SOD1 RNA interference agents

Abstract

Provided herein are SOD1 RNAi agents and compositions comprising a SOD1 RNAi agent. Also provided herein are methods of using the SOD1 RNAi agents or compositions comprising a SOD1 RNAi agent in reducing SOD1 expression and / or treating SOD1-mediated neurological disease.

Description

SOD1 RNA INTERFERENCE AGENTS SEQUENCE 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 “31211_WO” created August 26, 2025, and is 234 kilobytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety. BACKGROUND

[0002] Human superoxide dismutase 1 (SOD1) is a ubiquitously expressed enzyme that metabolizes free superoxide radicals in the body. SOD1 protein functions as a homodimer that binds copper and zinc ions and catalyzes the conversion of superoxide radicals into oxygen and hydrogen peroxide. SOD1 plays an important role in protecting cells from oxidative damages.

[0003] SOD1 mutations have been reported in both coding and non-coding regions. Such mutations in the SOD1 gene are associated with several neurological diseases such as Amyotrophic lateral sclerosis (ALS) and Down's syndrome (DS).

[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] 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. Antibodies directed to transferrin receptor (“TfR”) have been used for modulating BBB transport. 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 United States.

[0006] QALSODY (tofersen) is an antisense oligonucleotide indicated for the treatment of ALS in adults who have a mutation in the SOD1 gene under the accelerated approval by the FDA inthe United States. However, QALSODY has to be administered intrathecally, which could be associated with risk of inflammation or infection, elevated intracranial pressure, pain, and limited drug distribution.

[0007] There remains a need for therapeutic agents that can inhibit or adjust the expression of SOD1 for treating a SOD1-mediated neurological disease such as ALS, e.g., by utilizing RNAi, with improved administration route. There is also need for conjugates that can deliver SOD1 RNAi agents across the BBB into the CNS and achieve good distribution. SUMMARY OF INVENTION

[0008] Provided herein are SOD1 RNAi agents, including SOD1 RNAi agents capable of crossing BBB, and compositions comprising such SOD1 RNAi agent. Such SOD1 RNAi agent and composition can be administered intravenously or subcutaneously. Also provided herein are methods of using such SOD1 RNAi agents or compositions comprising a SOD1 RNAi agent for reducing SOD1 expression, and / or treating a SOD1-mediated neurological disease such as ALS in a subject.

[0009] In one aspect, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to SOD1 mRNA; wherein L is a linker, or absent; wherein P is a protein comprising one monovalent human TfR binding domain (“human TfR binding protein”); 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] In some embodiments, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to SOD1 mRNA; wherein L is a linker, or absent; 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 comprisesSEQ 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. 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. Exemplary sequences of human TfR binding domains and proteins are provided in Table 1a and 1b.

[0011] In some embodiments, L is a SMCC linker, OD linker, or MSPT linker (see Table 5). In some embodiments, L is a MSPT linker in Table 5.

[0012] Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human SOD1 mRNA are provided in Table 3a. In some embodiments, 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: 27, and the antisense strand comprises SEQ ID NO: 28, (b) the sense strand comprises SEQ ID NO: 29, and the antisense strand comprises SEQ ID NO: 30, (c) the sense strand comprises SEQ ID NO: 31, and the antisense strand comprises SEQ ID NO: 32, (d) the sense strand comprises SEQ ID NO: 52, and the antisense strand comprises SEQ ID NO: 53, (e) the sense strand comprises SEQ ID NO: 54, and the antisense strand comprises SEQ ID NO: 55, (f) the sense strand comprises SEQ ID NO: 56, and the antisense strand comprises SEQ ID NO: 57, (g) the sense strand comprises SEQ ID NO: 58, and the antisense strand comprises SEQ ID NO: 59, and (h) the sense strand comprises SEQ ID NO: 60, and the antisense strand comprises SEQ ID NO: 61, 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.

[0013] 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, 2’ deoxy nucleotide (DNA), or 2'-O-alkyl (e.g., 2’- O-C16alkyl) 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, 2’ deoxy nucleotide (DNA), or 2'-O-alkyl (e.g., 2’-O-C16 alkyl) modified nucleotide.

[0014] 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, at least one nucleotide of the sense strand is an unmodified RNA nucleotide. In some embodiments, at least one nucleotide of the sense strand is 2’ deoxy nucleotide (DNA). 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.

[0015] 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, at least one nucleotide of the sense strand is an unmodified RNA nucleotide. In some embodiments, at least one nucleotide of the sense strand is 2’ deoxy nucleotide (DNA). 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 antisense strand has three 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.

[0016] In some embodiments, the 5’ end of the antisense strand has a phosphate analog, e.g., 5’-vinylphosphonate (5’-VP).

[0017] In some embodiments, the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety.

[0018] In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is 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.

[0019] Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human SOD1 mRNA are provided in Table 3b. In some embodiments, 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: 33, and the antisense strand comprises SEQ ID NO: 34, (b) the sense strand comprises SEQ ID NO: 35, and the antisense strand comprises SEQ ID NO: 36, (c) the sense strand comprises SEQ ID NO: 37, and the antisense strand comprises SEQ ID NO: 38, (d) the sense strand comprises SEQ ID NO: 62, and the antisense strand comprises SEQ ID NO: 63, (e) the sense strand comprises SEQ ID NO: 64, and the antisense strand comprises SEQ ID NO: 65, (f) the sense strand comprises SEQ ID NO: 66, and the antisense strand comprises SEQ ID NO: 67, (g) the sense strand comprises SEQ ID NO: 68, and the antisense strand comprises SEQ ID NO: 69, and(h) the sense strand comprises SEQ ID NO: 70, and the antisense strand comprises SEQ ID NO: 71.

[0020] In some embodiments, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to SOD1 mRNA; wherein L is a linker, or absent; wherein P is a protein comprising one monovalent human TfR binding domain, and P is selected from TBP1, TBP2, TBP3, TBP4, or TBP5 in Table 1b; 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. In some embodiments, L is a linker in Table 5.

[0021] In some embodiments, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the dsRNA is any dsRNA in Table 3a or 3b; wherein L is a linker, or absent; wherein P is a protein comprising one monovalent human TfR binding domain; 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. In some embodiments, L is a linker in Table 5.

[0022] In some embodiments, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the dsRNA is any dsRNA in Table 3a or 3b; wherein L is a linker, or absent; wherein P is a protein comprising one monovalent human TfR binding domain, and P is selected from TBP1, TBP2, TBP3, TBP4, or TBP5 in Table 1b; 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. In some embodiments, L is a linker in Table 5.

[0023] In some embodiments, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to SOD1 mRNA; wherein L is a linker, or absent; wherein P is a protein comprising one monovalent human TfR binding domain; wherein the human TfR binding domain 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, and wherein n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, L is a linker in Table 5.

[0024] In some embodiments, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to SOD1 mRNA; wherein L is a linker, or absent; wherein P is a protein comprising one monovalent human TfR binding domain; wherein the human TfR binding domain 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, and wherein n is 1. In some embodiments, L is a linker in Table 5.

[0025] In another aspect, provided herein are methods of treating a SOD1-mediated neurological disease such as ALS in a patient in need thereof, and such the method comprises administering to the patient an effective amount of the SOD1 RNAi agent or a pharmaceutical composition described herein. The SOD1 RNAi agent or a pharmaceutical composition comprising SOD1 RNAi agent can be administered to the patient intravenously or subcutaneously.

