Specifically, for the TBP15-SNCA siRNA conjugate (DAR1), a 10 mg / kg siRNA dose induced 8% mRNA retention, a 5 mg / kg siRNA dose induced 10% mRNA retention, a 2.5 mg / kg siRNA dose induced 13% mRNA retention, and a 0.5 mg / kg siRNA dose induced 24% mRNA retention. For the TBP14-SNCA siRNA conjugate (DAR2), a 20 mg / kg siRNA dose induced 17% mRNA retention, a 10 mg / kg siRNA dose induced 20% mRNA retention, a 5 mg / kg siRNA dose induced 23% mRNA retention, and a 0.5 mg / kg siRNA dose induced 59% mRNA retention. Notably, a 10-fold siRNA drug dose reduction efficacy was demonstrated when comparing similar mRNA reductions with the 5 mg / kg TBP14-SNCA siRNA conjugate (DAR2) (23% retention) compared with the 0.5 mg / kg TBP15-SNCA siRNA conjugate (DAR1) (24% retention).This trend was also observed at higher doses, with similar mRNA reductions observed with 20 mg / kg TBP14-SNCA siRNA conjugate (DAR2) (17% remaining) compared with 2.5 mg / kg TBP15-SNCA siRNA conjugate (DAR1) (13% remaining).

[0373] Having demonstrated high efficacy of the human TfR binding protein-dsRNA conjugate DAR1 via the intravenous route, we evaluated the efficacy of the TBP15-SNCA siRNA conjugate (DAR1) delivered via a single subcutaneous (SC) injection at 5, 2, 0.5, and 0.25 mg / kg siRNA doses. For takedown, deeply anesthetized animals underwent cardiac perfusion 28 days after SC dosing, and brain tissue was then collected and processed for RT-qPCR in tissue homogenates. Data demonstrated similarly high efficacy of SC delivery at all doses evaluated, demonstrating 11% mRNA retention at the 5 mg / kg dose, 15% at the 2 mg / kg dose, 30% at the 0.5 mg / kg dose, and 42% at the 0.25 mg / kg dose (Figure 14E).

[0374] Sequence Listing

[0375] [Table 28-1]

[0376] (Continuation of the above table) [Table 28-2]

[0377] (Continuation of the above table) [Table 28-3]

[0378] (Continuation of the above table) [Table 28-4]

[0379] (Continuation of the above table) [Table 28-5]

[0380] (Continuation of the above table) [Table 28-6]

[0381] (Continuation of the above table) [Table 28-7]

[0382] (Continuation of the above table) [Table 28-8]

[0383] (Continuation of the above table) [Table 28-9]

[0384] (Continuation of the above table) [Table 28-10]

[0385] (Continuation of the above table) [Table 28-11]

[0386] (Continuation of the above table) [Table 28-12]

[0387] (Continuation of the above table) [Table 28-13]

[0388] (Continuation of the above table) [Table 28-14]

[0389] (Continuation of the above table) [Table 28-15]

[0390] (Continuation of the above table) [Table 28-16]

[0391] (Continuation of the above table) [Table 28-17]

[0392] (Continuation of the above table) [Table 28-18]

[0393] (Continuation of the above table) [Table 28-19]

[0394] (Continuation of the above table) [Table 28-20]

[0395] (Continuation of the above table) [Table 28-21]

Claims

1. 1. A protein comprising one monovalent human transferrin receptor (TfR) binding domain, wherein the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 21, HCDR3 comprises SEQ ID NO: 22, LCDR1 comprises SEQ ID NO: 23, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24, or (b) a protein wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO:

18.

2. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (b) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 7, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (c) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 8, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (d) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; or (e) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 14, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; or (f) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; or (g) The protein of claim 1, wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO:

18.

3. The VH and VL comprise the following sequences: (a) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; or (b) the VH comprises SEQ ID NO: 29 and the VL comprises SEQ ID NO: 28; or (c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; or (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 35; or (f) the VH comprises SEQ ID NO: 36 and the VL comprises SEQ ID NO: 37; or (g) The protein of claim 1 or 2, wherein VH comprises SEQ ID NO: 38 and VL comprises SEQ ID NO:

37.

