Conjugates comprising transferrin receptor binding protein

Abstract

Provided herein are compounds and conjugates comprising transferrin receptor binding protein, pharmaceutical compositions comprising such conjugates, and methods of treating CNS diseases (e.g., neurodegenerative disease) using such conjugates.

Description

CONJUGATES COMPRISING TRANSFERRIN RECEPTOR BINDING PROTEINSEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “30759_WO” created 18-Nov-2024 and is 158 kilobytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety.BACKGROUND

[0002] The blood brain barrier (BBB) is a selective semipermeable border of capillary endothelial cells that prevents solutes, including pathogens, from passing into the central nervous system (CNS). The BBB allows the passage of some small molecules by passive diffusion and the cells of BBB actively transport metabolic products crucial to neural function such as glucose and amino acids across the barrier using specific transport proteins. The BBB has neuroprotective function by tightly controlling access to the brain; but it also impedes access of therapeutic agents to CNS.

[0003] BBB shuttles for improving passage of the therapeutic agents across the blood brain barrier and into the CNS have been described. For example, W02003 / 009815 describes the use of antibodies directed to transferrin receptor (“TfR”) for modulating blood brain barrier transport and delivering therapeutic agents across BBB. However, attempts at using anti-TfR antibodies to shuttle therapeutic agents across the BBB have proven challenging. To date, there are no approved TfR shuttles or conjugates for the treatment of CNS diseases in the U.S.

[0004] RNA interference (RNAi) is a highly conserved regulatory mechanism in which RNA molecules are involved in sequence- specific suppression of gene expression by double-stranded RNA molecules (dsRNA) (Fire et al., Nature 391:806-811, 1998).

[0005] Therefore, there remains a need for compounds and conjugates comprising TfR binding protein that can deliver therapeutic agents, e.g., RNAi agent, across the BBB into the CNS for the treatment of various CNS diseases.SUMMARY OF INVENTION

[0006] Provided herein are compounds and conjugates comprising transferrin receptor binding protein, pharmaceutical compositions comprising such conjugates, and methods of treating CNS diseases (e.g., neurodegenerative disease) using such conjugates.

[0007] In one aspect, provided herein are conjugates of Formula (I): (A-L)n-P, wherein A is a therapeutic agent, wherein L is a linker comprising any one of the following formulae:wherein P is a protein comprising one monovalent human TfR binding domain, and wherein n is an integer of 1 to 3. One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P. Accordingly, in some embodiments, provided herein are conjugates of Formula (I): (A-L)n-P, wherein A is a therapeutic agent, wherein L is a linker comprising any one of the following formulae:wherein P is a protein comprising one monovalent human TfR binding domain, and wherein n is an integer of 1 to 3.

[0008] The conjugates provided herein have improved plasma stability when compared to similar conjugates comprising SMCC linker.

[0009] In some embodiments, provided herein are conjugates of Formula (I): (A-L)n-P, wherein A is a therapeutic agent, wherein L is a linker comprising any one of the following formulae:wherein P is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6, and wherein n is an integer of 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0010] One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P. Accordingly, in some embodiments, provided herein are conjugates of Formula (I): (A-L)n-P, wherein A is a therapeutic agent, wherein L is a linker comprising any one of the following formulae:wherein P is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6, and wherein n is an integer of 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0011] In some embodiments, the therapeutic agent (A) is selected from an oligonucleotide (e.g., antisense oligonucleotide), double stranded RNA (e.g., siRNA, saRNA), polypeptide, small molecule, nanoparticle, lipid nanoparticle, exosome, antibody or antigen binding fragment thereof, or a combination thereof. In some embodiments, the therapeutic agent is an oligonucleotide. In some embodiments, the therapeutic agent is a double stranded RNA (ds RNA). In some embodiments, A is a dsRNA comprising a sense stand and an antisense strand.

[0012] In some embodiments, L is connected to the 3’end of the sense strand of dsRNA, and wherein L comprises any one of the following formulae:One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P. Accordingly, in some embodiments, L is connected to the 3’end of the sense strand of dsRNA, and L comprises any one of the following formulae:

[0013] In some embodiments, L is connected to the 5 ’end of the sense strand of dsRNA, and wherein L comprises any one of the following formulae:One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P. Accordingly, in some embodiments, L is connected to the 5’end of the sense strand of dsRNA, and wherein L comprises any one of the following formulae:

[0014] In some embodiments, provided herein are conjugates comprising any one of the following formulae:wherein A is a therapeutic agent, and wherein P is a protein comprising one monovalent human TfR binding domain. One skilled in the art would recognize that the sulfur connection can bepart of a cysteine of P. Accordingly, in some embodiments, provided herein are conjugates comprising any one of the following formulae:wherein A is a therapeutic agent, and wherein P is a protein comprising one monovalent human TfR binding domain.

[0015] In some embodiments, the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 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.

[0016] In some embodiments, the dsRNA comprises an antisense strand complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE,BACE1, FMRI, LRRK2, HTT, S0D1, SCN10A, SCN9A or CACNA1B mRNA. In some embodiments, the dsRNA comprises an antisense strand complementary to SNCA mRNA.

[0017] Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human SNCA mRNA are provided in Table 5a. 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:(a) the sense strand comprises SEQ ID NO: 36, and the antisense strand comprises SEQ ID NO: 37;(b) the sense strand comprises SEQ ID NO: 38, and the antisense strand comprises SEQ ID NO: 37; and(c) the sense strand comprises SEQ ID NO: 39, and the antisense strand comprises SEQ ID NO: 40, 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: 36, and the antisense strand comprises SEQ ID NO: 37.

[0018] The dsRNA can include modifications. The modifications can be made to one or more nucleotides of the sense and / or antisense strand or to the internucleotide linkages. In some embodiments, one or more nucleotides of the sense strand and / or the antisense strand are independently modified nucleotides, which means 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, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-Ci6 alkyl) modified nucleotide. In some embodiments, each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, e.g., a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-Ci6 alkyl) modified nucleotide.

[0019] In some embodiments, the sense strand has four 2'-fhioro 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, theantisense 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.

[0020] 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. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 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.

[0021] In some embodiments, the 5’ end of the antisense strand has a phosphate analog, e.g., 5’-vinylphosphonate (5’-VP).

[0022] In some embodiments, the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety.

[0023] In some embodiments, the sense strand and the antisense strand have one or more modified intemucleotide linkages. In some embodiments, the modified internucleotide linkage is 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 has four or five phosphorothioate linkages. In some embodiments, the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.

[0024] Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human SNCA mRNA are provided in Table 5b.

[0025] In another aspect, provided herein are compounds comprising any one of the following formulae:wherein A is a therapeutic agent. Also provided herein are methods of generating conjugates by reacting any one of compounds of formula (X)-(XIII) with a protein comprising a cysteine.

[0026] In another aspect, provided herein are methods of treating a CNS disease, e.g., a neurodegenerative disease, in a patient in need thereof, and such the method comprises administering to the patient an effective amount of a conjugate or a pharmaceutical composition described herein.

[0027] In some embodiments, the neurodegenerative disease is Parkinson’ s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia.

[0028] The conjugate or a pharmaceutical composition can be administered to the patient intravenously or subcutaneously.

[0029] In another aspect, provided herein are conjugates described herein or pharmaceutical compositions comprising such conjugates for use in a therapy. Also providedherein are conjugates described herein or pharmaceutical compositions comprising such conjugates for use in the treatment of a neurodegenerative disease.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A shows an exemplary analytical anion exchange (aAEX) chromatogram of DAR profile for mTBPl-SMCC-dsRNA No. 4 conjugate before purification. Figure IB shows an exemplary aAEX chromatogram of DAR profile for mTBPl-SMCC-dsRNA No. 4 conjugate after purification.

[0031] Figure 2A shows an exemplary aAEX chromatogram of DAR profile for mTBPl-3’MSPT-dsRNA No. 4 conjugate before purification. Figure 2B shows an exemplary aAEX chromatogram of DAR profile for mTBPl-3’MSPT-dsRNA No. 4 conjugate after purification.

[0032] Figure 3A shows an exemplary aAEX chromatogram of DAR profile for mTBPl-3’OD-dsRNA No. 4 conjugate before purification. Figure 3B shows an exemplary aAEX chromatogram of DAR profile for mTBPl-3’OD-dsRNA No. 4 conjugate after purification.

[0033] Figure 4A shows an exemplary aAEX chromatogram of DAR profile for mTBPl-5’OD-dsRNA No. 5 conjugate before purification. Figure 4B shows an exemplary aAEX chromatogram of DAR profile for mTBPl-5’OD-dsRNA No. 5 conjugate after purification.

[0034] Figure 5A shows an exemplary aAEX chromatogram of DAR profile for mTBPl-5’MSPT-dsRNA No. 5 conjugate before purification. Figure 5B shows an exemplary aAEX chromatogram of DAR profile for mTBPl-5’MSPT-dsRNA No. 5 conjugate after purification.

[0035] Figure 6 shows conjugation kinetics across the different linkers to mTBPl in two hours at ambient temperature.

[0036] Figure 7 shows siRNA payload stability across conjugates in mouse plasma over 48 hours.

[0037] Figure 8 shows in vitro SNCA knockdown in primary mouse cortical neurons.

[0038] Figure 9A shows in vivo SNCA knockdown at 28 days following a single peripheral intravenous (IV) administration of mTBPl-linker-SNCA dsRNA at 0.25 mg / kgsiRNA dose. Figure 9B shows in vivo SNCA knockdown at 28 days following a single peripheral IV administration of mTBPl-linker-SNCA dsRNA at 4 mg / kg siRNA dose at 28 days. R values >0.0001=****; >0.001=***; >0.01=**; >0.05=*.

[0039] Figure 10A shows brain siRNA pharmacokinetics following a single peripheral IV administration of mTBPl-linker-SNCA dsRNA at 4 mg / kg siRNA dose. Figure 10B shows plasma conjugate exposure following a single peripheral IV administration of mTBPl-linker- SNCA dsRNA at 4 mg / kg siRNA dose. Figure 10C shows brain siRNA exposure following a single peripheral IV administration of mTBPl-linker-SNCA dsRNA at 0.25 mg / kg siRNA dose.Figure 10D shows plasma conjugate associated siRNA exposure across the different conjugation chemistries following a single peripheral IV administration of mTBPl-linker-SNCA dsRNA at 0.25 mg / kg siRNA dose. Figures 10E-10I show pharmacokinetic assessment of total IgG and mTBPl associated antisense strand of the indicated conjugates.

[0040] Figure 11A shows an exemplary aAEX chromatogram of DAR profile for TBP5- SMCC-dsRNA No. 4 conjugate after purification. Figure 11B shows an exemplary aAEX chromatogram of DAR profile for TBP5-3’MSPT-dsRNA No. 4 conjugate after purification.Figure 11C shows an exemplary aAEX chromatogram of DAR profile for TBP5-3’OD-dsRNA No. 4 conjugate after purification.

[0041] Figure 12 shows in vitro SNCA mRNA knockdown in SHSY-5Y cells.

[0042] Figure 13 shows in vivo SNCA mRNA knockdown in human TfR transgenic mice at 7, 28 and 84 days following a single peripheral intravenous (IV) administration of TBP- linker-SNCA dsRNA No. 4 at 1 mg / kg siRNA dose.

[0043] Figure 14A shows SNCA mRNA reduction in selected Cynomolgus monkey brain tissues one month after a single peripheral IV administration of TBP5-SMCC dsRNA No.4 at Img / kg siRNA. Figure 14B shows SNCA mRNA reductions in selected Cynomolgus monkey brain tissues one month after a single peripheral IV administration of TBP5-MSPT dsRNA No. 4 at Img / kg siRNA. Figure 14C shows SNCA mRNA reductions in selected Cynomolgus monkey brain tissues one month after a single peripheral IV administration of TBP5-OD dsRNA No. 4 at Img / kg siRNA.

[0044] Figure 15A shows plasma PK of conjugate associated antisense strand concentration following a single peripheral IV administration of TBP5-SMCC-dsRNA No. 4, TBP5-MSPT-dsRNA no. 4 and TBP5-OD-dsRNA No. 4. at 1 mg / kg siRNA in Cynomolgusmonkey. Figure 15B shows plasma PK of conjugate associated sense strand concentration following a single peripheral IV administration of TBP5-SMCC-dsRNA No. 4, TBP5-MSPT- dsRNA no. 4 and TBP5-OD-dsRNA No. 4. At 1 mg / kg siRNA in Cynomolgus monkey.

[0045] Figure 16A shows an exemplary aAEX chromatogram of DAR profile for TBP5- SMCC-dsRNA No. 8 conjugate after purification. Figure 16B shows an exemplary aAEX chromatogram of DAR profile for TBP5-3’MSPT-dsRNA No. 8 conjugate after purification. Figure 16C shows an exemplary aAEX chromatogram of DAR profile for TBP5-3’OD-dsRNA No. 8 conjugate after purification.

[0046] Figures 17 shows the in vivo efficacy of APP RNAi agents after a single IV dose of Img / kg (effective dsRNA concentration) in humanized-TfR mice after 7 days. APP mRNA knockdown for representative brain regions: Prefrontal cortex, Hippocampus, and Brain Stem by TBP5-dsRNA No. 8 are shown. One-way ANOVA with Dunnett's correction found significant APP mRNA reductions (p-values < 0.0001) relative to PBS control for all linker chemistries.