[0026] In another aspect, provided herein are SOD1 RNAi agents or pharmaceutical compositions comprising a SOD1 RNAi agent for use in a therapy. Also provided herein are SOD1 RNAi agents or pharmaceutical compositions comprising a SOD1 RNAi agent for use in the treatment of a SOD1-mediated neurological disease such as ALS. Also provided herein are uses of the SOD1 RNAi agent in the manufacture of a medicament for treating a SOD1-mediated neurological disease such as ALS. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figures 1A-C show an exemplary analytical anion exchange (AEX) chromatogram of DAR profile for TBP5-MSPT- dsRNA No.4 conjugate (1A), TBP5-MSPT- dsRNA No.5 conjugate (1B) and TBP5-MSPT- dsRNA No.6 conjugate (1C).

[0028] Figures 2A-C shows an exemplary analytical anion exchange (AEX) chromatogram of DAR profile for mTBP1-MSPT- dsRNA No.6 conjugate (2A), mTBP1- MSPT- dsRNA No.4 conjugate (2B) and mTBP1-MSPT- dsRNA No.5 conjugate (2C).DETAILED DESCRIPTION

[0029] Provided herein are SOD1 RNAi agents and compositions comprising a SOD1 RNAi agent. Also provided herein are methods of using the SOD1 RNAi agents or compositions comprising a SOD1 RNAi agent for reducing SOD1 expression and / or treating a SOD1-mediated neurological disease such as ALS in a subject.

[0030] In one aspect, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, wherein the antisense strand is complementary to SOD1 mRNA; wherein L is a linker, or absent; wherein P is a protein comprising one monovalent human TfR binding domain (“human TfR binding protein”); 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. For clarity, when L is absent, R is directly linked to P through a direct bond.

[0031] In some embodiments, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to SOD1 mRNA; wherein L is a linker, or absent; 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. In some embodiments, L is a linker in Table 5. For clarity, when L is absent, R is directly linked to P through a direct bond.

[0032] In some embodiments, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to SOD1 mRNA; wherein L is a linker, or absent; wherein P is a protein comprising one monovalent human TfR binding domain, and P is selected from TBP1, TBP2, TBP3, TBP4, or TBP5 in Table 1b; and wherein n is an integer of 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In someembodiments, n is 3. In some embodiments, L is a linker in Table 5. For clarity, when L is absent, R is directly linked to P through a direct bond.

[0033] In some embodiments, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the dsRNA is any dsRNA in Table 3a or 3b; wherein L is a linker, or absent; wherein P is a protein comprising one monovalent human TfR binding domain; 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. In some embodiments, L is a linker in Table 5. For clarity, when L is absent, R is directly linked to P through a direct bond.

[0034] In some embodiments, provided herein are SOD1 RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the dsRNA is any dsRNA in Table 3a or 3b; wherein L is a linker, or absent; wherein P is a protein comprising one monovalent human TfR binding domain, and P is selected from TBP1, TBP2, TBP3, TBP4, or TBP5 in Table 1b; 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. In some embodiments, L is a linker in Table 5. For clarity, when L is absent, R is directly linked to P through a direct bond.

[0035] In another aspect, provided herein are SOD1 RNAi agents comprising a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, wherein the sense stand and antisense strand sequences are selected from Table 3a or 3b. In some embodiments, SOD1 RNAi agents comprising any dsRNA in Table 3a or 3b. Human TfR binding proteins

[0036] The SOD1 RNAi agents described herein comprise a protein comprising one monovalent human TfR binding domain (“human TfR binding protein”). Human TfR binding protein of the SOD1 RNAi agents can bind TfR on BBB and transport the dsRNA into the CNS.

[0037] Exemplary sequences of human TfR binding domains and proteins are provided in Table 1a and 1b. 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, andLCDR3. 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 1a. Exemplary sequences of human TfR binding domains and proteins Region Sequence SEQ ID NO HCDR1 SYSMN 1Fab-VHH HC EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMN 11 WVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISOAH2 HC1 EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMN 16 (S124C) WVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTIS RDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNTable 1b. Exemplary sequences of human TfR binding proteins Human TfR HC1 LC1 HC2 LC2 binding r t in (TBP)n some em o men s, e monova en uman n ng oma n s 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.

[0039] In some embodiments, the human TfR binding protein further comprises a half- life extender, e.g., an immunoglobulin Fc region or a VHH that binds human serum albumin (HSA).

[0040] 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 IgG1 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 IgG1 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 hIgG1 effector null or hIgG1EN Fc region).

[0041] 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 / orcynomolgus monkey albumin. An exemplary VHH that binds human HSA is shown in Table 1c. 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: 12). In some embodiments, the VHH is linked to the C-terminus of the TfR binding domain. Table 1c. Exemplary sequences of VHH that binds human serum albumin (HSA) Region Sequence SEQ ID NO CDR1 ETAVA 20

[0042] 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 in WO2014150973, 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 1a. 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.

[0043] 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 atresidue 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).

[0044] 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).

[0045] 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).

[0046] In some embodiments, the human TfR binding protein is any one of the human TfR binding proteins in Table 1b, e.g., TBP1, TBP2, TBP3, TBP4, TBP5.

[0047] 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.

[0048] 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: 10.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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, NS0, HEK293 or COS cells according to techniques well known in the art.

[0053] 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

[0054] 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 Tables 2A and 2B. Such conjugates comprising a mouse TfR binding protein can serve as surrogate molecules in mouse models. Table 2A. Exemplary sequences of mouse TfR binding proteins Region Sequence SEQ ID NODDSISDAYFDLWGPGTLVTVSSASTKGPCVFPL APCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTTable 2B. Exemplary sequences of mouse TfR binding proteins Mouse TfR binding HC1 LC1 HC2 protein (mTBP)dsRNA

[0055] The SOD1 RNAi agents described herein comprise a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and wherein the antisense strand is complementary to SOD1 mRNA. After the antisense strand of the dsRNA is incorporated into the RNA-induced silencing complex (RISC), the RISC can bind and degrade target SOD1 mRNA.

[0056] 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, thedsRNA 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.

[0057] Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human SOD1 mRNA are provided in Table 3a.

[0058] In some embodiments, the sense strand comprises SEQ ID NO: 27, and the antisense strand comprises SEQ ID NO: 28. In some embodiments, the sense strand comprises SEQ ID NO: 29, and the antisense strand comprises SEQ ID NO: 30. In some embodiments, the sense strand comprises SEQ ID NO: 31, and the antisense strand comprises SEQ ID NO: 32. In some embodiments, the sense strand comprises SEQ ID NO: 52, and the antisense strand comprises SEQ ID NO: 53. In some embodiments, the sense strand comprises SEQ ID NO: 54, and the antisense strand comprises SEQ ID NO: 55. In some embodiments, the sense strand comprises SEQ ID NO: 55, and the antisense strand comprises SEQ ID NO: 57. In some embodiments, the sense strand comprises SEQ ID NO: 58, and the antisense strand comprises SEQ ID NO: 59. In some embodiments, the sense strand comprises SEQ ID NO: 60, and the antisense strand comprises SEQ ID NO: 61. Table 3a. Unmodified sequences of dsRNA targeting human SOD1 mRNA dsRNA Sense Strand (5' to 3') SEQ Antisense Strand (5' to 3') SE Start No. ID Q position of n 4.3 CUGAUAAACAUUAA 31 UCAGUGUUUAAUGUUU 32 613 ACACUGA AUCAGGATable 3b. Modified sequences of dsRNA targeting human SOD1 mRNA dsR Sense Strand (5' to 3') SE Antisense Strand (5' to 3') SEQ Start position NA Q ID of antisense t 1 .5 :13 mA*mU*mUmGmAmAmGmA 64 mU*fG*mAmGfAmUfCm 65 337 fUfUfCmUmGmUmGmAmUm AmCmAmGmAmAfUmCf * * A A A * A*vinylphosphonate (VP).

[0059] 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. Such 2’-modifications can be 2’-fluoro, 2’-O-methyl (i.e., 2’-methoxy), or 2'-O-alkyl (e.g., 2’-O-C16 alkyl).

