Compositions and methods for modulating synuclein expression

Abstract

Provided herein are SNCA antisense oligonucleotides (ASOs) and SNCA ASO conjugates for modulating expression of a SNCA target nucleic acid. Also provided herein are methods of use thereof.

Description

Compositions and Methods for Modulating Synuclein Expression CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Application No. 63 / 736,857, filed December 20, 2024, which is incorporated herein by reference.SEQUENCE LISTING

[0002] The Sequence Listing written in file DNL-047-02_SeqList.xml is 113,109 bytes in size, was created December 16, 2025, and is hereby incorporated by reference.BACKGROUND

[0003] Neurodegenerative diseases, including Parkinson's disease (PD), pose a significant and growing health challenge globally. PD is a complex progressive neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra, leading to motor dysfunction, tremors, rigidity, and postural instability. The exact etiology of PD remains elusive, but increasing evidence suggests that aberrant aggregation and accumulation of alpha-synuclein protein play a pivotal role in the pathogenesis of the disease.

[0004] Alpha-synuclein is intricately involved in the regulation of synaptic function and neurotransmitter release. However, under pathological conditions. SNCA undergoes misfolding and aggregation and forms insoluble fibrils known as Lewy bodies, which are the hallmark neuropathological features of PD and other neurodegenerative disorders such as multiple system atrophy (MSA). The abnormal aggregation of SNCA is believed to contribute to neuronal dysfunction and cell death, ultimately leading to the clinical manifestations observed in affected individuals.

[0005] Current therapeutic strategies for PD primarily focus on alleviating symptoms and enhancing dopaminergic neurotransmission; however, these approaches do not address the underlying mechanisms driving disease progression.

[0006] In vivo delivery of nucleic acid-based molecules, such as antisense oligonucleotides, often requires specific targeting to reach certain tissues or cell types. In particular, delivery to non-hepatic tissues remains an obstacle and has limited the use of such therapies. Delivery of oligonucleotides to the central nervous system (CNS) poses a distinct problem due to the blood brain barrier (BBB). One means to deliver oligonucleotides into the CNS is by intrathecal delivery. However, intrathecal delivery is invasive, has a higher risk of side-effects, and often leads to uneven distribution.

[0007] There is, therefore, a pressing need for innovative therapeutics that directly target the pathological processes associated with SNCA and its aggregation and the delivery of such therapeutics in vivo, particularly to CNS tissues.SUMMARY

[0008] Described herein are SNCA ASO conjugates comprising at least one SNCA antisense oligonucleotide (ASO) and a transferrin receptor (TfR)-targeting Fc polypeptide dimer, wherein an Fc polypeptide of the Fc polypeptide dimer is modified to bind to TfR. TfR is highly expressed on the blood-brain barrier (BBB), and TfR naturally moves transferrin from the blood into the brain. The described SNCA ASO conjugates bind TfR, are transported across the BBB. and transport the attached SNCA ASO across the BBB. In some embodiments, the TfR-targeting Fc polypeptide dimer further comprises at least one cysteine substitution to facilitate attachment of the SNCA ASO.

[0009] Described are SNCA antisense oligonucleotides (ASOs) comprising a nucleic acid sequence:(a) +[%C]$+A$+[%C]$dA*dT*dT$dG$dG$dA$dA*dC*dT*dG*dA*dG$dC$+A$+[%C]$+T (SEQ ID NO: 111);(b) +GS+TS+TSdA*dA*dA*dT*dCSdT$dASdGSdT*dT*dG$dT$+[%C]$+[%C]$+A(SEQ ID NO: 112);(c) +T$+[%C]$+T$dC*dT$dA$dT$dA*dT*dA*dA*dC*dA*dT$dC$+A$+[%C]$+T(SEQ ID NO: 113);(d) +AS+AS+[%C]SdT*dGSdC$dT$dT$dA$dG*dT*dG*dA*dT$dT$+[%C]$+[%C]S+A (SEQ ID NO: 114); or(e) +GS+GS+TSdASdASdC*dT*dT*dA*dG*dG*dA*dC*dASdAS+GS+G$+T(SEQ ID NO: 115);wherein +A, +[%C], +G, and +T are, respectively, locked adenosine, locked -methylcytosine, locked guanosine, and locked thymine nucleosides; dA, dC, dG, and dT are, respectively, deoxyadenosine, deoxycytidine, deoxyguanidine, and deoxythymidine nucleosides; each * indicates a phosphorothioate (PS) internucleoside linkage; and each $ is a stabilizing internucleoside linkage or a PS internucleoside linkage. In some embodiments, each $ is independently a PS internucleoside linkage, a phosphorodithioate (PS2) internucleoside linkage, an mesylphosphoramidate (MsPA) internucleoside linkage, or a phosphorylguanidine (PN) internucleoside linkage. In some embodiments, each internucleoside linkage is a PS internucleoside linkage. In some embodiments, one or more of $ is a stabilizing internucleosidelinkage. In some embodiments, one or more of $ is a PN linkage. In some embodiments, one or more of $ is a PS2 linkage. In some embodiments, one or more of $ is a MsPA linkage. In some embodiments, one or more of $ is a PS linkage. In some embodiments, two or more of $ is a stabilizing internucleoside linkage. In some embodiments, two or more of $ is a PN linkage. In some embodiments, two or more of $ is a PS2 linkage. In some embodiments, two or more of $ is a MsPA linkage. In some embodiments, two or more of $ is a PS linkage. In some embodiments, three or more of $ is a stabilizing internucleoside linkage. In some embodiments, three or more of $ is a PN linkage. In some embodiments, three or more of $ is a PS2 linkage. In some embodiments, three or more of $ is a MsPA linkage. In some embodiments, three or more of $ is a PS linkage. In some embodiments, four or more of $ is a stabilizing internucleoside linkage. In some embodiments, four or more of $ is a PN linkage. In some embodiments, four or more of $ is a PS2 linkage. In some embodiments, four or more of $ is a MsPA linkage. In some embodiments, four or more of $ is a PS linkage. In some embodiments, five or more of $ is a stabilizing internucleoside linkage. In some embodiments, five or more of $ is a PN linkage. In some embodiments, five or more of $ is a PS2 linkage. In some embodiments, five or more of $ is a MsPA linkage. In some embodiments, five or more of $ is a PS linkage. In some embodiments, six or more of $ is a stabilizing internucleoside linkage. In some embodiments, six or more of $ is a PN linkage. In some embodiments, six or more of $ is a PS2 linkage. In some embodiments, six or more of $ is a MsPA linkage. In some embodiments, six or more of $ is a PS linkage. In some embodiments, seven or more of $ is a stabilizing internucleoside linkage. In some embodiments, seven or more of $ is a PN linkage. In some embodiments, seven or more of $ is a PS2 linkage. In some embodiments, seven or more of $ is a MsPA linkage. In some embodiments, seven or more of $ is a PS linkage. In some embodiments, eight or more of $ is a stabilizing internucleoside linkage. In some embodiments, eight or more of $ is a PN linkage. In some embodiments, eight or more of $ is a PS2 linkage. In some embodiments, eight or more of $ is a MsPA linkage. In some embodiments, eight or more of $ is a PS linkage.

[0010] In some embodiments, 0, 1. 2, or 3 internucleoside linkages at the 5' end (linkages between nucleosides I and 2, 2 and 3, and 3 and 4) of the SNCA ASO are stabilizing internucleoside linkages. In some embodiments, 0, 1, 2, or 3 internucleoside linkages at the 5' end of the SNCA ASO are PN linkages. In some embodiments, 0, 1, 2, or 3 internucleoside linkages at the 5' end of the SNCA ASO are PS2 linkages. In some embodiments, 0, 1, 2, or 3 internucleoside linkages at the 5' end of the SNCA ASO are MsPA linkages.

[0011] In some embodiments, 0, 1, 2, 3, or 4 internucleoside linkages at the 3' end (linkages between nucleosides n and n-1. n-1 and n-2, n-2 and n-3. n-3, and n-4, wherein n is the number of nucleosides in the ASO) of the SNCA ASO are stabilizing internucleoside linkages. In some embodiments, 0, 1, 2, 3, or 4 internucleoside linkages at the 3' end of the SNCA ASO are PN linkages. In some embodiments, 0, 1, 2, 3, or 4 internucleoside linkages at the 3' end of the SNCA ASO are PS2 linkages. In some embodiments, 0. 1, 2. 3, or 4 internucleoside linkages at the 3' end of the SNCA ASO are MsPA linkages.

[0012] In some embodiments, the SNCA ASO contains 0, 1, 2, or 3 stabilizing internucleoside linkages between nucleotides 4 and n-4, wherein n is the number of nucleosides in the ASO. In some embodiments, the SNCA ASO contains 0, 1, 2, or 3 PN linkages between nucleotides 4 and n-4. In some embodiments, the SNCA ASO contains 0, 1, 2, or 3 PS2 linkages between nucleotides 4 and n-4. In some embodiments, the SNCA ASO contains 0, 1, 2, or 3 MsPA linkages between nucleotides 4 and n-4.

[0013] In some embodiments, the SNCA ASO comprises the sequence and modifications of any of the modified SNCA ASOs provided in Table 1.

[0014] Described are SNCA ASOs conjugated to a transferrin receptor (TfR)-targeting Fc polypeptide dimer. Any of the described SNCA ASOs can be conjugated to the TfR-targeting Fc polypeptide dimer.

[0015] In some embodiments, the TfR-targeting Fc polypeptide dimer comprises a first Fc polypeptide and a second Fc polypeptide, wherein the second Fc polypeptide comprises a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) (z.e., comprises modifications that create a TfR-binding site within the constant domain) and wherein the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide. In some embodiments, the TfR-targeting Fc polypeptide dimer comprises a first Fc polypeptide that does not contain a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) and a second Fc polypeptide comprising a modified constant domain that specifically binds to TfR, wherein first and second Fc polypeptide form an Fc dimer. In some embodiments, the first Fc polypeptide, the second Fc polypeptide, or both the first and second Fc polypeptides are modified to reduce effector function. In some embodiments, the first Fc polypeptide and the second Fc polypeptide are modified to reduce effector function. In some embodiments, the first Fc polypeptide comprises a C at position 239, an A at position 234, an A at position 235, and a S position 329, wherein the positions are according to EU numbering. In some embodiments, the second Fc polypeptide comprising an A at position 234, an A atposition 235, and S at position 329, wherein the positions are according to EU numbering and comprises a sequence having at least 90% sequence identity to SEQ ID NO: 12.

[0016] In some embodiments, the TfR-targeting Fc polypeptide dimer further comprises anon-targeting Fab (NTF) fused to the first Fc polypeptide via ahinge region to form a Fab-Fc fusion polypeptide. In some embodiments, the TfR-targeting Fc polypeptide dimer further comprises anon-targeting Fab (NTF) fused to the second Fc polypeptide via a hinge region to form a Fab-Fc fusion polypeptide.

[0017] In some embodiments, the TfR-targeting Fc polypeptide dimer comprises a first nontargeting Fab (NTF) fused to the first Fc polypeptide via a first hinge region to form a first Fab-Fc fusion polypeptide, and a second NTF fused to the second Fc polypeptide via a second hinge region to form a second Fab-Fc fusion polypeptide wherein the first and second Fab-Fc fusion polypeptides form a Fab-Fc dimer. In some embodiments, the first and second NTFs each comprise SEQ ID NO: 109 or 110. In some embodiments, the first and second NTFs each comprise a heavy chain segment comprising SEQ ID NO: 109 or 110 and a light chain comprising SEQ ID NO: 108. In some embodiments, the first and second NTFs each comprise a heavy chain segment comprising SEQ ID NO: 130 or 131 and a light chain comprising SEQ ID NO:129. In some embodiments, the hinge regions each comprise SEQ ID NO: 121.

[0018] In some embodiments, the TfR-targeting Fc polypeptide dimer further comprises anon-binding variable region (NVBR) fused to the first Fc polypeptide via a hinge region to form a NVBR-Fc fusion polypeptide. In some embodiments, the TfR-targeting Fc polypeptide dimer further comprises a NVBR fused to the second Fc polypeptide via a hinge region to form a NBVR-Fc fusion polypeptide. In some embodiments, the NVBRs each comprise the variable region of SEQ ID NO: 109 or 110. In some embodiments, the hinge regions each comprise SEQ ID NO: 121.

[0019] In some embodiments, the TfR-targeting Fc polypeptide dimer comprises a first NVBR fused to the first Fc polypeptide via a first hinge region to form a first NVBR-Fc fusion polypeptide and a second NVBR fused to the second Fc polypeptide via a second hinge region to form a second NVBR-Fc fusion polypeptide wherein the first and second NVBR-Fc fusion polypeptides form a NVBR-Fc dimer. In some embodiments, the first and second NVBRs each comprise SEQ ID NO: 109 or 110. In some embodiments, the hinge regions each comprise SEQ ID NO: 121.

[0020] Also described are nucleic acid sequences encoding the first Fc polypeptide and the second Fc polypeptide. Also described are nucleic acid sequences encoding the first Fab-Fc polypeptide, the second Fab-Fc polypeptide, and the NTF light chain. Also described arenucleic acid sequences encoding the first NVBR-Fc polypeptide and the second NVBR-Fc polypeptide.

[0021] The SNCA ASO is linked to the TfR-targeting Fc polypeptide dimer via a linking group. The linking group can be any linking group available in the art that is suitable for linking an oligonucleotide to a polypeptide. The SNCA ASO can be linked to the first Fc polypeptide, the second Fc polypeptide, the first NTF (if present), and / or the second NTF (if present). In some embodiments, the SNCA ASO is covalently linked to the first Fc polypeptide, the second Fc polypeptide, the first NTF (if present), and / or the second NTF (if present). In some embodiments, the SNCA ASO is linked to the first Fc polypeptide at a cysteine at position 239 (according to EU numbering). In some embodiments, the first Fc polypeptide comprises a CHI domain and the SNCA ASO is linked the first Fc polypeptide at a cysteine at position 114 (according to Kabat numbering) or position 124 (according to EU numbering (i.e., the CHI domain comprises a Al 14C substitution or an S124C substitution).

[0022] In some embodiments, the SNCA ASO in linked to the Fc polypeptide dimer via the 5' end of the SNCA ASO. In some embodiments, the SNCA ASO in linked to the first Fc polypeptide via the 5' end of the SNCA ASO. In some embodiments, the first Fc polypeptide is linked to the SNCA ASO via a linking group attached to the cysteine at position 239 and the 5' end of the SNCA ASO, wherein the linking group comprises:

[0023] In some embodiments, the modified constant domain of the second Fc polypeptide comprises: a glutamate (E), leucine (L), serine (S), valine (V), tryptophan (W), or tyrosine (Y) at position 153; a Y, phenylalanine (F), W, methionine (M), proline (P), or V at position 157; a threonine (T), an asparagine (N), or V at position 159; an E, Isoleucine (I), P, or V at position 160; a W at position 161; an alanine (A), I, V, serine (S), or T at position 162; aN, S, arginine (R), or T at position 163; a T, histidine (H), or S at position 186; an E. S, aspartate (D), glycine (G), T, P, glutamine (Q), or R at position 188; an E or R at position 1 9; a Q at position 191; a Q at position 192; aF, H, lysine (K), Y, orW at position 194; a S, T, orW at position 197; and a S, C, P, M, or W at position 199, and as numbered with reference to SEQ ID NO: 1.

[0024] In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E, L, S, V, W, or Y at position 380; a Y, F, W, M, P, or V at position 384; a T,N, or V at position 386; an E, I, P, or V at position 387; a W at position 388: an A, I, V, S, or T at position 389; aN, S. R, or T at position 390; a T, H, or S at position 413; an E, S, D, G, T, P, Q, or R at position 415; an E or R at position 416; a Q at position 418; a Q at position 419; a F, H, K, Y, or W at position 421; a S, T, or W at position 424; and a S, C, P, M, or W at position 426, according to EU numbering.

[0025] In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E at position 153; a Y at position 157; a T at position 159; an E at position 160; a W at position 161; a A at position 162; a N at position 163; a T at position 186; an E at position 188; an E at position!89; a Q at position 191; a Q at position 192; a F at position 194; a S at position 197; and a S at position 199, and as numbered with reference to SEQ ID NO: 1.

[0026] In some embodiments, the modified constant domain of the second Fc polypeptide comprises a E at position 380, a Y at position 384, a T at position 386, a E at position 387, a W at position 388, an A at position 389, an N at position 390, a T at position 413, a E at position 415, a E at position 416, and a F at position 421, according to EU numbering.

[0027] In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E at position 380; a Y at position 384; a T at position 386; an E at position 387; a W at position 388; an A at position 389; a N at position 390; a T at position 413; an E at position 415; an E at position 416; a Q at position 418; a Q at position 419; a F at position 421; a S at position 424; and a S at position 426, according to EU numbering.

[0028] In some embodiments, the second Fc polypeptide further comprises a W at position 366, according to EU numbering and the first Fc polypeptide further comprises a S a position 366, an A at position 368, and a V at position 407, according to EU numbering. In some embodiments, the second Fc polypeptide further comprises a S a position 366, an A at position 368, and a V at position 407, according to EU numbering, and the first Fc polypeptide further comprises a W at position 366, according to EU numbering.

[0029] In some embodiments, the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO:63 or 80. In some embodiments, the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 12, and the first Fc polypeptide compnses the amino acid sequence of SEQ ID NO:63 or 80.

[0030] In some embodiments, the first and second NTFs each comprise a heavy chain variable region comprising SEQ ID NO: 109 or 110 and a light chain variable region comprising SEQ ID NO: 108. In some embodiments, the first and second NTFs each comprise a heavy chainvariable region comprising SEQ ID NO: 130 or 131 and a light chain variable region comprising SEQ ID NO: 129.

[0031] In some embodiments, the first Fab-Fc fusion polypeptide comprises SEQ ID NO: 100 or 101 and the second Fab-Fc fusion polypeptide comprises SEQ ID NO:98 or 99.

[0032] Certain embodiments provide a pharmaceutical composition comprising a SNCA ASO conjugate as described herein and a pharmaceutically acceptable excipient.

[0033] Certain embodiments provide a pharmaceutical composition comprising a SNCA ASO conjugate as described herein and a pharmaceutically acceptable carrier or diluent.

[0034] Certain embodiments provide a method of generating a neuronal cell with decreased Synuclein expression, the method comprising delivering to the neuron cell a SNCA ASO conjugate as described herein, wherein the SNCA ASO decreases the expression level of an endogenous SNCA gene. The neuronal cell can be, but is not limited to, a brain cell, a deep brain cell, or a spinal cord cell.

[0035] Certain embodiments provide a method of modifying a neuronal cell to decrease Synuclein expression, the method comprising delivering to the neuron cell a SNCA ASO conjugate as described herein, wherein the SNCA ASO decreases the expression level of an endogenous SNCA gene. The neuronal cell can be, but is not limited to, a brain cell, a deep brain cell, or a spinal cord cell.

[0036] Certain embodiments provide a method of delivering a SNCA ASO to the CNS or cell of the CNS of a human subject in need thereof, comprising administering to the subject a SNCA ASO conjugate as described herein.

[0037] Delivery of a SNCA ASO to a neuronal cell using the described SNCA ASO conjugates can be used to treat a neurodegenerative disorder. Delivery of a described SNCA ASO to a neuronal cell or the can be used to treat a synucl ein-associated neurodegenerative disorder (z. e., a synucleinopathy). The neurodegenerative disorder can be, but is not limited to, Alzheimer’s disease, Parkinson's disease (PD), dementia with Lewy bodies (DLB, i.e., Lewy body dementia), pure autonomic failure (PAF), multiple system atrophy (MSA), and REM sleep behavior disorder (RBD).

[0038] Certain embodiments provide a method of reducing SNCA messenger ribonucleic acid (mRNA) expression or levels in a human subject in need thereof, the method comprising administering to the human subject an SNCA ASO conjugate as described herein.

[0039] Certain embodiments provide a method of reducing SNCA messenger ribonucleic acid (mRNA) expression or levels in the brain a human subject, the method comprising administering to the human subject an SNCA ASO conjugate as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1A-B. Graphs illustrating Multi dose PK and KD (plasma concentration) following intravenous delivery in hSNCA: TfR KI mice.

[0041] FIG. 1C-D. Graphs illustrating Multidose PK and KD (brain concentration) following intravenous delivery in hSNCA: TfR KI mice.