[0047] Figures 18A-18D show brain siRNA pharmacokinetics following a single peripheral IV administration of TBP5 -linker- APP dsRNA at 1 mg / kg siRNA dose. Figure 18A shows brain siRNA exposure following a single peripheral IV administration of TBP5-linker- APP dsRNA at 1 mg / kg siRNA dose. Figure 18B shows plasma exposure of total IgG and conjugate associated siRNA following a single peripheral IV administration of TBP5-SMCC- dsRNA No. 8 at 1 mg / kg siRNA dose. Figure 18C shows plasma exposure of total IgG and conjugate associated siRNA following a single peripheral IV administration of TBP5-MSPT- dsRNA No. 8 at 1 mg / kg siRNA dose. Figure 18D shows plasma exposure of total IgG and conjugate associated siRNA following a single peripheral IV administration of TBP5-OD- dsRNA No. 8 at 1 mg / kg siRNA dose.DETAILED DESCRIPTION

[0048] Provided herein are compounds and conjugates comprising transferrin receptor binding protein, pharmaceutical compositions comprising such conjugates, and methods of treating CNS diseases (e.g., neurodegenerative disease) using such conjugates.

[0049] In one aspect, provided herein are conjugates of Formula (I): (A-L)n-P, wherein A is a therapeutic agent, wherein L is a linker comprising any one of the following formulae:wherein P is a protein comprising one monovalent human TfR binding domain, and wherein n is an integer of 1 to 3. One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P. Accordingly, in some embodiments, provided herein are conjugates of Formula (I): (A-L)n-P, wherein A is a therapeutic agent, wherein L is a linker comprising any one of the following formulae:wherein P is a protein comprising one monovalent human TfR binding domain, and wherein n is an integer of 1 to 3.

[0050] The conjugates provided herein have improved plasma stability when compared to similar conjugates comprising SMCC linker.

[0051] In some embodiments, provided herein are conjugates of Formula (I): (A-L)n-P, wherein A is a therapeutic agent, wherein L is a linker comprising any one of the following formulae:wherein P is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6, and wherein n is an integer of 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. Such conjugates have improved plasma stability when compared to similar conjugates comprising SMCC linker.

[0052] One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P. Accordingly, in some embodiments, provided herein are conjugates of Formula (I): (A-L)n-P, wherein A is a therapeutic agent, wherein L is a linker comprising any one of the following formulae:wherein P is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6, and wherein n is an integer of 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. Such conjugates have improved plasma stability when compared to similar conjugates comprising SMCC linker.

[0053] In some embodiments, the therapeutic agent (A) is selected from an oligonucleotide (e.g., antisense oligonucleotide), a double stranded RNA (e.g., siRNA, saRNA), polypeptide, small molecule, nanoparticle, lipid nanoparticle, exosome, antibody or antigen binding fragment thereof, or a combination thereof. In some embodiments, the therapeutic agent is an oligonucleotide. In some embodiments, the therapeutic agent is a double stranded RNA (dsRNA). In some embodiments, A is a dsRNA comprising a sense stand and an antisense strand.

[0054] In some embodiments, L is connected to the 3’end of the sense strand of dsRNA, and wherein L comprises any one of the following formulae:One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P. Accordingly, in some embodiments, L is connected to the 3’end of the sense strand of dsRNA, and L comprises any one of the following formulae:

[0055] In some embodiments, L is connected to the 5’end of the sense strand of dsRNA, and wherein L comprises any one of the following formulae:One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P. Accordingly, in some embodiments, L is connected to the 5’end of the sense strand of dsRNA, and wherein L comprises any one of the following formulae:

[0056] In some embodiments, provided herein are conjugates comprising any one of the following formulae:wherein A is a therapeutic agent, and wherein P is a protein comprising one monovalent human TfR binding domain. One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P. Accordingly, in some embodiments, provided herein are conjugates comprising any one of the following formulae:wherein A is a therapeutic agent, and wherein P is a protein comprising one monovalent human TfR binding domain.

[0057] In some embodiments, the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 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.

[0058] In another aspect, provided herein are compounds comprising any one of the following formulae:wherein A is a therapeutic agent. Also provided herein are methods of generating conjugates by reacting any one of compounds of formula (X)-(XIII) with a protein comprising a cysteine.

[0059] In another aspect, provided herein are compounds comprising any one of the following formulae:Human TfR binding proteins

[0060] The conjugates described herein comprise a protein comprising one monovalent human TfR binding domain (“human TfR binding protein”). Human TfR binding protein can bind TfR on BBB and transport therapeutic agent into the CNS.

[0061] Exemplary sequences of human TfR binding domains and proteins are provided in Table la and lb. 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 complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, VH comprises SEQ ID NO: 7, and VL comprises SEQ ID NO: 8. In some embodiments, VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 7, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 8.Table la. Exemplary sequences of human TfR binding domains and proteinsTable lb. Exemplary sequences of human TfR binding proteins

[0062] In some embodiments, the monovalent human TfR binding domain is an antibody fragment, e.g., 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.

[0063] In some embodiments, the human TfR binding protein further comprises a halflife extender, e.g., an immunoglobulin Fc region or a VHH that binds human serum albumin (HSA).

[0064] In some embodiments, the human TfR binding protein further comprises an immunoglobulin Fc region, e.g., a modified human IgG4 Fc region, or a modified human IgGl Fc region. In some embodiments, the human TfR binding protein further comprises a modified human IgG4 Fc region comprising proline at residue 228, and alanine at residues 234 and 235 (all residues are numbered according to the EU Index numbering, also called hIgG4PAA Fc region). In some embodiments, the human TfR binding protein further comprises a modified human IgGl Fc region comprising alanine at residues 234, 235, and 329, serine at position 265, aspartic acid at position 436 (all residues are numbered according to the EU Index numbering, also called hlgGl effector null or hlgGlEN Fc region).

[0065] In some embodiments, the human TfR binding protein further comprise a VHH that binds human HSA. In some embodiments, the VHH also binds mouse, rat, and / or cynomolgus monkey albumin. An exemplary VHH that binds human HSA is shown in Table 2. In some embodiments, such a VHH comprises CDR1 comprising SEQ ID NO: 20, CDR2 comprising SEQ ID NO: 21, and CDR3 comprising SEQ ID NO: 22. In some embodiments, such a VHH comprises SEQ ID NO: 23. In some embodiments, the VHH is linked to the TfR binding domain through a peptide linker, e.g., (GGGGQ)4 (SEQ ID NO: 24). In some embodiments, the VHH is linked to the C-terminus of the TfR binding domain.Table 2. Exemplary sequences of VHH that binds human serum albumin (HSA)

[0066] In some embodiments, the human TfR binding protein is heterodimeric antibody that comprises a first arm comprising one monovalent human TfR binding domain and a second arm that is a null arm, e.g., an arm that does not bind any known human target (e.g., an isotype arm). Heterodimeric antibodies such as heteromab, orthomab or duobody have been described inWO2014150973, WO2016118742, WO2018118616, and WO2011131746. In some embodiments, the first arm comprises any monovalent human TfR binding domain described herein. In some embodiments, the second arm is a null arm that does not bind any known human target (e.g., an isotype arm) comprises the sequences in Table la. In some embodiments, the second arm comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 18, and the LC comprises SEQ ID NO: 19.

[0067] In some embodiments, the human TfR binding protein comprises heterodimeric mutations. In some embodiments, the human TfR binding protein comprises a modified Fc region comprising a first Fc CH3 domain comprising serine at residue 349, methionine at residue 366, tyrosine at residue 370, and valine at residue 409, and a second Fc CH3 domain comprising glycine at residue 356, aspartic acid at residue 357, glutamine at residue 364 and alanine at residue 407 (all residues are numbered according to the EU Index numbering). In some embodiments, the human TfR binding protein comprises a modified Fc region comprising a first Fc CH3 domain comprising leucine at residue 405, and a second Fc CH3 domain comprising arginine at residue 409 (all residues are numbered according to the EU Index numbering).

[0068] In some embodiments, the human TfR binding protein comprises one or more native cysteine residues, which can be used for conjugation. For example, in some embodiments, the human TfR binding protein comprises a native cysteine at position 220 of the light chain and / or a native cysteine at position 226 of the heavy chain, which can be used for conjugation (all residues according to the EU Index numbering).

[0069] In some embodiments, the human TfR binding protein comprises engineered cysteine residues for conjugation. The approach of including engineered cysteines as a means for conjugation has been described in WO 2018 / 232088. In some embodiments, the human TfR binding protein comprises a heavy chain comprising one or more cysteines at the following residues: 124, 157, 162, 262, 373, 375, 378, 397, 415 (all residues according to the EU Index numbering). In some embodiments, the human TfR binding protein comprises a light chain (e.g., a kappa light chain) comprising one or more cysteines at the following residues: 156, 171, 191, 193, 202, 208 (all residues according to the EU Index numbering). In some embodiments, the human TfR binding protein comprises a heavy chain constant region comprising cysteine at residue 124 (according to the EU Index numbering). In some embodiments, the human TfR binding protein comprises a light chain constant region comprising cysteine at residue 156(according to the EU Index numbering). In some embodiments, the human TfR binding protein comprises an immunoglobulin Fc region comprising cysteine at residue 378 (according to the EU Index numbering).

[0070] In some embodiments, the human TfR binding protein is any one of the human TfR binding proteins in Table lb, e.g., TBP1, TBP2, TBP3, TBP4, TBP5.

[0071] In some embodiments, the human TfR binding protein has a Fab format, e.g., TBP1. In some embodiments, the human TfR binding protein comprises one HC and one LC, and wherein the HC comprises SEQ ID NO: 9 and the LC comprises SEQ ID NO: 10.

[0072] In some embodiments, the human TfR binding protein has a Fab-VHH format, e.g., TBP2. In some embodiments, the human TfR binding proteins comprises one HC and one LC, wherein the HC comprises SEQ ID NO: 11 and the LC comprises SEQ ID NO: 12.

[0073] In some embodiments, the human TfR binding protein has a heterodimeric antibody format, e.g., TBP3. In some embodiments, the human TfR binding protein comprises two heavy chains HC1 and HC2 and two light chains LC1 and LC2, wherein HC1 comprises SEQ ID NO: 13, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 18, and LC2 comprises SEQ ID NO: 19.

[0074] In some embodiments, the human TfR binding protein has a one arm heteromab format, e.g., TBP4 or TBP5. In some embodiments, the human TfR binding protein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 14, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 15. In some embodiments, provided herein are human TfR binding proteins comprise two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 16, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 17.

[0075] The human TfR binding proteins 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 that encodes one or more signal peptides that facilitate secretion of the polypeptide(s) from a host cell. Expression vectors containing a polynucleotide of interest (e.g., a polynucleotide encoding a heavy chain or light chain of the TfR binding proteins) may be transferred into a host cell by well-known methods. Additionally, expression vectors may contain one or more selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to aide in detection of host cells transformed with the desired polynucleotide sequences.

[0076] A host cell includes cells stably or transiently transfected, transformed, transduced or infected with one or more expression vectors expressing all or a portion of the TfR binding proteins described herein. According to some embodiments, a host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing HC polypeptides and an expression vector expressing LC polypeptides of the TfR binding proteins described herein. In some embodiments, a host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing HC and LC polypeptides of the TfR binding proteins described herein. The TfR binding proteins may be produced in mammalian cells such as CHO, NSO, HEK293 or COS cells according to techniques well known in the art.

[0077] Medium, into which the TfR binding proteins has been secreted, may be purified by conventional techniques, such as mixed-mode methods of ion-exchange and hydrophobic interaction chromatography. For example, the medium may be applied to and eluted from a Protein A or G column using conventional methods; mixed- mode methods of ion-exchange and hydrophobic interaction chromatography may also be used. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. Various methods of protein purification may be employed, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).Mouse TfR binding proteins

[0078] Some conjugates used in the Examples below comprise a protein comprising one monovalent mouse TfR binding domain (“mouse TfR binding proteins” or mTBP). Exemplary sequences of mouse TfR binding proteins are provided in Table 3. Such conjugates comprising a mouse TfR binding protein can serve as surrogate molecules in mouse models. In some embodiments, the mouse TfR binding protein (e.g., mTBPl) comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 33, LC1 comprises SEQ ID NO: 34, HC2 comprises SEQ ID NO: 35.Table 3. Exemplary sequences of mouse TfR binding protein (mTBPl)Linker

[0079] In some embodiments, the therapeutic agent (A) is linked to the protein (P), e.g., human or mouse TfR binding protein (TBP), through a linker (L). In some embodiments, the linker is a SMCC linker, OD linker, or MS PT linker (structures of these linkers shown in Table 4). In some embodiments, the linker is an OD linker (e.g., an OD linker in Table 4). In some embodiments, the linker is a MSPT linker (e.g., a MSPT linker in Table 4).Table 4. Exemplary linker and conjugate structures* One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P.Therapeutic Agents

[0080] In some embodiments, the therapeutic agent (A) is selected from an oligonucleotide (e.g., antisense oligonucleotide), double stranded RNA (e.g., siRNA, saRNA), polypeptide, small molecule, nanoparticle, lipid nanoparticle, exosome, antibody or antigenbinding fragment thereof, or a combination thereof. In some embodiments, the therapeutic agent is an oligonucleotide. In some embodiments, the therapeutic agent is a double stranded RNA (dsRNA).

[0081] In some embodiments, the therapeutic agent is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand. In some embodiments, the dsRNA comprises an antisense strand complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMRI, LRRK2, HTT, SOD1, SCN10A, SCN9A or CACNA1B mRNA. In some embodiments, the dsRNA comprises an antisense strand complementary to SNCA mRNA.

[0082] In some embodiments, the sense strand and the antisense strand of the dsRNA are each 15-30 nucleotides in length, e.g., 20-25 nucleotides in length. In some embodiments, the dsRNA has a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides. In some embodiments, the sense strand and antisense strand of the dsRNA may have overhangs at either the 5’ end or the 3’ end (i.e., 5’ overhang or 3’ overhang). For example, the sense strand and the antisense strand may have 5’ or 3’ overhangs of 1 to 5 nucleotides or 1 to 3 nucleotides. In some embodiments, the antisense strand comprises a 3’ overhang of two nucleotides.