[0060] 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, at least one nucleotide of the sense strand is an unmodified RNA 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, 2’ deoxy nucleotide (DNA), or 2'- O-alkyl (e.g., 2’-O-C16alkyl) 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, 2’ deoxy nucleotide (DNA), or 2'-O-alkyl(e.g., 2’-O-C16 alkyl) modified nucleotide. In some embodiments, at least one nucleotide of the sense strand is 2’ deoxy nucleotide (DNA).

[0061] 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, at least one nucleotide of the sense strand is an unmodified RNA nucleotide. In some embodiments, at least one nucleotide of the sense strand is 2’ deoxy nucleotide (DNA). In some embodiments, the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides.

[0062] 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.

[0063] 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, at least one nucleotide of the sense strand is an unmodified RNA nucleotide. In some embodiments, at least one nucleotide of the sense strand is 2’ deoxy nucleotide (DNA). In some embodiments, the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides.

[0064] 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 antisense strand has three 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.

[0065] In some embodiments, the 5’ end of the antisense strand has a phosphate analog, e.g., 5’-vinylphosphonate (5’-VP).

[0066] 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 4.Table 4. Abasic or inverted abasic (iAb) moieties Structure ion of the sequences.

[0067] In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is 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.

[0068] Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human SOD1 mRNA are provided in Table 3b.

[0069] In some embodiments, the sense strand comprises SEQ ID NO: 33, and the antisense strand comprises SEQ ID NO: 34. In some embodiments, the sense strand comprises SEQ ID NO: 35, and the antisense strand comprises SEQ ID NO: 36. In some embodiments, the sense strand comprises SEQ ID NO: 37, and the antisense strand comprises SEQ ID NO: 38. In some embodiments, the sense strand comprises SEQ ID NO: 62, and the antisense strand comprises SEQ ID NO: 63. In some embodiments, the sense strand comprises SEQ ID NO: 64, and the antisense strand comprises SEQ ID NO: 65. In some embodiments, the sense strand comprises SEQ ID NO: 66, and the antisense strand comprises SEQ ID NO: 67. In some embodiments, the sense strand comprises SEQ ID NO: 68, and the antisense strand comprisesSEQ ID NO: 69. In some embodiments, the sense strand comprises SEQ ID NO: 70, and the antisense strand comprises SEQ ID NO: 71.

[0070] In some embodiments, the sense strand consists of SEQ ID NO: 33, and the antisense strand consists of SEQ ID NO: 34. In some embodiments, the sense strand consists of SEQ ID NO: 35, and the antisense strand consists of SEQ ID NO: 36. In some embodiments, the sense strand consists of SEQ ID NO: 37, and the antisense strand consists of SEQ ID NO: 38. In some embodiments, the sense strand consists of SEQ ID NO: 62, and the antisense strand consists of SEQ ID NO: 63. In some embodiments, the sense strand consists of SEQ ID NO: 64, and the antisense strand consists of SEQ ID NO: 65. In some embodiments, the sense strand consists of SEQ ID NO: 66, and the antisense strand consists of SEQ ID NO: 67. In some embodiments, the sense strand consists of SEQ ID NO: 68, and the antisense strand consists of SEQ ID NO: 69. In some embodiments, the sense strand consists of SEQ ID NO: 70, and the antisense strand consists of SEQ ID NO: 71.

[0071] 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 3a or 3b. 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 3a or 3b.

[0072] 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.

[0073] 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.

[0074] The RNAi agent described herein can be made by a variety of procedures known to one of ordinary skill in the art, some of which are illustrated in the preparations and examples below, e.g., in Examples 1-3. One of ordinary skill in the art recognizes that the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare the RNAi agent. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. The reagents and starting materials are readily available to one of ordinary skill in the art.

[0075] In some embodiments, the TfR binding protein with native or engineered cysteines described herein can be first treated with a reducing agent, e.g., DTT, and then re- oxidized with an oxidizing agent, e.g., DHAA. The resulting oxidized TfR binding protein is then incubated with a linker functionalized dsRNA, e.g., linker-dsRNA, to produce the conjugated RNAi agent. Linker

[0076] In some embodiments, the SOD1 RNAi agents described herein comprises a linker that links the human TfR binding protein to the dsRNA. Exemplary linker structures are shown in Table 5. In some embodiments, the linker comprises a SMCC linker, OD linker, or MSPT linker. In some embodiments, the linker is a SMCC linker, OD linker, or MSPT linker. In some embodiments, the linker is a SMCC linker. In some embodiments, the linker is a MSPT linker.

[0077] In some embodiments, the linker is conjugated to an engineered cysteine in the human TfR binding protein. In some embodiments, the linker is conjugated to the sense strand of the dsRNA, e.g., the 5’ end or 3’ end of the sense strand.Table 5. Exemplary linker structures Linker StructurePharmaceutical Composition

[0078] In another aspect, provided herein are pharmaceutical compositions comprising any of the SOD1 RNAi agents 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

[0079] In another aspect, provided herein are methods of treating a SOD1-mediated neurological disease in a patient in need thereof, and such method comprises administering to the patient an effective amount of the SOD1 RNAi agent or a pharmaceutical composition described herein. Exemplary SOD1-mediated neurological disease includes, but are not limited to, amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease), Down's syndrome (DS), Progressive Spastic Tetraplegia and Axial Hypotonia (STAHP).

[0080] The SOD1 RNAi agent or a pharmaceutical composition comprising SOD1 RNAi agent can be administered to the patient intrathecally, intravenously or subcutaneously.

[0081] SOD1 RNAi agent dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0082] 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.

[0083] In another aspect, provided herein are SOD1 RNAi agents or pharmaceutical compositions comprising a SOD1 RNAi agent for use in reducing SOD1 expression. Also provided herein are SOD1 RNAi agents or the pharmaceutical composition comprising a SOD1 RNAi agent for use in a therapy. Also provided herein are SOD1 RNAi agents or pharmaceutical compositions comprising a SOD1 RNAi agent for use in the treatment of a SOD1-mediated neurological disease such as ALS. Also provided herein are uses of SOD1 RNAi agents in the manufacture of a medicament for the treatment of a SOD1-mediated neurological disease such as ALS. Definitions

[0084] 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.

[0085] 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-C20alkyl” means a radical having 1-20 carbon atoms in a linear or branched arrangement.

[0086] 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., IgG1, IgG2, IgG3, IgG4).

[0087] 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 light chain variable region (VL) and a light chain constant region. The IgG isotype may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0088] The VH and VL regions can be further subdivided into regions of hyper- variability, 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 carboxyl- terminus 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 “LCDR1, LCDR2 and LCDR3”. 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-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available on at www.imgt.org; see Lefranc et al., Nucleic Acids Res.1999; 27:209-212).

[0089] 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.

[0090] 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.

[0091] The term “heterodimeric antibody”, as used herein, refers to an antibody that comprises two distinct antigen-binding domains.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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).

[0096] 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 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 protein or conjugate to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the protein or conjugate are outweighed by the therapeutically beneficial effects.

[0097] 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., IgG1, 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., IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is a human IgG Fc region, e.g., a human IgG1 Fc region, human IgG2 Fc region, human IgG3 Fcregion 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).

[0098] The term “knockdown” or “expression knockdown” refers to reduced mRNA or protein expression of a gene after treatment of a reagent.

