Transferrin receptor binding proteins and conjugates

Abstract

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

Description

[0001] Sequence Listing

[0002] This application is submitted together with a sequence listing in ST.26 XML format. The sequence listing is provided as a file named "30369_WO", created on July 18, 2023, and with a size of 667 kilobytes. The sequence listing information in ST.26 XML format is incorporated herein by reference in its entirety.

[0003] background

[0004] The blood-brain barrier (BBB) ​​is a selective semipermeable boundary of capillary endothelial cells that prevents solutes, including pathogens, from entering the central nervous system (CNS). The BBB allows some small molecules to pass through by passive diffusion, and the cells of the BBB use specific transporters to actively transport metabolites such as glucose and amino acids that are essential for neural function across the barrier. The BBB has a neuroprotective function by strictly controlling access to the brain; but it also hinders the access of therapeutic agents to the CNS.

[0005] BBB shuttles for improving the passage of therapeutic agents across the blood-brain barrier and into the CNS have been described. For example, WO2003 / 009815 describes the use of antibodies directed against the transferrin receptor ("TfR") for regulating blood-brain barrier transport. However, attempts to shuttle therapeutic agents across the BBB using anti-TfR antibodies have proven challenging. To date, there are no TfR shuttles or conjugates approved for the treatment of CNS diseases.

[0006] Therefore, there remains a need for TfR binding proteins and conjugates for the treatment of various CNS diseases that can deliver therapeutic agents across the BBB into the CNS. SUMMARY OF THE INVENTION

[0008] Provided herein are proteins comprising a monovalent human TfR binding domain ("human TfR binding protein"), proteins comprising a monovalent mouse TfR binding domain ("mouse TfR binding protein"), conjugates comprising such human or mouse TfR binding proteins, such as human TfR binding protein-dsRNA conjugates, pharmaceutical compositions comprising the human TfR binding protein or conjugate, and methods of using the human TfR binding protein or conjugate to treat CNS diseases (e.g., neurodegenerative diseases such as neurodegenerative synucleinopathy or tauopathy).

[0009] In one aspect, provided herein is a protein comprising one and only one 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), and the VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementary 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.

[0010] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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:

[0011] (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

[0012] (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.

[0013] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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:

[0014] (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;

[0015] (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;

[0016] (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;

[0017] (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;

[0018] (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;

[0019] (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

[0020] (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.

[0021] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein the VH and VL comprise the following sequences:

[0022] (a) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:27, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0023] (b) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:29, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0024] (c) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:30, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:31;

[0025] (d) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:32, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:33;

[0026] (e) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:34, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:35;

[0027] (f) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:36, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37; or

[0028] (g) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:38, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37.

[0029] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein the VH and VL comprise the following sequences:

[0030] (a) VH comprises SEQ ID NO:27 and VL comprises SEQ ID NO:28;

[0031] (b) VH comprises SEQ ID NO:29 and VL comprises SEQ ID NO:28;

[0032] (c) VH comprises SEQ ID NO:30 and VL comprises SEQ ID NO:31;

[0033] (d) VH comprises SEQ ID NO:32 and VL comprises SEQ ID NO:33;

[0034] (e) VH comprises SEQ ID NO:34 and VL comprises SEQ ID NO:35;

[0035] (f) VH comprises SEQ ID NO:36 and VL comprises SEQ ID NO:37; or

[0036] (g) VH comprises SEQ ID NO:38 and VL comprises SEQ ID NO:37.

[0037] In some embodiments, the monovalent human TfR binding domain is an antibody fragment, such as Fab, scFv, Fv or scFab (single-chain Fab). In some embodiments, the monovalent human TfR binding domain is Fab. In some embodiments, the human TfR binding domain further comprises a heavy chain constant region and / or a light chain constant region.

[0038] In some embodiments, the human TfR binding protein described herein further comprises a half-life extender, such as an immunoglobulin Fc region or a VHH that binds to human serum albumin (HSA).

[0039] 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.

[0040] In some embodiments, the human TfR binding protein described herein is any one of Tables 6a and 6b. In some embodiments, the human TfR binding protein described herein has a heavy chain (HC) and a light chain (LC), such as 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 form, such as TBP10, TBP11, TBP12 or TBP13.

[0041] In some embodiments, provided herein is a protein comprising a 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 346-364FGNMEGDCPSDWKTDSTCR (SEQ ID NO: 119), (b) residues 243-247FEDLY (SEQ ID NO: 162) and residues 345-364LFGNMEEGDCPSDWKTDSTCR) (SEQ ID NO: 163), or (c) residues 243-247FEDLY (SEQ ID NO: 162), residues 259-263AGKIT (SEQ ID NO: 164), and residues 532-538 (VEKLTLD) (SEQ ID NO: 165) of human TfR.

[0042] In another aspect, provided herein is a protein comprising a monovalent mouse TfR binding domain ("mouse TfR binding protein"). These mouse TfR binding proteins can serve as surrogate molecules for human TfR binding proteins in mouse models. In some embodiments, provided herein is a protein comprising a monovalent mouse TfR binding domain, wherein the mouse TfR binding domain comprises VH and VL, wherein the VH comprises a heavy chain complementary determining region HCDR1, HCDR2, and HCDR3, and the VL comprises a light chain complementary determining region LCDR1, LCDR2, and LCDR3, and 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 a 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.

[0043] Also provided herein are antibodies comprising a VH comprising a HCDR1, HCDR2, and HCDR3 selected from Table 1, and / or a VL comprising a LCDR1, LCDR2, and LCDR3 selected from Table 2. In some embodiments, such antibodies comprise a VH and / or VL selected from Table 3.

[0044] In another aspect, provided herein is a conjugate comprising a human or mouse TfR binding protein as described herein and a therapeutic agent. In some embodiments, the therapeutic agent is selected from double-stranded RNA (e.g., siRNA, saRNA), oligonucleotides (e.g., antisense oligonucleotides), peptides, small molecules, nanoparticles, lipid nanoparticles, exosomes, antibodies or their antigen-binding fragments, 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, 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. 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.

[0045] In some embodiments, the therapeutic agent is connected to the human or mouse TfR binding protein through a linker. In some embodiments, the linker is a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker (the structures of these linkers are shown in Table 8).

[0046] 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; wherein P is a protein comprising a monovalent human or mouse TfR binding domain; and wherein L is a linker, or optionally absent. In some embodiments, P is a human or mouse TfR binding protein as 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.

[0047] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human or mouse TfR binding domain; and wherein L is a linker, or is optionally absent, and wherein n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0048] 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; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, the human TfR binding domain described herein 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 the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences:

[0049] (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

[0050] (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.

[0051] 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; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, 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, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences:

[0052] (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;

[0053] (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;

[0054] (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;

[0055] (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;

[0056] (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;

[0057] (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

[0058] (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.

[0059] 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; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, the human TfR binding domain described herein comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the following sequences:

[0060] (a) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:27, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0061] (b) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:29, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0062] (c) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:30, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:31;

[0063] (d) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:32, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:33;

[0064] (e) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:34, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:35;

[0065] (f) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:36, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37; or

[0066] (g) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:38, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37.

[0067] 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; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, the human TfR binding domain described herein comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the following sequences:

[0068] (a) VH comprises SEQ ID NO:27 and VL comprises SEQ ID NO:28;

[0069] (b) VH comprises SEQ ID NO:29 and VL comprises SEQ ID NO:28;

[0070] (c) VH comprises SEQ ID NO:30 and VL comprises SEQ ID NO:31;

[0071] (d) VH comprises SEQ ID NO:32 and VL comprises SEQ ID NO:33;

[0072] (e) VH comprises SEQ ID NO:34 and VL comprises SEQ ID NO:35;

[0073] (f) VH comprises SEQ ID NO:36 and VL comprises SEQ ID NO:37; or

[0074] (g) VH comprises SEQ ID NO:38 and VL comprises SEQ ID NO:37.

[0075] In some embodiments, provided herein is Formula (II): (RL) n-P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, the human TfR binding domain described herein 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 the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the following sequences:

[0076] (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

[0077] (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,

[0078] And wherein n is 1 to 3.

[0079] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0080] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, 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, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the following sequences:

[0081] (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;

[0082] (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;

[0083] (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;

[0084] (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;

[0085] (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;

[0086] (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

[0087] (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,

[0088] And wherein n is 1 to 3.

[0089] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0090] In some embodiments, provided herein is a conjugate of formula (II): (RL)nP, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, wherein 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:

[0091] (a) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:27, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0092] (b) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:29, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0093] (c) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:30, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:31;

[0094] (d) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:32, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:33;

[0095] (e) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:34, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:35;

[0096] (f) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:36, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37; or

[0097] (g) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:38, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37,

[0098] And wherein n is 1 to 3.

[0099] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0100] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, wherein 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:

[0101] (a) VH comprises SEQ ID NO:27 and VL comprises SEQ ID NO:28;

[0102] (b) VH comprises SEQ ID NO:29 and VL comprises SEQ ID NO:28;

[0103] (c) VH comprises SEQ ID NO:30 and VL comprises SEQ ID NO:31;

[0104] (d) VH comprises SEQ ID NO:32 and VL comprises SEQ ID NO:33;

[0105] (e) VH comprises SEQ ID NO:34 and VL comprises SEQ ID NO:35;

[0106] (f) VH comprises SEQ ID NO:36 and VL comprises SEQ ID NO:37; or

[0107] (g) VH comprises SEQ ID NO: 38 and VL comprises SEQ ID NO: 37,

[0108] And wherein n is 1 to 3.

[0109] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0110] In some embodiments, the linker (L) is a Mal-Tet-TCO linker, a SMCC linker, or a GDM linker (see Table 8).

[0111] 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.

[0112] Exemplary unmodified sense and antisense strand sequences of dsRNAs 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:

[0113] (a) the sense strand comprises SEQ ID NO:81, and the antisense strand comprises SEQ ID NO:82;

[0114] (b) the sense strand comprises SEQ ID NO:83, and the antisense strand comprises SEQ ID NO:84;

[0115] (c) the sense strand comprises SEQ ID NO:85, and the antisense strand comprises SEQ ID NO:86;

[0116] (d) the sense strand comprises SEQ ID NO:87, and the antisense strand comprises SEQ ID NO:88;

[0117] (e) the sense strand comprises SEQ ID NO:89, and the antisense strand comprises SEQ ID NO:90; and

[0118] (f) the sense strand comprises SEQ ID NO:91, and the antisense strand comprises SEQ ID NO:92;

[0119] (g) the sense strand comprises SEQ ID NO: 116, and the antisense strand comprises SEQ ID NO: 82,

[0120] wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein 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.

[0121] Table 9b provides exemplary unmodified sense and antisense sequences of dsRNA targeting human MAPT mRNA. In some embodiments, the sense and antisense strands of dsRNA comprise a pair of nucleic acid sequences selected from the following:

[0122] (a) the sense strand comprises SEQ ID NO: 120, and the antisense strand comprises SEQ ID NO: 121;

[0123] (b) the sense strand comprises SEQ ID NO: 122, and the antisense strand comprises SEQ ID NO: 123; and

[0124] (c) the sense strand comprises SEQ ID NO: 124, and the antisense strand comprises SEQ ID NO: 125,

[0125] wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

[0126] dsRNA can include modification.Modification can be carried out for one or more nucleotides of sense strand and / or antisense strand or for internucleotide bonding.In some embodiments, one or more nucleotides of sense strand and / or antisense strand are independently modified nucleotides, which means that sense strand and antisense strand can have different modified nucleotides.In some embodiments, each nucleotide of sense strand is a modified nucleotide.In some embodiments, each nucleotide of 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'-O-alkyl modified nucleotide.In some embodiments, each nucleotide of sense strand and antisense strand is independently a modified nucleotide, such as a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O-alkyl modified nucleotide.

[0127] In some embodiments, the sense strand has four 2'-fluoro modified nucleotides, e.g., at positions 7, 9, 10, 11 from the 5' end of the sense strand. In some embodiments, the other nucleotides of 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, 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.

[0128] In some embodiments, the sense strand has three 2'-fluoro modified nucleotides, e.g., at positions 9, 10, 11 from the 5' end of the sense strand. In some embodiments, the other nucleotides of 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, 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, 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, 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.

[0129] In some embodiments, the 5' end of the antisense strand has a phosphate analog, for example, 5'-vinylphosphonate (5'-VP).

[0130] In some embodiments, the sense strand or the antisense strand comprises an abasic portion or an inverted abasic portion.

[0131] In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage. 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.

[0132] 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.

[0133] In another aspect, provided herein are methods for treating a CNS disease, such as a neurodegenerative disease, in a patient in need thereof, and such methods comprise administering to the patient an effective amount of a human TfR binding protein or conjugate or pharmaceutical composition described herein.

[0134] In a further aspect, provided herein are methods for treating a neurodegenerative synucleinopathy in a patient in need thereof, and such methods comprise 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 Lewy body dementia. The human TfR binding protein or conjugate or pharmaceutical composition can be administered to the patient intravenously or subcutaneously.

[0135] In a further aspect, provided herein are methods for treating tauopathy in a patient in need thereof, and such methods comprise administering to the patient an effective amount of a human TfR binding protein or conjugate or a 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 selected from Alzheimer's disease, frontotemporal dementia (FTD), chromosome 17-linked frontotemporal dementia with associated parkinsonism (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-oligoglossia (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (NFT) dementia, motor neuron dementia (MND) FTD, 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, diffuse neurofibrillary tangles with calcifications, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease disease), Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, tangled dementia, tangled 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 the patient.

[0136] In another aspect, provided herein is a human TfR binding protein or conjugate as described herein, or a pharmaceutical composition comprising such a human TfR binding protein or conjugate, for use in treatment. Also provided herein is a human TfR binding protein or conjugate as described herein, or a pharmaceutical composition comprising such a human TfR binding protein or conjugate (e.g., a TBP-SNCA siRNA conjugate as described herein, or a pharmaceutical composition comprising such a TBP-SNCA siRNA conjugate), for use in the treatment of neurodegenerative synuclein diseases, such as Parkinson's disease, Alzheimer's disease, multiple system atrophy, or Lewy body dementia. Also provided herein is a human TfR binding protein or conjugate as described herein, or a pharmaceutical composition comprising such a human TfR binding protein or conjugate (e.g., a TBP-MAPT siRNA conjugate as described herein, or a pharmaceutical composition comprising such a TBP-MAPT Pharmaceutical compositions of siRNA conjugates) for the treatment of tauopathies such as, for example, Alzheimer's disease, frontotemporal dementia (FTD), chromosome 17-linked frontotemporal dementia with associated parkinsonism (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-oligoglossia (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic granular dementia (AGD), British amyloidosis Tubular disease, 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-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

[0137] In another aspect, provided herein is the use of a human TfR binding protein or conjugate described herein in the preparation of a medicament for treating a CNS disease such as a neurodegenerative disease. In some embodiments, the neurodegenerative disease is a neurodegenerative synuclein disease, such as Parkinson's disease, Alzheimer's disease, multiple system atrophy, or Lewy body dementia. In some embodiments, the neurodegenerative disease is a tauopathy, such as Alzheimer's disease, frontotemporal dementia (FTD), chromosome 17-linked frontotemporal dementia with associated parkinsonism (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-oligoglossia (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic granular dementia (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-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, C Niemann-Pick disease (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT). BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1A An exemplary analytical anion exchange (aAEX) chromatogram of the DAR profile of the TBP11-dsRNA conjugate before purification is shown. Figure 1B An exemplary aAEX chromatogram of the DAR profile of the purified TBP14-dsRNA conjugate is shown. Figure 1C An exemplary aAEX chromatogram is shown for the DAR profile of the TBP15-dsRNA conjugate before purification. Figure 1DAn exemplary aAEX chromatogram of the DAR profile of the purified TBP15-dsRNA conjugate is shown. Figure 1E Exemplary diagrams of TBP-dsRNA conjugates of DAR2 (top) or DAR1 (bottom) are shown.

[0140] Figure 2 In vitro binding, internalization, and degradation of the indicated molecules in mouse cortical neurons are shown.

[0141] Figure 3 Shown are the in vitro efficacy of the indicated molecules for knocking down mouse SNCA in primary mouse cortical neurons.

[0142] Figure 4 In vitro binding, internalization and degradation assessment of the indicated molecules in SHSY5Y cells are shown.

[0143] Figure 5 The in vitro efficacy of the indicated molecules for knockdown of human SNCA in SH-SY5Y cells is shown.

[0144] Fig. 6A , 6B Figures 6 and 6C show proof-of-concept data in mice, which demonstrate the pharmacodynamic efficacy of the mTBP2-SNCA siRNA conjugate with multiple intravenous (IV) administrations at a single time point (28 days), showing reductions in SNCA mRNA and protein in the mouse brain ( Fig. 6A ) and spinal cord ( Figure 6B ) and lumbar dorsal root ganglia ( Figure 6C ) in the rats.

[0145] Fig. 7A and 7B Mouse proof-of-concept pharmacodynamic efficacy time course data of mTBP2-SNCA siRNA conjugate following a single IV dose, where mice were sacrificed at multiple time points (7, 28, 70, and 120 days) after dose, showing reductions in SNCA mRNA and protein in the mouse brain ( Fig. 7A ) and decreased SNCA mRNA and protein in the spinal cord ( Figure 7B )'s pharmacodynamic time course. Fig. 7A and 7B The error bars in are standard deviations, and statistical analysis was performed using one-way Anova with Dunnett's multiple comparison test for the PBS control group. Annotations indicate P values ​​> 0.0001 = ****; > 0.001 = ***; > 0.01 = **; > 0.05 = *.

[0146] Fig. 8AShown is the reduction of SNCA mRNA in cynomolgus monkey tissues 29 days after two consecutive single IV peripheral doses of TBP10-SNCA siRNA (dsRNA No. 8 in Table 11a) conjugate at 4.4 mg / kg siRNA, given two hours apart. Figure 8B Shown is the reduction of SNCA mRNA in cynomolgus monkey tissues 29 days after two consecutive single IV peripheral doses of 1.3 mg / kg siRNA (dsRNA No. 8 in Table 11a) conjugate at 1.3 mg / kg siRNA, given two hours apart. Fig. 8A and 8B The error bars in are standard errors of the mean, and statistical analysis was performed using one-way Anova with Dunnett's multiple comparison test for the PBS control group. Notes indicate P values ​​> 0.0001 to 0.05 = *. Figure 8C Shown is a comparison of mouse brain efficacy of mouse TfR binding protein conjugates at NHP equivalent siRNA doses adjusted for body weight. Figure 8C The error bars in are standard deviations, and statistical analysis was performed using one-way Anova with Dunnett's multiple comparison test for the PBS control group. Annotations indicate P values ​​> 0.0001 = ****; > 0.001 = ***; > 0.01 = **; > 0.05 = *.

[0147] Fig.9A Shown is the reduction of SNCA mRNA 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. Fig. 9B Shown is 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. Fig. 9C Shown is the reduction of SNCA mRNA 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. Fig.9D Shown is the reduction of α-synuclein protein 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. Fig.9EShown is a decrease in SNCA mRNA in gastrocnemius muscle following single or three consecutive monthly peripheral IV administrations of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate at 10 mg / kg siRNA.

[0148] Fig. 10A Shown is the reduction of MAPT mRNA 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. Fig. 10B Shown is 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.

[0149] Fig.11A Shown is the reduction of MAPT mRNA 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. Fig. 11B Shown is 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.

[0150] Fig. 12A Shown is the reduction of MAPT mRNA 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. Fig. 12B Shown is 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.

[0151] Fig.13A Shown is the reduction of SNCA mRNA 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. Fig. 13B and 13CShown is the reduction of SNCA mRNA 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) siRNA. Fig.13D Shown are the plasma PK of the conjugate-related siRNAs following a single peripheral IV administration of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR2) at 10 mg / kg siRNA or TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) at 10 mg / kg or 1 mg / kg siRNA. Fig.13E Shown are total siRNA concentrations in selected cynomolgus monkey brain tissues on day 29 following a single peripheral IV administration of TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) at 1 or 10 mg / kg siRNA.