[0083] Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human SNCA mRNA are provided in Table 5a.

[0084] Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human APP mRNA are provided in Table 5c.Table 5a. Unmodified Nucleic Acid Sequences of dsRNA targeting human SNCA mRNA (SNCA siRNA)Table 5c. Unmodified Nucleic Acid Sequences of dsRNA targeting human APP mRNA (APP siRNA)

[0085] 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:(a) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 36, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 37;(b) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 38, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 37; and(c) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 39, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 40, 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.

[0086] 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:(a) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 36, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 37;(b) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 38, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 37; and(c) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 39, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 40, 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.

[0087] 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:(a) the sense strand comprises SEQ ID NO: 36, and the antisense strand comprises SEQ ID NO: 37;(b) the sense strand comprises SEQ ID NO: 38, and the antisense strand comprises SEQ ID NO: 37; and(c) the sense strand comprises SEQ ID NO: 39, and the antisense strand comprises SEQ ID NO: 40, 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.

[0088] In some embodiments, the dsRNA targets APP mRNA. In some embodiments, the sense strand comprises SEQ ID NO: 53, and the antisense strand comprises SEQ ID NO: 54, 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.

[0089] The dsRNA can include modifications. The modifications can be made to one or more nucleotides of the sense and / or antisense strand or to the internucleotide linkages, which are the bonds between two nucleotides in the sense or antisense strand. For example, some 2’- modifications of ribose or deoxyribose can increase RNA or DNA stability and half-life. Such2’ -modifications can be 2’-fluoro, 2’-O-methyl (i.e., 2’-methoxy), or 2'-O-alkyl (e.g., 2’-O-Ci6 alkyl).

[0090] In some embodiments, one or more nucleotides of the sense strand and / or the antisense strand are independently modified nucleotides, which means 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, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-Ci6 alkyl) modified nucleotide. In some embodiments, each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, e.g., a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-Ci6 alkyl) modified nucleotide.

[0091] 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.

[0092] In some embodiments, the sense strand has three 2'-fhioro 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'-fhioro 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. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 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.

[0093] In some embodiments, the 5’ end of the antisense strand has a phosphate analog, e.g., 5’-vinylphosphonate (5’-VP).

[0094] In some embodiments, the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety, e.g., a moiety shown in Table 6.Table 6. Abasic or inverted abasic (iAb) moieties“5”’ and “3”’ indicate the 5’ to 3’ direction of the sequences.

[0095] In some embodiments, the sense strand and the antisense strand have one or more modified intemucleotide linkages. In some embodiments, the modified internucleotide linkage is 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 has four or five phosphorothioate linkages. In some embodiments, the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.

[0096] Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human SNCA mRNA are provided in Table 5b.

[0097] Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human APP mRNA are provided in Table 5d.

[0098] In some embodiments, the dsRNA comprises a sense strand that comprises a sequence that has 1, 2, or 3 differences from a sense stand sequence in Table 5a, 5b, 5c, 5d. In some embodiments, the dsRNA comprises an antisense strand that comprises a sequence that has 1, 2, or 3 differences from an antisense stand sequence in Table 5a, 5b, 5c, 5d.Table 5b: Modified Nucleic Acid Sequences of dsRNA targeting human SNCA mRNA (SNCA siRNA)Table 5d: Modified Nucleic Acid Sequences of dsRNA targeting human APP mRNA (APP siRNA)Note - The 5’ end of the AS may be substituted with 5’-vinylphosphonate.Abbreviations - “m” indicates 2’-0Me; “f” indicated 2’-fluoro; indicates phosphorothioate linkage;“VP” indicates 5’-vinylphosphonate; “Nth” indicates a 5 ’-amino group; “iAb” indicated an inverted abasic group; “S” means the sense strand; “AS” means the antisense strand.