[0099] As used herein, “modified internucleotide linkage” means an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage having a phosphodiester bond. A modified internucleotide linkage can be a non-naturally occurring linkage. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage. [000100] 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, 2’ deoxy nucleotide (DNA), or 2'-O-alkyl (e.g., 2’-O-C16 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 4. [000101] 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., riboseor 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). [000102] As used herein, a “null arm” means an antibody arm that does not bind any known human target. [000103] 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. [000104] 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. [000105] The term “patient”, as used herein, refers to a human patient. [000106] 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’ end of an oligonucleotide in place of a 5’-phosphate, which is sometimes 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. [000107] As used herein, “SOD1” refers to a human SOD1 mRNA transcript or a human SOD1 protein. The nucleotide sequence of the human SOD1 mRNA transcript can be found at NM_000454.5: 1 GCGTCGTAGT CTCCTGCAGC GTCTGGGGTT TCCGTTGCAG TCCTCGGAAC CAGGACCTCG 61 GCGTGGCCTA GCGAGTTATG GCGACGAAGG CCGTGTGCGT GCTGAAGGGC GACGGCCCAG 121 TGCAGGGCAT CATCAATTTC GAGCAGAAGG AAAGTAATGG ACCAGTGAAG GTGTGGGGAA 181 GCATTAAAGG ACTGACTGAA GGCCTGCATG GATTCCATGT TCATGAGTTT GGAGATAATA 241 CAGCAGGCTG TACCAGTGCA GGTCCTCACT TTAATCCTCT ATCCAGAAAA CACGGTGGGC 301 CAAAGGATGA AGAGAGGCAT GTTGGAGACT TGGGCAATGT GACTGCTGAC AAAGATGGTG 361 TGGCCGATGT GTCTATTGAA GATTCTGTGA TCTCACTCTC AGGAGACCAT TGCATCATTG 421 GCCGCACACT GGTGGTCCAT GAAAAAGCAG ATGACTTGGG CAAAGGTGGA AATGAAGAAA 481 GTACAAAGAC AGGAAACGCT GGAAGTCGTT TGGCTTGTGG TGTAATTGGG ATCGCCCAAT 541 AAACATTCCC TTGGATGTAG TCTGAGGCCC CTTAACTCAT CTGTTATCCT GCTAGCTGTA 601 GAAATGTATC CTGATAAACA TTAAACACTG TAATCTTAAA AGTGTAATTG TGTGACTTTT 661 TCAGAGTTGC TTTAAAGTAC CTGTAGTGAG AAACTGATTT ATGATCACTT GGAAGATTTG 721 TATAGTTTTA TAAAACTCAG TTAAAATGTC TGTTTCAATG ACCTGTATTT TGCCAGACTT 781 AAATCACAGA TGGGTATTAA ACTTGTCAGA ATTTCTTTGT CATTCAAGCC TGTGAATAAA 841 AACCCTGTAT GGCACTTATT ATGAGGCTAT TAAAAGAATC CAAATTCAAA CTAAA(SEQ ID NO: 39). The corresponding amino acid sequence of human SOD1 protein can be found at NP_000445.1: 1 MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE FGDNTAGCTS 61 AGPHFNPLSR KHGGPKDEER HVGDLGNVTA DKDGVADVSI EDSVISLSGD HCIIGRTLVV 121 HEKADDLGKG GNEESTKTGN AGSRLACGVI GIAQ (SEQ ID NO: 40). [000108] As used herein, the term “SOD1-mediated neurological disease” refers to a neurological disease or disorder mediated by SOD1 mutation. [000109] 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.[000110] 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. [000111] As used herein, “RNAi,” “RNAi agent,” “iRNA,” “iRNA agent,” or “RNA interference agent” means an agent that mediates sequence-specific degradation of a target mRNA by RNA interference, e.g., via RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agent has a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex (e.g., a double stranded RNA). [000112] As used herein, “strand” refers to a single, contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have two free ends (e.g., a 5’ end and a 3’ end). [000113] 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. [000114] The following examples are offered to illustrate, but not to limit, the claimed inventions. EXAMPLES Example 1: Generation and Characterization of TfR binding proteins Generation of human TfR binding proteins [000115] 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: 25, see Table 6) and mouse Transferrin Receptor protein with a His tag (mTfR-ECD-6His, SEQ ID NO: 24). 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. [000116] 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: 26, see Table 6). Antigen positive B-cells weresorted from pooled spleens. Binding of individual antibodies cloned from those B-cells to his- tagged hTfR-ECD was verified. Table 6. Sequences of the immunogens used to generate human or mouse TfR antibodies. Immunogen Sequence SEQ ID NO mTfR-ECD-6His HHHHHHCKRVEQKEECVKLAETEETDKSETMETEDV 24IPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTS ESKNVKLTVS[000117] Affinity variants of the generated human 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 and proteins are provided in Table 1a. [000118] Human TfR binding proteins were generated by recombinant DNA technology. Such human 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 [000119] 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+ (10mM Hepes pH 7.4 + 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 µM respectively by dilution of stock solution into running buffer. [000120] 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 µL / min for 140 seconds followed by return to buffer flow for 400 seconds to monitordissociation phase; (2) regeneration of chip surfaces with injection of 3M magnesium chloride, for 30 seconds at 100 µL / min over all cells; and (3) equilibration of chip surfaces with a 50 µL (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 7. Table 7. Binding Affinity of Exemplified human TfR binding proteins to human or cynomolgus TfR at 37 °C Human TfR Standard error Standard error ofExample 2: Synthesis and characterization of dsRNA targeting SOD1 [000121] Single strands (sense and antisense) of the dsRNA duplexes were typically synthesized on solid support via a K&A H-8 (K&A Labs GmbH) or a similar automated oligonucleotide synthesizer. The sense strands were synthesized using an appropriate CPG such as 3'-Cholesterol-TEG CNA CPG 500 (LGC Biosearch Technologies), 3’-TEG-Tocopherol (LGC Biosearch Technologies) or phthalamido amino C6 lcaa CPG 500 Å (Chemgenes) whereas the antisense strands used standard support (LGC Biosearch Technologies). The sequences of the sense and antisense strands were shown in Table 3a or 3b. [000122] Standard reagents were used in the oligo synthesis (Table 8), where 0.1M xanthane hydride in pyridine was used as the sulfurization reagent. All monomers (Table 9a) except for OMe U (0.1 M in 20% DMF / ACN) were made at 0.1M in ACN and contained a molecular sieves trap bag. [000123] The oligonucleotides were cleaved and deprotected (C / D) using AMA (1:1 mixture of concentrated ammonia and 40% wt methylamine in water) at room temperature for 2 hours. 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 Acrodisc®32mm syringefilter with 0.2 µm Supor®membrane. The CPG was back washed / rinsed with 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™. [000124] The crude oligonucleotides were purified via AKTA™ Pure purification system using anion-exchange (AEX). A Sepax Source 15Q column, 15 um, 10x250 mm was used at room temperature with mobile phase A (MPA): 20mM sodium phosphate buffer, 20% ACN, pH 7.0 and mobile phase B (MPB): 20mM sodium phosphate buffer, 1.5M NaBr, 20% ACN, pH 7.0. In all cases, fractions which contained a mass purity greater than 85% without impurities >5% where combined. [000125] The purified oligonucleotides were desalted using 15 mL 3K MWCO centrifugal spin tubes at 3500xg for ~30 min. The oligonucleotides were rinsed with RNAse free water until the eluent conductivity reached < 100 usemi / cm. After desalting was complete, approximately 1 -2 mL of sample was recovered and transferred to a 5 mL Eppendorf tube. The final desalted oligonucleotides were analyzed for concentration (nano drop at A260), characterized by IP-RP LCMS for mass purity and UV-purity. [000126] For the preparation of duplexes, 1 equivalent of sense strand and 1.03-1.05 equivalents of antisense strand were combined and monitored for their annealing via UPLC (ensuring no greater than 5% of excess antisense strand was present). Further integrity of the duplex was confirmed by LCMS using IP-RP. For in vivo analysis, the appropriate amount of duplex was either lyophilized or diluted with 1X PBS for rodent studies and a CSF for non- human primate studies. [000127] For in-vitro testing, cholesterol or tocopherol-conjugated oligonucleotides were annealed at this stage to give cholesterol or tocopherol conjugated dsRNA by mixing equimolar aliquots of sense and antisense strands at room temperature for 30 minutes. 