[0152] Fig.14A Shown are the plasma PK of conjugate-related siRNAs in human TfR transgenic mice following a single peripheral IV administration of 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. Fig. 14B Shown are brain tissue concentrations of total antisense siRNA at 24 hours in human TfR transgenic mice following a single peripheral IV administration of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR2) or TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) across various doses. Fig. 14C Shown are brain tissue concentrations of total siRNA in human TfR transgenic mice 24 hours after a single peripheral IV administration of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR2) or TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) across various siRNA doses. Fig.14DShown is a reduction in SNCA mRNA levels in whole brain homogenates at day 28 in human TfR transgenic mice following a single peripheral IV administration of TBP14-SNCA siRNA (dsRNA number 10 in Table 11a) conjugate (DAR2) or TBP15-SNCA siRNA (dsRNA number 10 in Table 11a) conjugate (DAR1) across various siRNA doses. Error bars are standard deviations, and statistical analysis was performed for the PBS control group using a one-way Anova with Dunnett's multiple comparison test. Annotations indicate P values ​​> 0.0001 = ****; > 0.001 = ***; > 0.01 = **; > 0.05 = *. Fig.14E Shown is a reduction in SNCA mRNA levels in whole brain homogenates at day 28 in human TfR transgenic mice following a single subcutaneous administration of TBP15-SNCA siRNA (dsRNA number 10 in Table 11a) conjugate (DAR1) across various siRNA doses. Error bars are standard deviations, and statistical analysis was performed using a one-way Anova with Dunnett's multiple comparison test against a PBS control group. Annotations indicate P values ​​> 0.0001 = ****; > 0.001 = ***; > 0.01 = **; > 0.05 = *.

[0153] Details

[0154] Provided herein are proteins comprising a monovalent human TfR binding domain ("human TfR binding protein"), proteins comprising a monovalent mouse TfR binding domain ("mouse TfR binding protein"), conjugates comprising such human or mouse TfR binding proteins, such as human TfR binding protein-dsRNA conjugates, pharmaceutical compositions comprising the human TfR binding protein or conjugate, and methods of using the human TfR binding protein or conjugate to treat CNS diseases (e.g., neurodegenerative diseases such as neurodegenerative synucleinopathy or tauopathy).

[0155] Human TfR binding protein

[0156] In one aspect, provided herein is a protein comprising a 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), and the VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementary 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. In some embodiments, the monovalent human TfR binding domain comprises a VL comprising LCDR1, LCDR2, and LCDR3 selected from Table 2. 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. In some embodiments, the monovalent human 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 protein described herein also binds to cynomolgus monkey TfR.

[0157] Table 1. Exemplary sequences of the heavy chain CDRs of the human TfR binding domain

[0158]

[0159] Table 2. Exemplary sequences of human TfR binding domain light chain CDRs

[0160]

[0161] Table 3. Exemplary sequences of human TfR binding domains VH and VL

[0162]

[0163] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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:

[0164] (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

[0165] (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.

[0166] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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 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 a monovalent human transferrin receptor (TfR) binding domain, wherein the human TfR binding domain comprises VH and 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 comprises SEQ ID NO:13, HCDR2 comprises SEQ ID NO:25, HCDR3 comprises SEQID NO:26, LCDR1 comprises SEQ ID NO:16, LCDR2 comprises SEQ ID NO:17, and LCDR3 comprises SEQ ID NO:18.

[0167] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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:

[0168] (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;

[0169] (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;

[0170] (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;

[0171] (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;

[0172] (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;

[0173] (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

[0174] (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.

[0175] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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 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 SEQID NO:5, and LCDR3 comprises SEQ ID NO:6. In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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 comprises SEQ ID NO:1, HCDR2 comprises SEQ ID NO:2, HCDR3 comprises SEQ ID NO:7, LCDR1 comprises SEQID NO:4, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:6. In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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 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 IDNO:6. In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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 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 a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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 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 a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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 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 a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and 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 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.

[0176] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein the VH and VL comprise the following sequences:

[0177] (a) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:27, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0178] (b) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:29, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0179] (c) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:30, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:31;

[0180] (d) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:32, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:33;

[0181] (e) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:34, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:35;

[0182] (f) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:36, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37; or

[0183] (g) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:38, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37.

[0184] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein the VH and VL comprise the following sequences:

[0185] (a) VH comprises SEQ ID NO:27 and VL comprises SEQ ID NO:28;

[0186] (b) VH comprises SEQ ID NO:29 and VL comprises SEQ ID NO:28;

[0187] (c) VH comprises SEQ ID NO:30 and VL comprises SEQ ID NO:31;

[0188] (d) VH comprises SEQ ID NO:32 and VL comprises SEQ ID NO:33;

[0189] (e) VH comprises SEQ ID NO:34 and VL comprises SEQ ID NO:35;

[0190] (f) VH comprises SEQ ID NO:36 and VL comprises SEQ ID NO:37; or

[0191] (g) VH comprises SEQ ID NO:38 and VL comprises SEQ ID NO:37.

[0192] In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein VH comprises SEQ ID NO: 27 and VL comprises SEQ ID NO: 28. In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein VH comprises SEQ ID NO: 29 and VL comprises SEQ ID NO: 28. In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein VH comprises SEQ ID NO: 30 and VL comprises SEQ ID NO: 31. In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein VH comprises SEQ ID NO: 32 and VL comprises SEQ ID NO: 33. In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein VH comprises SEQ ID NO: 34 and VL comprises SEQ ID NO: 35. In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein VH comprises SEQ ID NO: 36 and VL comprises SEQ ID NO: 37. In some embodiments, provided herein is a protein comprising a monovalent human TfR binding domain, wherein the human TfR binding domain comprises VH and VL, and wherein VH comprises SEQ ID NO: 38 and VL comprises SEQ ID NO: 37.

[0193] In some embodiments, the monovalent human TfR binding domain is an antibody fragment, such as Fab, scFv, Fv or scFab (single-chain Fab). In some embodiments, the monovalent human TfR binding domain is Fab. In some embodiments, the human TfR binding domain further comprises a heavy chain constant region and / or a light chain constant region.

[0194] In some embodiments, the human TfR binding protein described herein further comprises a half-life extender, such as an immunoglobulin Fc region or a VHH that binds to human serum albumin (HSA).

[0195] In some embodiments, the human TfR binding protein described herein further comprises 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 protein described herein further comprises a modified human IgG4 Fc region, which comprises a proline at residue 228 and an alanine at residues 234 and 235 (all residues are numbered according to the EU index number, also referred to as hIgG4PAAFc region). In some embodiments, the human TfR binding protein described herein further comprises a modified human IgG1 Fc region, which comprises 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 number, also referred to as hIgG1 effector null or hIgG1EN Fc region). In some embodiments, the human TfR binding protein described herein comprises a modified human IgG1 or IgG4 Fc region, wherein the Fc region comprises 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 the EU index numbering). In some embodiments, the 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 the EU index numbering).

[0196] In some embodiments, the human TfR binding protein described herein further comprises 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 VHHs include 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 VHHs include SEQ ID NO: 42. In some embodiments, the VHH is connected to the TfR binding domain via a peptide linker such as (GGGGQ) 4 (SEQ ID NO: 70).

[0197] Table 4. Exemplary sequences of VHHs that bind to human serum albumin (HSA)

[0198]

[0199] In some embodiments, the human TfR binding protein described herein is a heterodimeric antibody, which includes a first arm comprising a monovalent human TfR binding domain and a second arm as an empty arm, for example, an arm that does not bind to any known human target (e.g., an isotype arm). Heterodimer antibodies such as heteromabs, orthomabs, or bispecific antibodies (duobody) have been described in WO2014150973, WO2016118742, WO2018118616, and WO2011131746. In some embodiments, the first arm includes any monovalent human TfR binding domain described herein. In some embodiments, the second arm is an empty arm (e.g., an isotype arm) that does not bind to any known human target, comprising the sequence in Table 5. In some embodiments, the second arm comprises VH and VL, wherein the VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, and 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 VH and 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.

[0200] In some embodiments, the human TfR binding protein described herein comprises a heterodimer mutation. In some embodiments, the human TfR binding protein described herein comprises a modified Fc region comprising a first Fc CH3 domain and a second Fc CH3 domain, wherein the first Fc CH3 domain comprises a serine at residue 349, a methionine at residue 366, a tyrosine at residue 370, and a valine at residue 409, and the second Fc CH3 domain comprises 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 the EU index number). In some embodiments, the human TfR binding protein described herein comprises a modified Fc region comprising a first Fc CH3 domain and a second Fc CH3 domain, wherein the first Fc CH3 domain comprises a leucine at residue 405, and the second Fc CH3 domain comprises an arginine at residue 409 (all residues are numbered according to the EU index number).

[0201] Table 5. Exemplary sequences of isotype arms or empty arms (isotype Abs) that do not bind to any known target

[0202]

[0203] In some embodiments, the human TfR binding proteins described herein include one or more native cysteine ​​residues, which can be used for conjugation. For example, in some embodiments, the human TfR binding proteins described herein include 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 are numbered according to the EU index).

[0204] In some embodiments, human TfR binding proteins described herein include modified cysteine ​​residues for conjugation. Methods including modified cysteine ​​as a means for conjugation have been described in WO 2018 / 232088. In some embodiments, human TfR binding proteins described herein include heavy chains, which include one or more cysteines at the following residues: 124, 157, 162, 262, 373, 375, 378, 397, 415 (all residues are numbered according to the EU index). In some embodiments, human TfR binding proteins described herein include light chains (e.g., kappa light chains), which include one or more cysteines at the following residues: 156, 171, 191, 193, 202, 208 (all residues are numbered according to the EU index). In some embodiments, human TfR binding proteins described herein include heavy chain constant regions, which include cysteine ​​at residue 124 (numbered according to the EU index). In some embodiments, the human TfR binding protein described herein comprises a light chain constant region comprising a cysteine ​​at residue 156 (numbered according to the EU index). In some embodiments, the human TfR binding protein described herein comprises an immunoglobulin Fc region comprising a cysteine ​​at residue 378 (numbered according to the EU index).

[0205] In some embodiments, the human TfR binding protein described herein is any one of Tables 6a and 6b. In some embodiments, the human TfR binding protein described herein has a heavy chain (HC) and a light chain (LC), such as TBP1, TBP2, TBP3, TBP4, TBP5, TBP6, TBP7, TBP8 or TBP9 (see Table 6a).

[0206] In some embodiments, the human TfR binding protein described herein has a Fab-Fc form, such as TBP1, TBP2, TBP3, TBP4, TBP5, TBP6 or TBP7. In some embodiments, provided herein is a human TfR binding protein 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 is a human TfR binding protein 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 is a human TfR binding protein 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 is a human TfR binding protein 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.

[0207] In some embodiments, the human TfR binding protein described herein has a Fab form, such as TBP8. In some embodiments, provided herein is a human TfR binding protein comprising one HC and one LC, and wherein the HC comprises SEQ ID NO: 65 and the LC comprises SEQ ID NO: 59.

[0208] In some embodiments, the human TfR binding protein described herein has a Fab-VHH format, such as TBP9. In some embodiments, provided herein is a human TfR binding protein comprising one HC and one LC, wherein the HC comprises SEQ ID NO: 66 and the LC comprises SEQ ID NO: 67.

[0209] Table 6a. Exemplary sequences of human TfR binding proteins (one HC and one LC)

[0210]

[0211]

[0212]

[0213]

[0214] In some embodiments, the human TfR binding protein described herein has more than one heavy chain (HC) and / or more than one light chain (see Table 6b). 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 form, such as TBP10, TBP11, TBP12 or TBP13.

[0215] 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.

[0216] In some embodiments, the human TfR binding protein has two heavy chains (HC1 and HC2) and one light chain (LC1), such as 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.

[0217] Table 6b. Exemplary sequences of human TfR binding proteins (multiple HCs and / or LCs)

[0218]

[0219]

[0220]

[0221] In some embodiments, provided herein is a protein comprising a 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 346-364FGNMEGDCPSDWKTDSTCR (SEQ ID NO: 119), (b) residues 243-247FEDLY (SEQ ID NO: 162) and residues 345-364LFGNMEEGDCPSDWKTDSTCR) (SEQ ID NO: 163), or (c) residues 243-247FEDLY (SEQ ID NO: 162), residues 259-263AGKIT (SEQ ID NO: 164), and residues 532-538 (VEKLTLD) (SEQ ID NO: 165) of human TfR.

[0222] Also provided herein are antibodies comprising a VH comprising a HCDR1, HCDR2, and HCDR3 selected from Table 1, and / or a VL comprising a LCDR1, LCDR2, and LCDR3 selected from Table 2. In some embodiments, such antibodies comprise a VH and / or VL selected from Table 3.

[0223] The TfR binding protein or antibody described herein can be recombinantly produced in a host cell, for example, using an expression vector. For example, an expression vector may include a sequence encoding one or more signal peptides that promote the secretion of the polypeptide from the host cell. An expression vector containing a polynucleotide of interest (e.g., a polynucleotide encoding a heavy chain or light chain of a TfR binding protein or antibody) may be transferred into a host cell by well-known methods. In addition, the expression vector may contain one or more selection markers, such as tetracycline, neomycin, and dihydrofolate reductase, to help detect host cells transformed with the desired polynucleotide sequence.

[0224] Host cells include cells stably or transiently transfected, transformed, transduced or infected with one or more expression vectors, which express all or part of the TfR binding protein or antibody described herein. According to some embodiments, the host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing the HC polypeptide of the TfR binding protein or antibody described herein and an expression vector expressing the LC polypeptide. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing the HC and LC polypeptides of the TfR binding protein or antibody described herein. The TfR binding protein or antibody may be produced in mammalian cells such as CHO, NS0, HEK293 or COS cells according to techniques well known in the art.

[0225] The culture medium into which the TfR binding protein or antibody has been secreted may be purified by conventional techniques, such as a mixed mode method of ion exchange and hydrophobic interaction chromatography. For example, the culture medium may be applied to a protein A or G column and eluted therefrom using conventional methods; a mixed mode method of ion exchange and hydrophobic interaction chromatography may also be used. Soluble aggregates and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. Various protein purification methods may be employed, 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).

[0226] Mouse TfR binding protein

[0227] In another aspect, provided herein is a protein comprising a monovalent mouse TfR binding domain ("mouse TfR binding protein" or mTBP). These mouse TfR binding proteins can serve as surrogate molecules for the above-mentioned human TfR binding proteins in a mouse model. In some embodiments, the monovalent mouse TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH comprises a heavy chain complementary determining region HCDR1, HCDR2, and HCDR3, and the VL comprises a light chain complementary determining region LCDR1, LCDR2, and LCDR3. In some embodiments, the monovalent mouse TfR binding domain includes 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.

[0228] In some embodiments, provided herein is a protein comprising a monovalent mouse TfR binding domain, wherein the mouse TfR binding domain comprises VH and VL, wherein the VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, and 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 a 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.

[0229] In some embodiments, the mouse TfR binding protein described herein has one heavy chain (HC) and one light chain, such as mTBP1 in Table 7b. In some embodiments, the mouse TfR binding protein has two heavy chains (HC1 and HC2) and two light chains (LC1 and LC2), such as mTBP2 in Table 7b.

[0230] In some embodiments, provided herein is a protein comprising a 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.

[0231] In some embodiments, the mouse TfR binding protein described herein is a heterodimeric antibody comprising a first arm comprising a monovalent mouse TfR binding domain and a second arm that is an empty 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.

[0232] Also provided herein are antibodies comprising a VH comprising a HCDR1, HCDR2 and HCDR3 selected from Table 7a, and / or a VL comprising a LCDR1, LCDR2 and LCDR3 selected from Table 7a. In some embodiments, such antibodies comprise a VH and / or VL selected from Table 7a.

[0233] Table 7a. Exemplary sequences of mouse TfR binding domains

[0234]

[0235] Table 7b. Exemplary sequences of mouse TfR binding proteins

[0236]

[0237]

[0238] Conjugates containing human or mouse TfR binding protein

[0239] In another aspect, provided herein is a conjugate comprising a human or mouse TfR binding protein or antibody as described herein and a therapeutic agent. In some embodiments, the therapeutic agent is selected from double-stranded RNA (e.g., siRNA, saRNA), oligonucleotides (e.g., antisense oligonucleotides), peptides, small molecules, nanoparticles, lipid nanoparticles, exosomes, antibodies or their antigen-binding fragments, or a combination thereof. In some embodiments, the therapeutic agent is double-stranded RNA (dsRNA). In some embodiments, dsRNA includes sense strand and antisense strand, 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. In some embodiments, dsRNA includes sense strand and antisense strand, wherein the antisense strand is complementary to SNCAmRNA. In some embodiments, dsRNA includes sense strand and antisense strand, wherein the antisense strand is complementary to MAPT mRNA.

[0240] 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.

[0241] In some embodiments, the human TfR binding proteins described herein include one or more native cysteine ​​residues, which can be used for conjugation. For example, in some embodiments, the human TfR binding proteins described herein include 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 are numbered according to the EU index).

[0242] In some embodiments, human TfR binding proteins described herein include one or more modified cysteine ​​residues for conjugation. Methods including modified cysteine ​​as a means for conjugation have been described in WO 2018 / 232088. In some embodiments, human TfR binding proteins described herein include heavy chains, which include one or more cysteines at the following residues: 124, 157, 162, 262, 373, 375, 378, 397, 415 (all residues are numbered according to the EU index). In some embodiments, human TfR binding proteins described herein include light chains (e.g., kappa light chains), which include one or more cysteines at the following residues: 156, 171, 191, 193, 202, 208 (all residues are numbered according to the EU index). In some embodiments, human TfR binding proteins described herein include heavy chain constant regions, which include cysteine ​​at residue 124 (numbered according to the EU index). In some embodiments, the human TfR binding protein described herein comprises a light chain constant region comprising a cysteine ​​at residue 156 (numbered according to the EU index). In some embodiments, the human TfR binding protein described herein comprises an immunoglobulin Fc region comprising a cysteine ​​at residue 378 (numbered according to the EU index).

[0243] In some embodiments, the therapeutic agent is connected to the human or mouse TfR binding protein through a linker. In some embodiments, the linker is a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker (the structures of these linkers are shown in Table 8).

[0244] Table 8. Exemplary linker structures

[0245]

[0246]

[0247]

[0248] The conjugates described herein can be prepared by various procedures known to those of ordinary skill in the art, some of which are described in the preparations and examples below, such as in Example 3. One of ordinary skill in the art recognizes that the specific synthetic steps for each of the described pathways may be combined in different ways, or combined with steps from different schemes, to prepare the conjugates. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, grinding and crystallization. Reagents and raw materials are readily available to those of ordinary skill in the art.

[0249] In some embodiments, the TfR binding proteins described herein with native or modified cysteine ​​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.

[0250] Human TfR binding protein-dsRNA conjugate

[0251] 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; wherein P is a protein comprising a monovalent human or mouse TfR binding domain; and wherein L is a linker, or is optionally absent. In some embodiments, P is a human or mouse TfR binding protein as described herein. In some embodiments, the ratio of R to P is about 1 to 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.

[0252] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human or mouse TfR binding domain; and wherein L is a linker, or is optionally absent, and wherein n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0253] 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; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, the human TfR binding domain described herein 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 the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences:

[0254] (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

[0255] (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.

[0256] 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; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, 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, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences:

[0257] (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;

[0258] (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;

[0259] (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;

[0260] (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;

[0261] (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;

[0262] (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

[0263] (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.

[0264] 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; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, wherein 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:

[0265] (a) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:27, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0266] (b) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:29, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0267] (c) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:30, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:31;

[0268] (d) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:32, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:33;

[0269] (e) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:34, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:35;

[0270] (f) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:36, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37; or

[0271] (g) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:38, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37.

[0272] 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; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, wherein 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:

[0273] (a) VH comprises SEQ ID NO:27 and VL comprises SEQ ID NO:28;

[0274] (b) VH comprises SEQ ID NO:29 and VL comprises SEQ ID NO:28;

[0275] (c) VH comprises SEQ ID NO:30 and VL comprises SEQ ID NO:31;

[0276] (d) VH comprises SEQ ID NO:32 and VL comprises SEQ ID NO:33;

[0277] (e) VH comprises SEQ ID NO:34 and VL comprises SEQ ID NO:35;

[0278] (f) VH comprises SEQ ID NO:36 and VL comprises SEQ ID NO:37; or

[0279] (g) VH comprises SEQ ID NO:38 and VL comprises SEQ ID NO:37.

[0280] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, the human TfR binding domain described herein 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 the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the following sequences:

[0281] (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

[0282] (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,

[0283] And wherein n is 1 to 3.