[0099] 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:(a) the sense strand comprises SEQ ID NO: 41, and the antisense strand comprises SEQ ID NO: 42;(b) the sense strand comprises SEQ ID NO: 43, and the antisense strand comprises SEQ ID NO: 42; and(c) the sense strand comprises SEQ ID NO:44, and the antisense strand comprises 45. [000100] 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:(a) the sense strand consists of SEQ ID NO: 41, and the antisense strand consists of SEQ ID NO: 42;(b) the sense strand consists of SEQ ID NO: 43, and the antisense strand consists of SEQ ID NO: 42; and(c) the sense strand consists of SEQ ID NO: 44, and the antisense strand consists of SEQ ID NO: 45.[000101] In some embodiments, the dsRNA targets APP mRNA. In some embodiments, the sense strand comprises SEQ ID NO: 55, and the antisense strand comprises SEQ ID NO: 56. In some embodiments, the sense strand consists of SEQ ID NO: 55, and the antisense strand consists of SEQ ID NO: 56.[000102] The sense strand and antisense strand of dsRNA can be synthesized using any nucleic acid polymerization methods known in the art, for example, solid-phase synthesis by employing phosphoramidite chemistry methodology (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), H- phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, for example, MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems. Phosphorothioate linkages can be introduced using a sulfurizing reagent such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene) amino)-3H-l,2,4-dithiazaoline-3-thione). It is well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products to synthesize modified oligonucleotides or conjugated oligonucleotides.[000103] Purification methods can be used to exclude the unwanted impurities from the final oligonucleotide product. Commonly used purification techniques for single stranded oligonucleotides include reverse-phase ion pair high performance liquid chromatography (RP-IP-HPLC), capillary gel electrophoresis (CGE), anion exchange HPLC (AX-HPLC), and size exclusion chromatography (SEC). After purification, oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm. The sense strand and antisense strand can then be annealed to form a dsRNA.Pharmaceutical Composition[000104] In another aspect, provided herein are pharmaceutical compositions comprising any of the conjugates described herein and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can also comprise one or more pharmaceutically acceptable excipient, diluent, or carrier. Pharmaceutical compositions can 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).Method of Treatment and Therapeutic Use[000105] In another aspect, provided herein are methods of treating a CNS disease, e.g., a neurodegenerative disease, in a patient in need thereof, and such the method comprises administering to the patient an effective amount of the conjugate or a pharmaceutical composition described herein.[000106] In a further aspect, provided herein are methods of treating a neurodegenerative disease in a patient in need thereof, and such the method comprises administering to the patient an effective amount of the conjugate or a pharmaceutical composition described herein, e.g., a TBP-SNCA siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-SNCA siRNA conjugate. Exemplary neurodegenerative disease includes, but are not limited to, Parkinson’s disease; multiple system atrophy; Lewy body dementia or dementia with Lewy bodies; pure autonomic failure; Alzheimer’s disease; Lewy body dysphagia; and incidental Lewy body disease. In some embodiments, the neurodegenerative disease is selected from Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia. The human TfR binding protein conjugate or a pharmaceutical composition can be administered to the patient intravenously or subcutaneously.[000107] Human TfR binding protein conjugate dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolusmay be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. [000108] Dosage values may vary with the type and severity of the condition to be alleviated. It is 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.[000109] In another aspect, provided herein are human TfR binding protein conjugates described herein or pharmaceutical compositions comprising such conjugates for use in a therapy. Also provided herein are human TfR binding protein conjugates described herein or pharmaceutical compositions comprising such conjugates for use in the treatment of a neurodegenerative disease.Definitions[000110] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.[000111] As used herein, the term “alkyl” means saturated linear or branched-chain monovalent hydrocarbon radical, containing the indicated number of carbon atoms. For example, “C1-C20 alkyl” means a radical having 1-20 carbon atoms in a linear or branched arrangement.[000112] The term “antibody,” as used herein, refers to a molecule that binds an antigen. Embodiments of an antibody include a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, heterodimeric antibody, bispecific or multispecific antibody, or conjugated antibody. The antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA), and any subclass (e.g., IgGl, IgG2, IgG3, IgG4).[000113] An immunoglobulin G (IgG) type antibody comprised of four polypeptide chains: two heavy chains (HC) and two light chains (LC) that are cross-linked via inter-chain 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 primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is comprised of a lightchain variable region (VL) and a light chain constant region. The IgG isotype may be further divided into subclasses (e.g., IgGl, IgG2, IgG3, and IgG4).[000114] The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). The CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxylterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3” and the three CDRs of the light chain are referred to as “ECDR1, ECDR2 and ECDR3”. The CDRs contain most of the residues that form specific interactions with the antigen. Assignment of amino acid residues to the CDRs may be done according to the 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-Eazikani 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 Eoop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available on at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212).[000115] Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments that, as used herein, comprise at least a portion of an antibody retaining the ability to specifically interact with an antigen or an epitope of the antigen, such as Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fvs (sdFv), a Fd fragment.[000116] The term “antigen binding domain”, as used herein, refers to a portion of an antibody or antibody fragment that binds an antigen or an epitope of the antigen. For example, “TfR binding domain” refers to a portion of an antibody or antibody fragment that binds TfR or an epitope of TfR.[000117] The term “heterodimeric antibody”, as used herein, refers to an antibody that comprises two distinct antigen-binding domains.[000118] 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 an antisense strand. [000119] The terms “bind” and “binds” as used herein are intended to mean, unless indicated otherwise, the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in proximity of the two proteins or molecules as determined by common methods known in the art.[000120] As used herein, “complementary” means a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand, e.g., a hairpin) that permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. Complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. Likewise, two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity, as described herein.[000121] As used herein, “duplex,” in reference to nucleic acids or oligonucleotides, means a structure formed through complementary base pairing of two antiparallel sequences of nucleotides (i.e., in opposite directions), whether formed by two separate nucleic acid strands or by a single, folded strand (e.g., via a hairpin).[000122] An “effective amount” refers to an amount necessary (for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount of a compound, protein or conjugate may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound, protein or conjugate to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the compound, protein or conjugate are outweighed by the therapeutically beneficial effects.[000123] The term “Fc region” as used herein refers to a polypeptide comprising the CH2 and CH3 domains of a constant region of an immunoglobulin, e.g., IgGl, IgG2, IgG3, or IgG4. Optionally, the Fc region may include a portion of the hinge region or the entire hinge region of an immunoglobulin, e.g., IgGl, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is ahuman IgG Fc region, e.g., a human IgGl Fc region, human IgG2 Fc region, human IgG3 Fc region or human IgG4 Fc region. In some embodiments, the Fc region is a modified IgG Fc region with reduced or eliminated effector functions compared to the corresponding wild type IgG Fc region. The numbering of the residues 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: U.S. Dept, of Health and Human Services, Public Health Service, National Institutes of Health, 1991). The boundaries of the Fc region of an immunoglobulin heavy chain might vary, and the human IgG heavy chain Fc region is usually defined as the stretch from the N-terminus of the CH2 domain (e.g., the amino acid residue at position 231 according to the EU index numbering) to the C-terminus of the CH3 domain (or the C-terminus of the immunoglobulin) .[000124] The term “knockdown” or “expression knockdown” refers to reduced mRNA or protein expression of a gene after treatment of a reagent.[000125] As used herein, “modified internucleotide linkage” means an internucleotide linkage having one or more chemical modifications when compared with a reference intemucleotide linkage having a phosphodiester bond. A modified intemucleotide linkage can be a non-naturally occurring linkage. In some embodiments, the modified intemucleotide linkage is phosphorothioate linkage.[000126] As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide can have, for example, one or more chemical modification in its sugar, nucleobase, and / or phosphate group. Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-Ci6 alkyl) modified nucleotide. In some embodiments, the modified nucleotide has a phosphate analog, e.g., 5’-vinylphosphonate. In some embodiments, the modified nucleotide has an abasic moiety or inverted abasic moiety, e.g., a moiety shown in Table 6.[000127] As used herein, “nucleotide” means an organic compound having a nucleoside (a nucleobase, e.g., adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar, e.g., ribose or 2'-deoxyribose) linked to a phosphate group. A “nucleotide” can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).[000128] As used herein, a “null arm” means an antibody arm that does not bind any known human target.[000129] As used herein, “oligonucleotide” means a polymer of linked nucleotides, each of which can be modified or unmodified. An oligonucleotide is typically less than about 100 nucleotides in length. An oligonucleotide can be single stranded or double stranded.[000130] As used herein, “overhang” means the unpaired nucleotide or nucleotides that protrude from the duplex structure of a double stranded oligonucleotide. An overhang may include one or more unpaired nucleotides extending from a duplex region at the 5’ terminus or 3’ terminus of a double stranded oligonucleotide. The overhang can be a 3’ or 5’ overhang on the antisense strand or sense strand of a double stranded oligonucleotide.[000131] The term “patient”, as used herein, refers to a human patient.[000132] As used herein, “phosphate analog” means a chemical moiety that mimics 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 in place of a 5’- phosphate, which is often susceptible to enzymatic removal. A 5’ phosphate analog can include a phosphatase-resistant linkage. Examples of phosphate analogs include 5’ methylene phosphonate (5’-MP) and 5’-(E)-vinylphosphonate (5’-VP). In some embodiments, the phosphate analog is 5’ -VP.[000133] The term “% sequence identity” or “percentage 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 with the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp. 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. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percentage of “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the nucleic acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleotide, nucleoside, or nucleobase occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. The output is the percent identity of the subject sequence with respect to the query sequence.[000134] The term “polypeptide” or “protein”, as used herein, refers to a polymer of amino acid residues. The term applies to polymers comprising naturally occurring amino acids and polymers comprising one or more non-naturally occurring amino acids.[000135] As used herein, “strand” refers to a single, contiguous sequence of nucleotides linked together through intemucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have two free ends (e.g., a 5’ end and a 3’ end).[000136] As used herein, “SNCA” refers to an alpha- sy nuclein (SNCA) mRNA, protein, or polypeptide. The nucleic acid sequence of a human SNCA mRNA transcript can be found atNM-000345.4:1 GGCGACGACC AGAAGGGGCC CAAGAGAGGG GGCGAGCGAC CGAGCGCCGC GACGCGGAAG 61 TGAGGTGCGT GCGGGCTGCA GCGCAGACCC CGGCCCGGCC CCTCCGAGAG CGTCCTGGGC 121 GCTCCCTCAC GCCTTGCCTT CAAGCCTTCT GCCTTTCCAC CCTCGTGAGC GGAGAACTGG 181 GAGTGGCCAT TCGACGACAG TGTGGTGTAA AGGAATTCAT TAGCCATGGA TGTATTCATG 241 AAAGGACTTT CAAAGGCCAA GGAGGGAGTT GTGGCTGCTG CTGAGAAAAC CAAACAGGGT 301 GTGGCAGAAG CAGCAGGAAA GACAAAAGAG GGTGTTCTCT ATGTAGGCTC CAAAACCAAG 3 1 GAGGGAGTGG TGCATGGTGT GGCAACAGTG GCTGAGAAGA CCAAAGAGCA AGTGACAAAT 421 GTTGGAGGAG CAGTGGTGAC GGGTGTGACA GCAGTAGCCC AGAAGACAGT GGAGGGAGCA 481 GGGAGCATTG CAGCAGCCAC TGGCTTTGTC AAAAAGGACC AGTTGGGCAA GAATGAAGAA 541 GGAGCCCCAC AGGAAGGAAT TCTGGAAGAT ATGCCTGTGG ATCCTGACAA TGAGGCTTAT 601 GAAATGCCTT CTGAGGAAGG GTATCAAGAC TACGAACCTG AAGCCTAAGA AATATCTTTG 661 CTCCCAGTTT CTTGAGATCT GCTGACAGAT GTTCCATCCT GTACAAGTGC TCAGTTCCAA 721 TGTGCCCAGT CATGACATTT CTCAAAGTTT TTACAGTGTA TCTCGAAGTC TTCCATCAGC 781 AGTGATTGAA GTATCTGTAC CTGCCCCCAC TCAGCATTTC GGTGCTTCCC TTTCACTGAA 841 GTGAATACAT GGTAGCAGGG TCTTTGTGTG CTGTGGATTT TGTGGCTTCA ATCTACGATG 901 TTAAAACAAA TTAAAAACAC CTAAGTGACT ACCACTTATT TCTAAATCCT CACTATTTTT961 TTGTTGCTGT TGTTCAGAAG TTGTTAGTGA TTTGCTATCA TAT ATT AT AA GATTTTTAGG 1021 TGTCTTTTAA TGATACTGTC TA AG A AT A AT GACGTATTGT GAAATTTGTT AATATATATA 1081 ATACTTAAAA ATATGTGAGC ATGAAACTAT GCACCTATAA ATACTAAATA TGAAATTTTA 1141 CCATTTTGCG ATGTGTTTTA TTCACTTGTG TTTGTATATA AATGGTGAGA ATTAAAATAA 1201 AACGTTATCT CATTGCAAAA ATATTTTATT TTTATCCCAT CTCACTTTAA TAATAAAAAT 1261 CATGCTTATA AGCAACATGA ATTAAGAACT GACACAAAGG ACAAAAATAT AAAGTTATTA 1321 ATAGCCATTT GAAGAAGGAG GAATTTTAGA AGAGGTAGAG AAAATGGAAC ATTAACCCTA 1381 CACTCGGAAT TCCCTGAAGC AACACTGCCA GAAGTGTGTT TTGGTATGCA CTGGTTCCTT 1441 AAGTGGCTGT GATTAATTAT TGAAAGTGGG GTGTTGAAGA CCCCAACTAC TATTGTAGAG 1501 TGGTCTATTT CTCCCTTCAA TCCTGTCAAT GTTTGCTTTA CGTATTTTGG GGAACTGTTG 1561 TTTGATGTGT ATGTGTTTAT AATTGTTATA CATTTTTAAT TGAGCCTTTT ATTAACATAT 1621 ATTGTTATTT TTGTCTCGAA ATAATTTTTT AGTTAAAATC TATTTTGTCT GATATTGGTG 1681 TGAATGCTGT ACCTTTCTGA CAATAAATAA TATTCGACCA TGAATAAAAA AAAAAAAAAA 1741 GTGGGTTCCC GGGAACTAAG CAGTGTAGAA GATGATTTTG ACTACACCCT CCTTAGAGAG 1801 CCATAAGACA CATTAGCACA TATTAGCACA TTCAAGGCTC TGAGAGAATG TGGTTAACTT 1861 TGTTTAACTC AGCATTCCTC ACTTTTTTTT TTTAATCATC AGAAATTCTC TCTCTCTCTC 1921 TCTCTTTTTC TCTCGCTCTC TTTTTTTTTT TTTTTTTACA GGAAATGCCT TTAAACATCG 1981 TTGGAACTAC CAGAGTCACC TTAAAGGAGA TCAATTCTCT AGACTGATAA AAATTTCATG 2041 GCCTCCTTTA AATGTTGCCA AATATATGAA TTCTAGGATT TTTCCTTAGG AAAGGTTTTT 2101 CTCTTTCAGG GAAGATCTAT TAACTCCCCA TGGGTGCTGA AAATAAACTT GATGGTGAAA 2161 AACTCTGTAT AAATTAATTT AAAAATTATT TGGTTTCTCT TTTTAATTAT TCTGGGGCAT 2221 AGTCATTTCT AAAAGTCACT AGTAGAAAGT ATAATTTCAA G AC AG A AT AT TCTAGACATG 2281 CTAGCAGTTT ATATGTATTC ATGAGTAATG TGATATATAT TGGGCGCTGG TGAGGAAGGA 2341 AGGAGGAATG AGTGACTATA AGGATGGTTA CCATAGAAAC TTCCTTTTTT ACCTAATTGA 2401 AG AG AG ACTA CTACAGAGTG CTAAGCTGCA TGTGTCATCT TACACTAGAG AGAAATGGTA 2461 AGTTTCTTGT TTTATTTAAG TTATGTTTAA GCAAGGAAAG GATTTGTTAT TGAACAGTAT 2521 ATTTCAGGAA GGTTAGAAAG TGGCGGTTAG GATATATTTT AAATCTACCT AAAGCAGCAT 2581 ATTTTAAAAA TTTAAAAGTA TTGGTATTAA ATTAAGAAAT AGAGGACAGA ACTAGACTGA 2641 TAGCAGTGAC CT AG A AC A AT TTGAGATTAG GAAAGTTGTG ACCATGAATT TAAGGATTTA 2701 TGTGGATACA AATTCTCCTT TAAAGTGTTT CTTCCCTTAA TATTTATCTG ACGGTAATTT 2761 TTGAGCAGTG AATTACTTTA TATATCTTAA TAGTTTATTT GGGACCAAAC ACTTAAACAA 2821 AAAGTTCTTT AAGTCATATA AGCCTTTTCA GGAAGCTTGT CTCATATTCA CTCCCGAGAC 2881 ATTCACCTGC CAAGTGGCCT GAGGATCAAT CCAGTCCTAG GTTTATTTTG CAGACTTACA 2941 TTCTCCCAAG TTATTCAGCC TCATATGACT CCACGGTCGG CTTTACCAAA ACAGTTCAGA 3001 GTGCACTTTG GCACACAATT GGGAACAGAA CAATCTAATG TGTGGTTTGG TATTCCAAGT 3061 GGGGTCTTTT TCAGAATCTC TGCACTAGTG TGAGATGCAA ACATGTTTCC TCATCTTTCT 3121 GGCTTATCCA GTATGTAGCT ATTTGTGACA TA ATA A AT AT ATACATATAT GAAAATA(SEQ ID NO: 46).The amino acid sequence of a human SNCA protein can be found at NP_000336.1:1 MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK 61 EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP121 DNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 47).