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 8 - Oligonucleotide Synthesis Reagents ReagentsTable 9a- Phosphoramidites Phosphoramidite Abbreviation Supplier Catalog # CASDMT-2'-O-Me- mA Chemgenes ANP-5751 110782-31-5 A(Bz)-CETable 9b. Cholesterol or Tocopherol Conjugate Structures StructureExample 3: Generation of SOD1 RNAi agents [000128] Certain abbreviations are defined as follows: “ACN” refers to acetonitrile; “aAEX” refers to analytical anion exchange; “AS” refers to antisense strand; “DAR” refers to drug / siRNA to antibody / protein ratio; “DCM” refers to dichloromethane; “DHAA” refers to dehydroascorbic acid; “dsRNA” refers to double stranded ribonucleic acid; “DTT” refers todithiothreitol; “h” refers to hours; “HPLC” refers to high-performance liquid chromatography; “LC / MS” refers to liquid chromatography mass spectrometry; “LTQ / MS” refers to linear ion trap mass spectrometer; “min” refers to minutes; “MSPT” refers to 4-(5-methylsulfonyl-1H- tetrazole-1yl)phenol; “MW” refers to molecular weight; “MWCO” refers to molecular weight cut-off; “NHS” refers to N-hydroxysuccinimide; “OD” refers to 4-(5-(methylsulfonyl)-1,3,4- oxadiazol-2-yl)phenol; “PBS” phosphate-buffered saline; “PEG” refers to polyethylene glycol; “RNAi” refers to RNA interference; “rpm” refers to revolutions per minute; “SEC” refers to size exclusion chromatography; “siRNA” refers to small interfering RNA; “SMCC” refers to succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate; “SS” refers to sense strand; “TCO” refers to trans-cyclo-octene; “TfR” refers to transferrin receptor; “THF” refers to tetrahydrofuran; “TRIS” refers to tris(hydroxymethyl)aminomethane; “UPLC” refers to ultra performance liquid chromatography; and “UV” refers to ultraviolet. Scheme 1Scheme 1 depicts the synthetic route to the intermediates used to form the final MSPT and OD linkers shown in Table 5. 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 2DIAD and tributyl phosphine in a solvent such as THF to give compound (7). Step B depicts the phosphorylation 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 1 4-(5-(Methylthio)-1,3,4-oxadiazol-2-yl)phenol A solution of 4-(5-mercapto-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 NH4Cl (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 10 was prepared in a manner essentially analogous to that found in Preparation 1. Table 10 Prep Name Structure ES / MS m / z 2 4-(5-(Methylthio)-1H- 209 (M+H)tert-Butyl 2-(2-(2-(4-(5-(methylthio)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate In a pressure(5.5 g, 20 mmol) and potassium carbonate (4.2 g, 30 mmol) were added to 4-(5-(methylthio)-1,3,4-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 11 was prepared in a manner essentially analogous to that found in Preparation 3.Table 11 Prep Name Structure ES / MS m / z )p tert-Butyl 2-(2-(2-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate tert-Butyl 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 12 was prepared in a manner essentially analogous to that found in Preparation 5.Table 12 Prep Name Structure ES / MS m / z )Preparation 7 2-(2-(2-(4-(5-(Methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetic acid TFA (20 mL,2-(2-(2-(4-(5- (methylsulfonyl)-1,3,4-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-1-yl-2-(2-(2-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate EDCI (1.60(methylsulfonyl)- 1,3,4-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). Another20 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 13 was prepared in a manner essentially analogous to that found in Preparation 8. Table 13 Prep Name Structure ES / MS m / z )repara on 2-(((2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4- methoxytetrahydrofuran-2-yl)methyl)isoindoline-1,3-dione A solution of 1-(-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,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 11 2-Cyanoethyl ((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((1,3- dioxoisoindolin-2-yl)methyl)-4-methoxytetrahydrofuran-3-yl) diisopropylphosphoramiditeA solution of 2-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3- hydroxy-4-methoxytetrahydrofuran-2-yl)methyl)isoindoline-1,3-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, 81%).1H NMR (d6-DMSO) d 11.4 (br s, 1 H), 7.96-7.78 (m, 5H), 5.83 (dd, 1H), 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 12 3’ Oxadiazole linker-functionalized sense strandμmol) and 0.6 mL 20x borate buffer. 2,5-Dioxopyrrolidin-1-yl-2-(2-(2-(4-(5-(methylsulfonyl)- 1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (241 uL, 100 mg / mL stock solution inACN) was added followed by more ACN (3 mL). 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 de-salted using a 3K spin filter (Fisher biologics, 4500rpm, 3x 1 hour). The optical density measurement of the product solution (average of 3 measurements) was 165 OD / mL, total 7 mL, 37.79 mg. The compound in Table 14 below was prepared in a manner essentially analogous to that found in Preparation 12. Table 14 Prep Name Structure 13 3’ T tr z l -Linker-functionalization of SOD1 dsRNA A 40 mL Falcon tube was charged with SOD1-SS-C6Am (35.03 mg, 3.00 mL, 4.82 μmol) and 0.6 mL 20x borate buffer. 2,5-Dioxopyrrolidin-1-yl-2-(2-(2-(4-(5-(methylsulfonyl)- 1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (241 µL, 100 mg / mL stock solution in ACN) was added followed by more ACN (3 mL). 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 de-salted using a 3K spin filter (Fisher biologics, 4500rpm, 3x 1 hour). The optical density measurement of the product solution (average of 3 measurements) was 165 OD / mL, total 7 mL, 37.79 mg. The compound in Table 14 below was prepared in a manner essentially analogous to that found in Preparation 12. Linker-SOD1 Duplex The nanodrop concentrations of aqueous solutions of each strand (average of 3x) were measure as SS = 268.0µM and AS = 1138µM. The sense strand and antisense strand are annealed to form a dsRNA.10 mL of SS and 2.41 mL of AS are mixed and shook for 30 min at 20 °C. Theamount of residual SS strand was measured until completion. Removed endotoxins by filtering through a 0.45 µM filter. The resulting 12.41 mL of solution measured (Nanodrop™ Lite, 3x average) 76.2 OD / mL equating to 199.6µM and a total of 37.9 mg. LTQ / MS m / z 7457,7845; UV purity 95.1%. Conjugation of dsRNA to TfR binding proteins [000129] 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. [000130] Conjugation of dsRNA onto TfR binding proteins were done using the following methods. Conjugation Scheme [000131] The 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 linker[000132] Conjugation was monitored using analytical anion exchange chromatography. A ProPac™ SAX-10 HPLC Column, 10 µm particle, 4 mm diameter, 250 mm 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 + 1M NaCl, at 30 ºC. [000133] Drug / siRNA to antibody / protein ratio (DAR) was calculated based on peak area % from the analytical anion exchange (aAEX) chromatogram. [000134] 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 1X PBS pH 7.2 under an isocratic condition. Alternatively, anion exchange, e.g., ThermoFisher POROSTMXQ, 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 1M 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 (Table 15).Table 15. siRNA / drug to TBP / antibody ratio (DAR) Average DAR % of DAR0 % of DAR1 % of DAR2 TBP5-MSPT- 1.04 0.00 95.8 4.2Example 4: In vitro characterization of SOD1 RNAi agents Selected SOD1 RNAi agents were tested in SH-SY5Y cells and human iPSC-derived neurons. SH-SY5Y Cell Culture and RNAi Treatment and Analysis: The SH-SY5Y cells (CRL-2266) are neuroblastoma cell line (ATCC HTB-11). The growth culture medium was 1:1 mixture of Eagle MEM (Gibco 11090-81) and F12 medium (Corning 10-080-CV) and supplemented with 1x sodium pyruvate (Gibco 11360-070), 1x sodium bicarbonate (Gibco 25080-084), 1x NEAA (Gibco 11140-050), 10% heat-inactivated fetal bovine serum (Gibco 10082-147) and 1x penicillin-streptomycin (Gibco 15140-122). Cells incubated at 37 °C in a humidified atmosphere of 5% CO2. Passive uptake method was used for siRNA delivery. On day 1, 5000 cells per well were plated in fibronectin coated 96-well plate. On day 2, cell medium was changed to Accell siRNA delivery medium (Horizon B-005000- 500), and cells were treated with tocopherol-conjugated siRNAs for 72 hours, 7 points CRC was done at siRNA duplex final concentration starts at 1uM, then 1:5 serial dilution. On day 5, treated cells were washed with cold 1xPBS and lysed directly into the 96 well cell plate using TaqMan Fast Advanced Cell-to-Ct kit (Invitrogen A35378). cDNA was synthesized using the following steps in a thermocycler: 37oC for 30 minutes, 95oC for 5 minutes, and 4oC hold.Polymerase Chain Reaction (PCR) using the following cycles temperatures and times: 50oC for 2 minutes, 95oC for 20 seconds, 40 cycles of 95oC for 1 seconds and 60oC for 20 seconds. GraphPad Prism v9.0 was used to determine IC50 with a four-parameter logistic fit. The human SOD1 (ThermoFisher Hs00533490-M1) levels were normalized to human GAPDH (ThermoFisher 4352934E) and represented the relative knockdown of human SOD1 mRNA expression as compared to vehicle-treated control cells. The percentage knockdown and absolute IC50 of the exemplary SOD1 RNAi agents are shown in Tables 16. Table 16: In vitro activity of SOD1 RNAi agents in SH-SY5Y cells SOD1 RNAi Agent No.SHSY5Y cellAbsolute IC50 %KD @1 µM ( M)iPSC Glutamatergic Neuron Culture and RNAi Treatment and Analysis: iCell GlutaNeurons (FujiFilm 01279) are human iPSC-derived cortical glutamatergic neurons. They were cultured with Neural Base medium, Neural Supplement A / C, and Microglia supplement A / B (All supplied from FujiFilm).80K neurons were plated per well on 96 wellplates coated with 0.1% PEI and GelTrex (ThermoFisher A1569601) and incubated at 37 °C in a