[0284] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0285] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, 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, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the following sequences:

[0286] (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;

[0287] (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;

[0288] (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;

[0289] (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;

[0290] (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;

[0291] (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

[0292] (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,

[0293] And wherein n is 1 to 3.

[0294] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0295] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, wherein 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:

[0296] (a) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:27, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0297] (b) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:29, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:28;

[0298] (c) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:30, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:31;

[0299] (d) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:32, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:33;

[0300] (e) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:34, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:35;

[0301] (f) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:36, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37; or

[0302] (g) VH comprises a sequence having at least 95% sequence identity to SEQ ID NO:38, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO:37,

[0303] And wherein n is 1 to 3.

[0304] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0305] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is optionally absent, wherein 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:

[0306] (a) VH comprises SEQ ID NO:27 and VL comprises SEQ ID NO:28;

[0307] (b) VH comprises SEQ ID NO:29 and VL comprises SEQ ID NO:28;

[0308] (c) VH comprises SEQ ID NO:30 and VL comprises SEQ ID NO:31;

[0309] (d) VH comprises SEQ ID NO:32 and VL comprises SEQ ID NO:33;

[0310] (e) VH comprises SEQ ID NO:34 and VL comprises SEQ ID NO:35;

[0311] (f) VH comprises SEQ ID NO:36 and VL comprises SEQ ID NO:37; or

[0312] (g) VH comprises SEQ ID NO: 38 and VL comprises SEQ ID NO: 37,

[0313] And wherein n is 1 to 3.

[0314] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0315] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising one monovalent human TfR binding domain; and wherein L is a linker, or optionally absent, 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 complementary determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementary 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 wherein n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0316] In some embodiments, provided herein is Formula (II): (RL) n-P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising one monovalent human TfR binding domain; and wherein L is a linker, or optionally absent, wherein 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 wherein n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0317] In some embodiments, protein (P) also binds to cynomolgus monkey TfR. In some embodiments, the human TfR binding domain of protein (P) is Fab, scFv, Fv or scFab. In some embodiments, the human TfR binding domain of protein (P) is Fab. In some embodiments, the human TfR binding domain of protein (P) further comprises a heavy chain constant region, which comprises a cysteine ​​at residue 124 (numbered according to the EU index). In some embodiments, the human TfR binding domain of protein (P) further comprises a light chain constant region, which comprises a cysteine ​​at residue 156 (numbered according to the EU index).

[0318] In some embodiments, the protein (P) further comprises a half-life extender, such as an immunoglobulin Fc region or a VHH that binds to human serum albumin (HSA). In some embodiments, the protein (P) comprises an immunoglobulin Fc region, such as 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, which comprises a proline at residue 228 and an alanine at residues 234 and 235 (all residues are numbered according to the EU index, also referred to as the hIgG4PAA Fc region). In some embodiments, the protein (P) comprises a modified human IgG1 Fc region, which comprises 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, also referred to as the hIgG1 effector null or hIgG1ENFc region). In some embodiments, the protein (P) comprises a modified human IgG1 or IgG4 Fc region, wherein the Fc region comprises 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 the EU index). In some embodiments, the protein (P) 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 the EU index).

[0319] 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 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 VHH comprises SEQ ID NO: 42. In some embodiments, the VHH is connected to the TfR binding domain via a peptide linker such as (GGGGQ) 4 (SEQ ID NO: 70).

[0320] 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:

[0321] (a) HC comprises SEQ ID NO:53 and LC comprises SEQ ID NO:54;

[0322] (b) HC comprises SEQ ID NO:55 and LC comprises SEQ ID NO:54;

[0323] (c) HC comprises SEQ ID NO:56 and LC comprises SEQ ID NO:57;

[0324] (d) HC comprises SEQ ID NO:58 and LC comprises SEQ ID NO:59;

[0325] (e) HC comprises SEQ ID NO:60 and LC comprises SEQ ID NO:61;

[0326] (f) HC comprises SEQ ID NO:62 and LC comprises SEQ ID NO:63; or

[0327] (g) HC comprises SEQ ID NO:64 and LC comprises SEQ ID NO:63.

[0328] In some embodiments, the protein (P) comprises one HC and one LC, and wherein the HC comprises SEQ ID NO:65 and the LC comprises SEQ ID NO:59.

[0329] In some embodiments, the protein (P) comprises one HC and one LC, and wherein the HC comprises SEQ ID NO:66 and the LC comprises SEQ ID NO:67.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] In some embodiments, the protein (P) is a heterodimeric antibody comprising a first arm comprising a monovalent human TfR binding domain, and a second arm that is an empty arm, e.g., an arm that does not bind to any known human target (e.g., an isotype arm in Table 5).

[0334] 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:

[0335] (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;

[0336] (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;

[0337] (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

[0338] (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.

[0339] In some embodiments, a linker (L) is present and is selected from: a Mal-Tet-TCO linker, a SMCC linker, or a GDM linker (see Table 8). In some embodiments, a linker (L) is absent.

[0340] In some embodiments, the protein (P) is connected to the 3' end of the sense strand of the dsRNA. In some embodiments, the protein (P) is connected to the 5' end of the sense strand of the dsRNA. In some embodiments, the protein (P) is connected to an internal position of the sense strand of the dsRNA. In some embodiments, the protein (P) is connected to the 3' end of the antisense strand of the dsRNA. In some embodiments, the protein (P) is connected to an internal position of the antisense strand of the dsRNA.

[0341] 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.

[0342] In some embodiments, the sense strand and antisense strand of dsRNA are 15-30 nucleotides in length, for example, 20-25 nucleotides in length. In some embodiments, 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 dsRNA may have an overhang (i.e., 5' overhang or 3' overhang) at the 5' end 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 comprises a 3' overhang of two nucleotides.

[0343] Exemplary unmodified sense and antisense strand sequences of dsRNAs targeting human SNCA mRNA are provided in Table 9a. Exemplary unmodified sense and antisense strand sequences of dsRNAs targeting human MAPT mRNA are provided in Table 9b.

[0344] Table 9a. Unmodified nucleic acid sequences of dsRNA targeting human SNCA mRNA (SNCA siRNA)

[0345]

[0346]

[0347] Table 9b. Unmodified nucleic acid sequences of dsRNA targeting human MAPT mRNA (MAPT siRNA)

[0348]

[0349] In some embodiments, the dsRNA targets SNCA mRNA. In some embodiments, the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of:

[0350] (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;

[0351] (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;

[0352] (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;

[0353] (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;

[0354] (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;

[0355] (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

[0356] (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,

[0357] wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

[0358] In some embodiments, the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from:

[0359] (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;

[0360] (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;

[0361] (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;

[0362] (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;

[0363] (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;

[0364] (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

[0365] (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,

[0366] wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

[0367] In some embodiments, the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from:

[0368] (a) the sense strand comprises SEQ ID NO:81, and the antisense strand comprises SEQ ID NO:82;

[0369] (b) the sense strand comprises SEQ ID NO:83, and the antisense strand comprises SEQ ID NO:84;

[0370] (c) the sense strand comprises SEQ ID NO:85, and the antisense strand comprises SEQ ID NO:86;

[0371] (d) the sense strand comprises SEQ ID NO:87, and the antisense strand comprises SEQ ID NO:88;

[0372] (e) the sense strand comprises SEQ ID NO:89, and the antisense strand comprises SEQ ID NO:90;

[0373] (f) the sense strand comprises SEQ ID NO:91, and the antisense strand comprises SEQ ID NO:92; and

[0374] (g) the sense strand comprises SEQ ID NO: 116, and the antisense strand comprises SEQ ID NO: 82,

[0375] wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

[0376] In some embodiments, the dsRNA targets MAPT mRNA. In some embodiments, the sense strand and antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the following:

[0377] (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;

[0378] (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

[0379] (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,

[0380] wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

[0381] In some embodiments, the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from:

[0382] (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;

[0383] (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

[0384] (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,

[0385] wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

[0386] In some embodiments, the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from:

[0387] (a) the sense strand comprises SEQ ID NO: 120, and the antisense strand comprises SEQ ID NO: 121;

[0388] (b) the sense strand comprises SEQ ID NO: 122, and the antisense strand comprises SEQ ID NO: 123; and

[0389] (c) the sense strand comprises SEQ ID NO: 124, and the antisense strand comprises SEQ ID NO: 125,

[0390] wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

[0391] In some embodiments, provided herein is Formula (II): (RL) n-P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising one monovalent human TfR binding domain; and wherein L is a linker or absent, 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 complementary determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementary 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 wherein 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 from Table 8. In some embodiments, L is a SMCC linker from Table 8.

[0392] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising one monovalent human TfR binding domain; and wherein L is a linker or is absent, wherein 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 wherein 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.

[0393] In some embodiments, provided herein is Formula (II): (RL) n-P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising a monovalent human TfR binding domain, wherein 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; and wherein L is a linker or is absent, and wherein 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.

[0394] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising a monovalent human TfR binding domain, wherein 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; and wherein L is a linker or is absent, and wherein 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.

[0395] In some embodiments, provided herein is Formula (II): (RL) n-P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising one monovalent human TfR binding domain; and wherein L is a linker or absent, 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 complementary determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementary 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 wherein 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 from Table 8. In some embodiments, L is a SMCC linker from Table 8.

[0396] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising one monovalent human TfR binding domain; and wherein L is a linker or is absent, wherein 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 wherein 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.

[0397] In some embodiments, provided herein is Formula (II): (RL) n-P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising a monovalent human TfR binding domain, wherein 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; and wherein L is a linker or is absent, and wherein 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.

[0398] In some embodiments, provided herein is a conjugate of formula (II): (RL) n -P, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising a monovalent human TfR binding domain, wherein 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; and wherein L is a linker or is absent, and wherein 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.

[0399] In some embodiments, provided herein is Formula (II): (RL) n-P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising one monovalent human TfR binding domain; and wherein L is a linker or absent, 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 complementary determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementary 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 wherein 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 from Table 8. In some embodiments, L is a SMCC linker from Table 8.

[0400] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising one monovalent human TfR binding domain; and wherein L is a linker or is absent, wherein 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 wherein 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.

[0401] In some embodiments, provided herein is Formula (II): (RL) n-P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising a monovalent human TfR binding domain, wherein 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; and wherein L is a linker or is absent, and wherein 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.

[0402] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising a monovalent human TfR binding domain, wherein 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; and wherein L is a linker or is absent, and wherein 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.

[0403] In some embodiments, provided herein is Formula (II): (RL) n-P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising one monovalent human TfR binding domain; and wherein L is a linker or absent, 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 complementary determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementary 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 wherein 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 from Table 8. In some embodiments, L is a SMCC linker from Table 8.

[0404] In some embodiments, provided herein is a conjugate of formula (II): (RL) n -P, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising one monovalent human TfR binding domain; and wherein L is a linker or is absent, wherein 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 wherein 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.

[0405] In some embodiments, provided herein is Formula (II): (RL) n-P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising a monovalent human TfR binding domain, wherein 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; and wherein L is a linker or is absent, and wherein 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.

[0406] In some embodiments, provided herein is Formula (II): (RL) n -P conjugate, wherein R is a double-stranded RNA (dsRNA) comprising 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; wherein P is a protein comprising a monovalent human TfR binding domain, wherein 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; and wherein L is a linker or is absent, and wherein 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.

[0407] dsRNA can include modification.Modification can be carried out for one or more nucleotides of sense strand and / or antisense strand or for internucleotide bonding, and the internucleotide bonding is the bond between two nucleotides in sense strand or antisense strand.For example, some 2'-modifications of ribose or deoxyribose can increase the stability and half-life of RNA or DNA.Such 2'-modifications can be 2'-fluoro, 2'-O-methyl (i.e., 2'-methoxy) or 2'-O-alkyl.

[0408] In some embodiments, one or more nucleotides of the sense strand and / or antisense strand are independently modified nucleotides, which means that the sense strand and the antisense strand can 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'-O-alkyl modified nucleotide. In some embodiments, each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, such as a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, or a 2'-O-alkyl modified nucleotide.

[0409] In some embodiments, the sense strand has four 2'-fluoro modified nucleotides, e.g., at positions 7, 9, 10, 11 from the 5' end of the sense strand. In some embodiments, the other nucleotides of 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, 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.

[0410] In some embodiments, the sense strand has three 2'-fluoro modified nucleotides, e.g., at positions 9, 10, 11 from the 5' end of the sense strand. In some embodiments, the other nucleotides of 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, 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, 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, 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.

[0411] In some embodiments, the 5' end of the antisense strand has a phosphate analog, such as 5'-vinylphosphonate (5'-VP).

[0412] In some embodiments, the sense strand or the antisense strand comprises an abasic moiety or a reverse abasic moiety, such as those shown in Table 10. In some embodiments, the sense strand comprises an abasic moiety at position 10.

[0413] Table 10. Abasic or inverted abasic (iAb) moieties

[0414]

[0415] "5'" and "3'" indicate the 5' to 3' direction of the sequence.

[0416] In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage. 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.

[0417] 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.

[0418] In some embodiments, the dsRNA comprises a sense strand comprising a sequence having 1, 2, or 3 differences from a sense strand sequence in Table 9a or 11a. In some embodiments, the dsRNA comprises an antisense strand comprising a sequence having 1, 2, or 3 differences from an antisense strand sequence in Table 9a or 11a.

[0419] In some embodiments, the dsRNA comprises a sense strand comprising a sequence having 1, 2, or 3 differences from a sense strand sequence in Table 9b or 11b. In some embodiments, the dsRNA comprises an antisense strand comprising a sequence having 1, 2, or 3 differences from an antisense strand sequence in Table 9b or 11b.

[0420] Table 11a: Modified nucleic acid sequences of dsRNA targeting human SNCA mRNA (SNCA siRNA)

[0421]

[0422]

[0423]

[0424] Abbreviations - "m" indicates 2'-OMe; "f" indicates 2'-fluoro; "*" indicates phosphorothioate linkage; "VP" indicates 5'-vinylphosphonate; "iAb" indicates the inverted abasic portion in Table 10; "S" means sense strand; "AS" means antisense strand.

[0425] Table 11b: Modified nucleic acid sequences of dsRNA targeting human MAPT mRNA (MAPT siRNA)

[0426]

[0427]

[0428] Abbreviations—“m” indicates 2′-OMe; “f” indicates 2′-fluoro; “*” indicates phosphorothioate linkage; “VP” indicates 5′-vinylphosphonate; “S” means sense strand; “AS” means antisense strand.

[0429] In some embodiments, the dsRNA targets SNCA mRNA. In some embodiments, the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of:

[0430] (a) the sense strand comprises SEQ ID NO: 93 or 140, and the antisense strand comprises SEQ ID NO: 94;

[0431] (b) the sense strand comprises SEQ ID NO: 95 or 141, and the antisense strand comprises SEQ ID NO: 96;

[0432] (c) the sense strand comprises SEQ ID NO: 95 or 141, and the antisense strand comprises SEQ ID NO: 97;

[0433] (d) the sense strand comprises SEQ ID NO: 95 or 141, and the antisense strand comprises SEQ ID NO: 98;

[0434] (e) the sense strand comprises SEQ ID NO: 99 or 142, and the antisense strand comprises SEQ ID NO: 94;

[0435] (f) the sense strand comprises SEQ ID NO: 100 or 143, and the antisense strand comprises SEQ ID NO: 101;

[0436] (g) the sense strand comprises SEQ ID NO: 102 or 144, and the antisense strand comprises SEQ ID NO: 103;

[0437] (h) the sense strand comprises SEQ ID NO: 104 or 145, and the antisense strand comprises SEQ ID NO: 105;

[0438] (i) the sense strand comprises SEQ ID NO: 106 or 146, and the antisense strand comprises SEQ ID NO: 107;

[0439] (j) the sense strand comprises SEQ ID NO: 108 or 147, and the antisense strand comprises SEQ ID NO: 107;

[0440] (k) the sense strand comprises SEQ ID NO: 117 or 148, and the antisense strand comprises SEQ ID NO: 97; and

[0441] (l) the sense strand comprises SEQ ID NO:118 or 149, and the antisense strand comprises SEQ ID NO:97.

[0442] In some embodiments, the sense strand and the antisense strand of the dsRNA have a pair of nucleic acid sequences selected from the group consisting of:

[0443] (a) the sense strand consists of SEQ ID NO: 93 or 140, and the antisense strand consists of SEQ ID NO: 94;

[0444] (b) the sense strand consists of SEQ ID NO: 95 or 141, and the antisense strand consists of SEQ ID NO: 96;

[0445] (c) the sense strand consists of SEQ ID NO: 95 or 141, and the antisense strand consists of SEQ ID NO: 97;

[0446] (d) the sense strand consists of SEQ ID NO: 95 or 141, and the antisense strand consists of SEQ ID NO: 98;

[0447] (e) the sense strand consists of SEQ ID NO: 99 or 142, and the antisense strand consists of SEQ ID NO: 94;

[0448] (f) the sense strand consists of SEQ ID NO: 100 or 143, and the antisense strand consists of SEQ ID NO: 101;

[0449] (g) the sense strand consists of SEQ ID NO: 102 or 144, and the antisense strand consists of SEQ ID NO: 103;

[0450] (h) the sense strand consists of SEQ ID NO: 104 or 145, and the antisense strand consists of SEQ ID NO: 105;

[0451] (i) the sense strand consists of SEQ ID NO: 106 or 146, and the antisense strand consists of SEQ ID NO: 107;

[0452] (j) the sense strand consists of SEQ ID NO: 108 or 147, and the antisense strand consists of SEQ ID NO: 107;

[0453] (k) the sense strand consists of SEQ ID NO: 117 or 148, and the antisense strand consists of SEQ ID NO: 97; and

[0454] (l) the sense strand consists of SEQ ID NO: 118 or 149, and the antisense strand consists of SEQ ID NO: 97.

[0455] In some embodiments, the dsRNA targets MAPT mRNA. In some embodiments, the sense strand and antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the following:

[0456] (a) the sense strand comprises SEQ ID NO: 126 or 150, and the antisense strand comprises SEQ ID NO: 127;

[0457] (b) the sense strand comprises SEQ ID NO: 128 or 151, and the antisense strand comprises SEQ ID NO: 129;

[0458] (c) the sense strand comprises SEQ ID NO: 130 or 152, and the antisense strand comprises SEQ ID NO: 131;

[0459] (d) the sense strand comprises SEQ ID NO: 132 or 153, and the antisense strand comprises SEQ ID NO: 133;

[0460] (e) the sense strand comprises SEQ ID NO: 134 or 154, and the antisense strand comprises SEQ ID NO: 135; and

[0461] (f) the sense strand comprises SEQ ID NO: 136 or 155, and the antisense strand comprises SEQ ID NO: 137.

[0462] In some embodiments, the sense strand and the antisense strand of the dsRNA have a pair of nucleic acid sequences selected from the group consisting of:

[0463] (a) the sense strand consists of SEQ ID NO: 126 or 150, and the antisense strand consists of SEQ ID NO: 127;

[0464] (b) the sense strand consists of SEQ ID NO: 128 or 151, and the antisense strand consists of SEQ ID NO: 129;

[0465] (c) the sense strand consists of SEQ ID NO: 130 or 152, and the antisense strand consists of SEQ ID NO: 131;

[0466] (d) the sense strand consists of SEQ ID NO: 132 or 153, and the antisense strand consists of SEQ ID NO: 133;

[0467] (e) the sense strand consists of SEQ ID NO: 134 or 154, and the antisense strand consists of SEQ ID NO: 135; and

[0468] (f) the sense strand consists of SEQ ID NO: 136 or 155, and the antisense strand consists of SEQ ID NO: 137.

[0469] The sense and antisense strands of the dsRNA can be synthesized using any nucleic acid polymerization method known in the art, for example, by 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. An automated commercial synthesizer, such as the MerMade from LGC Biosearch Technologies, can be used. TM 12, or other synthesizers from BioAutomation or AppliedBiosystems. Thiothioate linkages can be introduced using thiolation reagents such as phenylacetyl disulfide or DDTT (((dimethylaminomethylene) amino)-3H-1,2,4-dithiazoline-3-thione). It is well known that similar techniques and commercially available modified phosphoramidites and controlled aperture glass (CPG) products are used to synthesize modified oligonucleotides or conjugated oligonucleotides.

[0470] Purification process can be used to exclude unwanted impurities from final oligonucleotide product. Common purification techniques for single-stranded oligonucleotide include reversed-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, oligonucleotide can be analyzed by mass spectrometry, and quantitatively by spectrophotometry at a wavelength of 260nm. Then sense strand and antisense strand can be annealed to form dsRNA.