[000137] The nucleic acid sequence of a mouse SNCA mRNA transcript can be found atNM_001042451.2; and the amino acid sequence of a mouse SNCA protein can be found atNP_001035916.1. The nucleic acid sequence of a rat SNCA mRNA transcript can be found atNM_019169.3; and the amino acid sequence of a rat SNCA protein can be found atNP_062042.1. The nucleic acid sequence of a monkey SNCA mRNA transcript can be found atXM_005555422.2; and the amino acid sequence of a monkey SNCA protein can be found atXP_005555479.1.[000138] As used herein, “TfR” refers to a transferrin receptor protein or polypeptide, e.g., a human or mouse transferrin receptor protein or polypeptide. The amino acid sequence of the human transferrin receptor protein (hTFR) can be found at NP_001121620.1 :1 MMDQARSAFS NLFGGEPLSY TRFSLARQVD GDNSHVEMKL AVDEEENADN NTKANVTKPK61 RCSGSICYGT IAVIVFFLIG FMIGYLGYCK GVEPKTECER LAGTESPVRE EPGEDFPAAR121 RLYWDDLKRK LSEKLDSTDF TGTIKLLNEN SYVPREAGSQ KDENLALYVE NQFREFKLSK181 VWRDQHFVKI QVKDSAQNSV IIVDKNGRLV YLVENPGGYV AYSKAATVTG KLVHANFGTK241 KDFEDLYTPV NGSIVIVRAG KITFAEKVAN AESLNAIGVL IYMDQTKFPI VNAELSFFGH301 AHLGTGDPYT PGFPSFNHTQ FPPSRSSGLP NIPVQTI SRA AAEKLFGNME GDCPSDWKTD361 STCRMVTSES KNVKLTVSNV LKEIKILNIF GVIKGFVEPD HYVWGAQRD AWGPGAAKSG421 VGTALLLKLA QMFSDMVLKD GFQPSRSI IF ASWSAGDFGS VGATEWLEGY LSSLHLKAFT481 YINLDKAVLG TSNFKVSASP LLYTLIEKTM QNVKHPVTGQ FLYQDSNWAS KVEKLTLDNA541 AFPFLAYSGI PAVSFCFCED TDYPYLGTTM DTYKELIERI PELNKVARAA AEVAGQFVIK601 LTHDVELNLD YERYNSQLLS FVRDLNQYRA DIKEMGLSLQ WLYSARGDFF RATSRLTTDF661 GNAEKTDRFV MKKLNDRVMR VEYHFLSPYV SPKESPFRHV FWGSGSHTLP ALLENLKLRK721 QNNGAFNETL FRNQLALATW TIQGAANALS GDVWDIDNEF (SEQ ID NO: 48).The amino acid sequence of the mouse transferrin receptor protein (mTFR) can be found atNP-001344227.1:1 MMDQARSAFS NLFGGEPLSY TRFSLARQVD GDNSHVEMKL AADEEENADN NMKASVRKPK61 RFNGRLCFAA IALVIFFLIG FMSGYLGYCK RVEQKEECVK LAETEETDKS ETMETEDVPT121 SSRLYWADLK TLLSEKLNSI EFADTIKQLS QNTYTPREAG SQKDESLAYY IENQFHEFKF181 SKVWRDEHYV KIQVKSSIGQ NMVTIVQSNG NLDPVESPEG YVAFSKPTEV SGKLVHANFG241 TKKDFEELSY SVNGSLVIVR AGEITFAEKV ANAQSFNAIG VLIYMDKNKF PWEADLALF301 GHAHLGTGDP YTPGFPSFNH TQFPPSQSSG LPNIPVQTIS RAAAEKLFGK MEGSCPARWN361 IDSSCKLELS QNQNVKLIVK NVLKERRILN IFGVIKGYEE PDRYVWGAQ RDALGAGVAA421 KSSVGTGLLL KLAQVFSDMI SKDGFRPSRS I IFASWTAGD FGAVGATEWL EGYLSSLHLK481 AFTYINLDKV VLGTSNFKVS ASPLLYTLMG KIMQDVKHPV DGKSLYRDSN WI SKVEKLSF 541 DNAAYPFLAY SGIPAVSFCF CEDADYPYLG TRLDTYEALT QKVPQLNQMV RTAAEVAGQL601 I IKLTHDVEL NLDYEMYNSK LLSFMKDLNQ FKTDIRDMGL SLQWLYSARG DYFRATSRLT661 TDFHNAEKTN RFVMREINDR IMKVEYHFLS PYVSPRESPF RHIFWGSGSH TLSALVENLK721 LRQKNITAFN ETLFRNQLAL ATWTIQGVAN ALSGDIWNID NEF (SEQ ID NO: 49).[000139] As used herein, “treatment” or “treating” refers to all processes wherein there may be a slowing, controlling, delaying, or stopping of the progression of the disorders or disease disclosed herein, or ameliorating disorder or disease symptoms, but does not necessarily indicate a total elimination of all disorder or disease symptoms. Treatment includes administration of a protein or nucleic acid or vector or composition for treatment of a disease or condition in a patient, particularly in a human.[000140] The following examples are offered to illustrate, but not to limit, the claimed inventions.EXAMPLESExample 1: Generation and Characterization of TfR binding proteinsGeneration of human or mouse TfR binding proteins[000141] Antibody against mouse TfR was 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: 50, see Table 7). mTfR antigen positive B-cells were sorted from peripheral blood and binding of individual antibodies cloned from those B-cells was verified on his-tagged mTfR.[000142] Antibody against human TfR was generated by immunizing AlivaMab® transgenic mice with the extracellular domains of human Transferrin Receptor 1 protein with a His tag (hTfR-ECD-6His, SEQ ID NO: 51, see Table 7) and mouse Transferrin Receptor protein (mTfR, SEQ ID NO: 49). Antigen positive B-cells were sorted from pooled spleens. Binding of individual antibodies cloned from those B-cells to his-tagged hTfR-ECD was verified.[000143] Additional antibody against human TfR was generated by immunizing AlivaMab® transgenic mice with the apical domain of human Transferrin Receptor 1 protein with a His tag (hTfR-ApD-6His, SEQ ID NO: 52, see Table 7). Antigen positive B-cells were sorted from pooled spleens. Binding of individual antibodies cloned from those B-cells to his- tagged hTfR-ECD was verified.Table 7. Sequences of the immunogens used to generate human or mouse TfR antibodies.[000144] Affinity variants of the generated human or mouse TfR antibodies were made by systematically introducing mutations into individual CDR of each antibody and the resulting variants were subjected to multiple rounds of selection with decreasing concentrations of antigen and / or increasing periods of dissociation to isolate clones with improved affinities. The sequences of individual variants were used to construct a combinatorial library which was subjected to an additional round of selection with increased stringency to identify additive or synergistic mutational pairings between the individual CDR regions. Individual combinatorial clones are sequenced. The heavy chain and light chain CDRs and VH / VL sequences of the human TfR binding domains are provided in Table la and Table lb. The heavy chain and light chain CDRs and VH / VL sequences of the mouse TfR binding protein (mTBPl) are provided in Table 3.[000145] Human or mouse TfR binding proteins were generated by recombinant DNA technology. Such TfR binding proteins can be expressed in a mammalian cell line such as HEK293 or CHO, either transiently or stably transfected with an expression system using an optimal predetermined HC:LC vector ratio or a single vector system encoding both HC and LC. Clarified media, into which the protein has been secreted, can be purified using the commonly used techniques.Binding affinity[000146] Binding affinity and binding stoichiometry of the exemplified human TfR binding proteins to human and cynomolgus TfR was characterized using a surface plasmon resonance assay on a Biacore 8K instrument primed with HBS-EP+ (lOmM Hepes pH7.4 + 150mM NaCl + 3mM EDTA + 0.05% (w / v) surfactant P20) running buffer and analysis temperature set at 37 °C. Target human and cynomologus TfR ECD’s were immobilized on a CM4 chip (Cytiva P / N 29104989) using standard NHS-EDC amine coupling. The TfR binding proteins were prepared at a final concentration of 0.3, 0.1, 0.033, 0.01, 0.0033, 0.001, 0.00033, 0.0001 pM respectively by dilution of stock solution into running buffer.[000147] Binding analysis was performed in a multi-cycle kinetics manner. Each analysis cycle consists of (1) injection of the lowest to highest concentration proteins over all Fc at 50 pL / min for 140 seconds followed by return to buffer flow for 400 seconds to monitor dissociation phase; (2) regeneration of chip surfaces with injection of 3M magnesium chloride, for 30 seconds at 100 pL / min over all cells; and (3) equilibration of chip surfaces with a 50 pL (30-sec) injection of HBS-EP+. Data were processed using standard double-referencing and fit to a 2-state binding model using Biacore 8K Evaluation software, to determine the association rate (kon, M-1s_1units), dissociation rate (kOff, s'1units), and Rmax (RU units). The equilibrium dissociation constant (KD) is calculated from the relationship KD = koff / kon, and is in molar units. Results are provided in Table 8.Table 8. Binding Affinity of Exemplified human TfR binding proteins to human or cynomolgus TfR at 37 °C[000148] The binding affinity and binding stoichiometry of the exemplified mouse TfR binding proteins to mouse TFR was determined using a surface plasmon resonance assay on a Biacore T200 instrument primed with HBS-EP+ (10 mM Hepes pH7.4 + 150 mM NaCl + 3 mM EDTA + 0.05% (w / v) surfactant P20) running buffer and analysis temperature set at 25°C. A human Fab capture kit (Cytiva P / N 28958325) was immobilized on a CM5 chip (Cytiva P / N 29104988) using standard NHS-EDC amine coupling on all four flow cells (Fc). Mouse TfR binding proteins were prepared at 10 pg / mL by dilution into running buffer. Target (mouse TFR-mlgGl-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.[000149] Each analysis cycle consists of (1) capturing antibody samples on separate flow cells (Fc2, Fc3 and Fc4); (2) injection of the respective concentration of TfR over all Fc at 100 pL / min for 60 seconds followed by return to buffer flow for 1800 seconds to monitor dissociation phase; (3) regeneration of chip surfaces with injection of 10 mM glycine, pH 1.5, for 30 seconds at 10 pL / min over all cells; and (4) equilibration of chip surfaces with a 10 pL (60- sec) injection of HBS-EP+. Data were processed using standard double-referencing and fit to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0.3, to determine the association rate (kon, M'1s'1units), dissociation rate (koff, s'1units), and Rmax (RU units). The equilibrium dissociation constant (KD) is calculated from the relationship KD = k0ff / k0n, and is in molar units. Results are provided in Table 9.Table 9: Binding Affinity of Exemplified mTfR Binding Proteins to mouse TFR at 25°C.Example 2: Synthesis and characterization of dsRNAs targeting SNCA[000150] Single strands (sense and antisense) of the dsRNA duplexes were synthesized on solid support via a MerMade™ 12 (LGC Biosearch Technologies). The sequences of the sense and antisense strands were shown in Table 5a, 5b, 5c, 5d. Sense strands conjugated via the 3’- terminus were synthesized using phthalamido amino C6 Icaa CPG 500 A (Chemgenes). Sense strands conjugated via the 5’-terminus were synthesized using a standard support (LGC Biosearch Technologies) and the Preparation 11 amidite for the final coupling. The antisense strands were synthesized using standard support (LGC Biosearch Technologies). The oligonucleotides were synthesized via phosphoramidite chemistry at either 5, 10, or 50 pmol scales.[000151] Standard reagents were used in the oligo synthesis (Table 10), where 0. IM xanthane hydride in pyridine was used as the sulfurization reagent and 20% DEA in ACN was used as an auxiliary wash post synthesis. All monomers (Table 11) were made at 0.1M in ACN and contained a molecular sieves trap bag.[000152] The oligonucleotides were cleaved and deprotected (C / D) at 45 °C for 20 hours. The sense strands were C / D from the CPG using cold 50% (methylamine / ammonia hydroxide 28-30%) at RT for 3 hrs, whereas 3% DEA in ammonia hydroxide (28-30%, cold) was used for the antisense strands. C / D was determined complete by IP-RP LCMS when the resulting mass data confirmed the identity of sequence. Dependent on scale, the CPG was filtered via 0.45 um PVDF syringeless filter, 0.22 um PVDF Steriflip® vacuum filtration or 0.22 um PVDF Stericup® Quick release. The CPG was back washed / rinsed with either 30% EtOH / RNAse free water then filtered through the same filtering device and combined with the first filtrate. This was repeated twice. The material was then divided evenly into 50 mL falcon tubes to remove organics via Genevac™. After concentration, the crude oligonucleotides were diluted back tosynthesized scale with RNAse free water and filtered either by 0.45 m PVDF syringeless filter, 0.22 |am PVDF Steriflip® vacuum filtration or 0.22 pm PVDF Stericup® Quick release.[000153] The crude oligonucleotides were purified via AKTA™ Pure purification system using anion-exchange (AEX). For AEX, an ES Industry Source™ 15Q column maintaining column temperature at 65 °C with MPA: 20mM NaH PCA, 15% ACN, pH 7.4 and MPB: 20 mM NaH2PO4, IM NaBr, 15% ACN, pH 7.4. Fractions which contained a mass purity greater than 85% without impurities >5% where combined.[000154] The purified oligonucleotides were desalted using 15 mL 3K MWCO centrifugal spin tubes at 35OO.yg for ~30 min. The oligonucleotides were rinsed with RNAse free water until the eluent conductivity reached < 100 usemi / cm. After desalting was complete, 2-3 mL of RNAse free water was added then aspirated lOx, the retainment was transferred to a 50 mL falcon tube, this was repeated until complete transfer of oligo by measuring concentration of compound on filter via nanodrop. The final oligonucleotide was then nano filtered 2x via 15 mL 100K MWCO centrifugal spin tubes at 35OO.vg for 2 min. The final desalted oligonucleotides were analyzed for concentration (nano drop at A260), characterized by IP-RP LC / MS for mass purity and UPLC for UV-purity.Table 10 Oligonucleotide Synthesis ReagentsTable 11 PhosphoramiditesExample 3: Generation of conjugates comprising TfR binding proteinsCertain abbreviations are defined as follows: “ACN” refers to acetonitrile; “AS” refers to antisense strand; “C / D” refers to cleaved and deprotected; “DAR” refers to drug / siRNA to antibody / protein ratio; “DCM” refers to dichloromethane; “DEA” refers to diethylamine; “DIAD” refers to diisopropyl azodicarboxylate; “DIEA” refers to N,N-diisopropylethylamine; “DMT” refers to dimethoxy trityl; “dsRNA” refers to double stranded ribonucleic acid; “EDCI” refers to l-ethyl-3-(3-dimethylaminopropyl)carbodiimide; “EtOAc” refers to ethyl acetate; “EtOH” refers to ethanol and ethyl alcohol; “h” refers to hours; “IP-RP” refers to ion-pair reverse phase; “LC / MS” refers to liquid chromatography mass spectrometry; “LTQ / MS” refers to linear ion trap mass spectrometer; “MeOH” refers to methanol and methyl alcohol; “min” refers to minutes; “MW” refers to molecular weight; “NHS” refers to N-hydroxy succinimide; “OD” refers to optical density; “PBS” phosphate-buffered saline; “rpm” refers to revolutions per minute; “siRNA” refers to small interfering RNA; “SMCC” refers to succinimidyl-4-(N- maleimidomethyl)cyclohexane- 1 -carboxylate; “SS” refers to sense strand; “TEA” refers to triethylamine; “TFA” refers to trifluoroacetic acid; “THF” refers to tetrahydrofuran; “UPLC” refers to ultra-performance liquid chromatography; and “UV” refers to ultraviolet.Scheme 1Q is 1,3,4-oxadiazole or 1 H-tetrazole.Scheme 1, step A depicts the methylation of the thiol on compound (1) using iodomethane and a suitable base such as DIEA in a solvent such as THF to give compound (2). Step B shows an alkylation of compound (2) with tert-butyl 2-(2-(2-bromoethoxy)ethoxy)acetate using a base such as potassium carbonate in a solvent such as acetone to give compound (3). Step C shows the oxidation of compound (3) with hydrogen peroxide and ammonium molybdate (VI) tetrahydrate in a solvent such as EtOH followed by an acidic deprotection using an acid such as TFA in a solvent such as DCM to give compound (4). Note that in the case of the 1H- tetrazole, the deprotection took place during the oxidation step. Step D depicts a coupling of compound (4) and 1 -hydroxypyrrolidine-2, 5-dione using EDCI in a solvent system such as DCM and THF to give compound (5).Scheme 2Scheme 2, step A shows the coupling of compound (6) and isoindoline- 1,3-dione usingDIAD and tributyl phosphine in a solvent such as THF to give compound (7). Step B depicts thephosphorylation of compound (7) with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite using a base such as DIEA in a solvent such as DCM to give compound (8).Preparation 14-(5-(Methylthio)-l,3,4-oxadiazol-2-yl)phenolA solution of 4-(5-mercapto-l,3,4-oxadiazol-2-yl)phenol (3.00 g, 15.4 mmol) in THF (50 mL) was cooled to 0 °C. DIEA (3.46 mL, 20.1 mmol) was added then stirred for 5 minutes before adding iodomethane (2.85 g, 20.1 mmol) dropwise over a period of 1 minute. The mixture was stirred at 0 °C for 5 minutes, and then stirred at ambient temperature for 2 hours. After this time, the mixture was diluted with DCM (100 mL) and washed with saturated aqueous NH4CI (pH was adjusted to ~5 by adding citric acid solution, 2 x 50 mL). The organic layer was separated, dried over