humidified atmosphere of 5% CO2. After seven days in culture they were treated with non- conjugated siRNAs encapsulated within lipid nanoparticles (Neuro9™ siRNA Spark™ Kit, Precision Nanosystems) at a siRNA concentration of 100nM (n=5 per condition) and incubated for another seven days. After which the RNA was isolated with RNAdvance Cell v2 (Beckman Coulter A47943). RT-qPCR was then performed with TaqMan probes for SOD1 (Hs00533490_m1) and normalized to GAPDH (4326317E) levels using the Luna® Universal Probe One-Step RT-qPCR Kit (NEB, E3006E) on the QuantStudio 6 instrument. Table 17 shows the % SOD1 mRNA knockdown relative to a non-targeting control siRNA. Non-targeting control siRNA in lipid nanoparticles, empty lipid nanoparticles, and non-treated condition controls had equivalent SOD1 mRNA levels. Table 17: In vitro activity of SOD1 RNAi agents in human iPSC glutamatergic neurons SOD1 RNAi Agent No. % SOD1 knockdownExample 5. In vivo characterization of mouse TBP-dsRNA conjugates in human SOD1 G93A transgenic mice [000135] To determine the efficacy of the mouse TfR binding proteins-dsRNA conjugates against SOD1, they were tested in human SOD1 G93A transgenic mice expressing human SOD1 with G93A mutation. mTBP1-MSPT-dsRNA No.4, mTBP1-MSPT- dsRNA No.5, or mTBP1- MSPT- dsRNA No.6 were dosed intravenously at 10 mg / kg siRNA concentration in hSOD1 G93A transgenic mice and compared to the PBS dosed control group (n=5 per group).28 days following initial dosing, mice were euthanized under carbon monoxide and sacrificed, then brain, spinal cord, samples were collected and processed for assessment of gene expression changes using RT-qPCR. SOD1 primer from Thermo Fisher (Hs00533490_m1) was used as the target primer and mGAPDH primer from Thermo Fisher (Mm99999915_g1) was used as the housekeeping gene primer. [000136] Table 18 shows SOD1 mRNA knockdown data. Table 18. In vivo activities of SOD1 RNAi agents%KD SOD1 SOD1 RNAi mRNA %KD SOD1 %KD SOD1 A L mRNA mRNA )[000137] SOD1 protein expression changes were also assessed from brain and spinal cord samples. The level of SOD1 protein in the protein lysate was measured using an in-house developed MSD assay. Briefly, the MSD GOLD 96-well Small Spot Streptavidin SECTOR Plate (Meso Scale Diagnostics, Rockville, Maryland) was simultaneously blocked with bovine serum albumin and coated with the capture antibody, the biotinylate-rabbit polyclonal anti-SOD1 antibody (ADI-SOD-100, Enzo Biochem, Inc., Farmingdale, NY) with shaking at room temperature for 1 hour. After washing, the wells on each plate were incubated with the protein lysate or the recombinant human SOD1 protein (Eli Lilly, Indianapolis, IN) in the presence of MSD Blocker A (Meso Scale Diagnostics), with shaking at room temperature for 2 hours. The plates were washed again, then incubated with the detection antibody, the SULFO-TAG- conjugated mouse monoclonal anti-SOD1 antibody (SAB4200807, clone SD-G6, Millipore Sigma, Saint Louis, MO) in the presence of MSD Blocker A with shaking at room temperature for 1 hour. After the incubation, the plates were washed, then added with 2X MSD Read Buffer T (Meso Scale Diagnostics). The electrochemiluminescence signal was then measured on an MSD SQ120MM plate reader within 5 minutes of addition of the MSD Read Buffer. [000138] Table 19 shows SOD1 protein knockdown data.Table 19. In vivo activities of SOD1 RNAi agents %KD SOD1 %KD SOD1 %KD SOD1 SOD1 RNAi Agent protein protein protein ) [00013NA conjugate against SOD1, they were dosed in human SOD1 G93A transgenic mice in dose response manner. mTBP1-MSPT- dsRNA No.5 was dosed intravenously at 0.1, 0.2, 0.7, 2.3, or 7.6 mg / kg siRNA concentration in hSOD1 G93A transgenic mice and compared to the PBS dosed control group (n=5 per group). In order to assess efficacy in subcutaneous route of administration, mTBP1-MSPT- dsRNA No.5 was dosed subcutaneously at 7.6 mg / kg siRNA concentration.28 days following initial dosing, mice were euthanized under carbon monoxide and sacrificed, then brain, spinal cord, samples were collected and processed for assessment of gene expression changes using method described above. [000139] Table 20 shows SOD1 mRNA knockdown data. Table 20. SOD1 mRNA knockdown data Dose Route of %KD k Ad i i t ti SOD1 %KD %KD SOD17.6 IV 75.5 73.6 77.5 7.6 SC 78.7 76.6 76.7[000140] A pharmacodynamic study was conducted to determine efficacy of human TfR binding protein-SOD1 siRNA conjugates after three monthly doses. A group of Cynomolgus monkeys weighing 2-3 kg were dosed monthly intravenously with i) PBS (n=4), ii) TBP5- MSPT-dsRNA No.4 (n=4) at 10 mg / kg effective siRNA concentration, or iii) TBP5-MSPT- dsRNA No.5 (n=4) at 10 mg / kg effective siRNA concentration. About 85 days after the first dose, deeply anesthetized animals underwent cardiac perfusion, then brain, spinal cord and peripheral tissues were collected. [000141] The brain and spinal cord were coronally sectioned, 4 mm punches were collected from indicated subregions to assess target mRNA and protein levels by RT-qPCR and ELISA respectively in tissue homogenates. To determine mRNA levels, the total RNA from NHP tissues were isolated using the RNadvance Tissue kit (Beckman Coulter, Indianapolis, IN) manually or on a Biomek i7 liquid handler (Beckman Coulter), following the manufacturer’s procedure with some modification. The expression of the respective gene targets in the cDNA was determined using TaqMan qPCR assays on the QuantStudio 7 Pro platform (Thermo Fisher Scientific). Gene expression levels of SOD1 were normalized by GAPDH using respective probes SOD1 (Mf04363557_m1) and GAPDH (Mf04392546_g1) (ThermoFisher) for brain and spinal cord. [000142] To determine SOD1 protein levels, frozen 4 mm-punches of neural tissue biopsies were mixed with cold RIPA buffer (Pierce #89901, Thermo Scientific, Waltham, MA), containing the protease and phosphatase Inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail, Thermo Scientific), at a ratio of 20 mL buffer to 1g tissue. The tissue-RIPA mixture was homogenized using a 5 mm stainless steel bead on a 2010 GenoGrinder (Spex SamplePrep, Metuchen, NJ). The homogenate was then centrifuged in a refrigerated centrifuge (Eppendorf, Hamburg, Germany), and the supernatant was transferred, made into multiple single-use aliquots, and stored in -80°C for further analysis. [000143] The level of SOD1 protein in the protein lysate was measured using an in-house developed MSD assay. Briefly, the MSD GOLD 96-well Small Spot Streptavidin SECTOR Plate (Meso Scale Diagnostics, Rockville, Maryland) was simultaneously blocked with bovine serumalbumin and coated with the capture antibody, the biotinylate-rabbit polyclonal anti-SOD1 antibody (ADI-SOD-100, Enzo Biochem, Inc., Farmingdale, NY) with shaking at room temperature for 1 hour. After washing, the wells on each plate were incubated with the protein lysate or the recombinant human SOD1 protein (Eli Lilly, Indianapolis, IN) in the presence of MSD Blocker A (Meso Scale Diagnostics), with shaking at room temperature for 2 hours. The plates were washed again, then incubated with the detection antibody, the SULFO-TAG- conjugated mouse monoclonal anti-SOD1 antibody (SAB4200807, clone SD-G6, Millipore Sigma, Saint Louis, MO) in the presence of MSD Blocker A with shaking at room temperature for 1 hour. After the incubation, the plates were washed, then added with 2X MSD Read Buffer T (Meso Scale Diagnostics). The electrochemiluminescence signal was then measured on an MSD SQ120MM plate reader within 5 minutes of addition of the MSD Read Buffer. [000144] The tissues analyzed for mRNA or protein levels include Lumbar Spinal Cord; Thoracic Spinal Cord; Cervical Spinal Cord and Motor Cortex. [000145] Three monthly peripheral IV administration of TBP5-MSPT-dsRNA No.4 and TBP5-MSPT-dsRNA No.5 at 10 mg / kg in NHPs led to significant reduction of SOD1 mRNA and protein in key brain regions and lumbar spinal cord compared to PBS treatment group at 85 days post first dose. Reduction of mRNA expression and protein level are shown below in Table 21 and Table 22 respectively. Table 21. In vivo activities of SOD1 RNAi agents in NHP (mRNA expression) %KD SOD1 %KD SOD1 %KD SOD1 SOD1 RNAi mRNA mRNA mRNA %KD SOD1 )Table 22. In vivo activities of SOD1 RNAi agents in NHP (Protein level) SOD1 RNAi %KD SOD1 %KD SOD1 %KD SOD1 %KD SOD1(Lumbar (Thoracic Spinal (Cervical (Motor Cortex) Spinal Cord) Cord) Spinal Cord)SEQUENCE LISTING SEQ Sequence ID F G F G G G F V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLG, wherein X is S or C. F V S C D D G F V S C D D L P S L S S K E A V A TIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFA EKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQS SGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKNVLKERRILNI IF D D P W S N II DI P F STCAGAGTTGC TTTAAAGTAC CTGTAGTGAG AAACTGATTT ATGATCACTT GGAAGATTTG TATAGTTTTA TAAAACTCAG TTAAAATGTC TGTTTCAATG ACCTGTATTT TGCCAGACTT AAATCACAGA TGGGTATTAA ACTTGTCAGA ATTTCTTTGT CATTCAAGCC TGTGAATAAA ASGS C P S F S A V P EmC*mU*mAmGmCmUmGmUfAfGfAmAmAmUmGmUmAmUmC*mC*mAmU*fG*mGmAfUmAfCmAmUmUmUmCmUfAmCfAmGmCmUmAmG*mC*mA