4. The protein of any one of claims 1 to 3, wherein the human TfR binding domain is a Fab, scFv, Fv, or scFab.

5. The protein of any one of claims 1 to 4, wherein the human TfR binding domain further comprises a heavy chain constant region comprising a cysteine ​​at residue 124 (according to EU index numbering).

6. The protein of any one of claims 1 to 5, wherein the human TfR binding domain further comprises a light chain constant region comprising a cysteine ​​at residue 156 (according to EU index numbering).

7. The protein of any one of claims 1 to 6, further comprising a half-life extender.

8. The protein of claim 7 , wherein the half-life extender is selected from a VHH that binds to an immunoglobulin Fc region or human serum albumin (HSA).

9. The protein of claim 7 or 8, wherein the half-life extender is an immunoglobulin Fc region.

10. The protein of claim 9 , wherein the Fc region is a modified human IgG4 Fc region.

11. 11. The protein of claim 10, wherein the modified human IgG4 Fc region comprises a proline at residue 228 and an alanine at residues 234 and 235 (all residues numbered according to EU index numbering).

12. 12. The protein of any one of claims 9 to 11, wherein the protein comprises an immunoglobulin Fc region comprising a cysteine ​​at residue 378 (according to EU index numbering).

13. The Fc region is (a) a first Fc CH3 domain comprising a serine at position 349, a methionine at position 366, a tyrosine at position 370, and a valine at position 409, and a second Fc CH3 domain comprising a glycine at position 356, an aspartic acid at position 357, a glutamine at position 364, and an alanine at position 407 (all residues are numbered according to EU index numbering); or 13. The protein of any one of claims 9 to 12, comprising (b) a first Fc CH3 domain comprising a leucine at residue 405, and a second Fc CH3 domain comprising an arginine at residue 409, all residues numbered according to EU index numbering.

14. The protein comprises one heavy chain (HC) and one light chain (LC), wherein the HC and LC comprise the following sequences: (a) the HC comprises SEQ ID NO: 53 and the LC comprises SEQ ID NO: 54; or (b) the HC comprises SEQ ID NO: 55 and the LC comprises SEQ ID NO: 54; or (c) the HC comprises SEQ ID NO: 56 and the LC comprises SEQ ID NO: 57; or (d) the HC comprises SEQ ID NO: 58 and the LC comprises SEQ ID NO: 59; or (e) the HC comprises SEQ ID NO: 60 and the LC comprises SEQ ID NO: 61; (f) the HC comprises SEQ ID NO: 62 and the LC comprises SEQ ID NO: 63; or (g) The protein of any one of claims 1 to 12, wherein the HC comprises SEQ ID NO: 64 and the LC comprises SEQ ID NO:

63.

15. 14. The protein of any one of claims 1 to 13, wherein the protein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 68, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO:

69.

16. 14. The protein of any one of claims 1 to 13, wherein the protein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 138, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO:

139.

17. 14. The protein of any one of claims 1 to 13, wherein the protein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 166, LC1 comprises SEQ ID NO: 54, and HC2 comprises SEQ ID NO:

167.

18. 5. The protein of any one of claims 1 to 4, wherein the protein comprises one heavy chain (HC) and one light chain (LC), wherein the HC comprises SEQ ID NO: 65 and the LC comprises SEQ ID NO:

59.

19. The protein of claim 7 or 8, wherein the half-life extender is a VHH that binds to HSA.

20. The protein of claim 19, wherein the VHH comprises a CDR1 comprising SEQ ID NO: 39, a CDR2 comprising SEQ ID NO: 40, and a CDR3 comprising SEQ ID NO:

41.

21. 21. The protein of claim 19 or 20, wherein the VHH comprises SEQ ID NO:

42.

22. 22. The protein of any one of claims 19 to 21, wherein the protein comprises one heavy chain (HC) and one light chain (LC), wherein the HC comprises SEQ ID NO: 66 and the LC comprises SEQ ID NO:

67.

23. The protein of any one of claims 1 to 14, wherein the protein is a heterodimeric antibody comprising a first arm comprising one monovalent human TfR-binding domain and a second arm that is a null arm.