sodium sulfate, and concentrated in vacuo to give the title compound as a pale-yellow solid (520 mg, 97%). ES / MS m / z: 209 (M+H).The compound in Table 12 was prepared in a manner essentially analogous to that found in Preparation 1.Table 12Preparation 3 tert-Butyl 2-(2-(2-(4-(5-(methylthio)- 1 ,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetateIn a pressure vessel, tert-butyl 2-(2-(2-bromoethoxy)ethoxy)acetate (5.5 g, 20 mmol) and potassium carbonate (4.2 g, 30 mmol) were added to 4-(5-(methylthio)-l,3,4-oxadiazol-2- yl)phenol (3.3 g, 15 mmol) in acetone (60 mL). The pressure vessel was sealed and heated at 70 °C for 5 hours with vigorous stirring. After this time, the mixture was cooled to ambient temperature. The mixture was filtered while washing through with EtOAc / DCM. The filtrate was concentrated in vacuo and purified via silica gel column chromatography eluting with 0- 100% EtOAc / DCM to give the title compound as a white solid (4.8 g, 74%). ES / MS m / z: 411 (M+H).The compound in Table 13 was prepared in a manner essentially analogous to that found in Preparation 3.Table 13Preparation 5 tert-Butyl 2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetatetert-Butyl 2-(2-(2-(4-(5-(methylthio)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate (5.20 g, 12.7 mmol) was dissolved in EtOH (100 mL) and cooled to 5-10 °C. Then, 30% hydrogen peroxide (10 mL, 97 mmol) was added, followed by ammonium molybdate (VI) tetrahydrate (501 mg, 0.405 mmol). After two hours of vigorous stirring, additional 30% hydrogen peroxide (15 mL, 145.5 mmol) and ammonium molybdate (VI) tetrahydrate (1 g, 0.910 mmol) were added. The mixture was stirred for 6 hours, then diluted with DCM (150 mL) and washed with saturated aqueous sodium chloride solution. The organic phase was separated, dried over sodium sulfate, and concentrated in vacuo. The resulting residue was triturated with MeOH to provide the first lot of the title compound. The solvent from the mother liquor was concentrated in vacuo and purified via silica gel column chromatography eluting with 0-100% EtOAc / DCM to give additional product as white solid. The recovered materials were combined to give the title compound as a white solid (5.3 g, 90%). ES / MS m / z: 387 (M+H-tBu).The compound in Table 14 was prepared in a manner essentially analogous to that found in Preparation 5.Table 14Note that the conditions were analogous, but the t-butyl group was removed in the process.Preparation 72-(2-(2-(4-(5-( Methyl sulfonyl)- 1.3.4-oxadiazol-2-yl) hcnoxy)cthoxyjcihoxy)acctic acidTFA (20 mL, 12.0 mmol) was added to a solution of tert-butyl 2-(2-(2-(4-(5- (methylsulfonyl)-l,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (5.60 g, 12.0 mmol) in DCM (60 mL). The mixture was stirred at ambient temperature for 2 hours, concentrated in vacuo, and purified via silica gel column chromatography eluting with 0-100% EtOAc / DCM to give the title compound (4.12g, 82%). ES / MS m / z 387 (M+H).Preparation 8 2,5-Dioxopyrrolidin-l-yl-2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetateEDCI (1.60 g, 10.3 mmol) was added to a solution of 2-(2-(2-(4-(5-(methylsulfonyl)- l,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetic acid (3.00 g, 7.38 mmol) and 1- hydroxypyrrolidine-2, 5-dione (1.19 g, 10.3 mmol) in DCM (50 mL) and THF (70 mL). Another 20 mL of DCM was added to bring the mixture into a solution followed by stirring at ambient temperature for 12 hours. After this time, concentrated in vacuo and purified via silica gel column chromatography eluting with 0-100% EtOAc / DCM to give the title compound (2.61g, 65%). ES / MS m / z: 484 (M+H).The compound in Table 15 was prepared in a manner essentially analogous to that found in Preparation 8.Table 15Preparation 10 2-(((2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3-hydroxy-4- methoxytetrahydrofuran-2-yl)methyl)isoindoline- 1 ,3-dioneA solution of l-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (30 g, 120 mmol), isoindoline- 1,3- dione (21 g, 140 mmol), DIAD (27 mL, 140 mmol), tributyl phosphine (36 mL, 150 mmol), and THF (300 mL) was stirred at ambient temperature for 12 h. The crude reaction was filtered, concentrated in vacuo, and purified via silica gel flash chromatography eluting with 0-100% EtOAc / hexanes to give the title compound as a white solid (6.0 g, 13%).Preparation 112-Cyanoethyl ((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-2-((l,3- dioxoisoindolin-2-yl)methyl)-4-methoxytetrahydrofuran-3-yl) diisopropylphosphoramiditeA solution of 2-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3- hydroxy-4-methoxytetrahydrofuran-2-yl)methyl)isoindoline-l, 3-dione (3.00 g, 7.74 mmol), 2- cyanoethyl-N,N-diisopropylchlorophosphoramidite (2.47 mL, 11.6 mmol), DIEA (4.05 mL, 23.2 mmol), and DCM (40 mL) was stirred at ambient temperature. After 1 hour, additional 2- cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.82 mL, 3.8 mmol) was added. After 1 hour, the crude reaction was poured into a slurry of silica gel (15 g) in 30 mL of 1% TEA / DCM, concentrated in vacuo to a dry powder, and purified via silica gel flash chromatography eluting with 40-100% EtOAc / hexanes (0.5% TEA) to give the title compound as a white foam (3.70 g,11.4 (br s, 1 H), 7.96-7.78 (m, 5H), 5.83 (dd, lH), 5.71 (dd, 1H), 4.46-3.47 (m, 9H), 3.39 (s, 1.5H), 3.35 (s, 1.5H), 2.82-2.73 (m, 2H), 1.16-0.97 (m, 12H).31P NMR (d6-DMSO) d 149.7, 149.4.Preparation 123’ Oxadiazole linker-functionalized sense strandA 40 mL Falcon tube was charged with SNCA-SS-6aM (7.66 mg, 3.00 mL, 1.07 pmol) and 3 mL of PBS 7.4 (lOx). 2,5-Dioxopyrrolidin-l-yl-2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4- oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (10.4 mg, 1.07 mL, 21.4 pmol) and ACN (1 mL) were added. The mixture was vortexed for 3 minutes, then shook at 900rpm at ambient temperature for another hour. After this time, the mixture was concentrated in vacuo then desalted using a 3K spin filter (Fisher biologies, 4500rpm, 3x 1 hour ). The OD measurement of the product solution (average of 3 measurements, 20x dilution) was 6.42 OD / mL, concentration = 628 mol / L, 4.72 mg / mL, total 1.65 mL, 7.80 mg.The compound in Table 16 below was prepared in a manner essentially analogous to that found in Preparation 12.Table 16*12.85 OD / mL, conccntration=626 umol / L, 4.71 mg / mL, total 1.65 mL, 7.80 mgPreparation 145’ Oxadiazole linker-functionalized sense strandA sense strand (0.0013 mmol in 0.470 mL water) synthesized using conditions found in the protocols below was added to 20X borate buffer (0.071 mL), then was treated with a solution of 2,5-dioxopyrrolidin-l-yl-2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate (0.0064 g, 0.0131 mmol) in MeCN (0.470 mL). The solution was shaken for 30 mins at 40 °C. The solution was then diluted to 20 mL using RNAse free water to bring concentration of organic solvent to < 10%. Excess NHS ester was removed using 20 mL 3K MWCO centrifugal spin tubes at 3500x for ~30 minutes. The oligonucleotides were rinsed with RNAse free water three times. After removing NHS ester, 1 mL of RNAse free water was added then aspirated lOx and the retentate was transferred to a 5 mL falcon tube. This was repeated until complete transfer of oligo by measuring concentration of compound on filter via nanodrop. The final oligonucleotide was analyzed for concentration (nano drop at A260),characterized by IP-RP, LCMS for mass purity, and UPLC for UV-purity. ES / MS (m / z): 7324.03 (M+H).The compound in Table 17 below was prepared in a manner essentially analogous to that found in Preparation 14.Table 17Linker-functionalization of SNCA dsRNAA freshly prepared solution of (2,5-dioxopyrrolidin-l-yl) 4-[(2,5-dioxopyrrol-l- 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 pM (-8.8 mg / mL)) in 0.2M phosphate buffer (44 mL). Vortexed vigorously for 2 minutes, and then shook at ambient temperature at 900 rpm for 2 h total. Analysis by LTQ showed about 94-95% conversion.Acidified to pH~4 with 20-30 drops of 5N HC1, and then removed organics in a Genevac concentrator. Desalted by centrifugal filtration on a 3K spin filter (4 x 4000 rpm, 30 min), and pooled the retentates. The OD measurement of the solution (average of 3 measurements, lOx dilution) was 266 equating to 1.3mM and a total of 316 mg. Extinction coefficient was 204.12. LTQ / MS m / z 7358.Linker-SNCA DuplexThe nanodrop concentrations of aqueous solutions of each strand (average of 3x) were measure as SS = 1322pM and AS = 1108pM. The sense strand and antisense strand are annealed to form a dsRNA. 32 mL of SS and 36.2 mL of AS are mixed and shook for 30 min at 30 °C. The amount of residual SS strand was measured until completion and required adding an additional 360 p L of AS. Removed endotoxins by filtering through a 0.45 pM filter. The resulting 75 mL ofsolution measured (Nanodrop™ Lite, 5x average, lOx dilution) 217 OD / mL equating to 575p M and a total of 653 mg. LTQ / MS m / z 7358,7825; UV purity 99+%.Conjugation ofdsRNA to TfR binding proteins[000155] Site-specific native or engineered cysteine amino acid residues in the TfR binding proteins were used to conjugate dsRNA. Cysteines can be engineered into the primary amino acid sequence of the TfR binding proteins. The approach of introducing cysteines as a means for conjugation has been described in WO 2018 / 232088, which is both incorporated by reference in its entirety and incorporated specifically in relation to conjugation via cysteine residues. For engineered cysteine conjugation, the TfR binding proteins were first reduced with 40 molar equivalents reducing agent dithiothreitol (DTT) at 37 °C for two hours, followed by desalting to remove reducing agent via dialysis or desalting columns. This is followed by re-oxidation of the TfR binding protein to reform the structural disulfides with 10 molar equivalent dehydroascorbic acid (DHAA) incubation at ambient temperature for two hours. A follow up desalting was performed to remove oxidizing agent.[000156] Conjugation of dsRNA onto TfR binding proteins were done using the following methods.Conjugation Scheme 1[000157] The first conjugation method utilized the 3’SS oxadiazole (OD) -functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding proteins. For this method, TfR binding protein was prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding proteins. This is followed by incubating the OD-dsRNA with the TfR binding proteins at 1.2 to 2 molar equivalents for overnight conjugation at ambient temperature.TfR binding protein conjugation with 3’ OD linkerConjugation Scheme 2[000158] The second conjugation method utilized the 5’SS oxadiazole (OD) -functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding proteins. For this method, TfR binding protein was prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding proteins. This is followed by incubating the OD-dsRNA with the TfR binding proteins at 1.2 to 2 molar equivalents for overnight conjugation at ambient temperature.TfR binding protein conjugation with 5’ OD linkerConjugation Scheme 3[000159] The third conjugation method utilized the 3’SS tetrazole (MSPT) -functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding proteins. For this method, TfR binding protein was prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding proteins. This is followed by incubating the MSPT-dsRNA with the TfR binding proteins at 1.2 to 2 molar equivalents for overnight conjugation at ambient temperature.TfR binding protein conjugation with 3’ MSPT linkerConjugation Scheme 4[000160] The fourth conjugation method utilized the 5’SS tetrazole (MSPT) -functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding proteins. For this method, TfR binding protein was prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding proteins. This is followed by incubating the MSPT-dsRNA with the TfR binding proteins at 1.2 to 2 molar equivalents for overnight conjugation at ambient temperature.TfR binding protein conjugation with 5’ MSPT linkerConjugation Scheme 5[000161] The fifth conjugation method utilized the SMCC-functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding proteins. For this method, TfR binding protein was prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding proteins. This is followed by incubating the SMCC-dsRNA with the TfR binding proteins at 1.2 to 2 molar equivalents for overnight conjugation at ambient temperature.[000162] Optionally, following conjugation, a maleimide hydrolysis step can be done to secure the linker-payload in terminal stage and avoid deconjugation during human body circulation via retro-Michael addition. This succinimide ring hydrolysis process was done by elevating the conjugate pH to 9.0 using 50mM Arginine (stock solution of 0.7M arginine, pH 9.0 was used) and incubating the solution at 37 °C for 20 hours. The hydrolysis state of the maleimide was confirmed by LCMS characterization of +18Da that is incurred by the water addition to the succinimide ring.TfR binding protein conjugation with SMCC linkerTfR binding protein conjugation with SMCC linker ring opening[000163] Conjugation was monitored using analytical anion exchange chromatography. A ProPac™ SAX-10 HPLC Column, 10pm particle, 4mm diameter, 250mm length was utilized with the following method. Flow rate of 1 mL / min, Buffer A: 20mM TRIS pH 7.0, Buffer B: 20 mM TRIS pH 7.0 + 1.5M NaCl, at ambient temperature.Table 18: HPLC gradient used to assess dsRNA conjugation to TfR binding protein[000164] Drug / siRNA to antibody / protein ratio (DAR) was calculated based on peak area % from the analytical anion exchange (aAEX) chromatogram. See Figures 1A, 2A, 3B, 4A, 5A. [000165] Post conjugation of dsRNA to the TfR binding protein, excess dsRNA and unconjugated protein was removed by further purification. Either preparative size exclusion chromatography (SEC) or preparative anion exchange chromatography was utilized for purification of the final conjugate. Preparative SEC was performed using Cytiva Superdex® 200 in IX PBS pH 7.2 under an isocratic condition. Alternatively, anion exchange, e.g., ThermoFisher POROS™ XQ, was used with starting buffer of 20mM TRIS pH 7.0 and eluting with 20 column volume gradient with a buffer containing 20mM TRIS pH 7.0 and IM NaCl. These resulted in purified TfR binding protein-dsRNA conjugate devoid of excess dsRNA and minimal unconjugated protein. The resulting conjugate profile was analyzed by analytical anion exchange for final DAR quantitation (see Figures IB, 2B, 3B, 4B, 5B; and Table 19).Table 19. siRNA / drug to TBP / antibody ratio (DAR)Conjugation kinetics[000166] Conjugation of SMCC, 3’OD, 5’ OD, 3’ MSPT, 5’ MSPT linker functionalized dsRNA to the engineered thiol on mTBPl was monitored by aAEX at 2 hours at ambient temperature at varying pH of 7.2 or 8.2. The results are shown in Figure 6.Example 4: In vitro Potency Assessment in Mouse Cortical Neurons[000167] Mouse primary cortical neurons were isolated from wild type C57BL6 mouse embryos at E18. Cells were plated in poly-D-lysine coated 96-well plates at a density of 40,000 cells / well and cultured in NbActivl (BrainBits, LLC) containing 1% Antibiotic / Antimycotic (Coming) for 7 days at 37°C in a tissue culture incubator in a humidified chamber with 5% CO2. On day 7, half of the medium was removed from each well and 2x concentration of mTBPl - SNCA siRNA (mTBPl -dsRNA conjugate, dsRNA linked to HC1 of mTBPl), in culture media with 2% FBS was added for treatment and incubated with cells for additional 7 days. At the end of treatment, RT-qPCR was performed to quantify targeted mRNA levels using TaqMan Fast Advanced Cell-to-CT kit. Specifically, cells were lysed, cDNA was generated on Mastercycler X50a (Eppendorf), and qPCR was carried out on QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Gene expression levels of the SNCA were normalized by [Lactin using respective probes (ThermoFisher).[000168] Results are provided in Figure 8 and Table 20. Results provided in Table 20 demonstrate the exemplified mouse TfR binding protein-siRNA conjugates (e.g., mTfRl-dsRNA conjugate) successfully knock down mouse SNCA.Table 20: In vitro potency of the indicated molecules for reducing mouse SNCA mRNA in mouse cortical neuronsExample 5: Ex vivo Plasma Stability[000169] The TBP-linker-dsRNA conjugates were subjected to ex vivo plasma stability assessment to evaluate stability of the conjugates and any dissociation of the siRNA from the TBP. The conjugates were incubated in mouse plasma at 37 °C at 0, 24 and 48 hours respectively with rotation at 5rpm. TBP was immunoprecipitated from the plasma sample using biotinylated goat anti-human IgG. Solution was then incubated with streptavidin beads at ambient temperature with rotation for 30 minutes. Following multiple washing step with IX PBS, the sample was eluted with 1% formic acid with 20% acetonitrile elution buffer by mixing at 2000rpm for 15 seconds followed by 5-minute static benchtop hold. The eluted sample was then injected into LC-MS for analysis.