[0471] Pharmaceutical composition

[0472] In another aspect, provided herein is a pharmaceutical composition comprising any human TfR binding protein or conjugate described herein and a pharmaceutically acceptable carrier. Such pharmaceutical compositions may also include one or more pharmaceutically acceptable excipients, diluents or carriers. The pharmaceutical composition 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).

[0473] Treatments and therapeutic uses

[0474] In another aspect, provided herein are methods for treating a CNS disease, such as a neurodegenerative disease, in a patient in need thereof, and such methods comprise administering to the patient an effective amount of a human TfR binding protein or conjugate or pharmaceutical composition described herein.

[0475] In a further aspect, provided herein are methods for treating a neurodegenerative synucleinopathy in a patient in need thereof, and such methods include administering to the patient an effective amount of a human TfR binding protein or 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 synucleinopathy include, but are not limited to, Parkinson's disease; multiple system atrophy; Lewy body dementia or Lewy body dementia; pure autonomic failure; Alzheimer's disease; Lewy body dysphagia; and sporadic Lewy body disease. In some embodiments, the neurodegenerative synucleinopathy is selected from Parkinson's disease, Alzheimer's disease, multiple system atrophy, or Lewy body dementia. The human TfR binding protein or conjugate or pharmaceutical composition can be administered to the patient intravenously or subcutaneously.

[0476] In a further aspect, provided herein are methods for treating tauopathy in a patient in need thereof, and such methods comprise administering to the patient an effective amount of a human TfR binding protein or conjugate or pharmaceutical composition described herein, such as 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), chromosome 17-linked frontotemporal dementia with associated parkinsonism (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-oligoglossia (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic granular dementia (AGD), British amyloid angiopathy, cerebral amyloid Vascular disease, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, epilepsy, Gerstmann-Straussler-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Nielsen disease type C Mann-Pick disease (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, tangled dementia, tangled dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART) or globular glial tauopathy (GGT). Human TfR binding protein or conjugate or pharmaceutical composition can be administered intravenously or subcutaneously to the patient.

[0477] The human TfR binding protein or conjugate dosage regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dosage can be proportionally reduced or increased as indicated by the urgency of the therapeutic situation.

[0478] Dosage values ​​may vary with the type and severity of the condition to be alleviated. It is to 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.

[0479] In another aspect, provided herein is a human TfR binding protein or conjugate as described herein, or a pharmaceutical composition comprising such a human TfR binding protein or conjugate, for use in treatment. Also provided herein is a human TfR binding protein or conjugate as described herein, or a pharmaceutical composition comprising such a human TfR binding protein or conjugate (e.g., a TBP-SNCA siRNA conjugate as described herein, or a pharmaceutical composition comprising such a TBP-SNCA siRNA conjugate), for use in treating neurodegenerative synuclein diseases, such as Parkinson's disease, Alzheimer's disease, multiple system atrophy, or Lewy body dementia.

[0480] 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-MAPT described herein). siRNA conjugates or pharmaceutical compositions comprising such TBP-MAPT siRNA conjugates) for the treatment of tauopathies, such as Alzheimer's disease, frontotemporal dementia (FTD), chromosome 17-linked frontotemporal dementia with associated parkinsonism (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-oligoglossia (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD D), 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-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dysplasia Malnutrition, Niemann-Pick disease type C (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

[0481] In another aspect, provided herein is the use of a human TfR binding protein or conjugate described herein in the preparation of a medicament for treating a CNS disease such as a neurodegenerative disease. In some embodiments, the neurodegenerative disease is a neurodegenerative synuclein disease, such as Parkinson's disease, Alzheimer's disease, multiple system atrophy, or Lewy body dementia. In some embodiments, the neurodegenerative disease is a tauopathy, such as Alzheimer's disease, frontotemporal dementia (FTD), chromosome 17-linked frontotemporal dementia with associated parkinsonism (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-oligoglossia (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic granular dementia (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-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, C Niemann-Pick disease (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

[0482] definition

[0483] As used herein, the terms "a", "an", "the" and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0484] As used herein, the term "alkyl" means a saturated linear or branched monovalent hydrocarbon radical containing a specified number of carbon atoms. 20"Alkyl" means a radical having 1 to 20 carbon atoms in a linear or branched arrangement.

[0485] As used herein, the term "antibody" refers to a molecule that binds to an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, heterodimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. The antibody can be any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0486] Immunoglobulin G (IgG) type 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 includes a variable region of about 100-125 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of each of the four polypeptide chains contains a constant region that is 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 can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0487] VH and VL regions can be further subdivided into highly variable regions called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). CDRs are exposed on the surface of the protein and are important regions of antibody for antigen binding specificity. Each VH and VL consists of three CDRs and four FRs arranged from amino terminal to carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this article, 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". CDR contains most of the residues that form specific interactions with the antigen. The assignment of amino acid residues to CDRs can be accomplished according to well-known schemes, including those described in 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, 274, 937-951 (1998)), and the like. Biology, 406, 228-256 (2011)), or IMGT (International ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27: 209-212).

[0488] Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments, which, as used herein, comprise at least a portion of an antibody that retains the ability to specifically interact with an antigen or antigenic epitope, such as Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fv (sdFv), Fd fragments.

[0489] As used herein, the term "antigen binding domain" refers to a portion of an antibody or antibody fragment that binds to an antigen or antigenic epitope. For example, a "TfR binding domain" refers to a portion of an antibody or antibody fragment that binds to TfR or a TfR epitope.

[0490] As used herein, the term "heterodimeric antibody" refers to an antibody comprising two different antigen binding domains.

[0491] As used herein, "antisense strand" means a single-stranded oligonucleotide that is complementary to a region of a target sequence. Likewise and as used herein, "sense strand" means a single-stranded oligonucleotide that is complementary to a region of the antisense strand.

[0492] Unless otherwise indicated, the terms "bind" and "binds" as used herein are intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule that results in the proximity of the two proteins or molecules as determined by common methods known in the art.

[0493] As used herein, "complementary" means a structural relationship between two nucleotides (e.g., on two relative nucleic acids or on relative regions of a single nucleic acid strand, such as a hairpin), which allows the two nucleotides to form base pairs with each other. For example, the purine nucleotides of a nucleic acid complementary to the pyrimidine nucleotides of the relative nucleic acid can be base-paired together by forming hydrogen bonds with each other. Complementary nucleotides can be base-paired in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. Similarly, two nucleic acids can have multiple nucleotide regions that complement each other to form a complementary region, as described herein.

[0494] As used herein, reference to a "duplex" of a nucleic acid or oligonucleotide refers to the structure formed by complementary base pairing of two antiparallel nucleotide sequences (i.e., in opposite orientations), whether formed by two separate nucleic acid strands or by a single folded strand (e.g., via a hairpin).

[0495] An "effective amount" refers to the amount necessary to achieve the desired therapeutic result (for a period of time and for the means of administration). The effective amount of a protein or conjugate may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein or conjugate to elicit a desired response in the individual. An effective amount is also an amount in which any toxic or deleterious effects of the protein or conjugate are offset by the therapeutically beneficial effects.

[0496] As mentioned herein, the term "epitope" refers to the amino acid residues in an antigen that are bound by an antibody. An epitope can be a linear epitope, a conformational epitope, or a mixed epitope. The term "epitope" can be used to refer to a structural epitope. According to some embodiments, a structural epitope may be used to describe an antigenic region covered by an antibody or antigen-binding protein. In some embodiments, a structural epitope can describe an amino acid residue of an antigen that is within a specified proximity (e.g., within a specified angstrom) of an amino acid residue of an antibody or antigen-binding protein. The term "epitope" can also be used to refer to a functional epitope. According to some embodiments, a functional epitope can be used to describe an amino acid residue of an antigen that interacts with an amino acid residue of an antibody or antigen-binding protein in a manner that contributes to the binding energy between an antigen and an antibody or antigen-binding protein.

[0497] Epitopes can be determined according to different experimental techniques (also referred to as "epitope mapping techniques"). It should be understood that the determination of epitopes may vary based on the different epitope mapping techniques used, and may also vary with the different experimental conditions used, such as due to conformational changes or cleavage of antigens induced by specific experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd Edition 2018), including but not limited to X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species exchange mutagenesis, alanine scanning mutagenesis, hydrogen-deuterium exchange (HDX) and cross-blocking assays.

[0498] 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 include a portion 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, such as 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, which has a reduced or eliminated effector function compared to the corresponding wild-type IgG Fc region. The residue numbering in the Fc region is based on the EU index as described in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: USDept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991). The boundaries of the Fc region of an immunoglobulin heavy chain may vary, and the human IgG heavy chain Fc region is usually defined as the stretch from the N-terminus of the CH2 domain (e.g., the amino acid residue at position 231 according to EU index numbering) to the C-terminus of the CH3 domain (or the C-terminus of the immunoglobulin).

[0499] The term "knockdown" or "expression knockdown" refers to a decrease in mRNA or protein expression of a gene following treatment with an agent.

[0500] As used herein, "modified internucleotide linkage" means an internucleotide linkage having one or more chemical modifications when compared to a reference internucleotide linkage having a phosphodiester linkage. The modified internucleotide linkage can be a non-naturally occurring linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage.

[0501] As used herein, "modified nucleotides" refer to nucleotides with one or more chemical modifications when compared to a corresponding reference nucleotide selected from the group consisting of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymine deoxyribonucleotides. The modified nucleotides may have, for example, one or more chemical modifications in their sugar, core base, and / or phosphate group. Additionally or alternatively, the modified nucleotides may have one or more chemical moieties conjugated to the corresponding reference nucleotides. In some embodiments, the modified nucleotides are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or 2'-O-alkyl-modified nucleotides. In some embodiments, the modified nucleotides have phosphate analogs, such as 5'-vinylphosphonates. In some embodiments, the modified nucleotides have a base-free moiety or a reverse base-free moiety, such as the moieties shown in Table 10.

[0502] 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 glial cells in the central and / or peripheral nervous systems.

[0503] As used herein, "nucleotide" means an organic compound having a nucleoside (a nucleobase, such as adenine, cytosine, guanine, thymine or uracil, and a pentose, such as ribose or 2'-deoxyribose) linked to a phosphate group. "Nucleotide" can serve as a monomer unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0504] As used herein, "empty arm" means an antibody arm that does not bind any known human target.

[0505] 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.

[0506] As used herein, "overhang" means one or more unpaired nucleotides protruding from the duplex structure of a double-stranded oligonucleotide. The overhang may include one or more unpaired nucleotides extending from the duplex region at the 5' end or 3' end of the double-stranded oligonucleotide. The overhang may be a 3' or 5' overhang on the antisense strand or sense strand of a double-stranded oligonucleotide.

[0507] As used herein, the term "patient" refers to a human patient.

[0508] As used herein, "phosphate analog" means a chemical moiety that simulates the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5' terminal nucleotide of an oligonucleotide, replacing the 5'-phosphate that is typically susceptible to enzymatic removal. A 5' phosphate analog can include phosphatase-resistant bonding. Examples of phosphate analogs include 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). In some embodiments, a phosphate analog is 5'-VP.

[0509] 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 those in a reference nucleic acid sequence, nucleosides or nucleobases, after the optimal alignment of the sequences and the introduction of spaces or overhangs, if necessary, to achieve maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., ed., 1987, Supplement 30, Section 7.7.18, Table 7.7.1), and including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Clustal W2.0 or Clustal X2.0 software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the sequence to be compared. The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, wherein the nucleic acid sequence fragments in the comparison window may contain additions or deletions (e.g., spaces 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 at which the same nucleotide, nucleoside, or nucleobase occurs in the two sequences to derive the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to derive the percentage of sequence identity. The output is the percentage identity of the subject sequence relative to the query sequence.

[0510] As used herein, the term "polypeptide" or "protein" refers to a polymer of amino acid residues. The term applies to polymers containing naturally occurring amino acids and polymers containing one or more non-naturally occurring amino acids.

[0511] As used herein, a "strand" refers to a single contiguous sequence of nucleotides linked together by internucleotide bonds (eg, phosphodiester bonds or phosphorothioate bonds). A strand may have two free ends (eg, a 5' end and a 3' end).

[0512] As used herein, "SNCA" refers to α-synuclein (SNCA) mRNA, protein or polypeptide. The nucleic acid sequence of human SNCA mRNA transcript can be found at NM_000345.4:

[0513]

[0514]

[0515] The amino acid sequence of human SNCA protein can be found at NP_000336.1:

[0516]

[0517] 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.

[0518] As used herein, "MAPT" refers to the human MAPT mRNA transcript encoding the microtubule-associated protein Tau. The nucleotide sequence of the human MAPT transcript variant and the amino acid sequence of the human Tau protein isoform can be found at:

[0519] i. MAPT transcript variant 1 → Tau protein isoform 1: NM_016835.5 (nucleotide sequence) → NP_058519.3 (amino acid sequence);

[0520] ii. MAPT transcript variant 2 → Tau protein isoform 2: NM_005910.6 (nucleotide sequence) → NP_005901.2 (amino acid sequence);

[0521] iii. MAPT transcript variant 3 → Tau protein isoform 3: NM_016834.5 (nucleotide sequence) → NP_058518.1 (amino acid sequence);

[0522] iv. MAPT transcript variant 4 → Tau protein isoform 4: NM_016841.5 (nucleotide sequence) → NP_058525.1 (amino acid sequence);

[0523] v.MAPT transcript variant 5→Tau protein isoform 5: NM_001123067.4 (nucleotide sequence)→NP_001116539.1 (amino acid sequence);

[0524] vi. MAPT transcript variant 6 → Tau protein isoform 6: NM_001123066.4 (nucleotide sequence) → NP_001116538.2 (amino acid sequence);

[0525] vii. MAPT transcript variant 7 → Tau protein isoform 7: NM_001203251.2 (nucleotide sequence) → NP_001190180.1 (amino acid sequence);

[0526] viii. MAPT transcript variant 8 → Tau protein isoform 8: NM_001203252.2 (nucleotide sequence) → NP_001190181.1 (amino acid sequence);

[0527] ix. MAPT transcript variant 9 → Tau protein isoform 9: NM_001377265.1 (nucleotide sequence) → NP_001364194.1 (amino acid sequence);

[0528] x.MAPT transcript variant 10→Tau protein isoform 10: NM_001377266.1 (nucleotide sequence)→NP_001364195.1 (amino acid sequence);

[0529] xi. MAPT transcript variant 11 → Tau protein isoform 11: NM_001377267.1 (nucleotide sequence) → NP_001364196.1 (amino acid sequence);

[0530] xii. MAPT transcript variant 12→Tau protein isoform 4: NM_001377268.1 (nucleotide sequence)→NP_001364197.1 (amino acid sequence).

[0531] The nucleotide sequence of human MAPT transcript variant 6 (encoding 2N4R Tau) can be found at NM_001123066.4:

[0532]

[0533]

[0534]

[0535] The corresponding amino acid sequence of human Tau protein isoform 6 can be found at NP_001116538.2:

[0536]

[0537] The nucleotide sequence of human MAPT transcript variant 5 (encoding 1N4R Tau) can be found at NM_001123067.4:

[0538]

[0539]

[0540]

[0541] The corresponding amino acid sequence of human Tau protein isoform 5 can be found at NP_001116539.1:

[0542]

[0543] The nucleotide sequence of human MAPT transcript variant 4 (encoding ON3R Tau) can be found at NM_016841.5:

[0544]

[0545]

[0546]

[0547] The corresponding amino acid sequence of human Tau protein isoform 4 can be found at NP_058525.1:

[0548]

[0549] As used herein, the term "tauopathy" refers to a disease associated with abnormal tau protein expression, secretion, phosphorylation, cleavage and / or aggregation.

[0550] As used herein, "TfR" refers to a transferrin receptor protein or polypeptide, such as a human or mouse transferrin receptor protein or polypeptide. The amino acid sequence of human transferrin receptor protein (hTFR) can be found at NP_001121620.1:

[0551]

[0552]

[0553] The amino acid sequence of mouse transferrin receptor protein (mTFR) can be found at NP_001344227.1:

[0554]

[0555] As used herein, "treatment" or "treating" refers to all processes in which there may be a slowing, control, delay or cessation of the progression of a condition or disease disclosed herein, or an amelioration of a condition or disease symptom, but does not necessarily indicate a complete elimination of all conditions 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.

[0556] The following examples are provided to illustrate but not to limit the present invention. Example

[0557] Example 1: Generation and characterization of TfR binding proteins

[0558] Generation of human or mouse TfR binding proteins

[0559] Antibodies against mouse TfR were generated by immunizing New Zealand white rabbits with the extracellular domain (ECD) of 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 each antibody cloned from these B cells was verified for mTfR with a His tag.

[0560] The cells were immunized with the extracellular domain of human transferrin receptor 1 protein with a His tag (hTfR-ECD-6His, SEQ ID NO: 114, see Table 12) and mouse transferrin receptor protein (mTfR, SEQ ID NO: 110). Transgenic mice were used to generate antibodies against human TfR. Antigen-positive B cells were sorted from pooled spleens. Binding of each antibody cloned from these B cells to His-tagged hTfR-ECD was verified.

[0561] By immunizing with the apical domain of human transferrin receptor 1 protein with a His tag (hTfR-ApD-6His, SEQ ID NO: 115, see Table 12) Transgenic mice were used to generate additional antibodies against human TfR. Antigen-positive B cells were sorted from pooled spleens. Individual antibodies cloned from these B cells were validated for binding to His-tagged hTfR-ECD.

[0562] Table 12. Sequences of immunogens used to generate human or mouse TfR antibodies.

[0563]

[0564]

[0565] By introducing mutations systematically into each CDR of each antibody, the affinity variants of the generated human or mouse TfR antibodies are prepared, and the resulting variants are subjected to multiple rounds of selection with reduced antigen concentration and / or increased dissociation period to separate clones with improved affinity. The sequence of each variant is used to construct a combinatorial library, which is subjected to another round of selection with increased stringency to identify additional or synergistic mutation pairings between each CDR region. Each combined clone is sequenced. The heavy chain and light chain CDR and VH / VL sequences of human TfR binding domains TBD1-7 are provided in Table 1-3. The heavy chain and light chain CDR and VH / VL sequences of mouse TfR binding protein (mTBP1) are provided in Table 7.

[0566] Human or mouse TfR binding proteins are generated by recombinant DNA technology. Such TfR binding proteins can be expressed in mammalian cell lines such as HEK293 or CHO, which are transiently or stably transfected using an expression system with an optimal predetermined HC:LC vector ratio or a single vector system encoding both HC and LC. The clarified culture medium into which the protein has been secreted can be purified using common techniques.

[0567] Binding affinity at 25°C

[0568] The binding affinity and binding stoichiometry of the exemplified mouse TfR binding protein to mouse TFR were determined using surface plasmon resonance assays on a Biacore T200 instrument pretreated with HBS-EP+(10mM Hepes pH7.4+150mM NaCl+3mM EDTA+0.05% (w / v) surfactant P20) running buffer and analysis temperature set at 25°C. 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). Mouse TfR binding protein was prepared at 10 μg / mL by diluting into running buffer. Target (mouse TFR-mlgG1-Fc) was prepared at final concentrations of 100.0, 25.0, 6.25, 1.56, 0.39, 0.097, 0.024 and 0 (blank) nM by dilution into running buffer.

[0569] Each analysis cycle consisted of: (1) capturing antibody samples on separate flow cells (Fc2, Fc3, and Fc4); (2) injecting the respective concentration of TfR on all Fcs at 100 μL / min for 60 seconds, followed by returning the buffer flow for 1800 seconds to monitor the dissociation phase; (3) injecting 10 mM glycine (pH 1.5) on all cells at 10 μL / min for 30 seconds to regenerate the chip surface; and (4) equilibrating the chip surface with a 10 μL (60 second) injection of HBS-EP+. Data were processed using standard double references 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 Unit), dissociation rate (k off ,s -1 Unit) and R max (RU units). Equilibrium dissociation constant (K D ) According to the relation K D =k off / k on Calculated and in molar units. The results are provided in Table 13.

[0570] Table 13: Binding affinity of exemplary mTfR binding proteins for mouse TFR at 25°C.

[0571]

[0572] These results demonstrate that the exemplified mouse TfR binding proteins and conjugates bind to mouse TfR with high affinity at 25°C.