24. The protein of claim 23, wherein the second arm comprises a VH and a VL, the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 43, HCDR2 comprises SEQ ID NO: 44, HCDR3 comprises SEQ ID NO: 45, LCDR1 comprises SEQ ID NO: 46, LCDR2 comprises SEQ ID NO: 47, and LCDR3 comprises SEQ ID NO:

48.

25. 25. The protein of claim 24, wherein the VH comprises SEQ ID NO: 49 and the VL comprises SEQ ID NO:

50.

26. The protein comprises two heavy chains HC1 and HC2 and two light chains LC1 and LC2, wherein HC1, LC1, HC2, and LC2 comprise the following sequences: (a) HC1 comprises SEQ ID NO: 64, LC1 comprises SEQ ID NO: 63, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52; or (b) HC1 comprises SEQ ID NO: 55, LC1 comprises SEQ ID NO: 54, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52; or (c) HC1 comprises SEQ ID NO: 56, LC1 comprises SEQ ID NO: 57, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52; or (d) A protein described in any one of claims 23 to 25, wherein HC1 comprises SEQ ID NO: 58, LC1 comprises SEQ ID NO: 59, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO:

52.

27. 1. A protein comprising one monovalent human transferrin receptor (TfR) binding domain, wherein the human TfR binding domain binds to an epitope comprising one or more residues of: (a) residues 346-364 FGNMEGDCPSDWKTDSTCR (SEQ ID NO: 119); (b) residues 243-247 FEDLY (SEQ ID NO: 162) and residues 345-364 LFGNMEEGDCPSDWKTDSTCR (SEQ ID NO: 163) of the human TfR; or (c) residues 243-247 FEDLY (SEQ ID NO: 162), residues 259-263 AGKIT (SEQ ID NO: 164), and residues 532-538 VEKLTLD (SEQ ID NO: 165) of the human TfR.

28. A protein comprising one monovalent mouse transferrin receptor (TfR) binding domain, wherein the mouse TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 71, HCDR2 comprises SEQ ID NO: 72, HCDR3 comprises SEQ ID NO: 73, LCDR1 comprises SEQ ID NO: 74, LCDR2 comprises SEQ ID NO: 75, and LCDR3 comprises SEQ ID NO:

76.

29. 29. The protein of claim 28, wherein the VH comprises SEQ ID NO: 77 and the VL comprises SEQ ID NO:

78.

30. 30. The protein of claim 28 or 29, wherein the protein comprises a heavy chain (HC) comprising SEQ ID NO: 79 and a light chain (LC) comprising SEQ ID NO:

80.

31. The protein of any one of claims 28 to 30, wherein the protein is a heterodimeric antibody comprising a first arm comprising one monovalent mouse TfR-binding domain and a second arm that is a null arm.

32. 32. The protein of any one of claims 28 to 31, wherein the protein comprises two heavy chains HC1 and HC2 and two light chains LC1 and LC2, wherein HC1 comprises SEQ ID NO: 79, LC1 comprises SEQ ID NO: 80, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO:

52.

33. A conjugate comprising the protein of any one of claims 1 to 32 and a therapeutic agent.

34. 34. The conjugate of claim 33, wherein the therapeutic agent is selected from a double-stranded RNA, an oligonucleotide, a peptide, a small molecule, a nanoparticle, a lipid nanoparticle, an exosome, an antibody or an antigen-binding fragment thereof, or a combination thereof.

35. 35. The conjugate of claim 33 or 34, wherein the therapeutic agent is linked to the protein via a linker.

36. The conjugate of any one of claims 33 to 35, wherein the therapeutic agent is double-stranded RNA (dsRNA).

37. 37. The conjugate of claim 36, wherein the dsRNA comprises a sense strand and an antisense strand, and the antisense strand is complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B mRNA.

38. 38. The conjugate of claim 37, wherein the antisense strand is complementary to SNCA mRNA.

39. 38. The conjugate of claim 37, wherein the antisense strand is complementary to MAPT mRNA.

40. The conjugate of any one of claims 35 to 39, wherein the linker is a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker.