[0042] FIG. 1E-F. Graphs illustrating Multidose PK and KD (target gene knockdown in brain) following intravenous delivery in hSNCA: T! R KI miceDETAILED DESCRIPTIONI. Definitions

[0043] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “an oligonucleotide” includes a plurality of oligonucleotides and the like. The conjunction “or” is to be interpreted in the inclusive sense, i.e., as equivalent to “and / or,” unless the inclusive sense would be unreasonable in the context.

[0044] In general, the term “about” indicates insubstantial variation in a quantity of a component of a composition not having any significant effect on the activity or stability of the composition. When the specification discloses a specific value for a parameter, the specification should be understood as alternatively disclosing the parameter at “about” that value. The terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations (e.g, standard margin of error of measurement (SEM)) from the value known to the skilled person in the art, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0045] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.

[0046] The use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” are not intended to be limiting and may include other elements not specifically recited. For example, a composition that “comprises” or “includes” an Fc polypeptide dimer may contain the Fc polypeptide dimer alone or in combination with other elements. It is to be understood that both the foregoing general description and detailed description are exemplar}' and explanatory only and are not restrictiveof the teachings. To the extent that any material incorporated by reference is inconsistent with the express content of this disclosure, the express content controls.

[0047] As used herein, the term “antibody’’ refers to a protein with an immunoglobulin fold that specifically binds to an antigen via its variable regions. The term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. The term “antibody,” as used herein, also includes antibody fragments that retain antigen-binding specificity, including but not limited to Fab, F(ab’)2, Fv, scFv, and bivalent scFv. Antibodies can contain light chains that are classified as either kappa or lambda. Antibodies can contain heavy chains that are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0048] The “EU numbering scheme” is generally used in the art when referring to a residue in an antibody heavy chain constant region. The EU numbering scheme is shown below with respect to SEQ ID NO:5 (Clone CH3C.35.23.2 knob):5 3 13 23 EU 230 240 250I I I PCPAPELLGGPSVFLFPPKPKDT5 33 43 53 63 73 EU 260 270 280 290 300I I I I I LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY5 83 93 103 113 123 EU 310 320 330 340 350I I I I I RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT5 133 143 153 163 173 EU 360 370 380 390 400I I I I I LPPSRDELTKNQVSLWCLVKGFYPSDLAVEWESYGTEWANYKTTPPVLDS5 183 193 203 213EU 410 420 430 440I l l i DGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLSLSPGK

[0049] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "‘variable light chain” (VL) and “variable heavy chain” (VH) refer to these light and heavy chains, respectively.

[0050] The term “variable region” or “variable domain” refers to a domain in an antibody heavy chain or light chain that is derived from a germline Variable (V) gene, Diversity7(D) gene, or Joining (J) gene (and not derived from a Constant (Cp and C5) gene segment), and that gives an antibody its specificity for binding to an antigen. Typically, an antibody variable region comprises four conserved “framework” regions interspersed with three hypervariable “complementarity7determining regions.”

[0051] The term “complementarity7determining region” or “CDR” refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy7chain variable regions. The CDRs are primarily responsible for antibody binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 or CDR-H3 is located in the variable region of the heavy chain of the antibody in which it is found, whereas a VL CDR1 or CDR-L1 is the CDR1 from the variable region of the light chain of the antibody in which it is found.

[0052] The “framework regions” or “FRs” of different light or heavy chains are relatively conserved within a species. The framework region of an antibody — the combined framework regions of the constituent light and heavy chains — serves to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBASE2” germline variable gene sequence database for human and mouse sequences.

[0053] The amino acid sequences of the CDRs and framework regions can be determined using various w ell-known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), AbM, and observed antigen contacts (“Contact”). In some embodiments, CDRs are determined according to the Contact definition. See, MacCallum et al.. J. Mol. Biol.262:732-745, 1996. In some embodiments, CDRs are determined by a combination of Kabat, Chothia, and / or Contact CDR definitions.

[0054] The term “Fd portion” refers to an N-terminal portion of an immunoglobulin heavy7chain. Ty pically, an Fd portion includes the heavy chain variable (VH) region and a heavy chain constant 1 (CHI) region.

[0055] The term “Fab” refers to an antigen-binding fragment consisting of a light chain variable region, a light chain constant region, a heavy chain variable region, and a heavy chain CHI constant region.

[0056] The term “single-chain variable fragment” or “scFv” refers to an antigen-binding fragment consisting of a heavy chain variable region and a light chain variable region linked together via a peptide linker. The linker can either connect the N-terminus of the VH with the C-terminus of the VL of the N-terminus of the VL with the C-terminus of the VH. An scFv lacks constant regions.

[0057] The term “epitope” refers to the area or region of an antigen to which a molecule, e.g., the CDRs of an antibody, specifically binds and can include a few amino acids or portions of a few amino acids, e.g., 5 or 6. or more, e.g., 20 or more amino acids, or portions of those amino acids. In some cases, the epitope includes non-protein components, e.g.. from a carbohydrate, nucleic acid, or lipid. In some cases, the epitope is a three-dimensional moiety. Thus, for example, where the target is a protein, the epitope can be comprised of consecutive amino acids (e.g, a linear epitope), or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g, a discontinuous or conformational epitope).

[0058] As used herein, the phrase “recognizes an epitope,” as used with reference to an antibody, means that the antibody CDRs interact with or specifically bind to the antigen at that epitope or a portion of the antigen containing that epitope.

[0059] The term “specifically binds” refers to a molecule (e.g, a Fab. or an scFv) that binds to an epitope or target with greater affinity, greater avidity, and / or greater duration to that epitope or target in a sample than it binds to another epitope or non-target compound (e.g, a structurally different antigen). In some embodiments, a Fab or an scFv that specifically binds to an epitope or target, is a Fab or an scFv that binds to the epitope or target with at least 5-fold greater affinity than other epitopes or non-target compounds, e.g, at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold, or greater affinity. The term “specific binding,” “specifically binds to,” or “is specific for” a particular epitope or target, as used herein, can be exhibited, for example, by a molecule having an equilibrium dissociation constant KD for the epitope or target to which it binds of, e.g., 10‘4M or smaller, e.g., 10‘5M, 10'6M, IO’7M, 10'8M, 10'9M, 10'10M, 10'11M, or 10'12M. It will be recognized by one of skill that a Fab or scFv that specifically binds to a target from one species may also specifically bind to orthologs of that target.

[0060] The term “binding affinity” is used herein to refer to the strength of a non-covalent interaction between two molecules, e.g., between a Fab or scFv and an antigen or epitope. Thus,for example, the term may refer to 1: 1 interaction between a Fab or scFv and an antigen, unless otherwise indicated or clear from context. Binding affinity’ may be quantified by measuring an equilibrium dissociation constant (KD), which refers to the dissociation rate constant (kd, time ') divided by the association rate constant (ka, time 'MKD can be determined by measurement of the kinetics of complex formation and dissociation, e.g., using Surface Plasmon Resonance (SPR) methods, e.g., a Biacore™ system; kinetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g.. using the ForteBio® Octet platform). As used herein, “binding affinity” includes not only formal binding affinities, such as those reflecting 1:1 interaction between a Fab or scFv and an antigen, but also apparent affinities for which Ki / s are calculated that may reflect avid binding.

[0061] Monoclonal antibodies and fragments thereof (including Fabs and conjugate), or other biological entities are typically provided in isolated form. An antibody (or fragment or conjugate thereof) is typically at least 50% w / w pure of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the antibody or Fab is combined with an excess of pharmaceutically acceptable carrier(s) or other vehicle intended to facilitate its use. Antibodies (or fragment or conjugate thereof) can be at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% w / w pure of interfering proteins and contaminants from production or purification. The isolated antibody (or fragment or conjugate thereof) can be the predominant macromolecular species remaining after its purification.

[0062] As used herein, the term “Fc region” refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide that is characterized by an Ig fold as a structural domain. An Fc polypeptide typically contains constant region sequences including at least the CH2 domain and / or the CH3 domain and may contain at least part of the hinge region. Two Fc peptides dimerize to from an Fc region or Fc fragment.

[0063] The terms “polypeptide” and “peptide” refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring ammo acid polymers and non-naturally occurring amino acid polymers. A “protein” can refer to either a polypeptide, a polypeptide dimer, or a polypeptide multimer. The single chain polypeptides of a protein dimer or multimer may be joined by a covalent bond (e.g., a disulfide bond) or by non-covalent interactions.

[0064] For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met,ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gin. his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Nonconservative substitutions constitute exchanging a member of one of these classes for a member of another.

[0065] The terms "identical” or percent "identity.” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one a sequence comparison algorithm or by manual alignment and visual inspection. For sequence comparison of polypeptides, typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment or using publicly available software using known algorithms to achieve maximal alignment. Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.

[0066] Percentage sequence identities are determined with antibody or Fc polypeptide sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain or Fc polypeptide) is being compared with the same region of a reference antibody, the percentage sequence identity between the subj ect and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.

[0067] Nucleic acid sequence identity’ can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspection, and the best alignment (i.e., resulting in the highestpercentage of sequence similarity' over a comparison window). Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of matched and mismatched positions not counting gaps in the window of comparison (z.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise indicated the window of comparison between two sequences is defined by the entire length of the shorter of the two sequences.

[0068] A modified internucleoside linkage is an internucleoside linkage other than a naturally occurring phosphate linkage. Shown below are modified internucleoside linkages linking the sugar groups (included in the structure) of nucleosides 5' and 3' of the modified internucleoside linkages.phosphorothioate (PS) phosphorodithioate (PS2) mesylphosphoramidate (MsPA)(a) phosphorylguanidine (b) phosphory Iguanidine (PN) As used herein, PN refers to structure (b).

[0069] As used herein “stabilizing internucleoside linkage” refers to a modified internucleoside linkage that is specifically introduced into the backbone of an oligonucleotide of an oligonucleotide polypeptide conjugate to remove a soft spot (e.g, nuclease sensitive site), increase resistance of the oligonucleotide to cleavage, or otherwise modulate one or more pharmacokinetic or pharmacodynamic properties of the oligonucleotide and / or oligonucleotidepolypeptide conjugate into which it is introduced. Stabilizing internucleoside linkages do not include phosphorothioates or phsophodiesters. Oligonucleotides and / or oligonucleotide polypeptide conjugates containing one or more stabilizing internucleoside linkages (stabilized oligonucleotides) have improved properties compared to an oligonucleotide or oligonucleotide polypeptide conjugate that has the same oligonucleotide sequence but does not contain the one or more stabilizing internucleoside linkages. Stabilized oligonucleotides and oligonucleotide polypeptide conjugates have improved properties over those having only phosphorothioate internucleoside linkages or a combination of phosphodiester and phosphorothioate internucleoside linkages. A stabilized oligonucleotide may contain phosphorothioate internucleoside linkage(s) and / or phosphodiester internucleoside linkage(s) in addition to the stabilizing internucleoside linkages. In some embodiments, one or more phosphorothioate and / or phosphodiester internucleoside linkages in an oligonucleotide (e.g., an ASO; e.g., a gapmer) are replaced by stabilizing internucleoside linkages. In some embodiments, the one or more phosphorothioate and / or phosphodi ester internucleoside linkages can be replaced by one or more stabilizing internucleoside linkages at or near a position identified in the oligonucleotide as being susceptible to cleavage (e.g., a soft spot). In some embodiments, one or more phosphorothioate and / or phosphodiester internucleoside linkages in a wing segment of a gapmer are replaced by stabilizing internucleoside linkages. In some embodiments, one or more phosphorothioate and / or phosphodiester internucleoside linkages in a gap segment of a gapmer are replaced by stabilizing internucleoside linkages. Examples of modified internucleoside linkages that can be used as a stabilizing internucleoside linkage include, but are not limited to, phosphorodithioate, phosphoramidates, mesylphosphoramidates, phosphonates, phosphotriesters, and phosphoryl guanidines (see: Nucleic Acids Res., 47, 5465-5479 (2019); Proc. Natl. Acad. Sci. U. S. A., 116. 1229-1234 (2019); Nucleic Acids Research, 50(10), 5401-5423 (2022); Vasquez G., Nucleic Acid Therapeutics, 32(1), 40-50 (2022); and Molecular Therapy: Nucleic Acids; 29: 176-188 (2022)). Additional examples of modified internucleoside linkages that can be used as a stabilizing internucleoside linkage include, but are not limited to, modified internucleoside linkage si, s2, s3, s4, s5, s6, s7, s8, s8, slO, si 1, sl2, s!3, sl4, s 15, s!6, s!7, or si 8 as described in WO 2017210647, the disclosure of which is incorporated by reference in its entirety. Further examples of modified internucleoside linkages that can be used as a stabilizing internucleoside linkage include, but are not limited to, modified internucleoside linkages nOOl, n002, n003, n004, n005, n006, n007, n008, n009, nOlO. n020, n025, or n026 as described in WO 2022099159, the disclosure of which is incorporated by reference in its entirety. In some embodiments, the stabilizinginternucleoside linkage are selected from the group consisting of PS2, MsPA, and PN internucleoside linkages.

[0070] “LNA” refers to a bicyclic nucleoside analogue which comprises a bridge between the 2' and 4' position in the ribose ring (2' to 4' bicyclic nucleotide analogue), and is known as “Locked Nucleic Acid” or “Locked Nucleoside.” As used herein, “LNA oligonucleotide,” refers to an oligonucleotide containing one or more such bicyclic nucleoside analogues. Biochemistry, 43(42): 13233-13240 (2004). In some embodiments, a LNA provided herein has the following structure, wherein “Base” is a nucleobase:

[0071] “Expression” refers to the transcription and / or translation of an endogenous gene, heterologous gene or nucleic acid segment, or a transgene in cells. For example, expression may refer to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein.

[0072] The term “pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

[0073] The terms “subject,” “individual,” and “patient,” refer to a mammal, including, but not limited to, a human, a non-human primate, a rodent (e.g, rat, mouse, and guinea pig), a rabbit, a cow, a pig, a horse, and other mammalian species. In some embodiments, the subject is a human. The term “subject” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0074] The term “disease” or “condition” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.

[0075] The terms “treat,” “treatment,” and the like, mean the methods or steps taken to provide relief from or alleviation of the number, severity, and / or frequency of one or more symptoms of a disease or condition in a subject. Treating generally refers to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily haveto be, prophylactic in terms of preventing or partially preventing a disease, symptom, or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom, or adverse effect attributed to the disease, disorder, or condition. The term treatment can include: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. Treating can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with disease or condition or those in which disease or condition is to be prevented. Treating can include inhibiting the disease, disorder, or condition, e.g.. impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the symptom without affecting or removing an underlying cause of the symptom. The treatment or amelioration of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment.

[0076] A '“pharmacologically effective amount,’7“therapeutically effective amount.” or simply “effective amount” refers to that amount (dose) of a described active pharmaceutical ingredient or pharmaceutical composition to produce the intended pharmacological, therapeutic, or preventive result. An “effective amount” can also refer to the amount of, for example an excipient, in a pharmaceutical composition that is sufficient to achieve the desired property of the composition. An effective amount can be administered in one or more administrations, applications, or dosages.II. Overview

[0077] Oligonucleotide therapies for CNS disorders caused by genetic abnormalities or increased protein accumulation are becoming an increasingly popular approach to modulate gene expression of such neurological disorders. The blood brain barrier (BBB) represents a challenge to the delivery’ of systemically administered oligonucleotides to the relevant sites of action within the CNS. Intrathecal (IT) delivery, in which drugs are administered directly into the cerebrospinal fluid (CSF) space, enables bypass of the BBB. However, one limitation ofthis approach is that delivery of these oligonucleotide therapies directly to the CSF via the IT approach does not achieve uniform distribution throughout the CNS.

[0078] Described are SNCA ASO conjugates that can be administered to a subject intravenously and facilitate transport of the SNCA ASO across the BBB and delivery of the SNCA ASO to CNS cells, where the SNCA ASO provides knockdown of SNCA expression in the brain and spinal cord. The SNCA ASO conjugates comprise a transferrin-targeting Fc polypeptide dimer linked to a SNCA ASO. Conjugation of a SNCA ASO to an Fc polypeptide dimer can be used to increase targeting efficacy of the molecule following administration into a subject. The Fc polypeptide dimer-cargo molecule conjugate has increased targeting efficacy compared to unconjugated molecule. The SNCA ASO comprises modifications that decrease in vivo plasma clearance and / or degradation / cleavage when the SNCA ASO is linked to the transferrin-targeting Fc polypeptide dimer.

[0079] In some embodiments, the described SNCA ASO conjugates provide for knockdown of SNCA expression across CNS regions including across brain regions that include deep brain regions, as well as the frontal lobe, parietal lobe, temporal lobe, occipital lobe, and cerebellum. In some embodiments, the described SNCA ASO conjugates provide knockdown of SNCA expression across multiple CNS cell types, including endothelial cells, neurons, astrocytes, oligodendrocytes, and microglia. In some embodiments, the described SNCA ASO conjugates provide for knockdown of SNCA expression in the spinal cord.

[0080] The described SNCA ASO conjugates provide for targeted delivery of therapeutic levels of SNCA ASOs to various tissues, including the CNS, following systemic administration. This targeted delivery allows for the use of a low er dose of the SNCA ASO compared with administration of the SNCA ASO alone, i.e., non-targeted delivery.

[0081] Described are SNCA ASO conjugates comprising a transferrin receptor (TfR)-targeting Fc polypeptide dimer and a SNCA antisense oligonucleotide (ASO), wherein:(a) the TfR-targeting Fc polypeptide dimer comprises:(i) a first Fc polypeptide comprising a C at position 239, an A at position 234, an A at position 235, and a S position 329, each according to EU numbering;(ii) a second Fc polypeptide comprising an A at position 234, an A at position 235, and S at position 329, each according to EU numbering, and a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) wherein the second Fc polypeptide comprises a sequence having at least 90% sequence identity to SEQ ID NO:11 or 12, wherein second Fc polypeptide forms an Fc dimer with the first Fc polypeptide; and(iii) a first non-targeting Fab (NTF) fused to the first Fc polypeptide via a first hinge region to form a first Fab-Fc fusion polypeptide and a second NTF fused to the second Fc polypeptide via a second hinge region to form a second Fab-Fc fusion polypeptide, wherein first and second NTFs each comprise SEQ ID NO: 109 or 110, wherein the hinge regions each comprise SEQ ID NO: 121; and(b) the SNCA ASO compnses: 5' +[%C]$+A$+[%C]$dA*dT*dT$dG$dG$dA SdA*dC*dT*dG*dA*dG$dC$+A$+[%C]$+T 3' (SEQ ID NO: 111)wherein+A, +[%C], and +T are locked adenosine, locked 5-methylcytosine, and locked thymine nucleosides, respectively,dA, dC. dG, and dT are deoxyadenosine, deoxycytidine, deoxy guanidine, and deoxythymidine nucleosides, respectively,each * indicates a phosphorothioate (PS) internucleoside linkage; and each $ is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkage; wherein the first Fc polypeptide is linked to the SNCA ASO via a linking group. In some embodiments, each $ is a stabilizing internucleoside linkage (e g., PS2, PN, or MsPA). In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E at position 380; a Y at position 384; a T at position 386; an E at position 387; a W at position 388; an A at position 389; a N at position 390; a T at position 413; an E at position 415; an E at position 416; a Q at position 418; a Q at position 419; a F at position 421; a S at position 424; and a S at position 426, according to EU numbering. In some embodiments, the SNCA ASO is linked to the cysteine at position 239 (EU numbering) of the first Fc polypeptide. In some embodiments, the 5' end of the SNCA ASO is linked to the cysteine at position 239 of the first Fc polypeptide, wherein the linking group is:In some embodiments, the NTF comprises SEQ ID NO: 109 or 110 and SEQ ID NO: 108. In some embodiments, the NTF comprises SEQ ID NO:130 or 131 and SEQ ID NO:129.