[0573] On a Biacore 8K instrument pretreated with HBS-EP+ (10mM Hepes pH7.4+150mM NaCl+3mM EDTA+0.05% (w / v) surfactant P20) running buffer and analysis temperature set at 25°C, surface plasmon resonance was used to determine the binding affinity and binding stoichiometry of the exemplified human TfR binding protein to human and cynomolgus monkey TfR. 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 TfRECD) were prepared at a final concentration of 500 μg / mL in running buffer. 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 solution into the running buffer.

[0574] Binding analysis was performed in a single-cycle kinetic manner. Each analysis cycle consisted of: (1) capturing target (human or cynomolgus monkey TfR ECD with His tag) samples on separate flow cells (Fc2, Fc3, and Fc4); (2) injecting the antibody or protein from the lowest to the highest concentration on all Fcs at 30 μL / min for a total of 900 seconds, followed by returning the buffer flow for 1800 seconds to monitor the dissociation phase; (3) injecting 10 mM glycine (pH 1.5) on all cells at 10 μL / min for a total of 30 seconds to regenerate the chip surface; and (4) equilibrating the chip surface with 10 μL (60 seconds) injection of HBS-EP+. Data were processed using standard double references and fitted to a 2-state binding model using Biacore 8K evaluation software to determine the binding rate (k on , M -1 s -1 Unit), dissociation rate (k off ,s -1 Unit) and R max (RU units). Equilibrium dissociation constant (K D ) According to the relation K D =k off / k on Calculations were performed and are in molar units. The results are provided in Table 14A.

[0575] Endogenously expressed human endothelial hCMEC-D3 (EMD Millipore SC066) and MDCK cell line (ATCC CCL-34) for expressing cynomolgus monkey TfR are used to evaluate the combination of antibody / protein and cell-bound TfR. Cells are grown and maintained at submaximal confluence, and Accutase cell detachment solution is used to detach from culture vessel (cultureware), washed, and 50000 cells per well are distributed for evaluating binding. Cells are treated with active dyes and then incubated on ice with titrated concentrations of TfR-binding proteins. Wash cells and detect the combination of test antibodies or proteins using PE-labeled secondary reagents. Then wash cells and use BioRad ZE5 cell counter readings on the same day. Perform post-acquisition analysis in FlowJo to analyze the fluorescence of single, vigorous, non-fragmented 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.

[0576] Table 14A: Binding affinity of exemplified human TfR binding proteins for human or cynomolgus monkey TfR at 25°C or 0°C.

[0577]

[0578]

[0579] Binding affinity at 37°C

[0580] The binding affinity and binding stoichiometry of the exemplified human TfR binding proteins to human and cynomolgus monkey TfR were further characterized using surface plasmon resonance measurements on a Biacore 8K instrument pretreated with HBS-EP+ (10mM Hepes pH7.4+150mM NaCl+3mM EDTA+0.05% (w / v) surfactant P20) running buffer and analysis temperature set at 37°C. The target human and cynomolgus monkey TfR ECD was immobilized on a CM4 chip (Cytiva P / N29104989) 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, 0.0001 μM, respectively, by diluting the stock solution into the running buffer.

[0581] Binding analysis was performed in a multi-cycle kinetic manner. Each analysis cycle consisted of: (1) injecting the antibody or protein from the lowest concentration to the highest concentration over all Fcs at 50 μL / min for 140 seconds, followed by returning the buffer flow for 400 seconds to monitor the dissociation phase; (2) injecting 3M magnesium chloride over all pools at 100 μL / min for 30 seconds to regenerate the chip surface; and (3) equilibrating the chip surface with a 50 μL (30 second) injection of HBS-EP+. Data were processed using standard double references and fitted to a 2-state binding model using Biacore 8K evaluation software to determine the association rate (k on , M -1 s -1 Unit), dissociation rate (k off ,s -1 Unit) and R max (RU units). Equilibrium dissociation constant (K D ) According to the relation K D =k off / k on Calculations were performed and are in molar units. The results are provided in Table 14B.

[0582] Table 14B. Binding affinity of exemplified human TfR binding proteins for human or cynomolgus monkey TfR at 37°C

[0583]

[0584] Epitope mapping by hydrogen deuterium exchange mass spectrometry (HDX-MS)

[0585] Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) was performed to determine the location of the exemplified TfR binding proteins binding to the human TfR extracellular domain (TfR-ECD).

[0586] Peptide identification for human TfR-ECD was performed on a Waters Synapt G2Si (Waters Corporation) instrument using 5 μg of human TfR-ECD protein (1:10 dilution in 0.1X phosphate-buffered saline in H2O) under zero exchange, using nepenthesin II (Nep II) for digestion, followed by online 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 and a scan time of 0.4 seconds. Data were processed using PLGS2.3.02 (Waters Corporation). For exchange experiments, complexes of human TfR-ECD protein with each TfR binding protein were prepared at a molar ratio of 1:1.2 in 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl (1xPBS buffer). The experiment was initiated by adding 2.5 μl of TfR-ECD (0.9 mg / mL) or TfR-ECD+protein complexes in 25 μL of D2O buffer containing 0.1x PBS at 15° C. for different amounts of time (0 sec, 10 sec, 2 min, 10 min, and 60 min) using a custom TECAN sample preparation system (Espada et al. 2019, J Am Soc Mass Spectrom. 2019 Dec; 30(12): 2580-2583). The reaction was quenched at 4° C. for two minutes using an equal volume of 0.32 M TCEP, 3 M guanidine hydrochloride, 0.1 M phosphate pH 2.5 and immediately frozen at –70° C. The sample injection system consisted of a UR3 robotic arm, a LEAP PAL3 HDX autosampler, and an HPLC system connected to a Waters Synapt G2Si (Waters Corporation), with modifications as described (Espada et al., 2019, J Am Soc Mass Spectrom. 2019 December; 30(12): 2580-2583.). The LC mobile phase consisted of water (A) and acetonitrile (B) each containing 0.2% formic acid. Each sample was thawed for 1 minute using 50 μL of 1.5 M guanidine hydrochloride, 0.1 M phosphate pH 2.5, and then injected onto a NepII column for digestion with mobile phase A at a flow rate of 250 μL / min at 4 ° C for 2.5 minutes.The resulting peptides were captured on a Waters BEH Vanguard Pre-column at 4°C and chromatographed using a Waters Acquity UPLC BEH C18 analytical column with a flow rate of 200 μL / min and a gradient of 3%–85% mobile phase B in 7 minutes at 4°C and introduced into a mass spectrometer for mass spectrometry analysis. Synapt G2Si was calibrated with Glu-fibrin peptide (Waters Corporation) prior to use. In HDMS mode, mass spectra were collected in the m / z range of 255 to 1950, with a lock mass m / z of 556.2771 (leucine enkephalin, Waters Corporation). Using the peptide list identified in DynamX 3.0 (Waters Corporation), the relative deuterium incorporation for each peptide was determined by processing the MS data for the deuterated sample together with the undeuterated control. The free state and the bound state of human TfR-ECD were compared for deuterium incorporation differences to identify protected regions indicating binding epitopes. The overall sequence coverage for human TFRECD was 90.4%.

[0587] For human TfR binding protein 1 (TBP1), reduced deuterium uptake after binding to human TfR-ECD was observed in residues 346-364FGNMEGDCPSDWKTDSTCR (SEQ ID NO: 119), pointing to possible epitope regions. For human TfR binding protein 13 (TBP13), reduced deuterium uptake after binding to human TfR-ECD was observed in residues 243-247 (FEDLY) (SEQ ID NO: 162) and 345-364 (LFGNMEEGDCPSDWKTDSTCR) (SEQ ID NO: 163), pointing to possible epitope regions. For human TfR binding protein 10 (TBP10), reduced deuterium uptake upon binding to human TfR-ECD 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), pointing to possible epitope regions.

[0588] Example 2: Synthesis and Characterization of dsRNA (eg, siRNA) Targeting SNCA

[0589] Via MerMade TMSingle strands (sense and antisense) of dsRNA duplexes were synthesized on a solid support using 12 (LGC Biosearch Technologies). The sequences of the sense and antisense strands are shown in Table 11. The sense strand used phthalamidoamino C6 lcaa CPG (Chemgenes), while the antisense strand used a standard support (LGCBiosearch Technologies). Oligonucleotides were synthesized via phosphoramidite chemistry at 5, 10, or 50 μmol scale.

[0590] Standard reagents were used in oligonucleotide synthesis (Table 16), with 0.1 M hydroxanthin in pyridine as the sulfurizing agent and 20% DEA in ACN as a post-synthesis auxiliary detergent. All monomers (Table 17) were prepared at 0.1 M in ACN and contained molecular sieve capture bags.

[0591] Oligonucleotides were cleaved and deprotected (C / D) at 45°C for 20 hours. The sense strand was C / D from CPG using cold 50% (methylamine / ammonium hydroxide 28-30%) at RT for 3 hours, while 3% DEA in ammonium hydroxide (28-30%, cold) was used for the antisense strand. When the quality data obtained confirmed the identity of the sequence, the C / D was determined to be complete by IP-RP LCMS. Depending on the scale, CPG was filtered through a 0.45um PVDF needleless filter, a 0.22um PVDF Vacuum filtration or 0.22um PVDF Quick release was used for filtration. The CPG was backwashed / rinsed with 30% EtOH / RNase-free water and then filtered through the same filtration unit and combined with the first filtrate. This was repeated twice. The material was then evenly divided into 50 mL falcon tubes for filtration via Genevac TM After concentration, the crude oligonucleotides were diluted back to synthesis scale with RNase-free water and passed through a 0.45 μm PVDF needle-free filter, a 0.22 μm PVDF Vacuum filtration or 0.22μm PVDF Quick release for filtration.

[0592] Crude oligonucleotides were purified by anion exchange (AEX) via AKTA TM Pure purification system for purification. For AEX, ES Industry Source TM15Q column maintaining the column temperature at 65°C with MPA: 20 mM NaH2PO4, 15% ACN, pH 7.4 and MPB: 20 ​​mM NaH2PO4, 1 M NaBr, 15% ACN, pH 7.4. Fractions containing mass purity greater than 85% and no impurities > 5% were pooled.

[0593] The oligonucleotide of purification uses 15mL 3K MWCO centrifugal spinning tube to desalt~30 minutes at 3500xg.Rinse oligonucleotide with water without RNA enzyme, until eluate conductivity reaches<100usemi / cm.After desalination is completed, add 2-3mL RNA enzyme-free water, then suction 10x, retentate is transferred in 50mL falcon pipe, this is repeated until the complete transfer of oligonucleotide, it is by dropping the compound concentration on the measurement filter via nanometer.Then, final oligonucleotide carries out 2x nanofiltration with 3500xg for 2 minutes via 15mL100K MWCO centrifugal spinning tube.Final desalted oligonucleotide is analyzed for concentration (nanometer drop at A260 place), characterizes (table 15) and characterizes for UV purity by UPLC by IP-RP LC / MS for mass purity.

[0594] Table 15: Exemplary LC / MS data

[0595]

[0596] Table 16 Oligonucleotide synthesis reagents

[0597] Reagents Activator solution (0.5 METT in ACN) Cap A (acetic anhydride, pyridine in THF, 1:1:8) Cap B (1-methylimidazole in THF, 16:84) Oxidizing solution (0.02 M iodine in THF / pyridine / water, 70:20:10) Deblocking solution, 3% TCA in DCM (w / v) Acetonitrile (anhydrous solution, maximum water content 10ppm) Hydrogenated xanthan gum (0.1M pyridine solution) Diethylamine (20% solution in acetonitrile)

[0598] Table 17 Phosphoramidites

[0599]

[0600] Example 3: Generation of TfR binding protein-dsRNA conjugates

[0601] Certain abbreviations are defined as follows: “ACN” refers to acetonitrile; “aAEX” refers to analytical anion exchange; “AS” refers to antisense; “DAR” refers to drug / siRNA to antibody / protein ratio; “DCM” refers to dichloromethane; “DHAA” refers to dehydroascorbic acid; “DIEA” refers to N,N-diisopropylethylamine; “DMF” refers to dimethylformamide; “dsRNA” refers to double-stranded RNA; “DTT” refers to dithiothreitol; “EtOAc” refers to ethyl acetate; “FEP” refers to fluorinated ethylene propylene; “FMI” refers to FluidMetering Inc; “h” means hour; “HATU” means tetramethyluranium hexafluorophosphate; “HPLC” means high performance liquid chromatography; “LC / MS” means liquid chromatography-mass spectrometry; “LTQ / MS” means linear ion trap-mass spectrometry; “min” means minute; “MTBE” means methyl tert-butyl ether; “MW” means molecular weight; “NHS” means N-hydroxysuccinimide; “OD” means optical density; “PBS” means phosphate-buffered saline; “PEG” means polyethylene glycol; “rpm” refers to revolutions per minute; “SEC” refers to size exclusion chromatography; “siRNA” refers to small interfering RNA; “SMCC” refers to succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate; “SS” refers to sense strand; “TCO” refers to trans-cyclooctene; “TEA” refers to triethylamine; “TFA” refers to trifluoroacetic acid; “TfR” refers to transferrin receptor; “THF” refers to tetrahydrofuran; “TRIS” refers to tris(hydroxymethyl)aminomethane; and “UV” refers to ultraviolet light.

[0602] Solution 1

[0603]

[0604] 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 shows the 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 in a solvent system such as DMF and THF, together with an appropriate base such as N,N-diisopropylamine, to give compound (3). One skilled in the art will recognize that a variety of coupling reagents, bases, and solvents may be used to perform amide couplings.

[0605] Solution 2

[0606]

[0607] Scheme 2, step A depicts the conversion of cis-olefin compound (4) to trans-olefin compounds (5) and (6) using irradiation and capture on a silver nitrate column adsorbed onto silica gel using a closed loop flow apparatus. Step B shows the reaction of compound (5) with N,N'-disuccinimidyl carbonate using a suitable base such as TEA in a solvent such as ACN to give compound (7).

[0608] Solution 3

[0609]

[0610] Scheme 3, step A depicts a 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 agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride together 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 perform amide couplings.

[0611]

[0612] Scheme 4, step A depicts the coupling of compound (10) and 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 a manner substantially similar to Scheme 1, step B.

[0613] Preparation 1

[0614] tert-Butyl 4-[2-(2,5-dioxopyrrol-1-yl)ethyl]piperazine-1-carboxylate

[0615]

[0616] 4-(2-aminoethyl)piperazine-1-carboxylic acid tert-butyl ester (3.00g, 13.1mmol) is dissolved in acetic acid (6mL). Furan-2,5-dione (1.28g, 13.1mmol) is added and stirred for 7 hours at ambient temperature. The mixture is then stored in a refrigerator for 18 hours. Most of the acetic acid is removed under vacuum at 50°C. Acetic anhydride (10mL, 106mmol) and sodium acetate (1.6g, 20mmol) are added and then heated to 80°C for 2 hours. Toluene is added and most of the acetic anhydride is removed under vacuum. The mixture is put into saturated aqueous ammonium chloride solution (60mL) and extracted with DCM (3×50mL). The combined organic layers are dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a crude product as a dark oil. Purification is carried out via silica gel chromatography eluted with EtOAc / hexane to give the title compound (2.1g, 52%). LC / MS m / z 310.3 (M+H).

[0617] Preparation 2

[0618] tert-Butyl 4-[3-(2,5-dioxopyrrol-1-yl)propyl]piperazine-1-carboxylate

[0619]

[0620] Furan-2,5-dione (789 mg, 7.97 mmol) was added to a solution of 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 under vacuum 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 Molecular sieves (8.8 g). The flask was equipped with a Dean-Stark separator, and the mixture was heated at 120 ° C for 48 hours. After cooling to ambient temperature, the solid was taken out by filtration and washed with DCM (40 mL). Volatiles were removed under reduced pressure to give a residue that 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).

[0621] Preparation 3

[0622] 1-[2-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyl]piperazin-1-yl]ethyl]pyrrole-2,5-dione

[0623]

[0624] 4-[2-(2,5-dioxopyrrole-1-yl)ethyl]piperazine-1-carboxylic acid tert-butyl ester (150mg, 0.485mmol) was dissolved in DCM (2mL). TFA (1mL, 13mmol) was added and stirred at ambient temperature for 1 hour. Concentrated under vacuum and further dried under high vacuum for 18 hours to give the intermediate product 1-(2-piperazine-1-ylethyl)pyrrole-2,5-dione trifluoroacetate. This material and methyl tetrazine-PEG4-acid (130mg, 0.283mmol) were dissolved in DMF (2.0mL) and THF (2mL). HATU (380mg, 0.969mmol) was then added, followed by N,N-diisopropylamine (0.45mL, 2.6mmol). Stirred for 2 hours at ambient temperature. Diluted with DCM (50mL) and washed with saturated aqueous ammonium chloride solution (30mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give the crude product as a red solid. Purification was performed via silica gel chromatography eluting with 0-20% MeOH / EtOAc to give the title compound as a red solid (150 mg, 49%). LC / MS m / z 628.6 (M+H).

[0625] Preparation 4

[0626] 1-[3-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyl]piperazin-1-yl]propyl]pyrrole-2,5-dione

[0627]

[0628] The title compound was prepared using tert-butyl 4-[3-(2,5-dioxopyrrol-1-yl)propyl]piperazine-1-carboxylate in an essentially analogous manner to the method found in Preparation 3. LC / MS m / z 642 (M+H).

[0629] Preparation 5

[0630] (1R,4E)-cyclooct-4-en-1-ol (axial) and (1R,4E)-cyclooct-4-en-1-ol (equatorial)

[0631]

[0632] A closed loop flow apparatus was assembled that allowed for irradiation of a solution of the cis-olefin and circulation of the solution through silver nitrate adsorbed onto a silica gel cartridge. Only the trans-olefin remained in the silica gel, so the cis-olefin was recycled back to the irradiation stage.

[0633] Equipment: (A) UV lamp (Pen-Ray 099912-1, 254nM), power supply 99-0055-01 lamp current 18mA / AC. According to the manufacturer's description, the lamp produces 254nM light with an intensity of 4400 to 4750 microwatts / cm^2 at 0.75". (B) FMI pump set at 10mL / min, which is from The reaction mixture was drawn into a round bottom flask (250 mL). It was connected to a FEP 1 / 16" tube surrounding a cold finger (7 mL loop total, air cooled). A UV lamp was placed in the center of the cold finger to irradiate the sample while air cooled. After irradiation, the sample tube continued into 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).

[0634] The following steps are performed. 50g silica gel cartridges are loaded on the top with 25g silver nitrate adsorbed on silica gel, covered with aluminum foil, and adjusted by pumping 1:1 hexane / diethyl ether solvent mixture for 1 hour. Racemic (4Z)-cyclooct-4-ene-1-ol (2.00g, 15.8mmol) and methyl benzoate (2.0mL, 16mmol) at the hydroxyl position are mixed in n-hexane (220mL) and diethyl ether (220mL), UV lamp is turned on, and the solution is circulated through the tube ring around the finger-shaped cold device at a flow rate of 10mL / min through the silica gel / silver nitrate cartridge and returned to the system for a total of 96 hours. The silica gel cartridge is rinsed with EtOAc (200mL) and air dried. Discard the filtrate. With concentrated NH4OH (150mL), the silica gel cartridge that is dried is rinsed with DCM (150mL) subsequently. Separate each layer and extract the aqueous layer with DCM (2×50mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification was performed via silica gel chromatography eluting with 0-45% MTBE / hexanes to give two products as clear liquids. Axial-(1R,4E)-cyclooct-4-ene-1-ol (569.8 mg, 28.5%). 1HNMR (CDCl3) 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).

[0635] Preparation 6

[0636] [(1R,4E)-Cyclooct-4-en-1-yl](2,5-dioxopyrrolidin-1-en-1-yl) carbonate

[0637]

[0638] N, N'-disuccinimidyl carbonate (2.79 g, 10.3 mmol) was added to a mixture of (1R, 4E)-cyclooct-4-ene-1-ol (axial) (569 mg, 4.50 mmol) and TEA (2.5 mL, 18 mmol) in ACN (25 mL) in small portions (~250-300 mg added each time, five minutes apart). The mixture was covered with aluminum foil and stirred at ambient temperature for 60 hours. The solvent was removed under reduced pressure to give an oil distributed between water (20 mL) and diethyl ether (50 mL). The layers were separated and the aqueous layer was extracted with diethyl ether (2×50 mL). The organic layers were combined and washed with saturated ammonium chloride, then washed with saturated sodium chloride aqueous solution, dried over MgSO4, filtered, and concentrated under reduced pressure. Silica gel chromatography was used for purification and eluted with 0-60% MTBE / hexanes to give the title compound as a colorless residue which formed a white solid (732 mg, 61%). LC / MS m / z 324 (M+H).