41. 41. The conjugate of any one of claims 33 to 40, wherein the ratio of therapeutic agent to protein is about 1:1 to 3:

1.

42. A conjugate of formula (I): R-L-P, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein containing one monovalent human TfR binding domain; L is a linker or optionally absent; The human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 21, HCDR3 comprises SEQ ID NO: 22, LCDR1 comprises SEQ ID NO: 23, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24, or (b) A conjugate wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO:

18.

43. 43. The conjugate of claim 42, wherein the ratio of R to P is about 1:1 to 3:

1.

44. Formula (II): (RL) ｎ -P conjugates, wherein R is double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; P is a protein containing one monovalent human TfR binding domain; L is a linker or optionally absent; The human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 21, HCDR3 comprises SEQ ID NO: 22, LCDR1 comprises SEQ ID NO: 23, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24, or (b) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; A conjugate wherein n is 1-3.

45. 45. The conjugate of claim 44, wherein n is 1.

46. 45. The conjugate of claim 44, wherein n is 2.

47. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (b) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 7, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (c) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 8, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (d) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 12; or (e) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 14, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; or (f) HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 15, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO: 18; or (g) The conjugate of any one of claims 42 to 46, wherein HCDR1 comprises SEQ ID NO: 13, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 16, LCDR2 comprises SEQ ID NO: 17, and LCDR3 comprises SEQ ID NO:

18.

48. The VH and VL comprise the following sequences: (a) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; or (b) the VH comprises SEQ ID NO: 29 and the VL comprises SEQ ID NO: 28; or (c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 33; or (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 35; or (f) the VH comprises SEQ ID NO: 36 and the VL comprises SEQ ID NO: 37; or (g) the conjugate of any one of claims 42 to 47, wherein the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO:

37.

49. 48. The conjugate of any one of claims 42 to 47, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 10, HCDR3 comprises SEQ ID NO: 11, LCDR1 comprises SEQ ID NO: 9, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO:

12.

50. The conjugate of any one of claims 42 to 48, wherein the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO:

33.

51. The conjugate of any one of claims 42 to 50, wherein the human TfR binding domain is a Fab, scFv, Fv, or scFab.

52. 52. The conjugate of any one of claims 42 to 51, wherein the human TfR binding domain further comprises a heavy chain constant region comprising a cysteine ​​at residue 124 (according to EU index numbering).

53. 53. The conjugate of any one of claims 42 to 52, wherein the human TfR binding domain further comprises a light chain constant region comprising a cysteine ​​at residue 156 (according to EU index numbering).

54. The conjugate of any one of claims 42 to 53, wherein the protein further comprises a half-life extender.

55. 55. The conjugate of claim 54, wherein the half-life extender is selected from a VHH that binds to an immunoglobulin Fc region or human serum albumin (HSA).

56. 56. The conjugate of claim 55, wherein the half-life extender is an immunoglobulin Fc region.

57. 57. The conjugate of claim 56, wherein the immunoglobulin Fc region is a modified human IgG4 Fc region.

58. 58. The conjugate of claim 57, wherein the modified human IgG4 Fc region comprises a proline at residue 228 and an alanine at residues 234 and 235 (all residues numbered according to EU index numbering).

59. 59. The conjugate of any one of claims 56 to 58, wherein the protein comprises an immunoglobulin Fc region comprising a cysteine ​​at residue 378 (according to EU index numbering).

60. The Fc region is (a) a first Fc CH3 domain comprising a serine at position 349, a methionine at position 366, a tyrosine at position 370, and a valine at position 409, and a second Fc CH3 domain comprising a glycine at position 356, an aspartic acid at position 357, a glutamine at position 364, and an alanine at position 407 (all residues are numbered according to EU index numbering); or (b) a first Fc CH3 domain comprising a leucine at residue 405, and a second Fc CH3 domain comprising an arginine at residue 409 (all residues numbered according to EU index numbering).