[000170] LCMS method:• Instrument: Sciex I.• Column: Agilent, PLRP-S 1000A 5pM 50x1.0 MM, PN: PL1312-1502.• Injection Volume: 20 pL.• Column Temperature: 80 °C.• Auto sampler Temperature: 5 °C.• Mobile Phase A: water with 0.05% TFA.• Mobile Phase B: acetonitrile with 0.05% TFA.• Gradient:• MS: m / z 2000-5000[000171] The results are shown in Figure 7, which shows the TBP-dsRNA conjugates with the 3’OD, 5’ OD, 3’ MSPT, 5’ MSPT linkers have increased plasma stability when compared to the TBP-dsRNA linker with the SMCC linker.Example 6: In vivo characterization of the mouse TfR binding proteins-dsRNA conjugates in the CNS with peripheral delivery[000172] To determine the efficacy of the mouse TfR binding protein-dsRNA conjugates with SMCC, OD and MSPT linkers, a study was performed with a single intravenous (IV) dose of 0.25 mg / kg or 4 mg / kg siRNA dose respectively and compared against the PBS control group (n=5 per group). 28 days following initial dosing, mice were perfused under anesthesia and sacrificed, then hemibrain was collected and processed for assessment of SNCA gene expression changes.[000173] As shown in Figures 9A and 9B, RT-qPCR data demonstrated significant reduction of SNCA in the hemibrain in both 0.25 mg / kg and 4 mg / kg dose groups, with comparable efficacy across SMCC, OD and MSPT linker chemistries.Example 7. Exposure response relationship characterization of the mouse TfR binding protein-dsRNA conjugates[000174] To understand exposure response relationships for the study described above, plasma pharmacokinetics (PK) and biodistribution of siRNA following a single IV dose, plasma samples from the above-mentioned study were collected and the exposure of the conjugate associated siRNA in plasma or the total siRNA in tissue was quantified by HP-LC / MS. Briefly,Liquid chromatography / mass spectrometry (LC / MS) was used to measure conjugate associated or total siRNA levels in mouse plasma and tissue samples. Plasma standards were prepared by adding in control mouse plasma. Tissue standards were prepared in control tissue homogenate. To control assay variability, an internal standard was added to all standards and samples. [000175] For conjugate associated siRNA, plasma standards and samples were incubated with a biotinylated polyclonal Goat Anti-Human IgG antibody (Southern Biotech, Birmingham, AL) followed by a second incubation with streptavidin beads (Promega, Madison, WI). The IgG-siRNA-streptavidin bead complex was isolated on a magnetic separator and the supernatant was discarded. Samples and standards were washed with phosphate buffered saline solution followed by conjugate-associated siRNA elution from the beads with triethylamine. The standards and samples were injected onto an LC / MS system.[000176] Tissue samples were homogenized in cell lysis buffer. For total siRNA measurements, tissue standards and samples were digested with proteinase K prior to being loaded onto an Oasis Wax micro-elution solid phase extraction (SPE) plate (Waters Inc, Milford, MA) for isolation. The SPE plate was washed with wash buffers and then analytes were eluted with elution buffer. Eluants from the SPE plates were dried, reconstituted, and injected onto an LC / MS system.[000177] The conjugate associated siRNA or total siRNA were 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). [000178] To understand the impact of linker chemistry on the plasma PK and biodistribution of siRNA following a single IV dose, mTBPl-linker-SNCA dsRNA conjugates were dosed in mice at 0.25 mg / kg or 4 mg / kg, and plasma samples were collected and the exposure of the conjugate-associated siRNA was quantified by HP-LC / MS at various times post dose through 1 month.[000179] Figure 10A shows brain tissue concentrations of total siRNA in mice over the course of 1 month following a single peripheral IV administration of mTBPl-linker-SNCA dsRNA at 4 mg / kg siRNA dose. Figure 10B shows plasma conjugate exposure from the same study.[000180] At 24 hours following a single peripheral IV administration of mTBPl -linker- SNCA dsRNA at 0.25 mg / kg siRNA dose, mTBPl-SMCC-SNCA dsRNA conjugate showed -20-30% lower AUC, compared to the OD and MSPT linker conjugates, mostly due to lower Cmax (Figure 10C). Figure 10D shows plasma conjugate associated siRNA exposure across the different conjugation chemistries from the same study.[000181] Total mTBPl (TIGG) and mTBPl associated siRNA (AAAS) exposure in plasma are shown in Figures 10E- 101, demonstrate conjugate-associated antisense strand exposure in the mTBPl-3’OD-dsRNA, mTBPl-5’OD-dsRNA, mTBPl-3’MSPT-dsRNA and mTBPl-5’MSPT- dsRNA conjugates is improved compared to the mTBPl-SMCC -dsRNA conjugates.Example 8: Generation of conjugates comprising TfR binding proteins and SNCA dsRNA [000182] In order to assess performance of the linker associated conjugates in humanized transferrin transgenic mice and non-human primates, TBP5-dsRNA No. 4 conjugates were generated using the conjugation schemes mentioned above. Conjugation scheme 1, conjugation scheme 3 and conjugation scheme 5 were used to generate TBP5-3’OD-dsRNA No. 4, TBP5- 3’MSPT-dsRNA No. 4 and TBP5-SMCC-dsRNA No. 4 conjugates respectively.[000183] Post conjugation of dsRNA to the TfR binding protein, excess dsRNA and unconjugated protein was removed by purification by method mentioned earlier. Drug / siRNA to antibody / protein ratio (DAR) of the final purified conjugate was calculated based on peak area % from the analytical anion exchange (aAEX) chromatogram and shown in Figure 11A-11C and Table 21.Table 21. siRNA / drug to TBP / antibody ratio (DAR)Example 9: Long-term high-temperature stability assessment of TBP-dsRNA conjugates [000184] Long-term high-temperature stability of the TBP-dsRNA conjugates were assessed to determine differences between SMCC, MSPT and OD linkers. TBP5-SMCC-dsRNA No. 4, TBP5-3’MSPT-dsRNA No. 4 and TBP5-3’OD-dsRNA No. 4 conjugates were analyzed for DAR profile by analytical anion exchange method as described earlier to get a baseline profile. Then, the conjugates were subjected to 35 °C incubation for 1 -month period as a stress test. The control samples were held at 4 °C during this time period. Following 1 -month incubation, samples were subjected to DAR profile analysis by analytical anion exchange. [000185] The difference in DAR loss compared to 1stday, over this one-month period is shown in Table 22 below. TBP5-SMCC-dsRNA showed a loss of 4% DARI at 35 °C held for a month, compared to 0.73% and 1.81% for TBP5-MSPT-dsRNA and TBP5-OD-dsRNA respectively.Table 22. High temperature DAR stability profile of TBP-dsRNA conjugatesExample 10: In vitro characterization of the TBP-SNCA dsRNA in SH-SY5Y cells [000186] In vitro SNCA knockdown of TBP5-dsRNA No. 4 conjugates were assessed in SH-SH5Y cells. SH-SY5Y cells (ATCC CRL-2266) were derived from the SK-N-SH neuroblastoma cell line (Ross, R. A., et al., 1983. J Natl Cancer Inst 71, 741-747). The base medium was composed of a 1:1 mixture of ATCC-formulated Eagle's Minimum EssentialMedium, (Cat No. 30-2003), and F12 Medium. The complete growth medium was supplemented with 10% fetal bovine serum, IX amino acids, IX sodium bicarbonate, and IX penicillinstreptomycin (Gibco) and cells incubated at 37 °C in a humidified atmosphere of 5% CO2. On Day One, SH-SY5Y cells were plated in 96 well fibronectin coated tissue culture plates and allowed to attach overnight. On Day Two, complete media was removed and replaced with RNAi agent in serum free media. Cells were incubated with RNAi agent for 72 hours, followed by media change with RNAi reagent for another 72 hours for a total of 144 hours of drug incubation before analysis of gene expression. Analysis of changes in gene expression in RNAi treated SH-SY5Y cells was measured using Cells-to-Cr Kits following the manufacturer’s protocol (ThermoFisher A35377). Predesigned gene expression assays (supplied as 20X mixtures) were selected from Applied Bio-systems (Foster City, CA, USA). The efficiencies of these assays (ThermoFisher Hs00240907_ml SNCA and Thermo Fisher Hs99999903_gl ACTB) were characterized with a dilution series of cDNA. RT-QPCR was performed in MicroAmp Optical 384-well reaction plates using QuantStudio 7 Flex system. The delta-delta CT method of normalizing to the housekeeping gene ACTB was used to determine relative amounts of gene expression. GraphPad Prism v9.0 was used to determine IC50 with a four parameter logistic fit. Results are shown in Figure 12 and Table 22.Table 22: In vitro activity of SNCA RNAi agentsExample 11: In vivo characterization of the human TfR binding proteins-SNCA dsRNA conjugates in the CNS of hTfR transgenic mice with peripheral delivery[000187] To determine the efficacy of the human TfR binding protein-dsRNA conjugates with SMCC, OD and MSPT linkers, a study was performed in human TfR transgenic knock-in mice, where the extracellular domain of transferrin-receptor has been humanized, with a single intravenous (IV) dose of 1 mg / kg siRNA dose and compared against the PBS control group (n=4 per group). 7, 28 and 84 days following initial dosing, mice were perfused under anesthesia and sacrificed, then hemibrain was collected and processed for assessment of SNCA gene expression changes.[000188] As shown in Figures 13 and Table 23, RT-qPCR data demonstrated significant reduction of SNCA in the hemibrain in all groups at 7, 28 and 84 days, with comparable efficacy across conjugates comprising SMCC, OD and MSPT linker chemistries.Table 23: In vivo activity of TBP-SNCA dsRNA conjugates in hTfR miceExample 12: In vivo characterization of the human TfR binding proteins-SNCA dsRNA conjugates in Non-human primate (NHP) with peripheral delivery[000189] Following demonstration of central efficacy with peripheral siRNA delivery in human TfR transgenic mice by human TfR binding proteins-SNCA siRNA conjugates, a 1- month efficacy with TBP-SNCA siRNA conjugates was conducted in NHP. Pharmacodynamic properties of human TfR binding protein-siRNA conjugates were assessed in NHPs according to the following. Cynomolgus monkeys (Macaca fascicularis) weighing 2-3 kg were dosed once intravenously in the Saphenous vein in the thigh with i) PBS (n=4), ii) TBP5-SMCC-dsRNA No. 4 (N=4) at 1 mg / kg effective siRNA concentration, iii) TBP5-MSPT-dsRNA No. 4 at Img / kg (N=4 each) effective siRNA, and iv) TBP5-OD-dsRNA No. 4 at Img / kg (N=4 each) effective siRNA and sacrificed 29 days after the first dose. For takedowns, deeply anesthetizedanimals underwent cardiac perfusion, then brain tissues were collected and processed for RT- qPCR in tissue homogenates.[000190] RT-qPCR data showed robust reduction of SNCA mRNA ranging from 40-70% in all key brain regions at 1 mg / kg siRNA dose demonstrating high efficacy of the TBP5-dsRNA conjugates (Figures 14A-14C).Example 13. Exposure response relationship characterization of the TBP binding protein- dsRNA conjugates in NHP[000191] To understand exposure response relationships for the studies described in Example 12 above, plasma pharmacokinetics (PK) and biodistribution of siRNA following a single IV dose, plasma samples from the above-mentioned study were collected and the exposure of the conjugate associated siRNA in plasma was quantified by HR-LC / MS (Figures 15A and 15B). Briefly, liquid chromatography / mass spectrometry (LC / MS) was used to measure conjugate associated or total siRNA levels in Cynomolgus plasma. Plasma standards were prepared by adding in control monkey plasma. To control assay variability, an internal standard was added to all standards and samples.[000192] For conjugate associated siRNA, plasma standards and samples were incubated with a biotinylated polyclonal Goat Anti-Human IgG antibody (Southern Biotech, Birmingham, AE) followed by a second incubation with streptavidin beads (Promega, Madison, WI). The IgG-siRNA-streptavidin bead complex was isolated on a magnetic separator and the supernatant was discarded. Samples and standards were washed with phosphate buffered saline solution followed by conjugate-associated siRNA elution from the beads with triethylamine. The standards and samples were injected onto an EC / MS system.[000193] The conjugate associated siRNA or total siRNA were 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). [000194] As shown in Figures 15A-15B, for the given dose, the plasma exposure across different linker chemistry was similar as seen based on antisense strand (Figure 15 A), as well as TBP-associated sense strand (Figure 15B).Example 14: Generation of conjugates comprising TfR binding proteins and APP dsRNA [000195] To assess performance of the linker associated conjugates in another target gene, TBP5-dsRNA conjugates targeting APP was generated. These conjugates were generated using the conjugation schemes mentioned earlier. Conjugation scheme 1, conjugation scheme 3 and conjugation scheme 5 were used to generate TBP5-OD-dsRNA No. 8, TBP5-MSPT-dsRNA No. 8 and TBP5-SMCC-dsRNA No. 8 conjugates respectively.[000196] Post conjugation of dsRNA to the TfR binding protein, excess dsRNA and unconjugated protein was removed by purification method mentioned earlier. Drug / siRNA to antibody / protein ratio (DAR) of the final purified conjugate was calculated based on peak area % from the analytical anion exchange (aAEX) chromatogram and shown in Figures 16A-16C and Table 24.Table 24. siRNA / drug to TBP / antibody ratio (DAR)Example 15. In vivo characterization of the human TfR binding proteins-dsRNA conjugates in the CNS of hTfR transgenic mice with peripheral delivery[000197] To determine the efficacy of the human TfR binding protein-dsRNA conjugates with SMCC, OD and MSPT linkers on another target gene, a study was performed in human TfR transgenic mice with a single intravenous (IV) dose of siRNA and compared against the PBS control group (n=3-5 per group). Specifically, human TfR transgenic knock-in mice where the extracellular domain of transferrin-receptor has been humanized, received a single 1 mg / kg (dsRNA) IV dose of human TfR binding proteins-dsRNA targeting APP conjugates: TBP5- SMCC-dsRNA No. 8, TBP5-MSPT-dsRNA No. 8, TBP5-OD-dsRNA No. 8, or a PBS (phosphate buffered saline) control. Animals were sacrificed 7 days after injection and brain samples were collected to assess pharmacodynamic efficacy. Mouse APP mRNA expression in brain were measured and analyzed by qPCR, APP probe (Mm00431829_ml). The delta-delta CTmethod of normalizing used include housekeeping genes, P-actin and GAPDH probes (Mm02619580_gl and Mm99999915_gl, respectively).[000198] Figure 17 demonstrates that TBP5-dsRNA No. 8 conjugates significantly (p-value <0.0001) reduced mouse APP mRNA levels by 70%, 69% and 67% in the brain (prefrontal cortex) with comparable efficacy across SMCC, MSPT, and OD linker chemistries. Significant reductions in mRNA levels were also observed for hippocampus and brain stem as well for all linker chemistries.Example 16. Exposure response relationship characterization of the IBP binding protein- dsRNA conjugates in hTfR transgenic mice[000199] To understand exposure response relationships for the study described above in Example 15, plasma pharmacokinetics (PK) and biodistribution of siRNA following a single IV dose, plasma samples from the study were collected and the exposure of the conjugate associated siRNA in plasma or the total siRNA in tissue was quantified by HR-LC / MS utilizing the method previously mentioned in Example 7.[000200] To understand the impact of linker chemistry on the plasma PK and biodistribution of siRNA following a single IV dose, TBP5 -linker- APP dsRNA conjugates were dosed in hTfR transgenic mice at 1 mg / kg, and plasma samples were collected and the exposure of the conjugate-associated siRNA was quantified by HR-LC / MS at various times post dose through 1 week.[000201] Figure 18A shows brain tissue concentrations of total siRNA in mice over the course of 1 week following a single peripheral IV administration of TBP5 -linker- APP dsRNA at 1 mg / kg siRNA dose. Figures 18B-18D show plasma conjugate exposure from the same study.[000202] Total TBP5 (TIGG) and TBP5 associated siRNA (AAAS) exposure in plasma are shown in Figures 18B-18D. TBP5-MSPT-dsRNA No. 8 and TBP5-OD-dsRNA No. 8 conjugates demonstrated improved conjugate-associated antisense strand exposure compared to the TBP5- SMCC-dsRNA No. 8 conjugate.SEQUENCE LISTING