[0082] Described are SNCA ASO conjugates comprising a transferrin receptor (TfR.)-targeting Fc polypeptide dimer and a SNCA antisense oligonucleotide (ASO), wherein:(a) the TfR-targeting Fc polypeptide dimer comprises(i) a first Fc polypeptide comprising a cysteine at position 239, an A at position 234. an A at position 235. and a S position 329, each according to EU numbering.(ii) a second Fc polypeptide comprising an A at position 234, an A at position 235, and S at position 329, each according to EU numbering, and a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) wherein the second Fc polypeptide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 11 or 12, wherein second Fc polypeptide forms an Fc dimer with the first Fc polypeptide,(iii) a first non-targeting Fab (NTF) fused to the first Fc polypeptide via a first hinge region to form a first Fab-Fc fusion polypeptide and a second NTF fused to the second Fc polypeptide via a second hinge region to form a second Fab-Fc fusion polypeptide, wherein first and second NTFs each comprise SEQ ID NO: 109 or 110, wherein the hinge regions each comprise SEQ ID NO: 121;(b) the SNCA ASO compnses: 5' +G$+T$+T$dA*dA*dA*dT*dC$dT$dA$dG $dT*dT*dG$dT$+[%C]$+[%C]$+A 3' (SEQ ID NO: 112)wherein+A, +[%C], +G, and +T are locked adenosine, locked 5-methylcytosine, locked guanosine, and locked thymine nucleosides, respectively,dA, dC, dG, and dT are deoxyadenosine, deoxycytidine, deoxyguanidine, and deoxythymidine nucleosides, respectively,each * indicates a phosphorothioate (PS) internucleoside linkage; and each $ is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkage; wherein the first Fc polypeptide is linked to the SNCA ASO via a linking group. In some embodiments, each $ is a stabilizing internucleoside linkage (e.g., PS2, PN. or MsPA). In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E at position 380; a Y at position 384; a T at position 386; an E at position 387; a W at position 388; an A at position 389; a N at position 390; a T at position 413; an E at position 415; an E at position 416; a Q at position 418; a Q at position 419; a F at position 421; a S at position 424; and a S at position 426, according to EU numbering. In some embodiments, the SNCA ASO is linked to the cysteine at position 239 (EU numbering) of the first Fc polypeptide. In some embodiments, the 5' end of the SNCA ASO is linked to the cysteine at position 239 of the first Fc polypeptide, wherein the linking group is:

[0083] Described are SNCA ASO conjugates comprising a transferrin receptor (TfR)-targeting Fc polypeptide dimer and a SNCA antisense oligonucleotide (ASO), wherein:(a) the TfR-targeting Fc polypeptide dimer comprises(i) a first Fc polypeptide comprising a cysteine at position 239, an A at position 234, an A at position 235, and a S position 329, each according to EU numbering, (ii) a second Fc polypeptide comprising an A at position 234, an A at position 235. and S at position 329, each according to EU numbering, and a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) wherein the second Fc polypeptide comprises a sequence having at least 90% sequence identity to SEQ ID NO:11 or 12, wherein second Fc polypeptide forms an Fc dimer with the first Fc polypeptide,(iii) a first non-targeting Fab (NTF) fused to the first Fc polypeptide via a first hinge region to form a first Fab-Fc fusion polypeptide and a second NTF fused to the second Fc polypeptide via a second hinge region to form a second Fab-Fc fusion polypeptide, wherein first and second NTFs each comprise SEQ ID NO: 109 or 110, wherein the hinge regions each comprise SEQ ID NO: 121;(b) the SNCA ASO comprises: 5' +T$+[%C]$+T$dC*dT$dA$dT$dA*dT*dA*dA *dC*dA*dT$dC$+A$+[%C]$+T 3' (SEQ ID NO: 113)wherein+A, +[%C], and +T are locked adenosine, locked 5-methylcytosine, and locked thymine nucleosides, respectively,dA, dC, and dT are deoxy adenosine, deoxycytidine, and deoxythymidine nucleosides, respectively,each * indicates a phosphorothioate (PS) internucleoside linkage; and each $ is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkage, wherein the first Fc polypeptide is linked to the SNCA ASO via a linking group. In some embodiments, each $ is a stabilizing internucleoside linkage (e.g., PS2, PN, or MsPA). In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E at position 380; a Y at position 384; a T at position 386; an E at position 387; a W at position 388; an A at position 389; a N at position 390; a T at position 413; an E at position415; an E at position 416; a Q at position 418; a Q at position 419; a F at position 421; a S at position 424; and a S at position 426, according to EU numbering. In some embodiments, the SNCA ASO is linked to the cysteine at position 239 (EU numbering) of the first Fc polypeptide. In some embodiments, the 5' end of the SNCA ASO is linked to the cysteine at position 239 of the first Fc polypeptide, wherein the linking group is:

[0084] Described are SNCA ASO conjugates comprising a transferrin receptor (TfR.)-targeting Fc polypeptide dimer and a SNCA antisense oligonucleotide (ASO), wherein:(a) the TfR-targeting Fc polypeptide dimer comprises(i) a first Fc polypeptide comprising a cysteine at position 239, an A at position 234, an A at position 235, and a S position 329, each according to EU numbering, (ii) a second Fc polypeptide comprising an A at position 234, an A at position 235, and S at position 329, each according to EU numbering, and a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) wherein the second Fc polypeptide comprises a sequence having at least 90% sequence identity to SEQ ID NO:11 or 12, wherein second Fc polypeptide forms an Fc dimer with the first Fc polypeptide,(iii) a first non-targeting Fab (NTF) fused to the first Fc polypeptide via a first hinge region to form a first Fab-Fc fusion polypeptide and a second NTF fused to the second Fc polypeptide via a second hinge region to form a second Fab-Fc fusion polypeptide, wherein first and second NTFs each comprise SEQ ID NO: 109 or 110, wherein the hinge regions each comprise SEQ ID NO: 121;(b) the SNCA ASO comprises: 5' +A$+A$+[%C]$dT*dG$dC$dT$dT$dA$dG *dT*dG*dA*dT$dT$+[%C]$+[%C]$+A 3' (SEQ ID NO: 114)wherein+A and +[%C] are locked adenosine and locked 5-methylcysodine nucleosides, respectively,dA, dC, dG, and dT are deoxyadenosine, deoxycytidine, deoxy guanidine, and deoxythymidine nucleosides, respectively,each * indicates a phosphorothioate (PS) internucleoside linkage; and each $ is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkage;wherein the first Fc polypeptide is linked to the SNCA ASO via a linking group. In some embodiments, each $ is a stabilizing internucleoside linkage (e.g., PS2, PN. or MsPA). In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E at position 380; a Y at position 384; a T at position 386; an E at position 387; a W at position 388; an A at position 389; a N at position 390; a T at position 413; an E at position 415; an E at position 416; a Q at position 418; a Q at position 419; a F at position 421; a S at position 424; and a S at position 426, according to EU numbering. In some embodiments, the SNCA ASO is linked to the cysteine at position 239 (EU numbering) of the first Fc polypeptide. In some embodiments, the 5' end of the SNCA ASO is linked to the cysteine at position 239 of the first Fc polypeptide, wherein the linking group is:

[0085] Described are SNCA ASO conjugates comprising a transferrin receptor (TfR)-targeting Fc polypeptide dimer and a SNCA antisense oligonucleotide (ASO), wherein:(a) the TfR-targeting Fc polypeptide dimer comprises(i) a first Fc polypeptide comprising a cysteine at position 239, an A at position 234, an A at position 235. and a S position 329, each according to EU numbering, (ii) a second Fc polypeptide comprising an A at position 234. an A at position 235, and S at position 329, each according to EU numbering, and a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) wherein the second Fc polypeptide comprises a sequence having at least 90% sequence identity to SEQ ID NO:11 or 12, wherein second Fc polypeptide forms an Fc dimer with the first Fc polypeptide,(iii) a first non-targeting Fab (NTF) fused to the first Fc polypeptide via a first hinge region to form a first Fab-Fc fusion polypeptide and a second NTF fused to the second Fc polypeptide via a second hinge region to form a second Fab-Fc fusion polypeptide, wherein first and second NTFs each comprise SEQ ID NO: 109 or 110, wherein the hinge regions each comprise SEQ ID NO: 121;(b) the SNCA ASO comprises: 5' +G$+G$+T$dA$dA$dC*dT*dT*dA*dG*dG *dA*dC*dA$dA$+G$+G$+T 3' (SEQ ID NO: 115)wherein+G and +T are locked guanosine and locked thymine nucleosides, respectively,dA, dC, dG, and dT are deoxyadenosine, deoxycytidine, deoxy guanidine, and deoxythymidine nucleosides, respectively,each * indicates a phosphorothioate (PS) internucleoside linkage; and each $ is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkage; wherein the first Fc polypeptide is linked to the SNCA ASO via a linking group. In some embodiments, each $ is a stabilizing internucleoside linkage (e.g., PS2, PN. or MsPA). In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E at position 380; a Y at position 384; a T at position 386; an E at position 387; a W at position 388; an A at position 389; a N at position 390; a T at position 413: an E at position 415; an E at position 416; a Q at position 418; a Q at position 419; aF at position 421; a S at position 424; and a S at position 426, according to EU numbering. In some embodiments, the SNCA ASO is linked to the cysteine at position 239 (EU numbering) of the first Fc polypeptide. In some embodiments, the 5' end of the SNCA ASO is linked to the cysteine at position 239 of the first Fc polypeptide, wherein the linking group is:

[0086] In some embodiments, 0, 1. 2, or 3 internucleoside linkages at the 5' end (linkages between nucleosides 1 and 2, 2 and 3, and 3 and 4) of the SNCA ASO are stabilizing internucleoside linkages. In some embodiments, 0, 1, 2, or 3 internucleoside linkages at the 5' end of the SNCA ASO are PN linkages. In some embodiments, 0, 1, 2, or 3 internucleoside linkages at the 5' end of the SNCA ASO are PS2 linkages. In some embodiments, 0, 1. 2, or 3 internucleoside linkages at the 5' end of the SNCA ASO are MsPA linkages.

[0087] In some embodiments, 0, 1, 2, or 3 of the internucleoside linkages between the first 4 nucleosides at the 5' end of the SNCA ASO are stabilizing internucleoside linkages. The stabilizing internucleoside linkages can be:(a) between nucleosides 1 and 2, between nucleosides 2 and 3, or between nucleosides 3 and 4;(b) between nucleosides 1 and 2 and 2 and 3, between nucleosides 1 and 2 and 3 and 4, or between nucleosides 2 and 3 and 3 and 4;(c) between nucleosides 1 and 2. 2 and 3, and 3 and 4.

[0088] In some embodiments, 3 internucleoside linkages at the 5' end (linkages between nucleosides 1 and 2, 2 and 3, and 3 and 4) of the SNCA ASO are stabilizing internucleoside linkages. In some embodiments, 3 internucleoside linkages at the 5' end of the SNCA ASO are PN linkages. In some embodiments, 3 internucleoside linkages at the 5' end of the SNCA ASO are PS2 linkages. In some embodiments, 3 internucleoside linkages at the 5' end of the SNCA ASO are MsPA linkages.

[0089] In some embodiments, 2 internucleoside linkages at the 5' end (linkages between nucleosides 1 and 2, and between nucleosides 2 and 3) of the SNCA ASO are stabilizing internucleoside linkages. In some embodiments, 2 internucleoside linkages at the 5' end of the SNCA ASO are PN linkages. In some embodiments, 2 internucleoside linkages at the 5' end of the SNCA ASO are PS2 linkages. In some embodiments, 2 internucleoside linkages at the 5' end of the SNCA ASO are MsPA linkages.

[0090] In some embodiments, 1 internucleoside linkage at the 5' end (linkage between nucleosides 1 and 2) of the SNCA ASO is a stabilizing internucleoside linkage. In some embodiments, 1 internucleoside linkage at the 5' end of the SNCA ASO is a PN linkage. In some embodiments, 1 internucleoside linkage at the 5' end of the SNCA ASO is a PS2 linkage. In some embodiments, 1 internucleoside linkage at the 5' end of the SNCA ASO is a MsPA linkage.

[0091] In some embodiments, 0, 1, 2, 3, or 4 internucleoside linkages at the 3' end (linkages between nucleosides n and n-1. n-1 and n-2, n-2 and n-3. n-3, and n-4. wherein n is the number of nucleosides in the ASO) of the SNCA ASO are stabilizing internucleoside linkages. In some embodiments, 0, 1, 2, 3, or 4 internucleoside linkages at the 3' end of the SNCA ASO are PN linkages. In some embodiments, 0, 1, 2, 3, or 4 internucleoside linkages at the 3' end of the SNCA ASO are PS2 linkages. In some embodiments, 0. 1, 2. 3, or 4 internucleoside linkages at the 3' end of the SNCA ASO are MsPA linkages.

[0092] In some embodiments, 0, 1, 2, 3, or 4 of the internucleoside linkages between the last 5 nucleosides at the 3' end of the SNCA ASO are stabilizing internucleoside linkages. The stabilizing internucleoside linkages can be:(a) between nucleosides n and n-1, between nucleosides n-1 and n-2, between nucleoside n-2 and n-3, or between nucleosides n-3 and n-4;(b) between nucleosides n and n-1 and n-1 and n-2, between nucleosides n and -1 and n-2 and n-3, between nucleosides n and n-1 and n-3 and n-4; between nucleosides n-1 and n-2 and n-2 and n-3. between nucleosides n-1 and n-2 and n-3 and n-4, or between nucleosides n-2 and n-3 and n-3 and n-4(c) between nucleosides n and n-1, n-1 and n-2, and n-2 and n-3, between nucleosides n and n-1, n-1 and n-2, and n-3 and n-4, or between nucleosides n-1 and n-2, n-2 and n-3, and n-3 and n-4; or(d) between nucleosides n and n-1, n-1 and n-2, n-2 and n-3, and n-3 and n-4.

[0093] In some embodiments, 4 internucleoside linkages at the 3' end (between nucleosides n and n-1, n-1 and n-2, n-2 and n-3, and n-3 and n-4) of the SNCA ASO are stabilizing internucleoside linkages. In some embodiments. 4 internucleoside linkages at the 3' end of the SNCA ASO are PN linkages. In some embodiments, 4 internucleoside linkages at the 3' end of the SNCA ASO are PS2 linkages. In some embodiments, 4 internucleoside linkages at the 3' end of the SNCA ASO are MsPA linkages.

[0094] In some embodiments. 3 internucleoside linkages at the 3' end (between nucleosides n and n-1, n-1 and n-2, and n-2 and n-3) of the SNCA ASO are stabilizing internucleoside linkages. In some embodiments, 3 internucleoside linkages at the 3' end of the SNCA ASO are PN linkages. In some embodiments, 3 internucleoside linkages at the 3' end of the SNCA ASO are PS2 linkages. In some embodiments, 3 internucleoside linkages at the 3' end of the SNCA ASO are MsPA linkages.

[0095] In some embodiments, 2 internucleoside linkages at the 3' end (between nucleosides n and n-1, and between nucleosides n-1 and n-2) of the SNCA ASO are stabilizing internucleoside linkages. In some embodiments. 2 internucleoside linkages at the 3' end of the SNCA ASO are PN linkages. In some embodiments, 2 internucleoside linkages at the 3' end of the SNCA ASO are PS2 linkages. In some embodiments, 2 internucleoside linkages at the 3' end of the SNCA ASO are MsPA linkages.

[0096] In some embodiments, 1 internucleoside linkage at the 3' end (between nucleosides n and n-1) of the SNCA ASO is a stabilizing internucleoside linkage. In some embodiments, 1 internucleoside linkage at the 3' end of the SNCA ASO is a PN linkage. In some embodiments, 1 internucleoside linkage at the 3' end of the SNCA ASO is a PS2 linkage. In some embodiments, 1 internucleoside linkage at the 3' end of the SNCA ASO is a MsPA linkage.

[0097] In some embodiments, the SNCA ASO contains 0. 1, 2, or 3 stabilizing internucleoside linkages between nucleotides 4 and n-4, wherein n is the number of nucleosides in the ASO. In some embodiments, the SNCA ASO contains 0, 1, 2, or 3 PN linkages between nucleotides 4 and n-4. In some embodiments, the SNCA ASO contains 0, 1, 2, or 3 PS2 linkages between nucleotides 4 and n-4. In some embodiments, the SNCA ASO contains 0, 1, 2, or 3 MsPA linkages between nucleotides 4 and n-4.

[0098] In some embodiments, the SNCA ASO contains 1 stabilizing internucleoside linkage between nucleotides 4 and n-4, wherein n is the number of nucleosides in the ASO. In some embodiments, the SNCA ASO contains 1 PN linkage between nucleotides 4 and n-4. In some embodiments, the SNCA ASO contains 1 or 4 PS2 linkage between nucleotides 4 and n-4. In some embodiments, the SNCA ASO contains 1 MsPA linkage between nucleotides 4 and n-4.

[0099] In some embodiments, the SNCA ASO contains 2 stabilizing internucleoside linkages between nucleotides 4 and n-4, wherein n is the number of nucleosides in the ASO. In some embodiments, the SNCA ASO contains 2 PN linkages between nucleotides 4 and n-4. In some embodiments, the SNCA ASO contains 2 PS2 linkages between nucleotides 4 and n-4. In some embodiments, the SNCA ASO contains 2 MsPA linkages between nucleotides 4 and n-4.

[0100] In some embodiments, the SNCA ASO contains 3 stabilizing internucleoside linkages between nucleotides 4 and n-4, wherein n is the number of nucleosides in the ASO. In some embodiments, the SNCA ASO contains 3 PN linkages between nucleotides 4 and n-4. In some embodiments, the SNCA ASO contains 3 PS2 linkages between nucleotides 4 and n-4. In some embodiments, the SNCA ASO contains 3 MsPA linkages between nucleotides 4 and n-4.

[0101] In some embodiments, an SNCA ASO contains 0-3 stabilizing internucleoside linkages at the 5' end of the SNCA ASO and 0-4 stabilizing internucleoside linkages at the 3' end of the SNCA ASO.

[0102] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO and 4 stabilizing internucleoside linkages at the 3' end of the SNCA ASO.

[0103] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO and 3 stabilizing internucleoside linkages at the 3' end of the SNCA ASO.

[0104] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO and 2 stabilizing internucleoside linkages at the 3' end of the SNCA ASO.

[0105] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO and 1 stabilizing internucleoside linkage at the 3' end of the SNCA ASO.

[0106] In some embodiments, an SNCA ASO contains 2 stabilizing internucleoside linkages at the 5' end of the SNCA ASO and 2 stabilizing internucleoside linkages at the 3' end of the SNCA ASO.

[0107] In some embodiments, an SNCA ASO contains 3 stabilizing internucleoside linkages at the 5' end of the SNCA ASO and 3 stabilizing internucleoside linkages at the 3' end of the SNCA ASO.

[0108] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO, 1 stabilizing internucleoside linkage at the 3' end of the SNCA ASO, and 1 stabilizing internucleoside linkage between nucleosides 4 and n-4 of the SNCA ASO. In some embodiments, all of the stabilizing internucleoside linkages are PS2 linkages. In some embodiments, all of the stabilizing internucleoside linkages are PN linkages. In some embodiments, all of the stabilizing internucleoside linkages are MsPA linkages.

[0109] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO, 1 stabilizing internucleoside linkage at the 3' end of the SNCA ASO, and 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO. In some embodiments, the 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO link adjacent nucleosides. In some embodiments, all of the stabilizing internucleoside linkages are PS2 linkages. In some embodiments, all of the stabilizing internucleoside linkages are PN linkages. In some embodiments, all of the stabilizing internucleoside linkages are MsPA linkages.

[0110] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO, 1 stabilizing internucleoside linkage at the 3' end of the SNCA ASO, and 3 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO. In some embodiments, the 3 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO link contiguous nucleosides. In some embodiments, all of the stabilizing internucleoside linkages are PS2 linkages. In some embodiments, all of the stabilizing internucleoside linkages are PN linkages. In some embodiments, all of the stabilizing internucleoside linkages are MsPA linkages. In some embodiments, the stabilizing internucleoside linkage at the 3' end of the SNCA ASO is a MsPA linkage and the stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO are PN linkages.

[0111] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO, 2 stabilizing internucleoside linkages at the 3' end of the SNCA ASO, and 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO. In some embodiments, the 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO link adjacent nucleosides. In some embodiments, all of the stabilizing internucleoside linkages are PS2 linkages. In some embodiments, all of thestabilizing internucleoside linkages are PN linkages. In some embodiments, all of the stabilizing internucleoside linkages are MsPA linkages.