61. The protein comprises one heavy chain (HC) and one light chain (LC), wherein the HC and LC comprise the following sequences: (a) the HC comprises SEQ ID NO: 53 and the LC comprises SEQ ID NO: 54; or (b) the HC comprises SEQ ID NO: 55 and the LC comprises SEQ ID NO: 54; or (c) the HC comprises SEQ ID NO: 56 and the LC comprises SEQ ID NO: 57; or (d) the HC comprises SEQ ID NO: 58 and the LC comprises SEQ ID NO: 59; or (e) the HC comprises SEQ ID NO: 60 and the LC comprises SEQ ID NO: 61; (f) the HC comprises SEQ ID NO: 62 and the LC comprises SEQ ID NO: 63; or (g) the conjugate of any one of claims 42 to 59, wherein the HC comprises SEQ ID NO: 64 and the LC comprises SEQ ID NO:

63.

62. 61. The conjugate of any one of claims 42 to 60, wherein the protein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 68, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO:

69.

63. 61. The conjugate of any one of claims 42 to 60, wherein the protein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 138, LC1 comprises SEQ ID NO: 59, and HC2 comprises SEQ ID NO:

139.

64. 61. The conjugate of any one of claims 42 to 60, wherein the protein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 166, LC1 comprises SEQ ID NO: 54, and HC2 comprises SEQ ID NO:

167.

65. 52. The conjugate of any one of claims 42 to 51, wherein the protein comprises one heavy chain (HC) and one light chain (LC), wherein the HC comprises SEQ ID NO: 65 and the LC comprises SEQ ID NO:

59.

66. 55. The conjugate of claim 54, wherein the half-life extender is a VHH that binds to HSA.

67. 67. The conjugate of claim 66, wherein the VHH comprises a CDR1 comprising SEQ ID NO: 39, a CDR2 comprising SEQ ID NO: 40, and a CDR3 comprising SEQ ID NO:

41.

68. 68. The conjugate of claim 66 or 67, wherein the VHH comprises SEQ ID NO:

42.

69. 69. The conjugate of any one of claims 66 to 68, wherein the protein comprises one heavy chain (HC) and one light chain (LC), wherein the HC comprises SEQ ID NO: 66 and the LC comprises SEQ ID NO:

67.

70. 61. The conjugate of any one of claims 42 to 60, wherein the protein is a heterodimeric antibody comprising a first arm comprising one monovalent human TfR binding domain and a second arm that is a null arm.

71. The conjugate of claim 70, wherein the second arm comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 43, HCDR2 comprises SEQ ID NO: 44, HCDR3 comprises SEQ ID NO: 45, LCDR1 comprises SEQ ID NO: 46, LCDR2 comprises SEQ ID NO: 47, and LCDR3 comprises SEQ ID NO:

48.

72. 72. The conjugate of claim 71, wherein the VH comprises SEQ ID NO: 49 and the VL comprises SEQ ID NO:

50.

73. The protein comprises two heavy chains HC1 and HC2 and two light chains LC1 and LC2, wherein HC1, LC1, HC2, and LC2 comprise the following sequences: (a) HC1 comprises SEQ ID NO: 64, LC1 comprises SEQ ID NO: 63, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52; or (b) HC1 comprises SEQ ID NO: 55, LC1 comprises SEQ ID NO: 54, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52; or (c) HC1 comprises SEQ ID NO: 56, LC1 comprises SEQ ID NO: 57, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO: 52; or (d) the conjugate of any one of claims 70 to 72, wherein HC1 comprises SEQ ID NO: 58, LC1 comprises SEQ ID NO: 59, HC2 comprises SEQ ID NO: 51, and LC2 comprises SEQ ID NO:

52.

74. 74. The conjugate of any one of claims 42 to 73, wherein the linker is a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker.

75. The conjugate of any one of claims 42 to 74, wherein the linker is an SMCC linker.

76. The conjugate of any one of claims 42 to 75, wherein P is linked to the 3' end of the sense strand of the dsRNA, optionally via the linker.

77. 77. The conjugate of any one of claims 42 to 76, wherein the antisense strand is complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B mRNA.

78. 78. The conjugate of claim 77, wherein the antisense strand is complementary to SNCA mRNA.