[0639] Preparation 7

[0640] (2,5-Dioxopyrrolidin-1-yl) 4-[[2-methyl-2-(2-pyridyldisulfanyl)propyl]amino]-4-oxo-butyrate

[0641]

[0642] 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. for 12 hours with vigorous stirring. 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. The mixture was stirred at ambient temperature for 5 minutes, then N-hydroxysuccinimide (126 mg, 1.07 mmol) was added in one portion and then stirred for 36 hours. The mixture was filtered and the resulting filtrate was directly loaded onto silica gel (2 g). Silica gel chromatography was used for purification and eluted with 75% EtOAc / hexanes to give the title compound as a light cloudy residue (100.2 mg, 24%). LC / MS m / z 426 (M+H) (hydrolyzed NHS ester).

[0643] TCO functionalized SNCA

[0644] In a set of 4 x 50mL Falcon TM In a tube, the sense strand of SNCA dsRNA with a hexylamine chain attached at the 3' end (SNCA_SS-3C6A) (measured SS concentration calculated as 412.5 OD / mL or 2mM, 120 mL, 0.24 mmol) and 20X borate buffer (6 mL) were aliquoted (10 mL each) and each was treated with 7.5 mL of [(1R,4E)-cyclooct-4-en-1-yl](2,5-dioxopyrrolidin-1-yl) carbonate solution (1.65 g, 6.17 mmol) dissolved in 1,4-dioxane (100 mL). Mix at 600 rpm for 30 minutes at 25°C. The remainder of the SS sample was separated and reacted in the same manner to give a total of 12 sample containers each containing ~150 mg of crude SS raw material. By placing the Falcon TM The tube was placed on a Genevac evaporator to remove dioxane. The remaining aqueous solution was combined and filtered to remove any suspended solids. AKTA was used with 13-45% ACN in 50 mM NaOAc(aq) and flow rate = 40 mL / min TM Purification was performed on a pure chromatography system. Appropriate fractions were combined and precipitated in a SpeedVac TM The organics were removed above the 4% HPLC-MS / MS column and then desalted and concentrated to give 214 mL with a measured OD / mL of 127.3 equivalent to 624 μM and 973 mg total. LTQ / MS m / z 7292; UV purity 99+%.

[0645] TCO-SNCA duplex

[0646] The nanodrop concentrations (5x averages) of the aqueous solutions for each chain 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 residual SS chain was measured until completion and an additional 21.9 mL of AS needed to be added. The resulting 345 mL solution was measured (Nanodrop TM Lite, 6x mean, 20x dilution) OD / mL of 159.5, equivalent to 421 μM and 2.19 g total. LTQ / MS m / z 7291, 7825; UV purity >99%.

[0647] SMCC functionalization of SNCA dsRNA

[0648] Freshly prepared solution of (2,5-dioxopyrrolidin-1-yl) 4-[(2,5-dioxopyrrolidin-1-yl)methyl]cyclohexanecarboxylate (185 mg, 0.542 mmol) in THF (50 mL) was added to SNCA_SS-3C6A (44 mL, 0.0528 mmol; OD / mL of 250.4, or ~1200 μM (~8.8 mg / mL)) in 0.2 M phosphate buffer (44 mL). Vortex vigorously for 2 minutes, then shake at 900 rpm for a total of 2 hours at ambient temperature. Analysis by LTQ showed approximately 94-95% conversion. Acidify to pH ~4 with 20-30 drops of 5N HCl, then remove organics in a Genevac concentrator. Desalt by centrifugal filtration on a 3K spin filter (4×4000 rpm, 30 minutes), and the retentates are combined. The OD of the solution was measured (mean of 3 measurements, 10x dilution) to be 266, equivalent to 1.3 mM and 316 mg total. The extinction coefficient was 204.12. LTQ / MS m / z 7358.

[0649] SMCC-SNCA duplex

[0650] The nanodrop concentration of the aqueous solution of each chain (average of 3x) was measured to be 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 chain was measured until completion and an additional 360 μL of AS needed to be added. Endotoxin was removed by filtering through a 0.45 μM filter. The resulting 75 mL solution was measured (Nanodrop TMLite, 5x mean, 10x dilution) 217 ​​OD / mL, equivalent to 575 μM and 653 mg total. LTQ / MS m / z 7358, 7825; UV purity 99+%.

[0651] GDM-functionalized SNCA

[0652] In 15mL Falcon TM In a tube, SNCA_SS-3C6A (SS concentration measured was calculated as 247.6 OD / mL or 1.21 mM, 3 mL, 0.0036 mmol) was diluted with 20X borate buffer (0.3 mL) and water (3 mL, 166.530 mmol), and then (2,5-dioxopyrrolidin-1-yl) 5-[[2-methyl-2-(2-pyridyldisulfanyl)propyl]amino]-5-oxo-pentanoate (3.6 mL, 0.75 M in dioxane) was added. Mix at 200 rpm for 1 hour. In a SpeedVac TM The organics were removed, desalted, and concentrated three times with water to give SNCA_SS-3C6A-GDM with an overall yield of 13.2 mL (OD / mL of 35.88, equivalent to 175.8 μM and 17.3 mg total). Extinction coefficient was 204.12. LTQ1 MS m / z 7449 (UV purity 95+%).

[0653] 2-Tris(2-carboxyethyl)phosphine hydrochloride (75 μL of 100 mM aqueous solution) was added to SNCA_SS-3C6A-GDM. Shake at 10°C for 4 hours and then shake at ambient temperature for 16 hours. Additional tris(2-carboxyethyl)phosphine hydrochloride (75 μL of 100 mM aqueous solution) was added and shaken for another 16 hours. Desalting was performed by centrifugal filtration on a 3K spin filter (3×40 minutes, 4000 rpm), and the retentates were combined to give 10 mL. The OD measurement of the solution (mean of 4 measurements, 10x 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+%.

[0654] GDM Annealing Steps

[0655] The nanodrop concentrations of the aqueous solution of each chain (average of 4x) were 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 residual SS chain was measured until completion and an additional 560 μL of AS needed to be added. Concentrated on a 3KMW cutoff filter (20 minutes), followed by a 50k spin filter and further concentrated through a 3K filter. The resulting 6 mL solution was measured (Nanodrop 400 μM). TM Lite, 5x mean, 20x dilution) 181.62 OD / mL, equivalent to 486 μM and 44.2 mg total. LTQ / MS m / z 7340, 7825; UV purity 99+%.

[0656] MAPT dsRNA functionalization and annealing can be performed in the same manner as described above for SNCA dsRNA.

[0657] Conjugation of dsRNA to TfR-binding proteins

[0658] Natural or modified site-specific cysteine ​​amino acid residues in TfR binding proteins are used to conjugate dsRNA. Cysteine ​​can be modified into the primary amino acid sequence of TfR binding proteins. The method of introducing cysteine ​​as a means for conjugation has been described in WO 2018 / 232088, which is incorporated by reference in its entirety and specifically incorporated in terms of conjugation via cysteine ​​residues. For modified cysteine ​​conjugation, the TfR binding protein is first reduced with 40 molar equivalents of the reducing agent dithiothreitol (DTT) at 37°C for two hours, followed by desalting via dialysis or a desalting column to remove the reducing agent. This is then incubated at room temperature for two hours with 10 molar equivalents of dehydroascorbic acid (DHAA) to re-oxidize the TfR binding protein to reform the structural disulfide. Subsequent desalting is performed to remove the oxidant.

[0659] Conjugation of dsRNA to TfR binding protein was accomplished using the following method.

[0660] Conjugation protocol 1

[0661] In the first approach, a bifunctional maleimide-methyl-tetrazine linker was conjugated to the 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. After this, a desalting step was 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.

[0662] Step 1a: Conjugation of TfR-binding protein using a maleimide-methyl-tetrazine linker

[0663]

[0664] Step 1b: Conjugation of the ring-opened TfR binding protein using a maleimide-methyl-tetrazine linker

[0665]

[0666] Step 2a: dsRNA conjugation with protein-linker intermediate

[0667]

[0668] Step 2b: dsRNA conjugation with protein-linker intermediate (opened circle)

[0669]

[0670] Conjugation Scheme 2

[0671] The second conjugation method utilized SMCC functionalized dsRNA for conjugation to the engineered cysteine ​​of the TfR binding protein. For this method, the TfR binding protein was prepared similarly to above to make the engineered thiol available for conjugation by subjecting the TfR binding protein to a reduction and oxidation process. This was followed by incubation of the SMCC-dsRNA with 4 molar equivalents of the TfR binding protein for overnight conjugation at 4°C.

[0672] Optionally, after conjugation, a maleimide hydrolysis step can be completed to ensure that the linker-payload is in the terminal stage and to avoid deconjugation during human circulation via reverse Michael addition. This succinimide ring hydrolysis process is completed by raising the pH of the conjugate to 9.0 using 50 mM arginine (using a stock solution of 0.7 M arginine, pH 9.0), and incubating the solution at 37 ° C for 20 hours. The hydrolysis state of the maleimide was confirmed by LCMS characterization of +18 Da, which is generated by adding water to the succinimide ring.

[0673] Step 1a: Conjugation of TfR-binding protein with SMCC linker

[0674]

[0675] Step 1b: Conjugation of TfR-binding protein with SMCC linker-opened ring

[0676]

[0677] Conjugation Scheme 3

[0678] The third conjugation method utilizes GDM functionalized dsRNA for being conjugated to the cysteine ​​of the transformation of TfR binding protein via disulfide bond.For this method, TfR binding protein is prepared similarly to the above, so that the thiol of transformation can be used for conjugation by making TfR binding protein experience reduction and oxidation process.Then, before dsRNA conjugation, disulfide bis (5-nitropyridine) is added to protein with 20 molar equivalents, to generate intermediate product.Excess disulfide bis (5-nitropyridine) is removed by desalination.In the second step, GDM functionalized dsRNA is added to protein intermediate product with 4 molar equivalents.Disulfide bis (5-nitropyridine) acts as a leaving group in this reaction, and is replaced by GDM-dsRNA.

[0679] Step 1: Conjugation of TfR-binding protein with dithiobis(5-nitropyridine) for intermediate generation

[0680]

[0681] Step 2: dsRNA conjugation with GDM-functionalized dsRNA

[0682]

[0683] Conjugation was monitored using analytical anion exchange chromatography. TM SAX-10 HPLC column, 10 μm particles, 4 mm diameter, 250 mm length. 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 at 30°C.

[0684] Table 18A: HPLC gradient used to evaluate conjugation of dsRNA to TfR binding proteins TBP10 and TBP11

[0685]

[0686] Table 18B: HPLC gradient used to evaluate the conjugation of dsRNA to the TfR binding protein TBP14

[0687]

[0688] The drug / siRNA to antibody / protein ratio (DAR) was calculated based on the peak area % from the analytical anion exchange (aAEX) chromatogram. An illustrative example of a chromatogram of TBP11-dsRNA conjugate before purification is shown in Figure 1A middle. Figure 1CAn exemplary aAEX chromatogram showing the DAR profile of the TBP15-dsRNA conjugate before purification is shown.

[0689] After conjugation of dsRNA to TfR binding protein, excess dsRNA and unconjugated protein are removed by further purification. Preparative size exclusion chromatography (SEC) or preparative anion exchange chromatography is used for purification of the final conjugate. Under isocratic conditions, use Cytiva Preparative SEC was performed in 1X PBS pH 7.2 at 200. Alternatively, an anion exchanger such as the ThermoFisher POROS ELISA Kit with a starting buffer of 20 mM TRIS pH 7.0 and a gradient elution of 20 column volumes with a buffer containing 20 mM TRIS pH 7.0 and 1 M NaCl was used. TM XQ. This produces a purified TfR binding protein-dsRNA conjugate free of excess dsRNA and minimal unconjugated protein. The resulting conjugate profile is analyzed by analytical anion exchange for final DAR quantification (see Figure 1B and 1D ; and Table 19).

[0690] An example of a chromatogram of the purified TBP14-dsRNA conjugate is shown in Figure 1B middle. Figure 1D An exemplary aAEX chromatogram showing the DAR profile of the purified TBP15-dsRNA conjugate.

[0691] Table 19. siRNA / drug to TBP / antibody ratio (DAR)

[0692]

[0693] Example 4: In vitro characterization of mouse TfR binding protein-dsRNA conjugates

[0694] In vitro Binding, Internalization, and Degradation Assessment in Mouse Cortical Neurons

[0695] Fluorescence signals corresponding to the total level and internalization of TfR binding protein or TfR binding protein-siRNA conjugate (ARC) were measured by performing high-content live cell imaging assays in primary mouse cortical neurons. 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% antibiotics / antimycotics (Corning) at 37°C in a humidified chamber with 5% CO2 in a tissue culture incubator for 7 days. On day 7, media was removed from each well and replaced with media with 5ug / ml (33nM) of either: (i) isotype Ab (isotype control antibody), (ii) mTBP2 (heterodimer antibody with monovalent mouse TfR binding arm and 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), together with DyLight 650 (Thermo Fisher #62266), DL650 together with BHQ3 dye (BioSearch Tech) in media with 6.7uM (1mg / ml) goat gamma globulin (Jackson Immuno #005-000-002). 10ug / ml (0.2uM) of anti-human IgG Fcγ fragment specific Fab fragment (Jackson Immuno #109-007-008) labeled with BHQ-3000S-5) or pHAb dye (Promega #G9845) was added and incubated with live cells grown in 96-well plates overnight at 37°C.

[0696] The next day, the cells were washed, incubated with NucBlue Hoechst dye (Thermo Fisher # R37605) for 20 minutes, washed again, and then imaged with a Cytation 5 high content imager (Biotek). The DyLight 650 signal measures the total TfR binding protein level, and the DyLight 650 plus BHQ3 signal measures the degradation signal, which increases the DyLight 650 fluorescence when the BHQ3 dye is released and the FRET quenching is lost, while the pHAb pH sensor dye signal only measures the internalized fluorescence. Excess goat gamma globulin was added to reduce the nonspecific binding and uptake of the antibody into the cell. The signal intensity in each well was divided by the number of nuclei stained with Hoechst to determine the signal intensity of each cell. The wells were analyzed in duplicate, and for each well, approximately 20,000 cells were analyzed from images taken with a 4x objective. The background signal was determined according to the human IgG isotype control and subtracted from the final value.

[0697] Results are shown in Figure 2 High-content imaging data confirmed the cellular activity (binding, internalization and degradation properties) of the exemplified mouse TfR binding proteins and isotype control antibodies. Isotype control antibodies and isotype control antibody-dsRNA conjugates lack activity, while binding, internalization and degradation activities of the exemplified mouse TfR binding proteins were confirmed in primary mouse cortical neurons. In addition, conjugation with dsRNA did not significantly change the activity of the exemplified mouse TfR binding proteins.

[0698] In vitro potency evaluation in mouse cortical neurons

[0699] 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 culture medium was removed from each well and one of the following was added at 2x concentration in culture medium with 2% FBS: (i) chol-teg-siSNCA (cholesterol-conjugated dsRNA No. 7); (ii) naked SNCA siRNA (unconjugated SNCA siRNA); (iii) isotype Ab-SNCA siRNA (isotype control antibody with dsRNA No. 7 linked to HC constant region 1), or (iv) mTBP2-SNCA siRNA (mTBP2-dsRNA No. 8 conjugate, dsRNA linked to HC constant region 1 of mTBP2) for treatment and incubated with cells for another 7 days. At the end of treatment, RT-qPCR was performed using the TaqMan Fast Advanced Cell-to-CT kit to quantify targeted mRNA levels. Specifically, cells were lysed, cDNA was generated on a Mastercycler X50a (Eppendorf), and qPCR was performed on a QuantStudio7Flex Real-Time PCR System (Applied Biosystems). The gene expression level of SNCA was normalized by β-actin using a separate probe (ThermoFisher).

[0700] The results are Figure 3 and are provided in Table 20. The results provided in Table 20 demonstrate that the exemplified mouse TfR binding protein-siRNA conjugates (e.g., mTfR2-dsRNA No. 8 conjugate) successfully target mouse SNCA and provide potency that is orders of magnitude greater than unconjugated siRNA (i.e., naked siRNA) and isotype Ab-SNCA siRNA, and is equivalent to or better than the potency of cholesterol-conjugated siRNA.

[0701] Table 20: In vitro efficacy of the indicated molecules for reducing mouse SNCA mRNA in mouse cortical neurons

[0702]

[0703] Example 5: In vitro characterization of human TfR binding protein-dsRNA conjugates

[0704] In vitro binding, internalization, and degradation evaluation in SHSY5Y cells

[0705] SH-SY5Y cells (ATCC CRL-2266, passage 5-20) were maintained in a medium consisting of: 225 ml MEM / EBSS (Hyclone: ​​SH30024.02; Gibco 11095-072), 10% heat-inactivated fetal bovine serum (Hyclone SH30071.03), 1X sodium pyruvate (100X, Hyclone: ​​SH30239.01), 1X non-essential amino acids (100X, Hyclone SH30238.01) and sodium bicarbonate (7.5%, Hyclone: ​​SH30033.01) and 225 mL HAMs 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, 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 medium with 5ug / ml (33nM) of any of the following: isotype control antibody (isotype Ab), TBP10, TBP11, or the above molecules conjugated to SNCA siRNA (dsRNA No. 8), together with 10ug / ml (0.2uM) of anti-human IgG Fcγ fragment specific Fab fragment (Jackson Immuno #005-000-002) labeled with DyLight650 (Thermo Fisher #62266), DL650 with BHQ3 dye (BioSearch Tech BHQ-3000S-5), or pHAb dye (Promega #G9845) in medium with 6.7uM (1mg / ml) goat gamma globulin (Jackson Immuno #005-000-002). Immuno #109-007-008) and incubated overnight at 37°C with live cells grown in 96-well plates.

[0706] The next day, the cells were washed, incubated with NucBlue Hoechst dye (Thermo Fisher # R37605) for 20 minutes, washed again, and then imaged with a Cytation 5 high content imager (Biotek). The DyLight 650 signal measures the total TfR binding protein level, and the DyLight 650 plus BHQ3 signal measures the degradation signal, which increases the DyLight 650 fluorescence when the BHQ3 dye is released and the FRET quenching is lost, while the pHAb pH sensor dye signal only measures the internalized fluorescence. Excess goat gamma globulin was added to reduce the nonspecific binding and uptake of the antibody into the cell. The signal intensity in each well was divided by the number of nuclei stained with Hoechst to determine the signal intensity of each cell. The wells were analyzed in duplicate, and for each well, approximately 20,000 cells were analyzed from images taken with a 4x objective. The background signal was determined according to the human IgG isotype control and subtracted from the final value.

[0707] Results are shown in Figure 4 In. High content imaging data confirmed the cellular activity (binding, internalization and degradation properties) of the exemplified human TfR binding protein and isotype control antibody. The isotype control antibody lacks basic activity, while the binding, internalization and degradation activities of the exemplified human TfR binding protein are confirmed on SH-SY5Y cells. In addition, conjugation with dsRNA does not reduce the activity of the exemplified human TfR binding protein.

[0708] In vitro potency evaluation in SYSY5Y cells

[0709] SH-SY5Y cells (ATCC CRL-2266, passages 5-20) were maintained as described above. On day 4, the culture medium was removed from each well and replaced with one of the following culture medium: isotype control antibody siRNA conjugate (isotype Ab-SNCA siRNA), TBP10-SNCA siRNA conjugate or TBP11-SNCA siRNA conjugate in a culture medium with 2% FBS was added for treatment and incubated with the cells for another 7 days. At the end of the treatment, RT-qPCR was performed using the TaqManFastAdvancedCell-to-CT kit to quantify the target mRNA level. Specifically, the cells were lysed, cDNA was generated on a Mastercycler X50a (Eppendorf), and qPCR was performed on a QuantStudio 7Flex Real-Time PCR System (Applied Biosystems). The gene expression level of SNCA was standardized by β-actin using separate probes (ThermoFisher).