[0112] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO, 3 stabilizing internucleoside linkages at the 3' end of the SNCA ASO, and 1 stabilizing internucleoside linkage between nucleosides 4 and n-4 of the SNCA ASO. In some embodiments, all of the stabilizing internucleoside linkages are PS2 linkages. In some embodiments, all of the stabilizing internucleoside linkages are PN linkages. In some embodiments, all of the stabilizing internucleoside linkages are MsPA linkages. In some embodiments, the stabilizing internucleoside linkages at the 3' end of the SNCA ASO are PN linkages and the stabilizing internucleoside linkage between nucleosides 4 and n-4 of the SNCA ASO is a PS2 linkage.

[0113] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO, 3 stabilizing internucleoside linkages at the 3' end of the SNCA ASO, and 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO. In some embodiments, the 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO link adjacent nucleosides. In some embodiments, all of the stabilizing internucleoside linkages are PS2 linkages. In some embodiments, all of the stabilizing internucleoside linkages are PN linkages. In some embodiments, all of the stabilizing internucleoside linkages are MsPA linkages. In some embodiments, the stabilizing internucleoside linkages at the 3' end of the SNCA ASO are PN linkages and the stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO are MsPA linkages. In some embodiments, the stabilizing internucleoside linkage at the 3' end (between nucleosides n and n-1) of the SNCA ASO is a MsPA linkage, the stabilizing internucleoside linkages at the between nucleosides n-1 and n-2 and between nucleosides n-2 and n-3 are PN linkages and the stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO are a MsPA linkages.

[0114] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO, 3 stabilizing internucleoside linkages at the 3' end of the SNCA ASO, and 3 stabilizing internucleoside linkage between nucleosides 4 and n-4 of the SNCA ASO. In some embodiments, the 3 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO link contiguous nucleosides. In some embodiments, all of the stabilizing internucleoside linkages are PS2 linkages. In some embodiments, all of the stabilizing internucleoside linkages are PN linkages. In some embodiments, all of the stabilizing internucleoside linkages are MsPA linkages. In some embodiments, the stabilizinginternucleoside linkages at the 3' end of the SNCA ASO are PN linkages and the stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO are a MsPA linkages. In some embodiments, the stabilizing internucleoside linkages at the 3' end of the SNCA ASO are MsPA linkages and the stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO are a PN linkages.

[0115] In some embodiments, an SNCA ASO contains 0 stabilizing internucleoside linkages at the 5' end of the SNCA ASO, 4 stabilizing internucleoside linkages at the 3' end of the SNCA ASO, and 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO. In some embodiments, the 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO link adjacent nucleosides. In some embodiments, all of the stabilizing internucleoside linkages are PS2 linkages. In some embodiments, all of the stabilizing internucleoside linkages are PN linkages. In some embodiments, all of the stabilizing internucleoside linkages are MsPA linkages. In some embodiments, the stabilizing internucleoside linkages at the 3' end of the SNCA ASO are PN linkages and the stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO are a PS2 linkages.

[0116] In some embodiments, an SNCA ASO contains 2 stabilizing internucleoside linkages at the 5' end of the SNCA ASO, 2 stabilizing internucleoside linkages at the 3' end of the SNCA ASO, and 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO. In some embodiments, the 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO link adjacent nucleosides. In some embodiments, all of the stabilizing internucleoside linkages are PS2 linkages. In some embodiments, all of the stabilizing internucleoside linkages are PN linkages. In some embodiments, all of the stabilizing internucleoside linkages are MsPA linkages. In some embodiments, the stabilizing internucleoside linkages at the 5' and 3' ends of the SNCA ASO are PN linkages and the stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO are a PS2 linkages.

[0117] In some embodiments, an SNCA ASO contains 3 stabilizing internucleoside linkages at the 5' end of the SNCA ASO, 3 stabilizing internucleoside linkages at the 3' end of the SNCA ASO, and 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO. In some embodiments, the 2 stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO link adjacent nucleosides. In some embodiments, the stabilizing internucleoside linkages at the 5' and 3' ends of the SNCA ASO are PN linkages and the stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO are a PS2 linkages. In some embodiments, the stabilizing internucleoside linkages at the 5' and 3'ends of the SNCA ASO are PN linkages and the stabilizing internucleoside linkages between nucleosides 4 and n-4 of the SNCA ASO are a MsPA linkages.

[0118] In some embodiments, the SNCA ASO comprises the sequence and modifications of any of the modified SNCA ASOs provided in Table 1.Table 1. Modified SNCA ASOs.Patent ID Sequence SEQ ID (ASO) NO: Parent l + [%C ] *+A*+ [%C ] *dA*dT*dT*dG*dG*dA*dA*dC*dT*dG*dA*dG*dC*+A*+ [%C] *+T 111 Parent_2 +G*+T*+T*dA*dA*dA*dT*dC*dT*dA*dG*dT*dT*dG*dT*+ [%C] *+ [%C] *+A 112 ASO_1 +G*+T*+T*dA*dA*dA*dT*dC*dTtdAtdG*dT*dT*dGndTn+ [%C] n+ [%C] n+A 112 ASO_2 +Gn+Tn+T*dA*dA*dA*dT*dC*dTtdAtdG*dT*dT*dG*dT*+ [%C] n+ [%C] n+A 112 ASO_3 +G*+T*+T*dA*dA*dA*dT*dC*dT*dAtdG*dT*dT*dG*dTn+ [%C] n+ [%C] n+A 112 ASO_5 +Gn+Tn+TndA*dA*dA*dT*dC*dTtdAtdG*dT*dT*dG*dTn+ [%C] n+ [%C] n+A 112 ASO_9 +G*+T*+T*dA*dA*dA*dT*dC*dT*dAtdG*dT*dT*dG*dT*+ [%C] *+ [%C] t+A 112 ASO_13 +G*+T*+T*dA*dA*dA*dT*dCndTndAndG*dT*dT*dG*dTu+ [%C] u+ [%C] u+A 112 ASO_16 +G*+T*+T*dA*dA*dA*dT*dC*dTudAudGudT*dT*dG*dTn+ [%C] n+ [%C] n+A 112 ASO_18 +Gn+Tn+TndA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dTn+ [%C] n+ [%C] n+A 112 ASO_20 +G*+T*+T*dA*dA*dA*dT*dCndTndAndG*dT*dT*dG*dT*+ [%C] *+ [%C] u+A 112 ASO_22 +G*+T*+T*dA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dTn+ [%C] n+ [%C] n+A 112 ASO_24 +G*+T*+T*dA*dA*dA*dT*dCudTudAudG*dT*dT*dG*dTn+ [%C] n+ [%C] n+A 112 ASO_26 +G*+T*+T*dA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dTn+ [%C] n+ [%C] u+A 112 ASO_28 +G*+T*+T*dA*dA*dA*dT*dC*dTndAndGndT*dT*dG*dT*+ [%C] *+ [%C] u+A 112 ASO_32 +G*+T*+T*dA*dA*dA*dT*dC*dT*dAudG*dT*dT*dG*dT*+ [%C] *+ [%C] u+A 112 ASO_33 +G*+T*+T*dA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dT*+ [%C] *+ [%C] u+A 112 ASO_34 +G*+T*+T*dA*dA*dA*dT*dC*dTndAndG*dT*dT*dG*dTn+ [%C] n+ [%C] n+A 112 ASO_38 +G*+T*+T*dA*dA*dA*dT*dC*dTndAndG*dT*dT*dG*dT*+ [%C] n+ [%C] n+A 112 Parent_3 +T*+ [%C] *+T*dC*dT*dA*dT*dA*dT*dA*dA*dC*dA*dT*dC*+A*+ [%C] *+T 113 Parent_4 +A*+A*+ [%C] *dT*dG*dC*dT*dT*dA*dG*dT*dG*dA*dT*dT*+ [%C ] *+ [%C] *+A 114 ASO_4 +A*+A*+ [%C] *dT*dG*dC*dTtdT*dA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C] n+A 114 ASO_6 +An+An+ [ %C ] ndT*dG*dC*dTtdTtdA*dG*dT*dG*dA*dT*dTn+ [ %C ] n+ [ %C ] n+A 114 ASO_7 +An+An+ [%C] *dT*dG*dC*dTtdTtdA*dG*dT*dG*dA*dT*dT*+ [%C] n+ [%C] n+A 114 ASO_8 +A*+A*+ [%C] *dT*dG*dC*dTtdTtdA*dG*dT*dG*dA*dTndTn+ [%C] n+ [%C] n+A 114 ASO_10 +A*+A*+ [%C] *dT*dG*dC*dTtdT*dA*dG*dT*dG*dA*dT*dT*+ [%C ] *+ [%C] t+A 114 ASO_11 +A*+A*+ [ %C ] *dT*dG*dCudTudTudA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C] n+A 114 ASO_12 +An+An+ [%C]ndT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C] n+A 114 ASO_14 +A*+A*+ [%C] *dT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C] n+A114ASO_15 +A*+A*+ [%C] MT*dGudCudTudT*dA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C] n+A 114 ASO_17 +A*+A*+ [%C] MT*dG*dC*dTndTndAndG*dT*dG*dA*dT*dT*+ [%C] *+ [%C]u+A 114 ASO_19 +A*+A*+ [%C] MT*dG*dCndTndTndA*dG*dT*dG*dA*dT*dT*+ [%C] *+ [%C]u+A 114 ASO_21 +A*+A*+ [%C] MT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C]u+A 114 ASO_23 +A*+A*+ [%C] *dT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C]u+A 114 ASO_25 +A*+A*+ [%C] *dT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C] n+A 114 ASO_27 +A*+A*+ [%C] *dT*dG*dC*dTudTudAudG*dT*dG*dA*dT*dTn+ [%C] n+ [%C] n+A 114 ASO_29 +An+An+ [%C]ndT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C] n+A 114 ASO_30 +A*+A*+ [%C] *dT*dG*dC*dTndTndAndG*dT*dG*dA*dT*dTu+ [%C]u+ [%C]u+A 114 ASO_31 +A*+A*+ [%C] *dT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dT*+ [%C] *+ [%C]u+A 114 ASO_35 +A*+A*+ [ %C ] *dT*dG*dCndTndT*dA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C] n+A 114 ASO_36 +A*+A*+ [%C ] *dT*dG*dC'udTudT*dA*dG*dT*dG*dA*dT*dTn+ [%C] n+ [%C]u+A 114 ASO_37 +A*+A*+ [%C ] *dT*dG*dC*dTndTndA*dG*dT*dG*dA*dT*dT*+ [%C] n+ [%C] n+A 114 ASO_39 +A*+A*+ [%C] *dT*dG*dCndTndT*dA*dG*dT*dG*dA*dT*dT*+ [%C] n+ [%C] n+A 114 Parent_5 +G*+G*+T*dA*dA*dC*dT*dT*dA*dG*dG*dA*dC*dA*dA*+G*+G*+T115A. Transferrin Receptor (TfR)-targeting Fc polypeptide dimer

[0119] In some embodiments, the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a human Fc peptide. In some embodiments, the first Fc polypeptide comprises a cysteine at position 239, 442, 330. and / or 289, according to EU numbering (z.e., the Fc domain comprises a S239C substitution, a S442C substitution, a A330C substitution, and / or a T289C substitution). In some embodiments, the first Fc polypeptide comprises a cysteine at position 239, according to EU numbering. In some embodiments, the first Fc polypeptide further comprises a CHI domain (z.e., a Fab-Fc fusion polypeptide), wherein the CHI domain comprises a cysteine at position 114 (according to Kabat numbering) or position 124 (according to EU numbering) (z.e., the first Fc polypeptide comprises an Al 14C substitution or an S 124C substitution). In some embodiments, the first Fc polypeptide comprises LALA-PS mutations (an A at position 234, an A at position 235, and a S position 329, each according to EU numbering). In some embodiments, the first Fc polypeptide comprises a cysteine at position 239 according to EU numbering, LALA-PS mutations, and either knob or hole mutations. In some embodiments, the first Fc polypeptide comprises: LALA-PS mutations, either knob or hole mutations, and further comprises a CHI domain, wherein the CHI domain comprises a cysteine at position 114 (according to Kabat numbering) or position 124 (according to EU numbering. In some embodiments, the first Fc polypeptide comprises a sequence having atleast 85%, 90%, 95%, 96%, 97%, 98% or 99% to any one of SEQ ID NOs: 47-48, 51-54, 61-63, 66, 73-77, and 80. In some embodiments, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO:63 or 80. In some embodiments, the first Fc polypeptide comprises a cysteine at position 239, an A at position 234, an A at position 235, and a S position 329 (each according to EU numbering) and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a SEQ ID NO:63 or 80. In some embodiments, the first Fc polypeptide comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 63 or 80.

[0120] In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E at position 380; a Y at position 384; a T at position 386; an E at position 387; a W at position 388; an A at position 389; a N at position 390; a T at position 413; an E at position 415; an E at position 416; a Q at position 418; a Q at position 419; a F at position 421; a S at position 424; and a S at position 426, according to EU numbering. In some embodiments, the second Fc polypeptide or Fab-Fc fusion polypeptide comprises an E at position 153, a Y at position 157. a T at position 159, an E at position 160, a W at position 161. an A at position 162, an N at position 163, a T at position 186, an E at position 188, an E at position 189, and an F at position 194, as numbered with reference to SEQ ID NO:5. In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E at position 380; a Y at position 384; a T at position 386; an E at position 387; a W at position 388; an A at position 389; aN at position 390; aT at position 413; an E at position 415; an E at position 416; a Q at position 418; a Q at position 419; a F at position 421; a S at position 424; and a S at position 426 (according to EU numbering) and has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identify to SEQ ID NO:4, 5, 11, or 12. In some embodiments, the second Fc polypeptide comprises an E at position 153, a Y at position 157, a T at position 159, an E at position 160, a W at position 161, an A at position 162, an N at position 163, a T at position 186, an E at position 188, an E at position 189, and an F at position 194 as numbered with reference to SEQ ID NO:5 and has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identify to SEQ ID NO:4, 5, 11, or 12. In some embodiments, the second Fc polypeptide comprises LALA-PS mutations (an A at position 234, an A at position 235, and a S position 329, each according to EU numbering). In some embodiments, the second Fc polypeptide comprises LALA-PS mutations, and either knob or hole mutations. In some embodiments, the modified constant domain of the second Fc polypeptide comprises: an E at position 380; a Y at position 384; a T at position 386; an E at position 387; a W at position 388; an A at position 389; a N at position 390; a T at position 413; an E at position 415; an E at position 416; a Q at position 418; a Q at position419; a F at position 421; a S at position 424; and a S at position 426 (according to EU numbering) and has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any of SEQ ID NOs:4-29, 36, and 43-44. In some embodiments, the second Fc polypeptide comprises an E at position 153, a Y at position 157, a T at position 159, an E at position 160, a W at position 161, an A at position 162, an N at position 163, a T at position 186, an E at position 188, an E at position 189, and an F at position 194 as numbered with reference to SEQ ID NO:5 and has at least 85%. 90%. 95%. 96%, 97%, 98% or 99% identify to any of SEQ ID NOs:4-29, 36, and 43-44.

[0121] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises one or more mutations or sets of mutations selected from the group consisting of: a knob mutation (e.g., T139W as numbered with reference to SEQ ID NO:5), hole mutations (e.g., T139S, L141 A, and Y180V as numbered with reference to SEQ ID NO:5), one or more mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)) as numbered with reference to SEQ ID NO:5), and / or one or more mutations that increase serum stability (e.g, (i) M25Y, S27T, and T29E as numbered with reference to SEQ ID NO:5, or (li) N207S with or without M201L as numbered with reference to SEQ ID NO:5). If the first Fc polypeptide contains a knob mutation, then the second Fc polypeptide contains hole mutations. If the second Fc polypeptide contains a knob mutation, then the first Fc polypeptide contains hole mutations.

[0122] In some embodiments, both the first and second Fc polypeptides contain one or more mutations that reduce effector function. In some embodiments, the first and second FC polypeptides each contain LALA mutations. In some embodiments, the first and second FC polypeptides each contain LALA-PG mutations. In some embodiments, the first and second FC polypeptides each contain LALA-PS mutations.

[0123] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a knob mutation (e.g., T139W as numbered with reference to SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a knob mutation and has at least 85% identity, at least 90% identify, at least 95% identify, at least 96% identify, at least 97% identity, at least 98% identity or at least 99% identify to the sequence of SEQ ID NO: 5 or 6. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:5 or 6.

[0124] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a knob mutation (e.g., T139W as numbered with reference to SEQ ID NO:5), and one or more mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S (e.g.. L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)) as numbered with reference toSEQ ID N0:5). In some embodiments, the second Fc polypeptide contains a knob mutation and one or more mutations that modulate effector function and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to the sequence of SEQ ID NO:7, 9, 11, 8, 10, or 12. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:7, 9 or 11. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 8, 10. or 12.

[0125] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a knob mutation (e.g., T139W as numbered with reference to SEQ ID NO:5), and one or more mutations that increase serum stability (e.g., M25Y, S27T, and T29E as numbered with reference to SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a knob mutation and one or more mutations that increase serum stability has at least 85% identity’, at least 90% identity, at least 95% identity', at least 96% identity', at least 97% identity, at least 98% identity or at least 99% identity to the sequence of SEQ ID NO: 13. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 13.

[0126] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a knob mutation (e.g., T139W as numbered with reference to SEQ ID NO:5) and one or more mutations that increase serum stability (e.g, N207S with or without M201L as numbered with reference to SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a knob mutation and one or more mutations that increase serum stability and has at least 85% identity, at least 90% identity, at least 95% identity', at least 96% identity', at least 97% identity, at least 98% identity or at least 99% identity' to the sequence of SEQ ID NO: 14. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 14.

[0127] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a knob mutation (e.g., T139W as numbered with reference to SEQ ID NO:5), one or more mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)) as numbered with reference to SEQ ID NO:5), and one or more mutations that increase serum stability (e.g., M25Y, S27T, and T29E as numbered with reference to SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains a knob mutation, one or more mutations that modulate effector function, one or more mutations that increase serum stability', and has at least 85% identity', at least 90% identity, at least 95% identity', at least 96% identity', at least 97% identity, at least 98% identity or at least 99% identity to the sequence of SEQ ID NO: 15 or 16. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 15 or 16.

[0128] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains a knob mutation (e.g.. T139W as numbered with reference to SEQ ID NO:5), one or more mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)) as numbered with reference to SEQ ID NO:5), and one or more mutations that increase serum stability (e.g., N207S with or without M201L as numbered with reference to SEQ ID NO:5). In some embodiments, the second Fc polypeptide contain a knob mutation, one or more mutations that modulate effector function, one or more mutations that increase serum stability, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to the sequence of SEQ ID NO: 17 or 18. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 17 or 18.

[0129] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains hole mutations (e.g., T139S, LI 41 A, and Y180V as numbered with reference to SEQ ID NO: 5). In some embodiments, the second Fc polypeptide contains hole mutations and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to the sequence of SEQ ID NO: 19. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 19.

[0130] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains hole mutations (e.g., T139S, L141A, and Y180V as numbered with reference to SEQ ID NO:5) and one or more mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)) as numbered with reference to SEQ ID NO:5). In some embodiments, the second Fc polypeptide may contains hole mutations and one or more mutations that modulate effector function, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity' or at least 99% identity to the sequence of SEQ ID NO:20 or 21. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:20 or 21.

[0131] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains hole mutations (e.g., T139S. L141 A, and Y180V as numbered with reference to SEQ ID NO:5) and one or more mutations that increase serum stability (e.g., M25Y, S27T, and T29E as numbered with reference to SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains hole mutations and one or more mutations that increase serum stability and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity or at least 99% identity' to the sequence of SEQ ID NO:22. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:22.

[0132] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains hole mutations (e.g., T139S, L141A, and Y180V as numbered with reference to SEQ ID NO:5) and one or more mutations that increase serum stability (e.g., N207S with or without M201L as numbered with reference to SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains hole mutations and one or more mutations that increase serum stability and has at least 85% identity, at least 90% identity7, at least 95% identity', at least 96% identity, at least 97% identity7, at least 98% identity7or at least 99% identity' to the sequence of SEQ ID NO:23. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:23.