79. The sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 81 and the antisense strand comprises SEQ ID NO: 82; (b) the sense strand comprises SEQ ID NO: 83 and the antisense strand comprises SEQ ID NO: 84; (c) the sense strand comprises SEQ ID NO: 85 and the antisense strand comprises SEQ ID NO: 86; (d) the sense strand comprises SEQ ID NO: 87 and the antisense strand comprises SEQ ID NO: 88; (e) the sense strand comprises SEQ ID NO: 89 and the antisense strand comprises SEQ ID NO: 90; (f) the sense strand comprises SEQ ID NO: 91 and the antisense strand comprises SEQ ID NO: 92; and (g) the sense strand comprises SEQ ID NO: 116 and the antisense strand comprises SEQ ID NO: 82; 79. The conjugate of any one of claims 42 to 78, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

80. 80. The conjugate of claim 79, wherein the sense strand comprises SEQ ID NO: 81 and the antisense strand comprises SEQ ID NO:

82.

81. 78. The conjugate of claim 77, wherein the antisense strand is complementary to MAPT mRNA.

82. The sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 120 and the antisense strand comprises SEQ ID NO: 121; (b) the sense strand comprises SEQ ID NO: 122 and the antisense strand comprises SEQ ID NO: 123; and (c) the sense strand comprises SEQ ID NO: 124 and the antisense strand comprises SEQ ID NO: 125; 82. The conjugate of any one of claims 42 to 77 and 81, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

83. The conjugate of any one of claims 42 to 82, wherein one or more nucleotides of the sense strand are modified nucleotides.

84. 84. The conjugate of claim 83, wherein each nucleotide of the sense strand is a modified nucleotide.

85. The conjugate of any one of claims 42 to 84, wherein one or more nucleotides of the antisense strand are modified nucleotides.

86. 86. The conjugate of claim 85, wherein each nucleotide of the antisense strand is a modified nucleotide.

87. 87. The conjugate of any one of claims 83 to 86, wherein the modified nucleotide is a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, or a 2'-O-alkyl modified nucleotide.

88. 88. The conjugate of any one of claims 83 to 87, wherein the sense strand has four 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5' end of the sense strand.

89. 89. The conjugate of claim 88, wherein nucleotides at positions other than 7, 9, 10, and 11 of the sense strand are 2'-O-methyl modified nucleotides.

90. 90. The conjugate of any one of claims 83 to 89, wherein the antisense strand has four 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand.

91. 91. The conjugate of claim 90, wherein nucleotides at positions other than 2, 6, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

92. 88. The conjugate of any one of claims 83 to 87, wherein the sense strand has three 2'-fluoro modified nucleotides at positions 9, 10, and 11 from the 5' end of the sense strand.

93. 91. The conjugate of claim 90, wherein nucleotides at positions other than 9, 10, and 11 of the sense strand are 2'-O-methyl modified nucleotides.

94. The conjugate of any one of claims 83 to 87, 92, and 93, wherein the antisense strand has five 2'-fluoro modified nucleotides at positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand.

95. 95. The conjugate of claim 94, wherein nucleotides at positions other than 2, 5, 7, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

96. The conjugate of any one of claims 83 to 87, 92, and 93, wherein the antisense strand has five 2'-fluoro modified nucleotides at positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand.

97. 97. The conjugate of claim 96, wherein nucleotides at positions other than 2, 5, 8, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

98. The conjugate of any one of claims 83 to 87, 92, and 93, wherein the antisense strand has five 2'-fluoro modified nucleotides at positions 2, 3, 7, 14, and 16 from the 5' end of the antisense strand.

99. 99. The conjugate of claim 98, wherein nucleotides at positions other than 2, 3, 7, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

100. 100. The conjugate of any one of claims 42 to 99, wherein the sense strand and the antisense strand have one or more modified internucleotide linkages.

101. 101. The conjugate of claim 100, wherein the modified internucleotide linkage is a phosphorothioate linkage.

102. 102. The conjugate of claim 100 or 101, wherein the sense strand has four or five phosphorothioate linkages.

103. The conjugate of any one of claims 100 to 102, wherein the antisense strand has four or five phosphorothioate linkages.