Claims

CLAIMS 1. A SOD1 RNAi agent comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, wherein the antisense strand is complementary to SOD1 mRNA; wherein L is a linker, or absent; and 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 SOD1 RNAi agent of claim 1, wherein n is 1.

3. The SOD1 RNAi agent of claim 1, wherein n is 2.

4. The SOD1 RNAi agent of any one of claims 1-3, 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: 27, and the antisense strand comprises SEQ ID NO: 28, (b) the sense strand comprises SEQ ID NO: 29, and the antisense strand comprises SEQ ID NO: 30, (c) the sense strand comprises SEQ ID NO: 31, and the antisense strand comprises SEQ ID NO: 32, (d) the sense strand comprises SEQ ID NO: 52, and the antisense strand comprises SEQ ID NO: 53, (e) the sense strand comprises SEQ ID NO: 54, and the antisense strand comprises SEQ ID NO: 55,(f) the sense strand comprises SEQ ID NO: 56, and the antisense strand comprises SEQ ID NO: 57, (g) the sense strand comprises SEQ ID NO: 58, and the antisense strand comprises SEQ ID NO: 59, and (h) the sense strand comprises SEQ ID NO: 60, and the antisense strand comprises SEQ ID NO: 61, 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.

5. The SOD1 RNAi agent of any one of claims 1-4, wherein VH comprises SEQ ID NO: 7 and VL comprises SEQ ID NO:

8.

6. The SOD1 RNAi agent of any one of claims 1-5, wherein the human TfR binding domain is a Fab, scFv, Fv, or scFab.

7. The SOD1 RNAi agent of any one of claims 1-6, wherein the human TfR binding domain further comprises a heavy chain constant region comprising cysteine at residue 124 (according to the EU Index numbering).

8. The SOD1 RNAi agent of any one of claims 1-7, wherein P further comprises a half-life extender.

9. The SOD1 RNAi agent of claim 8, wherein the half-life extender is an immunoglobulin Fc region or a VHH that binds human serum albumin (HSA).

10. The SOD1 RNAi agent of claim 8 or 9, wherein the half-life extender is an immunoglobulin Fc region.

11. The SOD1 RNAi agent of claim 10, wherein the immunoglobulin Fc region is a modified human IgG4 Fc region.

12. The SOD1 RNAi agent of claim 11, 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).

13. The SOD1 RNAi agent of any one of claims 10-12, wherein P comprises an immunoglobulin Fc region comprising cysteine at residue 378 (according to the EU Index numbering).

14. The SOD1 RNAi agent of any one of claims 10-13, wherein the immunoglobulin 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).

15. The SOD1 RNAi agent of any one of claims 1-7, 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.

16. The SOD1 RNAi agent of any one of claims 1-14, 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.

17. The SOD1 RNAi agent of any one of claims 1-14, 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.

18. The SOD1 RNAi agent of claim 8 or 9, wherein the half-life extender is a VHH that binds HSA.

19. The SOD1 RNAi agent of claim 18, wherein the VHH comprises CDR1 comprising SEQ ID NO: 20, CDR2 comprising SEQ ID NO: 21, and CDR3 comprising SEQ ID NO:

22.