Claims

CLAIMS1. A conjugate of Formula (I): (A-L)n-P, wherein A is a therapeutic agent, wherein L is a linker comprising any one of the following formulae:wherein P is a protein comprising one monovalent human TfR binding domain, wherein the human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6, and wherein n is an integer of 1 to 3.

2. The conjugate of claim 1, wherein A is an oligonucleotide.

3. The conjugate of claim 1 or 2, wherein A is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand.

4. The conjugate of claim 3, wherein L is connected to the 3’end of the sense strand, and wherein L comprises any one of the following formulae:The conjugate of claim 3, wherein L is connected to the 5’end of the sense strand, and wherein L comprises any one of the following formulae:

6. The conjugate of any one of claims 1-5, wherein n is 1.

7. The conjugate of any one of claims 1-5, wherein n is 2.

8. The conjugate of any one of claims 1-6, wherein the conjugate comprises any one of the following formulae:

9. The conjugate of any one of claims 1-8, wherein VH comprises SEQ ID NO: 7 and VL comprises SEQ ID NO: 8.

10. The conjugate of any one of claims 1-9, wherein the human TfR binding domain is a Fab, scFv, Fv, or scFab.

11. The conjugate of any one of claims 1-10, wherein the human TfR binding domain further comprises a heavy chain constant region comprising cysteine at residue 124 (according to the EU Index numbering).

12. The conjugate of any one of claims 1-11, wherein P further comprises a half-life extender.

13. The conjugate of claim 12, wherein the half-life extender is selected from an immunoglobulin Fc region or a VHH that binds human serum albumin (HSA).

14. The conjugate of claim 13, wherein the half-life extender is an immunoglobulin Fc region.

15. The conjugate of claim 14, wherein the immunoglobulin Fc region is a modified human IgG4 Fc region.

16. The conjugate of claim 15, wherein the modified human IgG4 Fc region comprises proline at residue 228, and alanine at residues 234 and 235 (all residues are numbered according to the EU Index numbering).

17. The conjugate of any one of claims 14-16, wherein P comprises an immunoglobulin Fc region comprising cysteine at residue 378 (according to the EU Index numbering).

18. The conjugate of any one of claims 14-17, 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 numbering); or(b) a first Fc CH3 domain comprising leucine at residue 405, and a second Fc CH3 domain comprising arginine at residue 409 (all residues are numbered according to the EU Index numbering).

19. The conjugate of any one of claims 1-18, wherein P comprises one heavy chain (HC) and one light chain (LC), wherein HC comprises SEQ ID NO: 9 and LC comprises SEQ ID NO: 10.

20. The conjugate of any one of claims 1-18, wherein P comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 14, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 15.

21. The conjugate of any one of claims 1-18, wherein P comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 16, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 17.

22. The conjugate of claim 13, wherein the half-life extender is a VHH that binds HSA.

23. The conjugate of claim 22, wherein P comprises one heavy chain (HC) and one light chain (LC), and wherein the HC comprises SEQ ID NO: 11 and the LC comprises SEQ ID NO: 12.

24. The conjugate of any one of claims 1-12, wherein P is a heterodimeric antibody that comprises a first arm comprising one monovalent human TfR binding domain and a second arm that is a null arm.

25. The conjugate of claim 24, wherein the second arm comprises one heavy chain (HC) and one light chain (LC), and wherein the HC comprises SEQ ID NO: 18 and the LC comprises SEQ ID NO: 19.

26. The conjugate of claim 24 or 25, wherein P comprises two heavy chains HC1 and HC2 and two light chains LC1 and LC2, wherein HC1 comprises SEQ ID NO: 13, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 18, and LC2 comprises SEQ ID NO: 19.

27. The conjugate of any one of claims 3-26, wherein the antisense strand is complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMRI, LRRK2, HTT, SOD1, SCN10A, SCN9A or CACNA1B mRNA.

28. The conjugate of claim 27, wherein the antisense strand is complementary to SNCA mRNA.

29. The conjugate of any one of claims 3-28, 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: 36, and the antisense strand comprises SEQID NO: 37;(b) the sense strand comprises SEQ ID NO: 38, and the antisense strand comprises SEQ ID NO: 37; and(c) the sense strand comprises SEQ ID NO: 39, and the antisense strand comprises SEQID NO: 40,wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more intemucleotide linkages of the sense strand and the antisense strand are modified intemucleotide linkages.

30. The conjugate of claim 27, wherein the antisense strand is complementary to APP mRNA.

31. The conjugate of claim 30, wherein the sense strand comprises SEQ ID NO: 53, and the antisense strand comprises SEQ ID NO: 54, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more intemucleotide linkages of the sense strand and the antisense strand are modified intemucleotide linkages.

32. The conjugate of claim 29 or 31, wherein one or more nucleotides of the sense strand are modified nucleotides.

33. The conjugate of claim 32, wherein each nucleotide of the sense strand is a modified nucleotide.

34. The conjugate of any one of claims 28-33, wherein one or more nucleotides of the antisense strand are modified nucleotides.

35. The conjugate of claim 34, wherein each nucleotide of the antisense strand is a modified nucleotide.

36. The conjugate of any one of claims 28-35, wherein the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide or 2’-O-Ci6 alkyl modified nucleotide.

37. The conjugate of any one of claims 28-36, wherein the sense strand has four 2'-fhioro modified nucleotides at positions 7, 9, 10, and 11 from the 5’ end of the sense strand.

38. The conjugate of claim 37, wherein nucleotides at positions other than positions 7, 9, 10, and 11 of the sense strand are 2'-O-methyl modified nucleotides.

39. The conjugate of any one of claims 28-36, 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.

40. The conjugate of claim 39, wherein nucleotides at positions other than positions 2, 6, 14 and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

41. The conjugate of any one of claims 28-36, wherein the sense strand has three 2'-fluoro modified nucleotides at positions 9, 10, and 11 from the 5’ end of the sense strand.

42. The conjugate of claim 41, wherein nucleotides at positions other than positions 9, 10, and 11 of the sense strand are 2'-O-methyl modified nucleotides.

43. The conjugate of any one of claims 28-38, 41, 42, 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.

44. The conjugate of claim 43, wherein nucleotides at positions other than positions 2, 5, 7, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

45. The conjugate of any one of claims 28-38, 41, 42, 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.

46. The conjugate of claim 45, wherein nucleotides at positions other than positions 2, 5, 8, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

47. The conjugate of any one of claims 28-38, 41, 42, wherein the antisense strand has five2'-fluoro modified nucleotides at positions 2, 3, 7, 14, and 16 from the 5’ end of the antisense strand.

48. The conjugate of claim 47, wherein nucleotides at positions other than positions 2, 3, 7, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

49. The conjugate of any one of claims 28-38, 41, 42, wherein the antisense strand has three 2'-fluoro modified nucleotides at positions 2, 14, and 16 from the 5’ end of the antisense strand.

50. The conjugate of claim 49, wherein nucleotides at positions other than positions 2, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

51. The conjugate of any one of claims 3-50, wherein the sense strand and the antisense strand have one or more modified internucleotide linkages.

52. The conjugate of claim 51, wherein the modified intemucleotide linkage is phosphorothioate linkage.

53. The conjugate of claim 52, wherein the sense strand has four or five phosphorothioate linkages.

54. The conjugate of claim52 or 53, wherein the antisense strand has four or five phosphorothioate linkages.

55. The conjugate of any one of claims 3-54, wherein the antisense strand has a phosphate analog at 5’ end.

56. The conjugate of claim 55, wherein the phosphate analog is 5’-vinylphosphonate.

57. The conjugate of any one of claims 3-56, wherein the sense strand comprises an abasic moiety or inverted abasic moiety.

58. The conjugate of any one of claims 28, 29, 32-57, 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: 41, and the antisense strand comprises SEQ ID NO: 42;(b) the sense strand comprises SEQ ID NO: 43, and the antisense strand comprises SEQ ID NO: 42; and(c) the sense strand comprises SEQ ID NO: 44, and the antisense strand comprises SEQ ID NO: 45.

59. The conjugate of any one of claims 28, 29, 32-58, wherein the sense strand and the antisense strand have a pair of nucleic acid sequences selected from the group consisting(a) the sense strand consists of SEQ ID NO: 41, and the antisense strand consists of SEQ ID NO: 42;(b) the sense strand consists of SEQ ID NO: 43, and the antisense strand consists of SEQ ID NO: 42; and(c) the sense strand consists of SEQ ID NO: 44, and the antisense strand consists of SEQ ID NO: 45.

60. The conjugate of any one of claims 30-57, wherein the sense strand comprises SEQ ID NO: 55, and the antisense strand comprises SEQ ID NO: 56.

61. The conjugate of any one of claims 30-58, wherein the sense strand consists of SEQ ID NO: 55, and the antisense strand consists of SEQ ID NO: 56.

62. A compound comprising any one of the following formulae:wherein A is a therapeutic agent.

63. A pharmaceutical composition comprising the conjugate of any one of claims 1-61, or the compound of claim 62, and a pharmaceutically acceptable carrier.

64. A method of treating a neurodegenerative disease in a patient in need thereof, the method comprising administering to the patient an effective amount of the conjugate of any one of claims 1-61.

65. The method of claim 64, wherein the neurodegenerative disease is Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia.

66. The method of claim 64 or 65, wherein the conjugate is administered to the patient intravenously or subcutaneously.

67. The conjugate of any one of claims 1-61, or the pharmaceutical composition of claim 63, for use in a therapy.

68. The conjugate of any one of claims 1-61, or the pharmaceutical composition of claim 63, for use in the treatment of a neurodegenerative disease.

69. The conjugate or pharmaceutical composition for use of claim 68, wherein the neurodegenerative disease is Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia.

70. Use of the conjugate of any one of claims 1-61, or the compound of claim 62, in the manufacture of a medicament for treating a neurodegenerative disease.

71. The use of claim 70, wherein the neurodegenerative disease is Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia.

72. A method of generating a conjugate, the method comprising reacting the compound of claim 62 with a protein comprising a cysteine.