[0133] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains hole mutations (e.g., T139S, LI 41 A, and Y180V as numbered with reference to SEQ ID NO: 5), one or more mutations that modulate effector function (e.g., L7A, L8A, and / orP102GorP102S (e.g., L7A and L8A; L7A, L8A, and P102G: or L7A, L8A, and P102S)) as numbered with reference to SEQ ID NO:5), and one or more mutations that increase serum stability (e.g., M25Y, S27T, and T29E as numbered with reference to SEQ ID NO: 5). In some embodiments, the second Fc polypeptide contains hole mutations, one or more mutations that modulate effector function, and one or more mutations that increase serum stability, and has at least 85% identity, at least 90% identity7, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity' to the sequence of SEQ ID NO:24 or 25. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 24 or 25.

[0134] In some embodiments, the first Fc polypeptide or the second Fc polypeptide contains hole mutations (e.g., T139S, L141 A, and Y 180V as numbered with reference to SEQ ID NO: 5), one or more mutations that modulate effector function (e.g., L7 A, L8 A, and / or P 102G or P 102S (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)) as numbered with reference to SEQ ID NO:5), and one or more mutations that increase serum stability (e.g., N207S with or without M201L as numbered with reference to SEQ ID NO:5). In some embodiments, the second Fc polypeptide contains hole mutations, one or more mutations that modulate effector function, and one or more mutations that increase serum stability', and has at least 85% identity, at least 90% identity', at least 95% identity', at least 96% identity', at least 97% identity, at least 98% identity7or at least 99% identity to the sequence of SEQ ID NO:26 or 27. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:26 or 27.

[0135] In some embodiments, the N-terminus of the second Fc polypeptide may include a hinge sequence or a portion of a hinge sequence (e.g.. SEQ ID NO: 49 or 50 for the second Fc polypeptide). In some embodiments, the N-terminus of the first Fc polypeptide is further joined to a CHI region (e.g., SEQ ID NOs:86, 87, or 94) or a NTF heavy chain sequence.

[0136] In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs:4, 5, 6, 7, 9, 11, 50, 86, and 87, wherein the polypeptide comprises an E at position 153, a Y at position 157, a T at position 159, an E at position 160, a W at position 161, an A at position 162, an N at position 163, a T at position 186, an E at position 188, an E at position 189, and an F at position 194, as numbered with reference to SEQ ID NO:5; and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of any one of SEQ ID NOs:54, 63, 66, and 90. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises a Q at position 192; a Q at position 193; a S at position 197; and a S at position 199, as numbered with reference to SEQ ID NO:5. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:7, and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:54. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:9, and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:66. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO: 11, and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:63.

[0137] In some embodiments, the N-terminus of the first and / or the second Fc polypeptide includes all or a portion of the hinge region (e.g., TCPPCP (SEQ ID NO: 121), DKTHTCP (SEQ ID NO:91), or DKTHTCPPCP (SEQ ID NO:92)). Thus, in some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:50, and the first Fc polypeptide comprises the sequence of SEQ ID NO:90.

[0138] In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs:6, 8, 10, 12, 43, 44, 49, and 94, wherein the polypeptide comprises a an E at position 153, a Y at position 157, a T at position 159, an E at position 160, a W at position 161, an A at position 162, an N at position 163, a T at position 186, an E at position 188. an E at position 189, and an F at position 194, as numbered with reference to SEQ ID NO:5; and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises thesequence of any one of SEQ ID NOs:48, 77, 78, 80, and 101. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises a Q at position 192; a Q at position 193; a S at position 197; and a S at position 199, as numbered with reference to SEQ ID NO:5. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:8, and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:77. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO: 10, and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:78. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO: 12, and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:80. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:49, and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:48. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:94, and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO: 101. In some embodiments, the N-terminus of the first and / or second Fc polypeptide includes a portion of the hinge region (e g., DKTHTCP (SEQ ID NO:91 or DKTHTCPPCP (SEQ ID NO:92)).

[0139] In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs:29, 36, and 44, wherein the polypeptide comprises an E at position 153, a Y at position 157, a T at position 159, an E at position 160, a W at position 161, an A at position 162, an N at position 163, a T at position 186, an E at position 188, an E at position 189, and an F at position 194 (as numbered with reference to SEQ ID NO:5), and an S at position 239 (according to EU numbering); and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of any one of SEQ ID NOs:47-48, 51-54, 61-63, 66, 73-78, 80, and 90. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises a Q at position 192; a Q at position 193; a S at position 197; and a S at position 199, as numbered with reference to SEQ ID NO:5. In some embodiments, the second polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:29, and the first polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:48, 53, 54, 74, or 77. In some embodiments, the second polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:36, and the first polypeptideor first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:61, 66, 75, or 78. In some embodiments, the second polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:44, and the first polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:74 or 77.

[0140] In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:44 or 49, wherein the polypeptide comprises an E at position 153, a Y at position 157, a T at position 159, an E at position 160, a W at position 161, an A at position 162, an N at position 163, a T at position 186, an E at position 188, an E at position 189, and an F at position 194, as numbered with reference to SEQ ID NO:5; and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of any one of SEQ ID NOs:48, 73-75, and 77. In some embodiments, the second Fc polypeptide or second Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:44, and the first Fc polypeptide or first Fab-Fc fusion polypeptide comprises the sequence of SEQ ID NO:77.

[0141] In some embodiments, the second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs:4, 5, 7, 9, 50, 86, and 87, wherein the polypeptide comprises an E at position 153, a Y at position 157, A T at position 159, an E at position 160, a W at position 161, an A at position 162, an N at position 163, a T at position 186, an E at position 188, an E at position 189, and an F at position 194, as numbered with reference to SEQ ID NO:5; and the first Fab-Fc fusion polypeptide comprises the sequence of any one of SEQ ID NOs:52, 53, and 61.

[0142] In some embodiments, the second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 6. 8, 10, 43, 49, and 94 wherein the polypeptide comprises an E at position 153, a Y at position 157, a T at position 159, an E at position 160, a W at position 161, an A at position 162, an N at position 163, A T at position 186, an E at position 188, an E at position 189, and a F at position 194, as numbered with reference to SEQ ID NO:5; and the first Fab-Fc fusion polypeptide comprises the sequence of any one of SEQ ID NOs:73-75.

[0143] In some embodiments, the second Fab-Fc fusion polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity any one of SEQ ID NOs: 6, 8, 10, 43, 49, and 94 wherein the polypeptide comprises an E at position 153, a Y at position 157, a T at position 159, an E at position 160, a W at position 161, an A at position 162, an N at position 163, a T at position 186, an E at position 188, an E at position 189, andan F at position 194, as numbered with reference to SEQ ID NO:5; and the first Fab-Fc fusion polypeptide comprises the sequence of any one of SEQ ID NOs:73-75.

[0144] In some embodiments, the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12. In some embodiments, the first Fc polypeptide comprises an A at position 234, an A at position 235, a S position 329, aE at position 380, a Y at position 384, a T at position 386, a E at position 387, aW at position 388, an A at position 389, anN at position 390, a T at position 413, a E at position 415, a E at position 416, and a F at position 421, according to EU numbering (each according to EU numbering) and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a SEQ ID NO: 11 or 12. In some embodiments, the first Fc polypeptide comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 11 or 12.B. Non-targeting Fab

[0145] A non-targeting Fab, (NTFs) comprises a light chain and a heavy chain, wherein the light chain comprises a VL region and a light chain constant region (CL) and the heavy chain comprises a VH region and a heavy chain CHI constant region. In some embodiments, a NTF does not specifically bind to a naturally occurring epitope in a subject. In some embodiments, a NTF does not specifically bind to an antigen expressed in a given mammal, mammalian tissue, or mammalian cell type. The antigen can be a mammalian antigen or an antigen found in the mammal such as from an infectious organism such as a virus, bacteria, fungus, or parasite. The mammal can be, but is not limited to, a non-human primate, a human, or a rodent (e.g., a mouse).

[0146] Specific binding of an antibody to an antigen means an affinity of at least 106M'1. Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Nonspecific binding is often the result of van der Waals forces. Non-targeting does not imply the NTF does not bind any antigen with any affinity. Rather, in some embodiments, a NTF does not exhibit specific binding to (a) any protein or epitope in mammalian cell, mammalian tissue, or mammal; (b) any surface accessible protein or epitope on a mammalian cell or mammalian tissue; or (c) any serum accessible protein or epitope in a mammalian tissue, or mammal.

[0147] NTF comprises three light chain CDRs and three heavy chain CDRs. In some embodiments, the heavy chain CDRs, CDR-H1, CDR-H2, and CDR-H3, comprise: SEQ ID NOs:105, 106 or 128, and 107. respectively, and the light chain CDRs, CDR-L1, CDR-L2, and CDR-L3, comprise SEQ ID NOs: 102, 103, and 104, respectively. In some embodiments, theheavy chain CDRs, CDR-H1, CDR-H2, and CDR-H3, comprise: SEQ ID NOs:125, 126 or 128, and 127, respectively, and the light chain CDRs. CDR-L1, CDR-L2, and CDR-L3, comprise SEQ ID NOs: 122, 123, and 124, respectively.

[0148] In some embodiments, an NTF comprises a heavy chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 109 or 110; and a light chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or 100% identify to the amino acid sequence of SEQ ID NO:108.

[0149] In some embodiments, an NTF comprises a heavy chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or 100% identify to the amino acid sequence of SEQ ID NO: 130 or 131; and a light chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or 100% identify to the amino acid sequence of SEQ ID NO: 129.

[0150] In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO: 109 or 110 and a light chain sequence comprising SEQ ID NO: 108. In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO: 109 and a light chain sequence comprising SEQ ID NO: 108. In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO: 110 and a light chain sequence comprising SEQ ID NO: 108.

[0151] In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO: 130 or 131 and a light chain sequence comprising SEQ ID NO: 129. In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO: 130 and a light chain sequence comprising SEQ ID NO: 129. In some embodiments, the NTF comprises a heavy chain sequence comprising SEQ ID NO: 131 and a light chain sequence comprising SEQ ID NO: 129.

[0152] In some embodiments, a NTF comprises a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 108, and a heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 109 or 110, and contains the CDR sequences of SEQ ID NO: 102 (CDR-L1), SEQ ID NO: 103 (CDR-L2), SEQ ID NO: 104 (CDR-L3), SEQ ID NO: 105(CDR-H1), SEQ ID NO:106 (CDR-H2) and SEQ ID NO:107 (CDR-H3) and maintains the non-targeting properties of a NTF comprising a light chain comprising SEQ ID NO: 108 and a heavy chain comprising SEQ ID NO: 109 or 110.

[0153] In some embodiments, a NTF comprises a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical to the amino acid sequence of SEQ ID NO: 129, and a heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 130 or 131, and contains the CDR sequences of SEQ ID NO: 122 (CDR-L 1 ), SEQ ID NO: 123 (CDR-L2), SEQ ID NO: 124 (CDR-L3), SEQ ID NO: 125 (CDR-H1). SEQ ID NO: 126 (CDR-H2) and SEQ ID NO: 127 (CDR-H3) and maintains the non-targeting properties of a NTF comprising a light chain comprising SEQ ID NO: 129 and a heavy chain comprising SEQ ID NO:130 or 131.

[0154] An NTF light chain and / or heavy chain can contain one or more modifications that facilitate conjugation to one or more cargo molecules. The modifications can be amino acid substitutions or insertions. The amino acid substitutions can be, but are not limited to, a lysine to cysteine substitution at positions 149 (EU numbering) of the light chain, a serine to cysteine substitution at position 156 (EU numbering) of the light chain, an alanine to cysteine substitution at position 118 (EU numbering (position 114 according to Kabat numbering)) of the heavy chain, or a serine to cysteine substitution at position 124 (EU numbering) of the heavy chain, or a combination thereof.

[0155] In some embodiments, the first Fab-Fc fusion polypeptide comprises a cysteine at position 239, an A at position 234, an A at position 235, and a S position 329 (each according to EU numbering) and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a SEQ ID NO: 100 or 101. In some embodiments, the first Fab-Fc fusion polypeptide comprises a cysteine at position 114 (according to Kabat numbering) or position 124 (according to EU numbering), and an A at position 234, an A at position 235. and a S position 329 (according to EU numbering). In some embodiments, the second Fab-Fc fusion polypeptide comprises an A at position 234, an A at position 235, a S position 329, a E at position 380, a Y at position 384, a T at position 386, a E at position 387, a W at position 388, an A at position 389, an N at position 390, a T at position 413, a E at position 415, a E at position 416, and a F at position 421, according to EU numbering (each according to EU numbering) and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a SEQ ID NO:98 or 99. In someembodiments, the first Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO: 100 or 101. In some embodiments, the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO:98 or 99. In some embodiments, the first Fab-Fc comprises an amino acid sequence of consisting of SEQ ID NO: 100 or 101 and the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO:98 or 99. In some embodiments, the first Fab-Fc comprises an amino acid sequence of consisting of SEQ ID NO: 100 and the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO:98. In some embodiments, the first Fab-Fc comprises an amino acid sequence of consisting of SEQ ID NO: 100 and the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO:99. In some embodiments, the first Fab-Fc comprises an amino acid sequence of consisting of SEQ ID NO: 101 and the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO:98. In some embodiments, the first Fab-Fc comprises an amino acid sequence of consisting of SEQ ID NO: 101 and the second Fab-Fc comprises an amino acid sequence consisting of SEQ ID NO:99.C. SNCA ASO

[0156] In some embodiments, the nucleobase sequence of the SNCA ASO comprises the nucleobase sequence of SEQ ID NO:111 (5' CACATTGGAACTGAGCACT 3'). In some embodiments, the nucleobase sequence of the SNCA ASO consists of the nucleobase sequence of SEQ ID NO:111.

[0157] In some embodiments, the nucleobase sequence of the SNCA ASO comprises the nucleobase sequence of SEQ ID NO: 112 (5' GTTAAATCTAGTTGTCCA 3'). In some embodiments, the nucleobase sequence of the SNCA ASO consists of the nucleobase sequence of SEQ ID NO: 112.

[0158] In some embodiments, the nucleobase sequence of the SNCA ASO comprises the nucleobase sequence of SEQ ID NO: 113 (5' TCTCTATATAACATCACT 3'). In some embodiments, the nucleobase sequence of the SNCA ASO consists of the nucleobase sequence of SEQ ID NO: 113.

[0159] In some embodiments, the nucleobase sequence of the SNCA ASO comprises the nucleobase sequence of SEQ ID NO: 114 (5' AACTGCTTAGTGATTCCA 3'). In some embodiments, the nucleobase sequence of the SNCA ASO consists of the nucleobase sequence of SEQ ID NO: 114.

[0160] In some embodiments, the nucleobase sequence of the SNCA ASO comprises the nucleobase sequence of SEQ ID NO: 115 (5' GGTAACTTAGGACAAGGT 3'). In someembodiments, the nucleobase sequence of the SNCA ASO consists of the nucleobase sequence of SEQ ID NO: 115.

[0161] In some embodiments, the SNCA ASO is linked to a delivery vehicle (e.g., a TfR-targeting Fc polypeptide dimer) via the 5' end of the oligonucleotide (e.g., the delivery vehicle is linked to the 5' terminus of the SNCA ASO).

[0162] Described are SNCA ASOs comprising any one of SEQ ID NOs:111-115 and having modifications that decrease non-specific plasma clearance and / or degradation / cleavage of the SNCA ASO when the SNCA ASO is linked to the peptide deliver}’ vehicle (e g., a TfR-targeting Fc polypeptide dimer). In some embodiments, the SNCA ASO is agapmer. In some embodiments, the SNCA ASO sequence consists of the nucleobase sequence of any one of SEQ ID NOs: 111-115.

[0163] In some embodiments, the SNCA gapmer comprises a 5' wing segment having 3, 4, or 5 nucleosides. In some embodiments, the SNCA gapmer comprises a 3' wing segment having 3, 4, or 5 nucleosides. In some embodiments, the SNCA gapmer comprises a 5' wing segment having 3. 4, or 5 nucleosides and a 3' wing segment having 3. 4, or 5 nucleosides.

[0164] In some embodiments, the SNCA gapmer comprises a 5' wing segment having 3 nucleosides. In some embodiments, the SNCA gapmer comprises a 3' wing segment having 3 nucleosides. In some embodiments, the SNCA gapmer comprises a 5' wing segment having 3 nucleosides and a 3' wing segment having 3 nucleosides.

[0165] In some embodiments, the SNCA gapmer comprises a gap segment comprising 8-16 nucleosides. In some embodiments, the SNCA gapmer comprises a gap segment comprising 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleosides. In some embodiments, the SNCA gapmer comprises a gap segment comprising 10-14 nucleosides. In some embodiments, the SNCA gapmer comprises a gap segment comprising 12 nucleosides. In some embodiments, the SNCA gapmer comprises a gap segment comprising 13 nucleosides. In some embodiments, the SNCA gapmer comprises a gap segment comprising 14 nucleosides. In some embodiments, each nucleoside in the gap segment comprises a 2'-deoxyribose sugar.

[0166] In some embodiments, a SNCA gapmer comprises: Xa-Yb-Zc, wherein a is an integer from 2 to 5, b is an integer from 8 to 16, and c is an integer from 2 to 5, each X nucleoside comprises a modified sugar, each Y nucleoside has a 2' deoxyribose, and each Z nucleoside comprises a modified sugar. In some embodiments, a SNCA gapmer comprises a 3-12-3 (X3-Y12-Z3), 3-13-3 (X3-Y13-Z3), or 3-14-3 (X3-Y14-Z3) gapmer. In some embodiments, a and c are each 3, b is 12. 13. or 14, and each X and Z nucleoside is an LNA.

[0167] In some embodiments, the wing nucleosides of the SNCA gapmer comprise at least one nucleoside having a modified sugar (modified ribose). In some embodiments, every nucleoside in a 5' wing segment, a 3' wing segment, or the 5' and 3' wing segments of a SNCA gapmer comprises a modified sugar. The modified sugar can be, but is not limited to, a 2'-O-methoxyethyl (MOE) sugar moiety, or a bicyclic sugar moiety, comprising a 2'-4' bridge. The 2'-4' bridge can be. but is not limited to, a -O-CH2- (locked nucleic acid or locked nucleoside (LNA)) or -Q-CH(CH)- (constrained ethyl (cEt)). In some embodiments, each wing nucleoside of a SNCA gapmer comprises a bicyclic sugar moiety, comprising a -O-CH2- 2'-4' bridge (LNA).i. Internucleoside linkages:

[0168] In some embodiments, every internucleoside linkage in a SNCA ASO comprises a modified linkage (i.e., a linkage other than a naturally occurring phosphate linkage).

[0169] In some embodiments, a SNCA ASO comprises at least two stabilizing internucleoside linkages at the end of the SNCA ASO opposite the end linked to the delivery vehicle. If the delivery vehicle is linked to the 5' end of the SNCA ASO, then at least two internucleoside linkages at the 3' end of the SNCA ASO comprise stabilizing internucleoside linkages. In some embodiments, the SNCA ASO further comprises stabilizing internucleoside linkages at the 5' end. In some embodiments, the stabilizing internucleoside linkages have increased stability or nuclease resistance relative to a PS internucleoside linkage. In some embodiments, the stabilizing internucleoside linkages are PN internucleoside linkages.

[0170] In some embodiments, a SNCA ASO comprises at least two PN linkages at the 3' end (i.e., PN linkages between nucleosides at positions n-2 (n minus 2) and n-1 and between nucleosides at positions n-1 and n, where n is 3' terminal nucleoside). In some embodiments, the SNCA ASO further comprises at least two PN linkages at the 5' end (i.e., PN linkages between nucleosides at positions 1 and 2 and between nucleosides at positions 2 and 3).

[0171] In some embodiments, a SNCA ASO comprises at least three PN linkages at the 3' end (i.e., PN linkages between nucleosides at positions n-3 and n-2, between nucleosides at positions n-2 and n-1. and between nucleosides at positions n-1 and n, where n is 3' terminal nucleoside). In some embodiments, the SNCA ASO further comprises at least three PN linkages at the 5' end (i.e., PN linkages between nucleosides at positions 1 and 2, between nucleosides at positions 2 and 3, and between nucleosides at positions 3 and 4).

[0172] In some embodiments, the SNCA ASO comprises at least two stabilizing internucleoside linkages between nucleosides at positions 6-9. In some embodiments, the stabilizing internucleoside linkages have increased stability or nuclease resistance relative to aPS internucleoside linkage. In some embodiments, the stabilizing internucleoside linkages are positioned at and / or near a site identified as a soft spot or site of catabolism of the SNCA ASO. A stabilizing internucleoside linkage can be, but is not limited to, a PS2 linkage, a MsPA linkage, or a PN linkage.