[0710] The results are shown in Table 21 and Figure 5 The results provided in Table 21 demonstrate that the exemplified human TFR binding protein-siRNA conjugates provide efficacy for knocking down the human SNCA gene, while the isotype control antibody conjugates showed low activity.

[0711] Table 21. In vitro efficacy for knocking down human SNCA mRNA in SH-SY5Y cells

[0712]

[0713] Example 6: In vivo proof-of-concept demonstration of the pharmacodynamic efficacy of mouse TfR binding protein-dsRNA conjugates in the CNS using peripheral delivery

[0714] In vivo pharmacodynamic evaluation in mice using multiple IV administrations

[0715] 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. PBS control, isotype Ab-SNCA siRNA, or mTBP2-SNCA siRNA (mTBP2 SNCA-dsRNA No. 8 conjugate) were intravenously administered at an effective siRNA concentration of 10 mg / kg in 8-week-old FVB mice, i) dosed once a week for four times and sacrificed 28 days after the first dose (see Fig. 6Aand 6B ), or ii) a single dose and sacrifice after 7, 28, 70 or 120 days (see Figure 6C and 6D). In addition, mouse anti-CD4 antibody (GK1.5) was administered at 10 mg / kg 2 to 3 days before the study to eliminate CD4-positive T cells to mitigate the undesirable pharmacokinetic consequences of false anti-drug antibody responses to the injected compounds. Mice at the designated time points were fully anesthetized and then subjected to cardiac perfusion (6 ml / min for 5 minutes) with cold PBS until the blood was completely drained to collect brain and spinal cord to evaluate target mRNA levels by RT-qPCR in tissue homogenates and target protein levels by ELISA. For RT-qPCR, RNA was isolated by using RNeasy Plus Universal Mini Kit (Qiagen 73404). Briefly, hemibrain, spinal cord and DRG tissue homogenates were prepared with FastPrep-24 Lysing MatrixD beads, and the tissues were homogenized for 40 seconds at 4°C with MP Fastprep 24 (MP Biomedical) at 6 m / s, and the vials were then centrifuged to collect the supernatant. RNA was then collected. After determining RNA quantity with A260 / 280 ratio on a spectrophotometer, cDNA was generated on a Mastercycler X50a (Eppendorf) and qPCR was performed on a QuantStudio 7Flex Real-Time PCR System (Applied Biosystems). The gene expression level of SNCA was normalized by β-actin using a separate probe (ThermoFisher).

[0716] Results are shown in Figures 6A-6C Multiple doses of IV administration of mTBP2 SNCA-siRNA in mice resulted in a robust 91% reduction in SNCA mRNA and 41% reduction in SNCA protein in the brain compared to PBS-dosed controls 28 days after the initial dose ( Fig. 6A Importantly, isotype Ab-SNCA siRNA did not induce a significant reduction in SNCA mRNA, confirming the need for active TfR-mediated transport to deliver the siRNA payload to the CNS, confirming BBB crossing and delivery to the brain. In addition, spinal cord evaluation 28 days after the initial dose also confirmed robust reductions in the cervical, thoracic, and lumbar regions with 79%, 79%, and 73% SNCA mRNA reductions, respectively, using mTBP2-SNCA siRNA compared to PBS-dosed controls ( Figure 6B There was also a significant 61% decrease in SNCA mRNA in the lumbar dorsal root ganglia (DRG) ( Figure 6C Interestingly, using isotype control (Isocontrol) Ab-SNCA siRNA, there was a low but significant level of SNCA mRNA reduction in the cervical and thoracic spinal cord and lumbar DRG, suggesting that without TfR-mediated delivery, there may be limited levels of spinal cord and DRG siRNA delivery.

[0717] Example 7: In vivo proof-of-concept demonstration of the pharmacodynamic time course of mouse TfR binding protein-dsRNA conjugates in the CNS using peripheral delivery

[0718] In vivo pharmacodynamic time course evaluation in mice using a single IV administration

[0719] The high efficacy of mTBP2-SNCA siRNA in the brain and spinal cord using multiple IV dosing suggests that significant efficacy is likely to exist using a single dose. Therefore, a subsequent proof-of-concept study was performed to determine the efficacy of a single IV dose and to determine the time course of pharmacodynamic efficacy with respect to SNCA mRNA and protein levels in order to provide a reference for subsequent study design in non-human primates. For each time point, 5 mice were sacrificed to collect tissues for analysis as described above.

[0720] like Fig. 7A As shown in , a single IV administration of mTBP2SNCA-siRNA in mice resulted in a robust reduction in SNCA in the brain compared to PBS-dosed controls, starting 7 days after dosing (60% mRNA reduction, 22% protein reduction), with a maximum reduction at 28 days (73% mRNA reduction, 41% protein reduction), followed by a sustained reduction at 70 days (34% mRNA reduction, 45% protein reduction), which returned to the PBS baseline group (6% mRNA reduction and 19% protein reduction) at 120 days.

[0721] In addition, if Figure 7B As shown in , a single IV administration of mTBP2 SNCA also resulted in robust SNCA reductions in the spinal cord compared to the PBS-dosed control group, starting 7 days after dosing for mRNA only (48% mRNA reduction, 3% protein reduction), reductions in both mRNA and protein at 28 days (48% mRNA reduction, 27% protein reduction), followed by sustained reductions at 70 days (32% mRNA reduction, 48% protein reduction), which returned to the PBS baseline group at 120 days (23% mRNA reduction and 14% protein reduction).

[0722] Example 8: In vivo characterization of human TfR binding protein-dsRNA conjugates

[0723] 8A. In vivo pharmacodynamic evaluation in non-human primates (NHPs) 29 days after a single dose of human TfR binding protein-SNCA siRNA conjugate.

[0724] After the solid proof-of-concept demonstration of peripheral siRNA delivery across the BBB into the CNS in mice, the pharmacodynamic properties of human TfR binding protein-SNCA siRNA conjugates were evaluated in NHPs as follows. Cynomolgus monkeys weighing 2-3 kg were intravenously administered in the saphenous vein in the thigh with: 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) (n=6) at an effective siRNA concentration of 2.6 mg / kg, and were sacrificed 29 days after the first dose. Deeply anesthetized animals were subjected to cardiac perfusion, and brain, spinal cord, and peripheral tissues were then collected. Brain is subjected to coronal sectioning, 3mm punch is collected from designated subregion and is frozen, and tissue is collected from spinal cord, liver and muscle, to evaluate target mRNA level by RT-qPCR in tissue homogenate.Use RNadvance Tissue test kit (Beckman Coulter, Indianapolis, IN) to separate total RNA from NHP tissue manually or on Biomek i7 liquid processor (Beckman Coulter), follow the manufacturer's program, with some modifications.In brief, frozen tissue section is mixed with a 5mm stainless steel ball, lysis buffer and proteinase K, on ​​2010GenoGrinder (SPEX SamplePrep, Metuchen, NJ), 5 cycles of 30 seconds of homogenization with 1200rpm, with 20 seconds intervals between cycles.Before homogenization, tissue from some regions is scraped on dry ice.Make homogenate incubate 1 hour at 37C, then extract with isopyknic 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 for 15 ng / 10 uL PCR, digested again with ezDNase (ds-DNA specific), and then reverse transcribed using the SSIV VILO kit (Thermo Fisher Scientific, Waltham, MA). The expression of the respective gene targets in the cDNA was determined using the TaqMan qPCR assay on the QuantStudio 7Pro platform (Thermo Fisher Scientific). Gene expression levels of SNCA were normalized by β-actin using respective probes (ThermoFisher).The tissues analyzed and their acronyms are: liver; gastrocnemius muscle; AN, arcuate nucleus; MedEm, median eminence; LSC, lumbar spinal cord; medulla oblongata; 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.

[0725] Peripheral IV administration of TBP10-SNCA siRNA at 8.8 mg / kg in NHP resulted in a significant decrease in SNCA mRNA in key brain regions and lumbar spinal cord compared to the PBS-treated group 29 days after dosing. Fig. 8A As shown in ( ), significant SNCA mRNA reduction was demonstrated in the liver (48%), arcuate nucleus (58%), lumbar spinal cord (82%), medulla oblongata (71%), pons (77%), midbrain (56%), substantia nigra (76%), caudate nucleus (81%), putamen (76%), hypothalamus (64%), hippocampus (83%), prefrontal cortex gray matter (74%), prefrontal cortex white matter (76%). Fig. 8A ), other brain regions and tissues evaluated did not demonstrate significant reductions in SNCA mRNA.

[0726] Peripheral IV administration of TBP11-SNCA siRNA at a lower dose of 2.6 mg / kg in NHP also resulted in a significant decrease in SNCA mRNA in key brain regions and lumbar spinal cord compared to the PBS-treated group 29 days after dosing. Figure 8B As shown in ( ), significant SNCA mRNA reduction was demonstrated in the lumbar spinal cord (62%), medulla oblongata (63%), pons (48%), substantia nigra (66%), caudate nucleus (59%), hippocampus (72%), and prefrontal cortical gray matter (39%). Figure 8B ), other brain regions and tissues evaluated did not demonstrate significant reductions in SNCA mRNA.

[0727] To determine the expected level of brain SNCA mRNA reduction at NHP-equivalent doses, mouse cohorts received a single IV dose with equivalent 8.8 mg / kg and 2.6 mg / kg concentrations of mTBP2-SNCA siRNA and were treated as described above to assess the transferability of mRNA KD efficacy by RT-qPCR.

[0728] Mice dosed 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 administration at an effective siRNA concentration of 2.6 mg / kg demonstrated a 53% reduction in SNCA mRNA in the brain, demonstrating similar efficacy translation from rodents to NHPs ( Figure 8C).

[0729] 8B. In vivo pharmacodynamic evaluation in NHPs 85 days after a single dose or three monthly doses of human TfR binding protein-SNCA siRNA conjugate.

[0730] Pharmacodynamic studies were performed to determine the efficacy of human TfR binding protein-SNCA siRNA conjugates 3 months after a single dose or three monthly doses. Cynomolgus monkeys weighing 2-3 kg were administered a single intravenous dose of 10 mg / kg of TBP14-SNCA siRNA (dsRNA number 10 in Table 11a) conjugate (n=5) in the saphenous vein in the thigh, or three monthly intravenous doses of the following: i) PBS (n=5), or ii) 10 mg / kg of TBP14-SNCA siRNA (dsRNA number 10 in Table 11a) conjugate (n=5) in the saphenous vein in the thigh. All groups were administered anti-CD4 antibody at 30 mg / kg immediately after the dose of the test article to reduce anti-drug antibody responses. After a single dose or 85 days after the first dose in the three monthly dosing regimen, deeply anesthetized animals were subjected to cardiac perfusion, and brain, spinal cord and peripheral tissues were then collected.

[0731] The brain is subjected to coronal sectioning, 4mm punches are collected from designated subregions and frozen, and tissues are collected from spinal cord, liver and muscle to evaluate target mRNA and protein levels by RT-qPCR and ELISA in tissue homogenates, respectively. In order to determine mRNA levels, the total RNA from NHP tissues is separated manually or on a Biomeki7 liquid processor (Beckman Coulter) using RNadvance Tissue test kit (Beckman Coulter, Indianapolis, IN), following the manufacturer's procedure with some modifications. In brief, frozen tissue sections are mixed with a 5mm stainless steel ball, lysis buffer and proteinase K, and 5 cycles of 30 seconds of homogenization at 1200rpm are performed on a 2010GenoGrinder (SPEX SamplePrep, Metuchen, NJ), with 20 seconds intervals between cycles. Before homogenization, tissues from some regions are scraped on dry ice. The homogenate is incubated at 37°C for 1 hour, 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 for 15 ng / 10 uL PCR, digested again with ezDNase (ds-DNA specific), and then reverse transcribed using the SSIV VILO kit (Thermo Fisher Scientific, Waltham, MA). The expression of the respective gene targets in the cDNA was determined using the TaqMan qPCR assay on the QuantStudio 7Pro platform (Thermo Fisher Scientific). Gene expression levels of SNCA were normalized by β-actin using respective probes for the CNS region (ThermoFisher) and GAPDH for gastrocnemius (ThermoFisher).

[0732] To determine α-synuclein protein levels, 4 mm punches of frozen neural tissue biopsies were mixed with cold RIPA buffer (Pierce #89901, Thermo Scientific, Waltham, MA) containing protease and phosphatase inhibitors (Halt TMProtease and Phosphatase Inhibitor Cocktail, Thermo Scientific). The tissue-RIPA mixture was homogenized using 5 mm stainless steel beads on a 2010 GenoGrinder (SpexSamplePrep, Metuchen, NJ). The homogenate was then centrifuged in a refrigerated centrifuge (Eppendorf, Hamburg, Germany), and the supernatant was transferred, prepared into multiple single-use aliquots, and stored at -80°C for further analysis.

[0733] Follow the manufacturer's instructions for use with Pierce TM BCA Protein Assay Kit (Thermo Scientific) was used to determine the protein concentration in the protein lysate. Specifically, serially diluted bovine serum albumin (BSA) standards were analyzed in duplicate; at the same time, each protein lysate sample was diluted 10-fold or 20-fold in water and then analyzed in singleplex. The protein concentration in the undiluted sample was then obtained by averaging the protein concentrations derived from the 10-fold dilution and the protein concentrations derived from the 20-fold dilution.

[0734] α-Synuclein protein levels in protein lysates were measured using an in-house developed sandwich ELISA. Briefly, half of a 96-well flat-bottom UV-transparent microplate (Corning, Corning, NY) was coated with capture antibody (α-Synuclein: anti-Synuclein antibody, Syn42, Eli Lilly, Indianapolis, IN) overnight at 4°C with agitation. The wells were incubated with phosphate-buffered saline Tween20 at room temperature (RT). TM The plate was blocked with 2% bovine serum albumin (BSA) (Thermo Scientific) in PBST (Thermo Scientific) for 60 minutes. After washing, protein lysates or recombinant human α-synuclein protein (α-synuclein: rPeptide, Watkinsville, GA) diluted in PBST containing 2% BSA were added to the wells on each plate for a-SynELISA. The plate was incubated overnight at 4°C with agitation.

[0735] The plates for a-Syn ELISA were washed and then incubated with detection antibody (rabbit pAb anti-a-synuclein, USBiological, Salem, MA) in PBST containing 2% BSA for 3 hours at RT. The plates were washed again and then incubated with anti-rabbit HRP-linked antibody in PBST containing 2% BSA for 1 hour at RT.

[0736] In order to minimize the change, 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. After deducting the plate blank value, the arithmetic mean of the OD450 from duplicate was used for further calculation. Using JMP software (SAS Institute, Cary, NY), by fitting the respective OD450 (Y-axis) and protein concentration (X-axis) of the serially diluted protein standards with the logistic 4P nonlinear regression model, the standard curve on each ELISA plate was created. Then based on the standard curve, the concentration of the respective protein in each diluted sample was calculated from the respective OD450 reverse calculations. The α-synuclein protein level in each sample is standardized for the level of total protein, and the remaining α-synuclein protein expression in the treatment group is calculated as the average expression of the protein in the aCSF or PBS control group, and the percentage of the remaining α-synuclein protein expression in the treatment group.

[0737] The tissues analyzed for mRNA or protein levels and their acronyms are: gastrocnemius; LSC, lumbar spinal cord; SN, substantia nigra; caudate nucleus; PUT, putamen; H, hippocampus; PFC, prefrontal cortical gray matter and LDRG, lumbar DRG.

[0738] At 85 days after the first dose, three monthly peripheral IV administrations of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate at a dose of 10 mg / kg in NHP resulted in a significant decrease in SNCA mRNA in key brain regions and the lumbar spinal cord compared to the PBS-treated group. Fig.9A As shown in , significant SNCA mRNA reductions were demonstrated in the lumbar spinal cord (72%), substantia nigra (76%), caudate nucleus (81%), putamen (66%), hippocampus (76%), and prefrontal cortical gray matter (73%). Fig. 9B Significant reductions in alpha-synuclein protein in key brain regions and the lumbar spinal cord were demonstrated 85 days after the first dose compared with the PBS-treated group. Fig. 9B As shown in Figure 2, significant decreases 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%).

[0739] A single peripheral IV administration of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate at a dose of 10 mg / kg in NHP resulted in a significant decrease in SNCA mRNA in key brain regions compared to the PBS-treated group 85 days after dosing. Fig. 9C As shown in ( Fig. 9C ), no significant decrease in SNCA mRNA was demonstrated in other brain regions and tissues evaluated. Fig.9D Significant reductions in alpha-synuclein protein in key brain regions and the lumbar spinal cord were demonstrated 85 days after the first dose compared with the PBS-treated group. Fig.9D As shown in ( Fig.9D ), other brain regions and tissues evaluated did not demonstrate significant reductions in α-synuclein protein.

[0740] Fig.9E Figure 2 shows a decrease in SNCA mRNA in gastrocnemius muscle after single or three monthly dosings.

[0741] 8C. In vivo pharmacodynamic evaluation in NHPs after three monthly doses of human TfR binding protein-MAPT siRNA conjugate.

[0742] Pharmacodynamic studies were conducted to determine the efficacy of human TfR binding protein-MAPT siRNA conjugates after three monthly doses. A group of cynomolgus monkeys weighing 2-3 kg were intravenously administered once a month in the saphenous vein in the thigh with the following: i) PBS (n = 5), or ii) TBP14-MAPT siRNA (dsRNA number 38 in Table 11b) (n = 5) at an effective siRNA concentration of 10 mg / kg. A separate group of cynomolgus monkeys weighing 2-3 kg were intravenously administered once a month in the saphenous vein in the thigh with the following: i) PBS (n = 5), ii) TBP14-MAPT siRNA (dsRNA number 39 in Table 11b) (n = 5), or iii) TBP14-MAPT siRNA (dsRNA number 40 in Table 11b) (n = 5) at an effective siRNA concentration of 10 mg / kg. All groups were dosed with anti-CD4 antibody at 30 mg / kg immediately after the test article dose to mitigate anti-drug antibody responses. Approximately 85 days after the first dose, deeply anesthetized animals underwent cardiac perfusion followed by collection of brain, spinal cord, and peripheral tissues.

[0743] Brain is carried out coronal section, 4mm punch is collected from designated sub-region and freezing, and tissue is collected from spinal cord, liver and muscle, to evaluate target mRNA and protein level by RT-qPCR and ELISA respectively in tissue homogenate.In order to determine mRNA level, use RNadvance Tissue test kit (Beckman Coulter, Indianapolis, IN) manually or on Biomeki7 liquid processor (Beckman Coulter) separate total RNA from NHP tissue, follow the manufacturer's program, with some modifications.In brief, frozen tissue section is mixed with a 5mm stainless steel ball, lysis buffer and proteinase K, on ​​2010GenoGrinder (SPEX SamplePrep, Metuchen, NJ) with 5 circulations of 1200rpm homogenization 30 seconds, with 20 seconds intervals between circulations.Before homogenization, tissue from some regions is scraped on dry ice.Make homogenate incubate 1 hour at 37C, then extract with isopyknic phenol-chloroform. RNA in the supernatant was purified with RNadvance tissue kit, including 30 minutes of digestion with DNA enzyme. The concentration and purity (A260 / A280) of RNA eluate were determined by spectrophotometry. RNA was standardized for 15ng / 10uL PCR, digested again with ezDNase (ds-DNA specificity), and then reverse transcribed using SSIV VILO kit (Thermo Fisher Scientific, Waltham, MA). The expression of the respective gene targets in the cDNA was determined using the TaqMan qPCR assay on the QuantStudio 7Pro platform (Thermo Fisher Scientific). The gene expression level of MAPT was standardized by β-actin using the respective probes (ThermoFisher) for CNS regions and for GAPDH (ThermoFisher) for gastrocnemius.

[0744] To determine Tau protein levels, 4 mm punches of frozen neural tissue biopsies were mixed with cold RIPA buffer (Pierce #89901, Thermo Scientific, Waltham, MA) containing protease and phosphatase inhibitors (Halt TMProtease and Phosphatase Inhibitor Cocktail, Thermo Scientific). 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 transferred, prepared into multiple single-use aliquots, and stored at -80°C for further analysis.

[0745] Follow the manufacturer's instructions for use with Pierce TM BCA Protein Assay Kit (Thermo Scientific) was used to determine the protein concentration in the protein lysate. Specifically, serially diluted bovine serum albumin (BSA) standards were analyzed in duplicate; at the same time, each protein lysate sample was diluted 10-fold or 20-fold in water and then analyzed in singleplex. The protein concentration in the undiluted sample was then obtained by averaging the protein concentrations derived from the 10-fold dilution and the protein concentrations derived from the 20-fold dilution.