104. The conjugate of any one of claims 42 to 103, wherein the antisense strand has a phosphate analog at the 5' end.

105. 105. The conjugate of claim 104, wherein the phosphate analog is a 5'-vinyl phosphonate.

106. 106. The conjugate of any one of claims 42 to 105, wherein the sense strand comprises an abasic portion or an inverted abasic portion.

107. The conjugate of any one of claims 42 to 106, wherein the sense strand comprises an abasic moiety at position 10.

108. The sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 93 or 140, and the antisense strand comprises SEQ ID NO: 94; (b) the sense strand comprises SEQ ID NO: 95 or 141, and the antisense strand comprises SEQ ID NO: 96; (c) the sense strand comprises SEQ ID NO: 95 or 141, and the antisense strand comprises SEQ ID NO: 97; (d) the sense strand comprises SEQ ID NO: 95 or 141, and the antisense strand comprises SEQ ID NO: 98; (e) the sense strand comprises SEQ ID NO: 99 or 142, and the antisense strand comprises SEQ ID NO: 94; (f) the sense strand comprises SEQ ID NO: 100 or 143, and the antisense strand comprises SEQ ID NO: 101; (g) the sense strand comprises SEQ ID NO: 102 or 144, and the antisense strand comprises SEQ ID NO: 103; (h) the sense strand comprises SEQ ID NO: 104 or 145, and the antisense strand comprises SEQ ID NO: 105; (i) the sense strand comprises SEQ ID NO: 106 or 146, and the antisense strand comprises SEQ ID NO: 107; (j) the sense strand comprises SEQ ID NO: 108 or 147, and the antisense strand comprises SEQ ID NO: 107; (k) the sense strand comprises SEQ ID NO: 117 or 148 and the antisense strand comprises SEQ ID NO: 97; and (l) The conjugate of any one of claims 42 to 80 or 83 to 107, wherein the sense strand comprises SEQ ID NO: 118 or 149 and the antisense strand comprises SEQ ID NO:

97.

109. The sense strand and the antisense strand have a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand consists of SEQ ID NO: 93 or 140, and the antisense strand consists of SEQ ID NO: 94; (b) the sense strand consists of SEQ ID NO: 95 or 141, and the antisense strand consists of SEQ ID NO: 96; (c) the sense strand consists of SEQ ID NO: 95 or 141, and the antisense strand consists of SEQ ID NO: 97; (d) the sense strand consists of SEQ ID NO: 95 or 141, and the antisense strand consists of SEQ ID NO: 98; (e) the sense strand consists of SEQ ID NO: 99 or 142, and the antisense strand consists of SEQ ID NO: 94; (f) the sense strand consists of SEQ ID NO: 100 or 143, and the antisense strand consists of SEQ ID NO: 101; (g) the sense strand consists of SEQ ID NO: 102 or 144, and the antisense strand consists of SEQ ID NO: 103; (h) the sense strand consists of SEQ ID NO: 104 or 145, and the antisense strand consists of SEQ ID NO: 105; (i) the sense strand consists of SEQ ID NO: 106 or 146, and the antisense strand consists of SEQ ID NO: 107; (j) the sense strand consists of SEQ ID NO: 108 or 147, and the antisense strand consists of SEQ ID NO: 107; (k) the sense strand consists of SEQ ID NO: 117 or 148 and the antisense strand consists of SEQ ID NO: 97; and (l) The conjugate of any one of claims 42 to 80 or 83 to 107, wherein the sense strand consists of SEQ ID NO: 118 or 149 and the antisense strand consists of SEQ ID NO:

97.

110. The sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 126 or 150, and the antisense strand comprises SEQ ID NO: 127; (b) the sense strand comprises SEQ ID NO: 128 or 151, and the antisense strand comprises SEQ ID NO: 129; (c) the sense strand comprises SEQ ID NO: 130 or 152, and the antisense strand comprises SEQ ID NO: 131; (d) the sense strand comprises SEQ ID NO: 132 or 153, and the antisense strand comprises SEQ ID NO: 133; (e) the sense strand comprises SEQ ID NO: 134 or 154 and the antisense strand comprises SEQ ID NO: 135; and (f) the conjugate of any one of claims 42 to 77 or 81 to 107, wherein the sense strand comprises SEQ ID NO: 136 or 155 and the antisense strand comprises SEQ ID NO:

137.