[0173] In some embodiments, a SNCA ASO comprises at least two PS2 internucleoside linkages between nucleosides at positions 6-9. In some embodiments, the SNCA ASO comprises PS2 internucleoside linkages between nucleosides at positions 6 and 7 and between nucleosides at positions 7 and 8.

[0174] In some embodiments, a SNCA ASO comprises at least two MsPA internucleoside linkages between nucleosides at positions 6-9. In some embodiments, the SNCA ASO comprises MsPA internucleoside linkages between nucleosides at positions 6 and 7 and between nucleosides at positions 7 and 8. In some embodiments, the SNCA ASO comprises MsPA internucleoside linkages between nucleosides at positions 6 and 7, between nucleosides at positions 7 and 8, and between nucleosides and positions 8 and 9.

[0175] In some embodiments, a SNCA ASO comprises at least two PN internucleoside linkages between nucleosides at positions 6-9. In some embodiments, the SNCA ASO comprises PN internucleoside linkages between nucleosides at positions 7 and 8 and between nucleosides at positions 8 and 9. In some embodiments, the SNCA ASO comprises PN internucleoside linkages between nucleosides at positions 6 and 7, between nucleosides at positions 7 and 8, and between nucleosides and positions 8 and 9.

[0176] In some embodiments, a SNCA ASO comprises:Xa-Yb-Ze,wherein(a) a is an integer from 3 to 5, b is an integer from 8 to 16. and c is an integer from 3 to 5;(b) each X is a nucleoside comprising a modified sugar, each Y is a nucleoside having a 2' deoxyribose, and each Z is nucleoside comprising a modified sugar;(c) each internucleoside linkage between X nucleosides comprises a PS linkage or a PN linkage and each internucleoside linkage between Z nucleosides comprises a PN linkage;(d) the internucleoside linkage between X and Y nucleosides and the internucleoside linkage between Y and Z nucleosides independently comprise a PS linkage or a PN linkage;(e) each internucleoside linkage between Y nucleosides comprises a modified linkage wherein Yb comprises two to three contiguous PS2 linkages, two to three contiguous MsPAlinkages, of two to three contiguous PN linkages, and wherein the other internucleoside linkages between Y nucleosides comprise PS linkages.

[0177] In some embodiments, a and c are each 3. In some embodiments, a and c are each 3, and b is 10-14. In some embodiments, a and c are each 3 and b is 12.Gapmer with PN linkage wings

[0178] In some embodiments, a and c are each 3 and b is 12, and each internucleoside linkage between X nucleosides comprises a PS linkage.

[0179] In some embodiments, a and c are each 3 and b is 12, and each internucleoside linkage between X nucleosides comprises a PN linkage.

[0180] In some embodiments, a and c are each 3 and b is 12, each internucleoside linkage between X nucleosides comprises a PS linkage, the internucleoside linkage between X and Y nucleosides comprises a PS linkage, and the internucleoside linkage between Y and Z nucleosides comprises a PS linkage.

[0181] In some embodiments, a and c are each 3 and b is 12, each internucleoside linkage between X nucleosides comprises a PS linkage, the internucleoside linkage between X and Y nucleosides comprises a PS linkage, and the internucleoside linkage between Y and Z nucleosides comprises a PN linkage.

[0182] In some embodiments, a and c are each 3 and b is 12, each internucleoside linkage between X nucleosides comprises a PN linkage, the internucleoside linkage between X and Y nucleosides comprises a PS linkage and the internucleoside linkage between Y and Z nucleosides comprise a PS linkage.

[0183] In some embodiments, a and c are each 3 and b is 12, and each internucleoside linkage between X nucleosides comprises a PN linkage, and the internucleoside linkage between X and Y nucleosides comprises a PN linkage and the internucleoside linkage between Y and Z nucleosides comprise a PN linkage.Gapmer with PS2, MsPA, or PN linkages in gap

[0184] In some embodiments, a and c are each 3 and b is 12, and Yb comprises two to three contiguous PS2 linkages. In some embodiments, Yb comprises two contiguous PS2 linkages. In some embodiments, the two contiguous PS2 linkages are betw een the 3rdand 4thand between the 4thand 5thnucleosides of Yb. In some embodiments, Yb comprises three contiguous PS2 linkages.

[0185] In some embodiments, a and c are each 3 and b is 12-14, and Yb comprises two to three contiguous MsPA linkages. In some embodiments. Yb comprises two contiguous MsPA linkages. In some embodiments, the two contiguous MsPA linkages are between the 3rdand 4thand between the 4thand 5thnucleosides of Yb. In some embodiments, Yb comprises three contiguous MsPA linkages. In some embodiments, the three contiguous MsPA linkages are between the 3rdand 4th, between the 4thand 5th, between the 5thand 6thnucleosides of Yb.

[0186] In some embodiments, a and c are each 3 and b is 12-14, and Yb comprises two to three contiguous PN linkages. In some embodiments, Yb comprises two contiguous PN linkages. In some embodiments, the two contiguous PN linkages are between the 4thand 5thand between the 5thand 6thnucleosides of Yb. In some embodiments, Yb comprises three contiguous PN linkages.

[0187] In some embodiments, at least one C nucleoside in Xaor Zc, if present, comprises a 5-methylcytosine. In some embodiments, any C nucleoside in Xa or Zc, if present, comprises a 5-methylcytosine.ii. Specific Combinations

[0188] In some embodiments, a and c are each 3 and b is 12-14, each internucleoside linkage between X nucleosides comprises a PS linkage, and any C nucleoside in Xaor Zc, if present, comprises a 5-methylcytosine.

[0189] In some embodiments, a and c are each 3 and b is 12-14, each internucleoside linkage between X nucleosides comprises a PS linkage, the internucleoside linkage between X and Y nucleosides comprises a PS linkage, the internucleoside linkage between Y and Z nucleosides comprises a PS linkage, the internucleoside linkages linking the 4thand 5thand the 5thand 6thnucleosides of Yb comprise PN linkages, and any C nucleoside in Xaor Zc, if present, comprises a 5-methylcytosine.D. Linking Group

[0190] The SNCA ASO can be linked to the first Fc polypeptide, the second Fc polypeptide, the first NTF, and / or the second NTF. In some embodiments, the SNCA ASO is covalently linked to the first Fc polypeptide, the second Fc polypeptide, the first NTF, and / or the second NTF. The first Fc polypeptide, the second Fc polypeptide, the first NTF, and / or the second NTF can contain an amino acid substitution (e.g., a cysteine substitution) to facilitate attachment of the SNCA ASO.

[0191] In some embodiments, the SNCA ASO is linked to the first Fc polypeptide. In some embodiments, the SNCA ASO is linked to a cysteine residue in the first Fc polypeptide. In some embodiments, the cysteine residue in the first Fc polypeptide comprises a cysteine substitution at position 239 (according to EU numbering). In some embodiments, the first Fc polypeptide comprises a cysteine at position 239, 442, 330, and 289, according to EUnumbering. In some embodiments, the cysteine residue in the first Fab-Fc fusion polypeptide comprises a cysteine substitution at position 114 (according to Kabat numbering) or position 124 (according to EU numbering).

[0192] The SNCA ASO can be linked to the first Fc polypeptide, the second Fc polypeptide, or the first NTF, and / or the second NTF via any linking group available in the art suitable for linking an oligonucleotide (e.g., an ASO, e.g., a gapmer) to a polypeptide (eg., a Fc polypeptide or a Fab polypeptide). In some embodiments, the linking group comprises any known linking group that is a bifunctional linker capable of covalently linking an oligonucleotide to a polypeptide.

[0193] Oligonucleotide polypeptide conjugates can be generated using well-known chemical cross-linking reagents and protocols that covalently link an oligonucleotide and a polypeptide through a linking group. For example, there are a large number of chemical cross-linking agents that are known to those skilled in the art and useful for cross-linking a protein with an agent of interest. For example, the cross-linking agents can be heterobifunctional cross-linkers, which can be used to link molecules in a stepwise manner. Heterobifunctional cross-linkers provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrences of unwanted side reactions such as homo-protein polymers. A wide variety of heterobifunctional cross-linkers are known in the art, including, but not limited to, N-hydroxysuccinimide (NHS) or its water soluble analog N-hydroxysulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC). m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl) aminobenzoate (SI AB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Those cross-linking agents having N-hydroxysuccinimide moieties can be obtained as the N-hydroxysulfosuccinimide analogs, which generally have greater water solubility. In addition, those cross-linking agents having disulfide bridges within the linking chain can be synthesized instead as the alkyl derivatives to reduce the amount of linker cleavage in vivo. In addition to the heterobifunctional cross-linkers, there exist a number of other cross-linking agents including homobifunctional and photoreactive cross-linkers. Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH) and dimethylpimelimidate.2HCl (DMP) are examples of useful homobifunctional cross-linking agents, and bis-[B-(4-azidosalicylamido)ethyl]disulfide(BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenyl-amino)hexanoate (SANPAH) are examples of useful photoreactive cross-linkers.

[0194] Non-limiting examples of conjugate linkers include those described in WO2023 / 279099 and WO2023 / 056388, each of which is incorporated by reference in its entirety. In some embodiments, the linking group comprises a val-cit linker as described in U. S. 6,214.345, which is incorporated herein by reference. In some embodiments, the linking group comprises those described in W02020 / 028840, and WO2022 / 212886, each of which is incorporated by reference in its entirety. Other linkers can include those described in Bioconjugate Chemistry 2023 34 (11), 2096-2111.

[0195] The linking group may be attached to any region of the polypeptide, (e g., to the N-terminal region, to the C-terminal region, or to an amino acid within the protein, such as a cysteine residue or a glutamine residue), so long as the oligonucleotide does not prevent binding of the second Fc polypeptide to TfR.. Similarly, the linking group may be attached to any region of the oligonucleotide (e.g, the 5' end, the 3' end or to a nucleic acid residue within the molecule), so long as the polypeptide does not interfere with the functionality of the oligonucleotide (e.g, complementary binding to a target nucleic acid). For example, the linker may be attached to the oligonucleotide through any number of synthetically feasible points located throughout the oligonucleotide, such as at the 3' or 5' terminal residues of the oligo; at a sugar moiety; at a base moiety; or at a residue located within the backbone. In some embodiments, the linker is attached to the oligonucleotide at the 5' terminal residue of the oligonucleotide.

[0196] In some embodiments, the linking group comprises a spacer. The spacer can be, but is not limited to, a hydrophilic spacer. The hydrophilic spacer can be, but is not limited to, polyethylene glycol (PEG).

[0197] The linking group can be a cleavable linking group or a non-cleavable linking group. In some embodiments, the linking group is cleavable. A cleavable linking group contains a cleavable linker. Cleavable linkers include, but are not limited to, nuclease-cleavable linkers, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, and disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U. S. Pat. No.5,208,020).

[0198] In some embodiments, the linking group comprises:As used herein a wavy line "■ / vv” that intersects a bond in a chemical structure indicates the point of attachment of the bond that the wavy bond intersects in the chemical structure to the remainder of a molecule.NUCLEIC ACIDS

[0199] Described herein are nucleic acids encoding any of the heavy and light chains of any of the described Fc peptides, Fc polypeptide dimers, NTFs, and / or Fab-Fc fusion polypeptides. Optionally, such nucleic acids further encode a signal peptide. Coding sequences of nucleic acids can be operably linked to one or more regulatory sequences to facilitate expression of the coding sequences in a host cell. Such regulatory sequence includes, but are not limited to, a promoter, an enhancer, a ribosome binding site, a transcription termination signal, and the like. The nucleic acids encoding heavy and light chains can occur in isolated form or can be cloned into one or more vectors. The nucleic acids can be synthesized by, for example, solid state synthesis or PCR of overlapping oligonucleotides. Nucleic acids encoding heavy and light chains can be joined as one contiguous nucleic acid, e.g, within an expression vector, or can be separate, e.g., each cloned into its own expression vector.

[0200] In some embodiments, nucleic acids encoding the Fc polypeptides, Fc polypeptide dimers. NTFs, or Fab-Fc fusion polypeptides comprise nucleotide sequences encoding the amino acid sequences of any of the described Fc peptides. Fc polypeptide dimers. NTFs, and / or Fab-Fc fusion polypeptides. The nucleic acid sequences can be provided in expression vectors to facilitate expression of the heavy and / or light chains in a host cell. The nucleic acid or expression vector containing the nucleic acid is transformed into a host cell. The host cell can then be used to produce the heavy and / or light chains of the NTF.

[0201] In some embodiments, nucleic acids encoding a TfR-targeting Fc polypeptide dimer comprise a first nucleic acid sequence encoding the first Fc fusion polypeptide, and a second nucleic acid sequence encoding the second Fab-Fc fusion polypeptide. In some embodiments the first nucleic acid sequence encodes any one of SEQ ID NOs:l. 47-48, 51-54, 61-63, 66, 73-78, 80, and 90; and the second nucleic acid sequence encodes any one of SEQ ID NOs:4-29, 36, 43-44, and 49-50.

[0202] In some embodiments, nucleic acids encoding a TfR-targeting Fc polypeptide dimer comprise a first nucleic acid sequence encoding a first Fab-Fc fusion polypeptide, a second nucleic acid sequence encoding a second Fab-Fc fusion polypeptide and a third nucleic acid sequence encoding a NTF light chain. In some embodiments the first nucleic acid sequence encodes any one of SEQ ID NOs: 100-101 and 130-131; the second nucleic acid sequence encodes any one of SEQ ID NOs:98-99, and the third nucleic acid sequence encodes any one of SEQ ID NOs: 108 and 129.

[0203] In some embodiments, the first nucleic acid sequence comprises SEQ ID NO: 133 or a sequence having at least 75% identity to SEQ ID NO: 133; the second nucleic acid sequence comprises SEQ ID NO: 132 or a sequence having at least 75% identity to SEQ ID NO: 132; and the third nucleic acid sequence comprises SEQ ID NO: 134 or a sequence having at least 75% identity' to SEQ ID NO: 134. In some embodiments the first nucleic acid sequence comprises SEQ ID NO:133; the second nucleic acid sequence comprises SEQ ID NO:132, and the third nucleic acid sequence comprises SEQ ID NO: 134.

[0204] Methods of preparing the described Fc polypeptides, Fc polypeptide dimers. NTFs, Fab-Fc fusion polypeptides, or antibodies include, but are not limited to, expressing in a plurality of host cells one or more nucleic acids encoding the Fc polypeptide, Fc polypeptide dimer, NTF, Fab-Fc fusion polypeptide, or heavy and light chains of a NTF, propagating the cells under conditions suitable for expression of the Fc polypeptide, Fc polypeptide dimer, NTF. Fab-Fc fusion polypeptide, or heavy and light chains of the NTF in the cells, and purifying the Fc polypeptide, Fc polypeptide dimer, NTF, Fab-Fc fusion polypeptide, or heavy and light chains of the NTF. The Fc polypeptide, Fc polypeptide dimer, Fab-Fc fusion polypeptide, purified NTF can be conjugated to one or more of the described SNCA ASOs.

[0205] Described are cells containing nucleic acids encoding the described Fc polypeptides, Fc polypeptide dimers, NTFs, Fab-Fc fusion polypeptides, or heavy and light chains of the Fab. The cell can be a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. The cell can be used to express the Fc polypeptide, Fc polypeptide dimer, a NTF, or aFc polypeptide-NTF heavy chain fusion polypeptide. The expressed polypeptides can then be isolated and optionally purified from the cell.METHODS OF USE

[0206] A SNCA ASO conjugate as described herein may be used for a variety of purposes, including therapeutic indications.

[0207] In some embodiments, the SNCA ASO conjugate is used to deliver a SNCA ASO to a target cell type that expresses the transferrin receptor. In some embodiments, a SNCA ASO conjugate may be used to transport a SNCA ASO across an endothelium, e.g, the blood-brain barrier, to be taken up by the brain.

[0208] In some embodiments, methods of reducing the expression of a SNCA gene in a subject are described, the methods comprising administering an effective amount of a SNCA ASO conjugate or composition thereof as described herein to the subject. In some embodiments, SNCA ASO conjugates or compositions thereof for use in reducing the expression of a SNCA gene in a cell of subject are provided. In some embodiments, the SNCA ASO binds to a SNCA transcript and recruits RNase H, which degrades the transcript. In some embodiments, administration of a described SNCA ASO conjugate or composition thereof to a cell or subject reduces expression of the SNCA gene in the cell or subject. In some embodiments, the cell in the CNS. Expression can be reduced by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%, compared to the expression in a control (e.g, a cell or subject that was not administered the SNCA ASO, SNCA ASO conjugate or composition as described herein) or compared to the level of expression of SNCA in the cell or subject prior to administration of the SNCA ASO.

[0209] For example, certain embodiments provide a method for transcytosis of a SNCA ASO across an endothelium, the method comprising contacting the endothelium (e.g, blood-brain barrier (BBB)) with a SNCA ASO conjugate as described herein. Thus, certain embodiments provide a method of transporting a SNCA ASO across the BBB of a subject in need thereof, comprising administering a SNCA ASO conjugate as described herein to the subject. In some embodiments, SNCA ASO conjugates as described herein for use in transporting a SNCA ASO across the BBB of a subject in need thereof are provided. In some embodiments, provided herein are methods of delivering SNCA ASOs to the CNS. In some embodiments, provided herein are methods of delivering a SNCA ASO to deep brain regions (e.g, cortex, brainstem, hippocampus, striatum, cerebellum, thalamus, caudate putamen, and substantia nigra). In some embodiments, provided herein are methods of delivering a SNCA ASO to deep brain regions and spinal cord (e.g, cervical spinal cord, lumbar spinal cord). In some embodiments, provided herein are methods of delivering a SNCA ASOs to the CNS and muscle (e.g, cardiac and skeletal). In some embodiments, provided herein are methods of delivering a SNCA ASOs to the CNS, peripheral nerves (e.g. retina, sciatic nerve), muscle (e.g. quadricep), and other peripheral organs (e.g, heart, diaphragm, spleen, intestine, lung, liver, and kidney).

[0210] Certain embodiments also provide methods of modulating the expression SNCA in a subject in need thereof, comprising administering an effective amount of a SNCA ASO conjugate as described herein to the subject. In some embodiments, the SNCA ASO conjugates as described herein are for use in modulating the expression of SNCA is a subject. In some embodiments, the SNCA is expressed in a cell in the brain of a subject.

[0211] A SNCA ASO or a SNCA ASO conjugate as described herein may be administered to a subject at a therapeutically effective amount or dose. The dosages, however, may be varied according to several factors, including the chosen route of administration, the formulation of the composition, patient response, the severity of the condition, the subject’s weight, and the judgment of the prescribing physician. The dosage can be increased or decreased over time, as required by an individual patient.

[0212] In various embodiments, a SNCA ASO or a SNCA ASO conjugate as described herein is administered parenterally. In some embodiments, the SNCA ASO or SNCA ASO conjugate is administered intravenously. Intravenous administration can be by infusion, e.g, over a period of from about 10 to about 30 minutes, or over a period of at least 1 hr, 2 hr. or 3 hr. In some embodiments, the SNCA ASO or SNCA ASO conjugate is administered as an intravenous bolus. Combinations of infusion and bolus administration may also be used.

[0213] In some parenteral embodiments, a SNCA ASO or a SNCA ASO conjugate is administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, the SNCA ASO or SNCA ASO conjugate is administered intradermally or intramuscularly. In some embodiments, the SNCA ASO or SNCA ASO conjugate is administered intrathecally, such as by epidural administration, or intracerebroventricularly.

[0214] In other embodiments, a SNCA ASO or SNCA ASO conjugate as described herein may be administered orally, by pulmonary administration, intranasal administration, intraocular administration, or by topical administration. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0215] The SNCA ASOs and SNCA ASO conjugates described herein may also be used to treat, prevent, or ameliorate diseases, disorders, or conditions associated with Synuclein (e.g, a synucleinopathy). Thus, in some embodiments, provided herein are methods of treatment, prevention, or amelioration of diseases, disorders, or conditions associated with Synuclein in a subject in need thereof. In some embodiments, a synuclein associated disease is a synuclein associated neurodegenerative disorder. In some embodiments, synuclein associated diseases include, but are not limited to, Alzheimer's Disease, Parkinson’s disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic TraumaticEncephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, Dravet’s Syndrome, multiple systems atrophy (MSA), Lewy body dementia, pure autonomic failure (PAF), and REM sleep behavior disorder (RBD).