[0746] Tau protein levels in protein lysates were measured using an in-house developed sandwich ELISA. Briefly, half of a 96-well flat-bottom UV-transparent microplate (Corning, Corning, NY) was coated with capture antibody (Tau: anti-human Tau antibody, Tau5, Eli Lilly, Indianapolis, IN) overnight at 4°C with agitation. The wells were incubated with phosphate-buffered saline Tween 20 at room temperature (RT). TM The plate was blocked with 2% bovine serum albumin (BSA) (Thermo Scientific) in PBST (Thermo Scientific) for 60 minutes. After washing, protein lysate or recombinant human Tau protein (Tau: Tau441, Eli Lilly) diluted in PBST containing 2% BSA and detection antibody (Tau: anti-human Tau antibody, biotinylated DA9, Eli Lilly) were added to the wells on each plate. The plate was incubated overnight at 4°C with agitation.

[0747] On the second day, the plates were washed and then incubated with Pierce TMHigh-sensitivity streptavidin protein conjugated horseradish peroxidase (HRP) (Thermo Scientific) was incubated together in PBST containing 2% BSA for 30 minutes. The HRP enzymatic 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) on an Envision plate reader (PerkinElmer, Waltham, MA).

[0748] In order to minimize the variation, 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. After subtracting the plate blank value, the arithmetic mean of the OD450 from the duplicates was used for further calculations. Using JMP software (SAS Institute, Cary, NY), the standard curve on each ELISA plate was created by fitting the OD450 (Y-axis) and protein concentration (X-axis) of the serially diluted protein standards with a logistic 4P nonlinear regression model. Then, based on the standard curve, the concentration of each protein in each diluted sample was reversely calculated from the respective OD450. The Tau protein level in each sample was standardized for the level of total protein, and the remaining Tau protein expression in the treatment group was calculated as the percentage of the remaining Tau protein expression in the treatment group relative to the average expression of the protein in the aCSF or PBS control group.

[0749] Tissues analyzed for mRNA or protein levels and their acronyms are: LSC, lumbar spinal cord; SN, substantia nigra; caudate nucleus; PUT, putamen; H, hippocampus, and PFC, prefrontal cortical gray matter.

[0750] At 85 days after the first dose, three monthly peripheral IV administrations of TBP14-MAPT siRNA (dsRNA No. 38 in Table 11b) at 10 mg / kg in NHP resulted in significant reductions in MAPT mRNA and protein in key brain regions and the lumbar spinal cord compared to the PBS-treated group. Fig. 10A As shown in ( ), significant MAPT mRNA reduction was demonstrated in the lumbar spinal cord (24%), caudate nucleus (31%), putamen (38%), hippocampus (41%), and prefrontal cortical gray matter (40%). Fig. 10A ), other brain regions and tissues evaluated did not demonstrate a significant decrease in MAPT mRNA. Fig. 10BSignificant reductions in Tau in key brain regions and the lumbar spinal cord were demonstrated 85 days after the first dose compared to the PBS-treated group. Fig. 10B As shown in ( Fig. 10B ), no significant reduction of Tau protein was demonstrated in other brain regions and tissues evaluated.

[0751] At 85 days after the first dose, three monthly peripheral IV administrations of TBP14-MAPT siRNA (dsRNA No. 39 in Table 11b) conjugate at 10 mg / kg in NHP resulted in a significant decrease in MAPT mRNA in key brain regions and lumbar spinal cord compared to the PBS treated group. Fig.11A As shown in , significant MAPT mRNA reduction was demonstrated in the lumbar spinal cord (41%), substantia nigra (41%), caudate nucleus (67%), putamen (67%), hippocampus (57%), and prefrontal cortical gray matter (65%). Fig. 11B Significant reductions in Tau in key brain regions and the lumbar spinal cord were demonstrated 85 days after the first dose compared to the PBS-treated group. Fig. 11B As shown in Figure 2, 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%).

[0752] At 85 days after the first dose, three monthly peripheral IV administrations of TBP14-MAPT siRNA (dsRNA No. 40 in Table 11b) conjugate at 10 mg / kg dose in NHP resulted in a significant decrease in MAPT mRNA in key brain regions and lumbar spinal cord compared to the PBS treated group. Fig. 12A As shown in , significant MAPT mRNA reductions were demonstrated in the lumbar spinal cord (37%), substantia nigra (35%), caudate nucleus (61%), putamen (54%), hippocampus (36%), and prefrontal cortical gray matter (61%). Fig. 12B Significant reductions in Tau protein in key brain regions and the lumbar spinal cord were demonstrated 85 days after the first dose compared to the PBS-treated group. Fig. 12B As shown in Figure 2, 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%).

[0753] 8D. In vivo pharmacodynamic evaluation in NHPs 1 month after a single dose of a BBB-penetrating antibody targeting SNCA human TfR binding protein-SNCA siRNA conjugate (DAR1)

[0754] After demonstrating central efficacy using peripheral siRNA delivery in cynomolgus monkeys (Macaca fascicularis) using DAR2 average human TfR binding protein-SNCA siRNA conjugates, a 1-month efficacy using DAR1 average human TfR binding protein-SNCA siRNA conjugate was performed to determine the difference in efficacy. The pharmacodynamic properties of human TfR binding protein-siRNA conjugates were evaluated in NHPs as follows. Cynomolgus monkeys weighing 2-3 kg were dosed intravenously once in the saphenous vein in the thigh with: 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 (each N=4), and were sacrificed 29 days after the first dose. For takedowns, deeply anesthetized animals underwent cardiac perfusion, and brain tissue was then collected and processed for RT-qPCR in tissue homogenates.

[0755] RT-qPCR data showed a robust reduction of SNCA mRNA ranging from 60-80% in all key brain regions at 1 mg / kg siRNA dose, confirming the high efficacy of the DAR1 conjugate ( Fig.13A and 13B Increasing the dose tenfold to 10 mg / kg triggered only an additional 5-10% decrease in mRNA from 1 mg / kg, suggesting that TfR-mediated drug delivery had been saturated ( Fig. 13C ).

[0756] 8E. Exposure Response Relationship

[0757] To understand the exposure-response relationship for the studies described in Examples 8B and 8D above, plasma pharmacokinetics (PK) after a single IV dose, and biodistribution of siRNA, plasma samples from the above-mentioned studies were collected and the exposure of conjugate-related siRNA in plasma or total siRNA in tissues was quantified by HR-LC / MS ( Fig.13D and 13E). Briefly, liquid chromatography / mass spectrometry (LC / MS) was used to measure conjugate-related or total siRNA levels in cynomolgus monkey plasma and tissue samples. Plasma standards were prepared by adding control monkey plasma. Tissue standards were prepared in control tissue homogenates. To control for assay variability, internal standards were added to all standards and samples.

[0758] For the siRNA associated with the conjugate, plasma standards and samples were incubated with biotinylated polyclonal goat anti-human IgG antibodies (Southern Biotech, Birmingham, AL), followed by a secondary incubation with streptavidin protein beads (Promega, Madison, WI). IgG-siRNA-streptavidin protein bead complexes were separated on a magnetic separator and the supernatant was discarded. Samples and standards were washed with phosphate buffered saline solution, and the siRNA associated with the conjugate was subsequently eluted from the beads with triethylamine. Standards and samples were injected onto the LC / MS system.

[0759] Tissue samples were homogenized in cell lysis buffer. For total siRNA measurements, tissue standards and samples were digested with proteinase K and then loaded onto Oasis Wax microelution solid phase extraction (SPE) plates (Waters Inc, Milford, MA) for separation. The SPE plates were washed with wash buffer and the analytes were then eluted with elution buffer. The eluate from the SPE plates was dried, reconstituted and injected onto the LC / MS system.

[0760] 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).

[0761] For the TBP15-SNCA conjugate (DAR1), plasma PK appeared to be greater than dose proportional (7.8 μM*hr vs. 111.6 μM*hr) between 1 and 10 mg / kg siRNA doses based on AUC (0-168 hr) ( Fig.13D ). This plasma PK is consistent with TMDD-mediated clearance. For the DAR2 conjugate, TBP14-SNCA siRNA (DAR2) at 10 mg / kg siRNA, the AUC (0-72 hr) was 78 μM*hr, which is approximately 1 / 1.8 of the exposure observed for TBP15-SNCA siRNA (DAR1) at the same dose.

[0762] For TBP15-SNCA siRNA (DAR1), dose-dependent plasma PK translated into brain distribution, albeit with an even less dose-proportional profile compared to plasma exposure ( Fig.13E ). For a given dose, exposure across different brain regions was similar. Brain exposure for TBP14-SNCA (DAR2) was undetectable at 3 months, consistent with lower plasma exposure (data not shown).

[0763] Example 9. Further characterization of human TfR binding protein-dsRNA conjugates in human TfR (hTfR) transgenic mice

[0764] To understand the effect 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 dosed at 10 mg / kg in hTfR transgenic mice, and plasma samples were collected and the exposure of conjugate-associated siRNA was quantified by HR-LC / MS at different times up to 1 month after dosing ( Fig.14A Based on AUC (0-168hr), the plasma PK for DAR1 was 3.5-fold higher than that for DAR2 (323 μM*hr vs. 92 μM*hr). This plasma PK is consistent with TMDD-mediated clearance. This dose-dependent plasma PK translates to the brain, where 3.6-fold exposure was observed for DAR1 relative to DAR2 ( Fig. 14B ).

[0765] Fig. 14C Shown are brain tissue concentrations of total siRNA in human TfR transgenic mice 24 hours after a single peripheral IV administration of TBP14-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR2) or TBP15-SNCA siRNA (dsRNA No. 10 in Table 11a) conjugate (DAR1) across various siRNA doses.

[0766] The pharmacodynamic efficacy relationships of DAR1 and DAR2 of human TfR binding protein-dsRNA conjugates were evaluated at various doses to determine the effect of dose reduction when matching antibody and siRNA concentrations. Fig.14DAs indicated in, TBP15-SNCA siRNA conjugates (DAR1) and TBP14-SNCA siRNA conjugates (DAR2) were administered in hTfR transgenic mice by a single IV injection of 20, 10, 5, 2.5, and 0.5 mg / kg siRNA compared to the PBS-administered group (n=4 each). For sampling, deeply anesthetized animals were subjected to cardiac perfusion 28 days after IV administration, and brain tissues were then collected and processed for RT-qPCR in tissue homogenates. Fig.14D As shown in, TBP15-SNCA siRNA conjugate (DAR1) demonstrated higher SNCAmRNA KD efficacy at all matched dose levels compared to TBP14-SNCA siRNA conjugate (DAR2). Specifically, for TBP15-SNCA siRNA conjugate (DAR1), 10 mg / kg siRNA dose triggered 8% mRNA residual, 5 mg / kg siRNA dose triggered 10% mRNA residual, 2.5 mg / kg siRNA dose triggered 13% mRNA residual, and 0.5 mg / kg siRNA dose triggered 24% mRNA residual. For TBP14-SNCA siRNA conjugate (DAR2), 20 mg / kg siRNA dose triggered 17% mRNA residual, 10 mg / kg siRNA dose triggered 20% mRNA residual, 5 mg / kg siRNA dose triggered 23% mRNA residual, and 0.5 mg / kg siRNA dose triggered 59% mRNA residual. In particular, a 10-fold siRNA drug dose reduction efficacy was demonstrated when comparing TBP14-SNCA siRNA conjugate at 5 mg / kg (DAR2) (23% residual) to similar mRNA reduction at 0.5 mg / kg TBP15-SNCA siRNA conjugate (DAR1) (24% residual). This trend was also observed at higher doses when comparing TBP14-SNCA siRNA conjugate at 20 mg / kg (DAR2) (17% residual) to similar mRNA reduction at 2.5 mg / kg TBP15-SNCA siRNA conjugate (DAR1) (13% residual).

[0767] The high efficacy of DAR1 of the human TfR binding protein-dsRNA conjugate via the intravenous administration route has been demonstrated, and the efficacy of the TBP15-SNCA siRNA conjugate (DAR1) delivered by a single subcutaneous (SC) administration at 5, 2, 0.5 and 0.25 mg / kg siRNA doses was evaluated. In order to obtain the material, 28 days after SC administration, the deeply anesthetized animals were subjected to cardiac perfusion, and then the brain tissue was collected and processed for RT-qPCR in the tissue homogenate. The data indicated similar high efficacy of SC delivery at all doses evaluated, confirming 11% mRNA residual at a dose of 5 mg / kg, 15% residual at a dose of 2 mg / kg, 30% mRNA residual at a dose of 0.5 mg / kg, and 42% mRNA residual at a dose of 0.25 mg / kg ( Fig.14E ).

[0768] Sequence Listing

[0769]

[0770]

[0771]

[0772]

[0773]

[0774]

[0775]

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782]

Claims

1. A protein comprising a 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 complementary determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementary 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) 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 protein of claim 1, 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: 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; (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; (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; (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; (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; (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.

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

37.

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

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

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

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

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

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

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

13. The protein of any one of claims 9-12, wherein the Fc region comprises: (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 the EU index); 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 are numbered according to the EU index).

14. The protein according to any one of claims 1-12, wherein the protein 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; (b) HC comprises SEQ ID NO:55 and LC comprises SEQ ID NO:54; (c) HC comprises SEQ ID NO:56 and LC comprises SEQ ID NO:57; (d) HC comprises SEQ ID NO:58 and LC comprises SEQ ID NO:59; (e) HC comprises SEQ ID NO:60 and LC comprises SEQ ID NO:61; (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.

15. The protein according to any one of claims 1-13, 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. The protein according to any one of claims 1-13, 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. The protein according to any one of claims 1-13, 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. The protein of any one of claims 1-4, wherein the protein comprises one heavy chain (HC) and one light chain (LC), and wherein the HC comprises SEQ ID NO: 65 and the LC comprises SEQ ID NO:

59.

19. The protein according to claim 7 or 8, wherein the half-life extender is a VHH binding 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. The protein of claim 19 or 20, wherein the VHH comprises SEQ ID NO:

42.

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

67.

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

24. The protein of claim 23, wherein the second arm comprises VH and 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 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. The protein of claim 24, wherein the VH comprises SEQ ID NO:49 and the VL comprises SEQ ID NO:

50.

26. The protein according to any one of claims 23-25, wherein 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; (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; (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.

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

28. A protein comprising a 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 complementary determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, and 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. The protein of claim 28, wherein the VH comprises SEQ ID NO: 77 and the VL comprises SEQ ID NO:

78.

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 28-30, wherein the protein is a heterodimeric antibody comprising a first arm comprising a monovalent mouse TfR binding domain and a second arm that is an empty arm.

32. The protein of any one of claims 28-31, 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 1-32 and a therapeutic agent.

34. The conjugate of claim 33, wherein the therapeutic agent is selected from 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. 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 33-35, wherein the therapeutic agent is double-stranded RNA (dsRNA).

37. The conjugate of claim 36, wherein the dsRNA comprises a sense strand and an antisense strand, 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.

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

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

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

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

1.

42. A conjugate of formula (I): RLP, Wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is 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 complementary determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, 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) 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. The conjugate of claim 42, wherein the ratio of R to P is about 1:1 to 3:

1.

44. A formula (II): (RL) n -P conjugate, Wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand; wherein P is a protein comprising a monovalent human TfR binding domain; and wherein L is a linker, or is 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 complementary determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, 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) 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; And wherein n is 1 to 3.

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

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

47. The conjugate of any one of claims 42-46, 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: 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; (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; (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; (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; (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; (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.

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

37.

49. The conjugate of any one of claims 42-47, 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 42-48, wherein VH comprises SEQ ID NO: 32 and VL comprises SEQ ID NO:

33.

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

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

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

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

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

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

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

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

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

60. The conjugate of any one of claims 56-59, wherein the Fc region comprises: (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 the EU index); 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 are numbered according to the EU index).

61. The conjugate of any one of claims 42-59, wherein the protein 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; (b) HC comprises SEQ ID NO:55 and LC comprises SEQ ID NO:54; (c) HC comprises SEQ ID NO:56 and LC comprises SEQ ID NO:57; (d) HC comprises SEQ ID NO:58 and LC comprises SEQ ID NO:59; (e) HC comprises SEQ ID NO:60 and LC comprises SEQ ID NO:61; (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.

62. The conjugate of any one of claims 42-60, 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. The conjugate of any one of claims 42-60, 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. The conjugate of any one of claims 42-60, 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. The conjugate of any one of claims 42-51, wherein the protein comprises one heavy chain (HC) and one light chain (LC), and wherein the HC comprises SEQ ID NO: 65 and the LC comprises SEQ ID NO:

59.

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

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

42.

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

67.

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

71. The conjugate of claim 70, wherein the second arm comprises VH and 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 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. The conjugate of claim 71, wherein the VH comprises SEQ ID NO: 49 and the VL comprises SEQ ID NO:

50.

73. The conjugate of any one of claims 70-72, wherein 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; (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; (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.

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

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

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

77. The conjugate of any one of claims 42-76, 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. The conjugate of claim 77, wherein the antisense strand is complementary to SNCA mRNA.

79. The conjugate of any one of claims 42-78, wherein 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, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

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

82. The conjugate of any one of claims 42-77 and 81, wherein 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, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein 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 42-82, wherein one or more nucleotides of the sense strand are modified nucleotides.

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 42-84, wherein one or more nucleotides of the antisense strand are modified nucleotides.

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

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

88. The conjugate of any one of claims 83-87, 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. The conjugate of claim 88, wherein the nucleotides at positions other than positions 7, 9, 10 and 11 of the sense strand are 2'-O-methyl modified nucleotides.

90. The conjugate of any one of claims 83-89, 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. The conjugate of claim 90, wherein nucleotides at positions other than positions 2, 6, 14 and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

92. The conjugate of any one of claims 83-87, 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. The conjugate of claim 90, wherein nucleotides at positions other than positions 9, 10, and 11 of the sense strand are 2'-O-methyl modified nucleotides.

94. The conjugate of any one of claims 83-87, 92, 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. The conjugate of claim 94, wherein nucleotides at positions other than positions 2, 5, 7, 14 and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

96. The conjugate of any one of claims 83-87, 92, 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. The conjugate of claim 96, wherein nucleotides at positions other than positions 2, 5, 8, 14 and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

98. The conjugate of any one of claims 83-87, 92, 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. The conjugate of claim 98, wherein nucleotides at positions other than positions 2, 3, 7, 14 and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

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

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

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 100-102, wherein the antisense strand has four or five phosphorothioate linkages.

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

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

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

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

108. The conjugate of any one of claims 42-80 or 83-107, wherein the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from: (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.

109. The conjugate of any one of claims 42-80 or 83-107, wherein 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 sense strand consists of SEQ ID NO: 118 or 149, and the antisense strand consists of SEQ ID NO:

97.

110. The conjugate of any one of claims 42-77 or 81-107, wherein 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 sense strand comprises SEQ ID NO: 136 or 155, and the antisense strand comprises SEQ ID NO:

137.

111. The conjugate of any one of claims 42-77 or 81-107, wherein 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 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 the protein according to any one of claims 1-27 or the conjugate according to any one of claims 33-111, and a pharmaceutically acceptable carrier.

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

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

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

117. The method of claim 116, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, frontotemporal dementia (FTD), chromosome 17-linked frontotemporal dementia with associated parkinsonism (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-oligoglossia (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic granular dementia (AGD), British amyloidosis Tubular disease, 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-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

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

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

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

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. The conjugate of any one of claims 39-77, 81-107, 110, 111 or the pharmaceutical composition of claim 108 for use in treating tauopathy.

123. The conjugate or pharmaceutical composition for use according to claim 122, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with chromosome 17 and associated parkinsonism (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-oligoglossia (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (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, diffuse neurofibrillary tangles with calcifications, Down syndrome, epilepsy, Gerstmann-Straussler-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy Poor, Niemann-Pick disease type C (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, 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 33-111 in the preparation of a medicament for the treatment of a CNS disease.

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

126. The use according to 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 the conjugate according to any one of claims 39-77, 81-107, 110, 111 in the preparation of a medicament for treating tauopathy.

128. The method of claim 127, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with chromosome 17 and associated parkinsonism (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-oligoglossia (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic granular dementia (AGD), amyloidosis of the British type. Tubular disease, 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-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

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