111. The sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand consists of SEQ ID NO: 126 or 150, and the antisense strand consists of SEQ ID NO: 127; (b) the sense strand consists of SEQ ID NO: 128 or 151, and the antisense strand consists of SEQ ID NO: 129; (c) the sense strand consists of SEQ ID NO: 130 or 152, and the antisense strand consists of SEQ ID NO: 131; (d) the sense strand consists of SEQ ID NO: 132 or 153, and the antisense strand consists of SEQ ID NO: 133; (e) the sense strand consists of SEQ ID NO: 134 or 154 and the antisense strand consists of SEQ ID NO: 135; and (f) the conjugate of any one of claims 42 to 77 or 81 to 107, wherein the sense strand consists of SEQ ID NO: 136 or 155 and the antisense strand consists of SEQ ID NO:

137.

112. A pharmaceutical composition comprising a protein according to any one of claims 1 to 27 or a conjugate according to any one of claims 33 to 111, and a pharmaceutically acceptable carrier.

113. 112. A method of treating a CNS disorder in a patient in need thereof, said method comprising administering to said patient an effective amount of a conjugate of any one of claims 33 to 111, or a pharmaceutical composition of claim 112.

114. 112. A method of treating a neurodegenerative synucleinopathy in a patient in need thereof, said method comprising administering to said patient an effective amount of a conjugate of any one of claims 38, 40-80, 83-109, or a pharmaceutical composition of claim 112.

115. 115. The method of claim 114, wherein the neurodegenerative synucleinopathy is selected from Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies.

116. 112. A method of treating a tauopathy in a patient in need thereof, said method comprising administering to said patient an effective amount of a conjugate of any one of claims 39-77, 81-107, 110, 111, or a pharmaceutical composition of claim 112.

117. The tauopathy may be Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PPA-S), or primary logopenic progressive aphasia. (PPA-L), multisystem tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), amyloid angiopathy of the British type, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration ( CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down's syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Gibberish syndrome with neurofibrillary tangles 117. The method of claim 116, wherein the disease is selected from Amanian motor neuron disease, post-encephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

118. 118. The method of any one of claims 113 to 117, wherein the conjugate is administered to the patient intravenously or subcutaneously.

119. A conjugate according to any one of claims 33 to 111 or a pharmaceutical composition according to claim 112 for use in therapy.

120. A conjugate according to any one of claims 38, 40 to 80, 83 to 109, or a pharmaceutical composition according to claim 112 for use in the treatment of a neurodegenerative synucleinopathy.

121. 121. The conjugate or pharmaceutical composition for use according to claim 120, wherein the neurodegenerative synucleinopathy is selected from Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies.

122. A conjugate according to any one of claims 39 to 77, 81 to 107, 110, 111, or a pharmaceutical composition according to claim 108, for use in the treatment of a tauopathy.

123. The tauopathy may be Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PPA-S), primary logopenic progressive aphasia (PPA -L), multisystem tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), amyloid angiopathy of the British type, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease Feldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down's syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, 123. The conjugate or pharmaceutical composition for use according to claim 122, wherein the disease is selected from post-encephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

124. Use of a conjugate according to any one of claims 33 to 111 in the manufacture of a medicament for treating a CNS disorder.

125. Use of a conjugate according to any one of claims 38, 40 to 80, 83 to 109 in the manufacture of a medicament for treating a neurodegenerative synucleinopathy.

126. 126. The use of claim 125, wherein the neurodegenerative synucleinopathy is selected from Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies.

127. Use of a conjugate according to any one of claims 39 to 77, 81 to 107, 110, 111 in the manufacture of a medicament for treating a tauopathy.

128. The tauopathy may be Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PPA-S), or primary logopenic progressive aphasia. (PPA-L), multisystem tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), amyloid angiopathy of the British type, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration ( CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down's syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Gibberish syndrome with neurofibrillary tangles 128. The use of claim 127, wherein the disease is selected from Amanian motor neuron disease, post-encephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

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