[0216] Accordingly, certain embodiments provide methods of treating a synuclein-associated neurodegenerative disorder in a subject in need thereof, the method comprising administering to the subject a SNCA ASO, a SNCA ASO conjugate or a composition as described herein.

[0217] In some embodiments, the synuclein-associated neurodegenerative syndrome is Parkinson’s Disease. Thus, certain embodiments provide a method of treating Parkinson’s disease, the method comprising administering to a subject in need thereof, a SNCA ASO, a SNCA ASO conjugate or a composition as described herein. The subject may be diagnosed with Parkinson's disease, diagnosed with one or more symptoms of Parkinson’s disease, or be at risk of developing Parkinson’s disease or one or more symptoms associated with Parkinson’s disease.

[0218] Described are methods of targeting delivery' of an SNCA ASO to CNS tissue in a patient comprising administering to the subject any of the described SNCA ASO conjugates or pharmaceutical compositions. In some embodiments, the SNCA ASO is distributed throughout the CNS. In some embodiments, the SNCA ASO is distributed across brain regions. Brain regions include, but are not limited to, frontal lobe, parietal lobe, temporal lobe, occipital lobe, and cerebellum. In some embodiments, the SNCA ASO is distributed to a deep brain region. In some embodiments, the SNCA ASO is distributed to the spinal cord. In some embodiments, the SNCA ASO modulates the expression of a target gene. In some embodiments, modulation of target gene expression is inhibition of gene expression (i.e., gene knockdow n).

[0219] In some embodiments, the subject is a human subject.PHARMACEUTICAL COMPOSITIONS AND KITS

[0220] In some embodiments, pharmaceutical compositions and kits comprising a SNCA ASO conjugate as described herein are provided.A. Pharmaceutical compositions

[0221] Guidance for preparing formulations for use as described herein can be found in any number of handbooks for pharmaceutical preparation and formulation that are known to those of skill in the art.

[0222] In some embodiments, a pharmaceutical composition comprises a SNCA ASO conjugate as described herein and further comprises one or more pharmaceutically acceptable carriers and / or excipients.

[0223] In some embodiments, the composition comprises a plurality of SNCA ASO conjugates as described herein, which can be the same or different (e.g.. a mixture of different SNCA ASO conjugates).

[0224] In some embodiments, an SNCA ASO conjugate as described herein comprises 1-8 (1, 2, 3, 4, 5, 6, 7, or 8) SNCA ASOs linked to one or more amino acids of the SNCA ASO conjugate. In some embodiments, an SNCA ASO conjugate comprises one (1) SNCA ASO linked to an ammo acid of the SNCA ASO conjugate. In some embodiments, an SNCA ASO conjugate comprises two (2) SNCA ASOs linked to two amino acids of the SNCA ASO conjugate. In some embodiments, an SNCA ASO conjugate comprises two (2) SNCA ASOs linked to one amino acid of the SNCA ASO conjugate. In some embodiments, an SNCA ASO conjugate comprises four (4) SNCA ASOs linked to four amino acids of the SNCA ASO conjugate. In some embodiments, an SNCA ASO conjugate comprises four (4) SNCA ASOs linked to two amino acids of the SNCA ASO conjugate (2 SNCA ASOs linked to each of two amino acids). In some embodiments, an SNCA ASO conjugate comprises four (4) SNCA ASOs linked to one amino acid of the SNCA ASO conjugate.

[0225] In some embodiments, a single SNCA ASO is attached to the TfR-targeting Fc dimer or Fab-Fc dimer. In some embodiments, two or more SNCA ASOs are attached to the TfR-targeting Fc dimer or Fab-Fc dimer. Two or more SNCA ASOs can be linked to a single linking group or two or more SNCA ASOs can also be linked to the TfR-targeting Fc dimer or Fab-Fc dimer via two or more linking groups. The two or more linked groups can be the same or different. In some embodiments, two SNCA ASOs are attached to the TfR-targeting Fc dimer or Fab-Fc dimer. In some embodiments, four SNCA ASOs are attached to the TfR-targeting Fc dimer or Fab-Fc dimer.

[0226] In some embodiments, 1 SNCA ASO is attached to a single linking group (L). In some embodiments, 2 SNCA ASOs are attached to a single linking group (L). In some embodiments, two or more SNCA ASOs are linked in tandem to a single linker (L). For tandem linkage, L may be linked to the 5' end of a first SNCA ASO and a second SNCA ASO is linked to the 3' end of the first SNCA ASOs. Alternatively, for tandem linkage, L may be linked to the 3' end of a first SNCA ASO and a second SNCA ASO is linked to the 5' end of the first SNCA ASOs. The first and second SNCA ASO may be linked to each other via a nucleic acid linker or a non-nucleic acid cleavable linker.

[0227] In some embodiments, two or more SNCA ASOs are linked to a single branched linker (L). The linker can be a branched linking group wherein 2 or more oligonucleotides are attached separately to a single linking group (L) (i.e., y is 2 or more).

[0228] In some embodiments, the ratio of oligonucleotide to protein in the composition is about 1:1 to about 4:1. In some embodiments, the ratio of oligonucleotide to protein in the composition is about 1:1 to about 2:1. In some embodiments, the ratio of oligonucleotide to protein in the composition is about 1.23. In some embodiments, the ratio of oligonucleotide to protein in the composition is about 2:1 to about 3:1. In some embodiments, the ratio of oligonucleotide to protein in the composition is about 2.5.

[0229] As used herein, the term pharmaceutically acceptable earner includes any solvents, dispersion media, or coatings that are physiologically compatible and that preferably does not interfere with or otherwise inhibit the activity of the active agent. Various pharmaceutically acceptable excipients are well-known. In some embodiments, the carrier is suitable for intravenous, intrathecal, intracerebroventricular, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compounds that act, for example, to stabilize the composition or to increase or decrease the absorption of the SNCA ASO conjugate. Physiologically acceptable compounds can include, for example: carbohydrates (e.g., glucose, sucrose, or dextrans), antioxidants (e.g, ascorbic acid or glutathione), chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, excipients, stabilizers, and / or buffers. Other pharmaceutically acceptable carriers and their formulations are also available in the art.

[0230] The pharmaceutical compositions described herein can be manufactured in a manner that is known to those of skill in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. The following methods and excipients are merely exemplary and are in no way limiting.

[0231] For administration to a subject, a SNCA ASO conjugate as described herein can be formulated by combining it with pharmaceutically acceptable carriers that are well-known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, and the like. Pharmaceutical preparations for administration to a subject use can be obtained by mixing the SNCA ASO conjugates with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / orpolyvinylpyrrolidone. If desired, disintegrating agents can be added, such as a cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0232] As disclosed above, a SNCA ASO conjugate as described herein can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. For injection, the SNCA ASO conjugates can be formulated into preparations by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives. In some embodiments, SNCA ASO conjugates can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks’s solution, Ringer’s solution, or physiological saline buffer. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0233] Typically, a pharmaceutical composition for use in in vivo administration is sterile. Sterilization can be accomplished according to methods known in the art, e.g., heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0234] Dosages and desired drug concentration of pharmaceutical compositions as described herein may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of one in the art. Suitable dosages are also described above.B. Kits

[0235] In some embodiments, kits comprising a SNCA ASO conjugate as described herein are provided. In some embodiments, the kits are for use in modulating the expression of a target gene or sequence (e.g., a target gene expressed in the brain or central nervous system (CNS)). In some embodiments, the kits are for use in in modulating the expression of a target gene.

[0236] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises a SNCA ASO conjugate as described herein and further comprises one or more additional therapeutic agents. In some embodiments, the kit further comprises instructional materials containing directions (i.e., protocols) for the practice of the methods described herein (e.g., instructions for using the kit for administering a composition across the blood-brain barrier). While the instructional materials typically comprise written or printed materials, they are not limited to such. Anymedium capable of storing such instructions and communicating them to an end user is contemplated herein. Such media include, but are not limited to. electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD-ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.Table 2. SequencesSEQSequence Description ID NO.1 PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD Wild-type human Fc GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE sequence:KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ amino acids 1-3 PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK (PCP) are from a SLSLSPGK hinge region 2 NSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPV Human TfR apical NGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHL domain GTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSD WKT D S TO RMVT S E S KNVKL TVS3 MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTK Human transferrin ANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTES receptor protein 1 PVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGS (TFR1) QKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYL VENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGS IVIVRAGKITFA EKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNH TQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESK NVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVWGAQRDAWGPGAAKSGVGTA LLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLK AFTYINLDKAVLGTSNFKVSAS PLLYTL IEKTMQNVKHPVTGQFL YQDSNWAS KVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPEL NKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADTKEMG LSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPY VS PRE S PFRHVFWGSGSHTL PALLENLKLRKQNNGAFNETL FRNQLALATWT I QGAANAL S GDVWD I DNE F4 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C 35232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES YGTEWA NYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LSPGK5 PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD Clone CH3C.35.23.2 GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE with knob mutation KTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESYGT amino acids 1-3 EWANYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQK (PCP) are from a SLSLSPGK hinge region 6 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI with knob mutation SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA NYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LSPG7 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPTEKTI with knob and LALA SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA mutations NYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LSPGK8 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C 35232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI with knob and LALASKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA mutationsNYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PG APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTT with knob and SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA LALAPG mutations NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI with knob and SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA LALAPG mutations NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PG APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTI with knob and SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA LAL APS mutations NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.23.2 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTI with knob and SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA LALAPS mutations, NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS w / lysine truncation LS PG APELLGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with knob and YLE SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDTAVEWES YGTEWA mutations NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPTEKTT with knob and SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDTAVEWES YGTEWA M201L and N207S NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLS mutations LS PGK APEAAGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with knob, LAL A, SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA and YLE mutations NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APEAAGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTT with knob, LALAPG, SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA and YLE mutations NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C 35232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with knob, LAL A, SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA and M201L and NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLS N207S mutations LS PGK APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI with knob, LALAPG, SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA and M201L and NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLS N207S mutations LS PGK APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.23.2 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with hole mutations S KAKGQ P RE PQVYT L P P S RD E L TKNQVS L S C AVKGF Y P S D I AVEWE S YGT EWA NYKTT PPVLDSDGS FFLVSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with hole and LALAS KAKGQ P RE PQVYT L P P S RD E L TKNQVS L S C AVKGF Y P S D T AVEWE S YGT EWA mutationsNYKTT PPVLDSDGS FFLVSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI with hole and S KAKGQ P RE PQVYT L P P S RD E L TKNQVS L S C AVKGF Y P S D I AVEWE S YGT EWA LALAPG mutations NYKTT PPVLDSDGS FFLVSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APELLGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with hole and YTE S KAKGQ P RE PQVYT L P P S RD E L TKNQVS L S C AVKGF Y P S D I AVEWE S YGT EWA mutations NYKTT PPVLDSDGS FFLVSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with hole and M201L S KAKGQ P RE PQVYT L P P S RD E L TKNQVS L S C AVKGF Y P S D I AVEWE S YGT EWA and N207S mutations NYKTT PPVLDSDGS FFLVSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLS LS PGK APEAAGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.23.2 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with hole, LALA, and S KAKGQ P RE PQVYT L P P S RD E L TKNQVS L S C AVKGF Y P S D L AVEWE S YGT EWA YTE mutations NYKTT PPVLDSDGS FFLVSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APEAAGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI with hole, LALAPG, S KAKGQ P RE PQVYT L P P S RD E L TKNQVS L S C AVKGF Y P S D I AVEWE S YGT EWA and YTE mutations NYKTT PPVLDSDGS FFLVSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with hole, LALA, and S KAKGQ P RE PQVYT L P P S RD E L TKNQVS L S C AVKGF Y P S D I AVEWE S YGT EWA M201L and N207S NYKTT PPVLDSDGS FFLVSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLS mutations LS PGK APEAAGGPSVFLFPPKPKDTLMLSRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI with hole, LALAPG, S KAKGQ P RE PQVYT L P P S RD E L TKNQVS L S C AVKGF Y P S D I AVEWE S YGT EWA and M201L and NYKTT PPVLDSDGS FFLVSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLS N207S mutations LS PGK APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with M201L and SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES YGTEWA N207S mutations NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLS LS PGK APEAAGGPCVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C 35232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with knob, LALA, SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA and S239C mutations NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APEAAGGPCVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI with knob, LALAPG, SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA and S239C mutations NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PGK APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.23.2 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES YGTEWA NYKTT PPVLDSDGS FFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LS PG APEAAGGPCVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Clone CH3C.35.232 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTI with knob, LALA,SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWA and S239C mutationsNYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LSPG APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Wild-type human Fc VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI sequence SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN amino acids 1-3 NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS (PCP) are from a LSPG hinge region DKTHTCPPCPAPEAAGGPCVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV Fc sequence with KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNK portion of human ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAV IgGl hinge sequence EWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEAL and hole, LALA, and HNHYTQKSLSLSPG S239C mutations DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEV Clone 35.23.2 with KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNK portion of human ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAV IgGl hinge sequence EWESYGTEWANYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEAL and knob and LALA HNHYTQKSLSLSPG mutations DKTHTCPPCPAPEAAGGPCVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV Fc sequence with KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNK portion of human ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAV IgGl hinge sequence EWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEAL and hole, LALA, and HNHYTQKSLSLSPGK S239C mutations DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV Clone 35.23.2 with KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNK portion of human ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAV IgGl hinge sequence EWESYGTEWANYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEAL and knob and LALA HNHYTQKSLSLSPGK mutations APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI knob and LALA SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN mutations NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI hole mutations SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI hole and LALA SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN mutations NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK APEAAGGPCVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPLEKTL hole, LALA, and SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN S239C mutations NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI hole and LALAPG SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN mutations NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTI hole and LALAPS SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN mutations NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK APEAAGGPCVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALSAPTEKTI hole, LALAPS, andSKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN S239C mutationsNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK APEAAGGPCVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI hole, LALAPG, and SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN S239C mutations NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI hole mutations SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI hole and LALA SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN mutations NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI hole and LALAPG SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDTAVEWESNGQPEN mutations NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTI hole and LALAPS SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN mutations NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG APEAAGGPCVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI hole, LALA, and SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN S239C mutations NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG APEAAGGPCVFLFPPKPKDTLMTSRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI hole, LALAPG, and SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN S239C mutations w / NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS lysine truncation LSPG APEAAGGPCVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE Fc sequence with VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTI hole, LALAPS, and SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN S239C mutations w / NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS lysine truncation LSPG ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF Heavy constant PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT region sequence with HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN 35.23.2, knob, and WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP LALA, mutations APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWE S YGTEWANYKT T P PVL DS DGS F FL YS KL TVTKEEWQQGFVF S C S VMHEALHNH YTQKSLSLSPGK ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF Heavy constant PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT region sequence with HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN 35.23.2, knob, and WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALG LALAPG, mutations APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWE S YGTEWANYKT T P PVL DS DGS F FL YS KL TVTKEEWQQGFVF S C S VMHEALHNH YTQKSLSLSPGK ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF Heavy constant PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT region sequence with HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN 35.23.2, knob, and WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALG LALAPG, mutationsAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWES YGTEWANYKT T P PVL DS DGS F FL YS KL TVTKEEWQQGFVF S C S VMHEALHNH YTQKSLSLSPG DKTHTCP Portion of human IgGl hinge sequence DKTHTCPPCP Portion of human IgGl hinge sequence EPKSCDKTHTCPPCP Human IgG1 hinge amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ Kappa constant ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE region sequence C QVQLQESGPGLVKPSETLSLTCAVSGYS ITSDYAWGWIRQPPGKGLEWIGSMS Fab-Fc polypeptide YSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWG 35.23.2 LAL APS QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG Knob ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALSAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFY PSDIAVEWESYGTEWANYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCS VMHEALHNHYT QKS L S L S PGKcaagtgcaactgcaggaaagtgggcctggtttggtcaaacccagtgaaacact Fab-Fc polypeptide tagtcttacttgcgctgtatctggatactctattacaagcgattatgcctggg 35.23.2 LAL APS ggtggattcggcagccccctggaaaaggcttggagtggatcggttcaatgagt Knob nucleic acid tatagtggtagtacatactacaacccttccctgaagtctcgcgtcacaataag sequence cgtagatacaagtaagaatcaattttccctgaaacttagcagtgtaactgccg ctgataccgcagtctactattgtgccagaggatggcccctggcctactggggt cagggcaccctcgtgaccgtatcatcagctagcaccaaaggtcccagcgtgtt cccactcgccccgagttcaaaatcaacttctggaggcaccgccgccctgggtt gcctggtaaaggactacttcccagagcccgtgaccgtgagctggaactccggg gcactgacatctggcgttcatactttcccggccgtgctccagtcttcaggtct gtatagtctctcctctgtggtcactgtcccatctagctctctgggcacccaaa cctacatatgcaacgttaatcacaagccgagcaatactaaagttgacaaaaag gtggaacccaagtcttgtgacaagacccacacgtgtcccccctgcccggctcc tgaggctgcaggcggccccagcgtctttctctttcccccaaagccaaaagata ccttgatgatcagcagaactcccgaggtgacatgcgtcgtcgtggacgtaagc catgaagatcccgaggttaagttcaactggtatgtcgatggcgtggaagtcca taatgctaagactaaacctcgcgaagagcagtacaattcaacttaccgggtcg tttccgttctgaccgtgctgcatcaggactggctgaatggtaaagagtacaag tgcaaagtgtctaacaaggcactcagcgccccaattgagaagactatctccaa agctaaagggcaaccaagagagccccaggtctacaccctgcccccctcaaggg atgagcttactaagaaccaggttagtctctggtgcttggttaaaggattttat ccaagcgatattgctgtggagtgggagtcctatggcacggagtgggccaacta taaaaccaccccccctgttcttgacagtgacggtagtttcttcctgtattcca aactgaccgtcacaaaggaggagtggcaacagggatttgtgttcagctgctcc gtgatgcatgaggcgctccataatcattacacacaaaaaagtttgtccctgag cccaggcaagQVQLQESGPGLVKPSETLSLTCAVSGYS ITSDYAWGWIRQPPGKGLEWIGSMS Fab-Fc polypeptide YSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWG 35.23.2 LAL APS QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG Knob w / lysine ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK truncation VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALSAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFY PSDIAVEWESYGTEWANYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCS VMHEALHNHYT QKS L S L S PG QVQLQESGPGLVKPSETLSLTCAVSGYS ITSDYAWGWIRQPPGKGLEWIGSMS Fab-Fc polypeptide YSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWG LALAPS S239C QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG HoleALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPCVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALSAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFY PSDTAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYT QKS L S L S PGKcaagtgcaactgcaggaaagtgggcctggtttggtcaaacccagtgaaacact Fab-Fc polypeptide tagtcttacttgcgctgtatctggatactctattacaagcgattatgcctggg LALAPS S239C ggtggattcggcagccccctggaaaaggcttggagtggatcggttcaatgagt Hole nucleic acid tatagtggtagtacatactacaacccttccctgaagtctcgcgtcacaataag sequence cgtagatacaagtaagaatcaattttccctgaaacttagcagtgtaactgccg ctgataccgcagtctactattgtgccagaggatggcccctggcctactggggt cagggcaccctcgtgaccgtatcatcagctagcaccaaaggtcccagcgtgtt cccactcgccccgagttcaaaatcaacttctggaggcaccgccgccctgggtt gcctggtaaaggactacttcccagagcccgtgaccgtgagctggaactccggg gcactgacatctggcgttcatactttcccggccgtgctccagtcttcaggtct gtatagtctctcctctgtggtcactgtcccatctagctctctgggcacccaaa cctacatatgcaacgttaatcacaagccgagcaatactaaagttgacaaaaag gtggaacccaagtcttgtgacaagacccacacgtgtcccccctgcccggctcc tgaggctgcaggcggcccctgcgtctttctctttcccccaaagccaaaagata ccttgatgatcagcagaactcccgaggtgacatgcgtcgtcgtggacgtaagc catgaagatcccgaggttaagttcaactggtatgtcgatggcgtggaagtcca taatgctaagactaaacctcgcgaagagcagtacaattcaacttaccgggtcg tttccgttctgaccgtgctgcatcaggactggctgaatggtaaagagtacaag tgcaaagtgtctaacaaggcactcagcgccccaattgagaagactatctccaa agctaaagggcaaccaagagagccccaggt ct acaccctgcccccctcaaggg atgagcttactaagaaccaggttagtctcagctgcgcggttaaaggattttat ccaagcgatattgctgtggagtgggagtccaacggccagcctgagaacaatta taaaaccaccccccctgttcttgacagtgacggtagtttcttcctggtttcca aactgaccgtcgataagagcagatggcaacagggaaatgtgttcagctgctcc gtgatgcatgaggcgctccataatcattacacacaaaaaagtttgtccctgag cccaggcaagQVQLQESGPGLVKPSETLSLTCAVSGYS ITSDYAWGWTRQPPGKGLEWTGSMS Fab-Fc polypeptide YSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWG LALAPS S239C QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG Hole w / lysine ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK truncation VEPKSCDKTHTCPPCPAPEAAGGPCVFLFPPKPKDTLMTSRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALSAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYT QKS L S L S PG RASQSVSSYLAWY NTF1 CDR-L1 DASNLAT NTF1 CDR-L2 QQRSNWPPIT NTF1 CDR-L3 GYSITSDYAWG NTF1 CDR-H1 SMSYSGSTYYNPSLKS NTF1 CDR-H2 ARGWPLAY NTF1 CDR-H3 SASSSVS YMYWY NTF2 CDR-L1 DTSNLAS NTF2 CDR-L2 QQWSSYPPIT NTF2 CDR-L3 GYSITSDYAWN NTF2 CDR-H1 YMSYSGSTRYNPSLRS NTF2 CDR-H2 ARGWPLAY NTF2 CDR-H3 X1MSYSGSTX2YNPSLX3S NFT CDR-H2 Wherein: X1=Y or S; X2= R or Y; and X3= R or K consensus ETVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASN NTFl light chain LATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPITFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDST YSLS STLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNR GECgaaattgtcctcacccagagcccagccactctttccctctctccaggggaaag NTF1 light chainagcaacactgagttgccgggcctcccagagtgtaagttcctacctcgcatggt nucleic acid sequenceatcaacagaagcctggtcaagcaccccggcttctgatttacgatgccagcaat ctcgccaccggaatccccgcaaggttttcagggagcggttcagggactgactt taccctgaccataagcagtcttgagcctgaggattttgcagtgtattactgcc aacagagaagtaactggccccccataacctttggacaggggactaaggtggag attaaacgtacggtggcagctccatcagtttttatcttcccaccaagcgacga gcaattgaagtccggcactgcctctgtggtgtgccttctgaacaacttctatc caagggaggccaaggtccagtggaaggtcgataatgcgctgcagagcgggaac agccaagagtcagtgaccgagcaggactcaaaagatagcacatactctctgag ttccaccctgaccctgtcaaaggctgactacgaaaagcataaggtatacgcat gcgaagtgacccatcagggtctctcatctcccgtaaccaaatcttttaatagaggagaatgc109 QVQLQESGPGLVKPSETLSLTCAVSGYS ITSDYAWGWIRQPPGKGLEWIGSMS NTF1 heavy chain YSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTH110 QVQLQESGPGLVKPSETLSLTCAVSGYS ITSDYAWGWIRQPPGKGLEWIGSMS NTF1 heavy chain YSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWG with portion of hinge QGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG region ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCP129 EIVMTQSPATLSLSPGERATLSCSASSSVSYMYWYQQKPGQAPRLLIYDTSNL NTF2 light chain ASGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQWSSYPPITFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC130 QVQLQESGPGLVKPSETLSLTCAVSGYS ITSDYAWNWIRQPPGKGLEWI GYMS NTF2 heavy chain YSGSTRYNPSLRSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPK131 QVQLQESGPGLVKPSETLSLTCAVSGYS ITSDYAWNWIRQPPGKGLEWI GYMS NTF2 heavy chain YSGSTRYNPSLRSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWG with portion of hinge QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG region ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCP111 C AC AT TGGAAC T GAGC AC T SNCA ASO1 112 GT T AAAT CTAGTTGTCCA SNCA ASO2 113 T C T C TAT AT AT AAC AT C AC T SNCA ASO3 114 AACTGCTTAGTGATTCCA SNCA ASO4 115 GGTAACT TAGGACAAGGT SNCA ASO5 116 ATTGGAACTGAGC SNCA ASO1 gap sequence117 AAATCTAGTTGT SNCA ASO2 gap sequence118 C TATATATAAC AT C SNCA ASO3 gap sequence119 TGCTT AGT GATT SNCA ASO4 gap sequence120 AACTTAGGACAA SNCA ASO5 gap sequence121 TCPPCP Hinge regionsequenceEXAMPLESExample 1. Soft Spot Identification Assay.