20. The SOD1 RNAi agent of claim 18 or 19, wherein the VHH comprises SEQ ID NO:

23.

21. The SOD1 RNAi agent of any one of claims 18-20, 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:

10.

22. The SOD1 RNAi agent of any one of claims 1-14, 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.

23. The SOD1 RNAi agent of claim 22, 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.

24. The SOD1 RNAi agent of claim 22 or 23, 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.

25. The SOD1 RNAi agent of any one of claims 1-24, wherein L is a SMCC linker, OD linker, or MSPT linker.

26. The SOD1 RNAi agent of any one of claims 1-25, wherein L is a MSPT linker.

27. The SOD1 RNAi agent of any one of claims 1-26, wherein P is linked to the 3’ end of the sense strand of dsRNA via the linker.

28. The SOD1 RNAi agent of any one of claims 1-27, wherein one or more nucleotides of the sense strand are modified nucleotides.

29. The SOD1 RNAi agent of claim 28, wherein each nucleotide of the sense strand is a modified nucleotide.

30. The SOD1 RNAi agent of any one of claims 1-29, wherein one or more nucleotides of the antisense strand are modified nucleotides.

31. The SOD1 RNAi agent of claim 30, wherein each nucleotide of the antisense strand is a modified nucleotide.

32. The SOD1 RNAi agent of any one of claims 28-31, wherein the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, 2’ deoxy nucleotide (DNA), or 2'-O-C16 alkyl modified nucleotide.

33. The SOD1 RNAi agent of any one of claims 28-32, wherein the sense strand has four 2'- fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5’ end of the sense strand.

34. The SOD1 RNAi agent of claim 33, wherein nucleotides at positions other than positions 7, 9, 10, and 11 of the sense strand are 2'-O-methyl modified nucleotides.

35. The SOD1 RNAi agent of any one of claims 28-34, 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.

36. The SOD1 RNAi agent of claim 35, wherein nucleotides at positions other than positions 2, 6, 14 and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

37. The SOD1 RNAi agent of any one of claims 28-32, wherein the sense strand has three 2'- fluoro modified nucleotides at positions 9, 10, and 11 from the 5’ end of the sense strand.

38. The SOD1 RNAi agent of claim 37, wherein nucleotides at positions other than positions 9, 10, and 11 of the sense strand are 2'-O-methyl modified nucleotides.

39. The SOD1 RNAi agent of any one of claims 28-34, 37, 38, 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.

40. The SOD1 RNAi agent of claim 39, wherein nucleotides at positions other than positions 2, 5, 7, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

41. The SOD1 RNAi agent of any one of claims 28-34, 37, 38, 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.

42. The SOD1 RNAi agent of claim 41, wherein nucleotides at positions other than positions 2, 5, 8, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

43. The SOD1 RNAi agent of any one of claims 28-34, 37, 38, wherein the antisense strand has five 2'-fluoro modified nucleotides at positions 2, 3, 7, 14, and 16 from the 5’ end of the antisense strand.

44. The SOD1 RNAi agent of claim 43, wherein nucleotides at positions other than positions 2, 3, 7, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

45. The SOD1 RNAi agent of any one of claims 28-34, 37, 38, wherein the antisense strand has three 2'-fluoro modified nucleotides at positions 2, 14, and 16 from the 5’ end of the antisense strand.

46. The SOD1 RNAi agent of claim 45, wherein nucleotides at positions other than positions 2, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

47. The SOD1 RNAi agent of any one of claims 1-46, wherein the sense strand and the antisense strand have one or more modified internucleotide linkages.

48. The SOD1 RNAi agent of claim 47, wherein the modified internucleotide linkage is phosphorothioate linkage.

49. The SOD1 RNAi agent of claim 47 or 48, wherein the sense strand has four or five phosphorothioate linkages.

50. The SOD1 RNAi agent of any one of claims 47-49, wherein the antisense strand has four or five phosphorothioate linkages.

51. The SOD1 RNAi agent of any one of claims 1-50, wherein the antisense strand has a phosphate analog at the 5’ end.

52. The SOD1 RNAi agent of claim 51, wherein the phosphate analog is 5’- vinylphosphonate.

53. The SOD1 RNAi agent of any one of claims 1-52, wherein the sense strand or antisense strand comprises an abasic moiety or inverted abasic moiety.

54. The SOD1 RNAi agent of any one of claims 1-53, 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: 33, and the antisense strand comprises SEQ ID NO: 34, (b) the sense strand comprises SEQ ID NO: 35, and the antisense strand comprises SEQ ID NO: 36, (c) the sense strand comprises SEQ ID NO: 37, and the antisense strand comprises SEQ ID NO: 38, (d) the sense strand comprises SEQ ID NO: 62, and the antisense strand comprises SEQ ID NO: 63, (e) the sense strand comprises SEQ ID NO: 64, and the antisense strand comprises SEQ ID NO: 65,(f) the sense strand comprises SEQ ID NO: 66, and the antisense strand comprises SEQ ID NO: 67, (g) the sense strand comprises SEQ ID NO: 68, and the antisense strand comprises SEQ ID NO: 69, and (h) the sense strand comprises SEQ ID NO: 70, and the antisense strand comprises SEQ ID NO:

71.

55. The SOD1 RNAi agent of any one of claims 1-54, wherein the sense strand and the antisense strand consist of a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand consists of SEQ ID NO: 33, and the antisense strand consists of SEQ ID NO: 34, (b) the sense strand consists of SEQ ID NO: 35, and the antisense strand consists of SEQ ID NO: 36, (c) the sense strand consists of SEQ ID NO: 37, and the antisense strand consists of SEQ ID NO: 38, (d) the sense strand consists of SEQ ID NO: 62, and the antisense strand consists of SEQ ID NO: 63, (e) the sense strand consists of SEQ ID NO: 64, and the antisense strand consists of SEQ ID NO: 65, (f) the sense strand consists of SEQ ID NO: 66, and the antisense strand consists of SEQ ID NO: 67, (g) the sense strand consists of SEQ ID NO: 68, and the antisense strand consists of SEQ ID NO: 69, and (h) the sense strand consists of SEQ ID NO: 70, and the antisense strand consists of SEQ ID NO:

71.

56. A pharmaceutical composition comprising the SOD1 RNAi agent of any one of claims 1- 55 and a pharmaceutically acceptable carrier.

57. A method of treating a SOD1-mediated neurological disease in a patient in need thereof, the method comprising administering to the patient an effective amount of the SOD1 RNAi agent of any one of claims 1-55, or the pharmaceutical composition of claim 56.

58. The method of claim 57, wherein the SOD1-mediated neurological disease is amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease), Down's syndrome (DS), or Progressive Spastic Tetraplegia and Axial Hypotonia (STAHP).

59. The method of any one of claims 57-58, wherein the SOD1 RNAi agent is administered to the patient intrathecally, intravenously or subcutaneously.

60. The SOD1 RNAi agent of any one of claims 1-55, or the pharmaceutical composition of claim 56, for use in a therapy.

61. The SOD1 RNAi agent of any one of claims 1-55, or the pharmaceutical composition of claim 56, for use in the treatment of a SOD1-mediated neurological disease.

62. The SOD1 RNAi agent or pharmaceutical composition for use of claim 61, wherein the SOD1-mediated neurological disease is amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease), Down's syndrome (DS), or Progressive Spastic Tetraplegia and Axial Hypotonia (STAHP).

63. Use of the SOD1 RNAi agent of any one of claims 1-55 in the manufacture of a medicament for treating a SOD1-mediated neurological disease.

64. The use of claim 63, wherein the SOD1-mediated neurological disease is amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease), Down's syndrome (DS), or Progressive Spastic Tetraplegia and Axial Hypotonia (STAHP).

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