[0237] In order to determine the catabolism and biotransformation of oligonucleotides (ASOs) and to further characterize the oligonucleotides and oligonucleotide polypeptide conjugates described herein, a stability assay to identify likely sites of catabolism (clipping sites) of ASOs (referred to herein as "soft spots") was developed. The catabolism assay, described in detail below, was used to identify soft spots for potential modification in order to design and provide more stable molecules. Previous assay methods for in vitro analysis of oligonucleotides are described, for example, by Basiri et al. in Molecular Therapy: Nucleic Acids; 21: 725-736 (2020).

[0238] Tissue Homogenization and Incubation — Frozen mouse livers were homogenized at 200 mg / mL tissue concentration in PBS buffer (pH 7.4) containing 1% NP-40. 200 μL of the homogenate was spiked with ASOs at 1-2 mM concentration and incubated at 37 °C for 48 hr.

[0239] Sample Preparation: 200 mL of 10% phosphoric acid was added to 200 mL incubated tissue homogenates and were vortexed for 5 min. 600 mL of the Clarity OTX Lysis-Loading Buffer (Phenomenex, PN ALO-8579) was added to each tube and were vortexed for 5 min followed by centrifugation at 3200 rpm at 4° C for 10 min. Clarity OTX SPE plates were used.Table 3 shows the detailed procedure.Table 3. Sample PreparationSPE Step SolventCondition 1 mL MethanolEquilibrate 1 mL 50 mM NaH2PO4, 2 mM NaN3, 10 mM K2EDTA in water (pH = 5.5) Load 0.4 mL pretreated sampleEquilibrate 1 mL 50 mM NaH2PO4. 2 mM NaN3, 10 mM K2EDTA in water (pH = 5.5) Wash 1 1 mL 50 mM NaH2PO4 in 50:50 (v:v) water: acetonitrile (pH = 5.5)Wash 2 1 mL 50 mM NH4HCO3 in 50:50 (v:v) water: acetonitrile (pH = 5.5)Elute 2×0.5 mL 100 mM NH4HCO3, 10 mM TCEP in 50:40:10 (v:v:v)water:acetonitrile:tetrahydrofuran (pH = 9.8)Evaporation Under nitrogen dryer (60°C) until dryReconstitute Reconstitute with 100 μL 5% MeOH in waterAnalysis 10-20 μL samples injected into the LC-HRMS for analysis

[0240] Sample Analyses were conducted by liquid chromatography and mass spectrometry'.

[0241] Liquid chromatography (LC): The ASO separation was carried out by ion pairing chromatography at 70 °C with a Waters BEH oligonucleotide 2.1 / 50 mm column. Mobile phase buffer A and B are water and methanol with 100 mM hexafluoroisopropanol (HFIP) and 15 mM N, N-diisopropylethylamine (DIEA). Chromatography was performed at 0.3 mL / min under the following gradient condition (min-%B) 0-5, 1-5. 6-50, 6.1-95, 7.1-95, 7.2-5, 10-5.The total run time was 10 min and the LC output was diverted to waste from 0-1 min and 7-10 min.

[0242] Mass spectrometry (MS): A high resolution MS with IDA method was used to identify catabolites.

[0243] Identified Soft Spots: The table below shows primary soft spots that were identified according to the methods provided herein. Once one or more soft spots are identified, including secondary soft spots, the relevant internucleoside linkage(s) is (are) independently replaced by a stabilizing internucleoside linkage, e.g, aPS2, MsPA, or PN internucleoside linkage. In some embodiments, one or more internucleoside linkages adjacent to the soft spot may be replaced with stabilizing internucleoside linkages. Table 4A lists primary soft spots (soft spots identified in a first round of soft spot analysis). The process may be repeated as necessary after stability testing in order to identify secondary soft spots and improve the stability of an ASO.Table 4A. Primary Soft Spots Identified in Exemplary' ASO Sequences.SEQ ID Modified Sequence (5' — > 3')NO111 + [%C] *+A*+ [%C] *A*T*T*G*$G*$A*A*dC*T*G*A*G*C*+A*+ [ %C ] *+T 112 + G*+T*+T*A*A*A*T*dC*T*A*G*T*T*G*T*+ [ %C] *+ [%C] *$+A113 +T*+ [ %C] *+T*C*T*$A*$T*A*T*A*T*A*A*dC*A*T*C*+A*+ [ %C ] *+T 114 +A*+A*+ [ %C] *T*G*C*T*T*A*G*T*G*A*T*T*+ [%C] *+ [ %C] *$+A115 +G*+G*+T*A*$A*dC*T*T*A*G*G*A*C*A*A*+G*+G*+T*+ = LNA (locked nucleoside)[%C] = 5'-methylcytosine* = phosphorothioate linkage$ = soft spot and is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkageTable 4B. Primary and Secondary Soft Spots Identified in Exemplary ASO Sequences. SEQ ID Modified Sequence (5' — > 3')NO112 +G*+T*+T*A*A*A*T*dC*T$A$G*T*T*G*T*+ [ %C] *+ [%C] *$+A +A*+A*+ [ %C] *T*G*C$T$T$A*G*T*G*A*T*T*+ [ %C] *+ [ %C] *$+A114+ = LNA (locked nucleoside)[%C] = 5'-methylcytosine* = phosphorothioate linkage$ = soft spot and is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkageExample 2. Construction ofOTV: SNCA ASO Conjugates.

[0244] i. Fctb-Fc dimer fusions design, cloning. Fab-Fc dimer fusions were designed that contain a first Fc polypeptide comprising a S239C cysteine substitution and hole and LALAPS mutations (SEQ ID NO:63), a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide and comprises a modified constant domain that specifically binds to a transferrin receptor (35.23.2), knob and LALAPS mutations (SEQ ID NO: 11), and wherein the first and second Fc polypeptide are each fused to a non-targeting Fab (SEQ ID NO: 108 and 109). Therefore, the first Fab-Fc polypeptide fusion has a sequence of SEQ ID NO: 100 and the second Fab-Fc polypeptide fusion has a sequence of SEQ ID NO:98.

[0245] Additionally, as described herein, the C-terminal lysine residue of an Fc polypeptide may be fully or partially removed by the cellular machinery during protein production. Therefore, a first Fc polypeptide may comprise SEQ ID NO: 80 and a second Fc may comprise SEQ ID NO: 12. Similarly, the first Fab-Fc polypeptide fusion may have a sequence of SEQ ID NO: 101 and the second Fab-Fc polypeptide fusion may have a sequence of SEQ ID NO:99.

[0246] Constructs were cloned by gene synthesis and Gibson assembly into a mammalian expression vector pRK5.

[0247] ii. Fab-Fc dimer fusions protein expression, and purification. Vectors were cotransfected to Expi293 cells along with the corresponding light chain vector in the ratio knob:hole:light chain of 1:1:2. The expressed protein was purified from conditioned media by loading the supernatant over a Protein A column. The column was washed with 10 column volumes of PBS, pH 7.4. The proteins were eluted with 50 mM sodium citrate, pH 3.0 containing 150 mM NaCl, and immediately neutralized with 200 mM arginine, 137 mM succinic acid, pH 5.0. The proteins were further purified by size-exclusion chromatography (SEC) (GE Superdex200) using 200 mM arginine, 137 mM succinic acid, pH 5.0 as running buffer. The purified proteins were confirmed by intact mass LC / MS, and purity of > 95% was confirmed by SDS-PAGE and analytical HPLC-SEC.

[0248] Hi. Synthesis of 5 ’-maleimide modified ASO

[0249] Preparation of 5’ -amino-modified ASO

[0250] Solid phase oligonucleotide synthesis. Oligonucleotide synthesis was performed on a MerMade 12 (LGC) DNA / RNA synthesizer at 100 pmol scale following standard solid-phase oligonucleotide synthesis protocols. All locked nucleic acid (LNA) and deoxyribonucleic acid (DNA) phosphorami dites were purchased from Hongene Biotech Corporation, including LNA-A(Bz), LNA-5MeC(Bz), LNA-T, LNA-G(dmf), and dA(Bz), dC(Ac), dT, dG(dmf). LNA-5MeC(Bz) was dissolved in a mixed solvent of DCM / acetonitrile (1:1, v / v), while all other phosphoramidites were dissolved in acetonitrile and molecular sieves (3 A) were added. Theparent antisense oligonucleotide (ASO) sequences were first assembled on UnyLinker CPG solid support, followed by attachment of amino linker on 5 '-end using 6-(Trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N, N-diisopropyl)-phosphoramidite (CAS: 133975-85-6; Glen Research Cat. # 10-1916). The synthesis cycle for adding one nucleotide (or non-nucleic acid) unit consists of four individual steps, detritylation, coupling, oxidation (or sulfurization), and capping. 5-Ethylthio-lH-tetrazole (ETT, 0.25 M in acetonitrile) was used as activator solution. A 0.2 M solution of PADS (phenylacetyl disulfide) in 50% pyridine / 50% acetonitrile was employed to introduce phosphorothioate linkages. Detailed protocols for 100 pmol scale ASO synthesis are summarized in Table 5A-5D below.Table 5A. Generic 100 pmol scale ASO synthesis parameters.Process Reagents Parameters Solid support UnyLinker CPG. 500 A, 80 pmol / g 100 pmol scale Wash Anhydrous acetonitrile (moisture content < 10 ppm) 10 mL, 20 s Detritylation 3% Trichloroacetic acid (TCA) in DCM 10 mL, 45 s 0.1 M phosphoramidites in ACN or ACN / DCM or DCE (1:1,Coupling v / v) 2.5 mL amidite,5 mL ETT, 6 min 0.25 M ETT in acetonitrileOxidation 0.02 M I₂ in THF / Pyridine / H₂O (7:2:1) 10 mL, 2 min 0.2 M PADS in 50% pyridine / 50% acetonitrileSulfurization or 10 mL, 4 min ADTT (0.2 M, 120 cq.) in pyridine, 10 minCap Mix A: THF / Pyridine / AceO (8:1:1) 2.5 mL Cap A, Capping 2.5 mL Cap B, Cap Mix B: 16% Methyl imidazole in THF40 sTable 5B. Example synthesis of oligo containing MsPA backbone modifications.Synthesizer AKTA oligopilot plus 100 (CV=6.3 mL)CPG Universal CPG (1000 A. 40 pmol / g)Scale 100 pmol0.2 M amidite, 6.0 eq.; 1.0 M DCI / 0.1 MNMI, 45 eq., 17 min (cycled Evice Coupling 1 (recycle)for the 1stmonomer; one time for all other amidites) Oxidation for PS bonds 0.02 M I₂ in MeCN / pyridine / H₂O, 75 / 20 / 5 (v / v / v), 5 eq., 5 min Oxidation for MsPA methanesulfonyl azide (0.5 M, 75 eq.) in MeCN, 30 min; repeated 2 times, bonds 60 min in totalSulfurization ADTT (0.2 M, in pyridine), 10 CV. 126 eq., 10 minTable 5C. Example synthesis of oligo containing PN backbone modifications.Synthesizer AKTA oligopilot plus 100 (CV=6.3 mL)CPG Universal CPG (1000 A. 40 pmol / g)Scale 100 pmol0.2 M amidite, 6.0 eq.; 1.0 M DCI / 0.1 M NMI, 45 eq., 17 min (cycled tw ice Coupling 1 (recycle)for the 1stmonomer; one time for all other amidites) Oxidation for PS bonds 0.02 M I₂ in MeCN / pyridine / H₂O. 75 / 20 / 5 (v / v / v), 5 eq., 5 min2-Azido-l,3-dimethylimidazolinium Hexafluorophosphate (0.5 M, in Oxidation for PN bonds MeCN), 10 mL. 50 eq., 120 minSulfurization ADTT (0.2 M, in pyridine), 10 CV, 126 eq., 10 minTable 5D. Example synthesis of oligo containing PS2 and PN backbone modifications.Synthesizer AKTA oligopilot plus 100 (CV=6.3 mL)CPG Universal CPG (500 A, 24 pmol / g)Scale 100 |imolLNA-C in 4:1 MeCN / DCM, other amidites in MeCN at 0.2 M, 6.0 eq.; 10.6 Coupling for PS and PN M ETT, 27 eq.. 19 min (cycled twice for the 1stmonomer; one time for all other amidites)Oxidation for PS bonds 0.02 M I₂ in MeCN / pyridine / H₂O. 75 / 20 / 5 (v / v / v), 5 eq., 5 min2-Azido-l,3-dimethylimidazolinium Hexafluorophosphate (0.5 M, in Oxidation for PN bondsMeCN), 10 mL, 50 eq., 120 minCoupling for PS2PS2 amidite 0.2 M in DCE, 6 eq.; 0.6 M ETT, 27 eq.. 19 min; one time monomersSulfurization ADTT (0.2 M, in pyridine), 10 CV, 126 eq., 10 min

[0251] Post-synthetic manipulations. Upon completion of solid-phase oligonucleotide synthesis, the phosphate protecting group (2-cyanoethyl group) was removed by a 20% solution of diethylamine (DEA) in acetonitrile for 1 hr. Cleavage from solid support and nucleobase deprotection (C& D) were performed in NH₄OH / EtOH (3:1) at 45 °C for 20 hr. The crude oligonucleotide solution was concentrated by centrifugal evaporation under reduced pressure, and the solid residue was reconstituted in water for prep-HPLC purification.

[0252] Purification of 5'-amino ASO by prep-HPLC. The crude 5'-amino ASO was purified by ion-pairing reverse phase HPLC. The detailed parameters were summarized in Table 6. The appropriate fractions were pooled and lyophilized to give the purified 5'-amino ASO.Table 6 Prep-HPLC purification conditions.Column Waters Xbridge BEH C18 19*150 mmMobile phase A 240 mM HFIP and 7 mM TEA in 95% water / 5% MeOH Mobile phase B 240 mM HFIP and 7 mM TEA in MeOHFlow rate 15 mL / minColumn temperature 50 °C0-3 min (10% B), 3-25 min (10% - 30%), 100% B hold time: Gradient2 minAmount per injection 100 mg crude ASO

[0253] Synthesis of 5'-amino-modified ASO. 5'-amino modified ASOs shown in Tables 1, 4A and 4B (above) were synthesized in accordance with the general methods mentioned above.

[0254] Preparation of 5'-maleimide modified ASO. General procedure: To a solution of ASO amine TEA salt (30 mg, by OD) in PBS buffer (TEKNOVA 10x PBS stock solution, pH 6.0, 2.5 mL) was added a solution of 3-maleimidopropionic acid N-hydroxysuccinimide ester (MCOSu, 10 eq.) in DMF (2.5 mL) at room temperature. The resulting solution was shaken at room temperature for 18 hr. Upon completion, the solution was desalted by passing through a sephadex G25 column (2.5x40 cm) on AKTA pure 25 M, eluting with Milli Q water at 3 mL / min, monitored by UV 260 / 280 and conductivity. The appropriate fractions were pooled and lyophilized to give desired 5'-maleimide ASO.

[0255] iv. Bioconjugation. The Fab-Fc dimer fusions generated above containing the S239C cysteine modification for conjugation was first reduced using 30 molar equivalents of TCEP and 2 mM EDTA, 37 °C for 1 hr. Reduction was confirmed by LC / MS. Post reduction, remaining TCEP was removed by dialysis using 1 xPBS, pH 6.8 with 2 mM EDTA (purification by e.g.. dialysis) and the Fab-Fc dimer fusions were reoxidized with 50 molar equivalents of dHAA at room temperature for 3 hr. Oxidation was confirmed by LC / MS of dHAA. For the bioconjugation, 1.2 molar equivalents of the 5’-maleimide modified ASOs generated above were added to the oxidized Fab-Fc dimer fusions at room temperature for 1 hr. The resulting SNCA ASO conjugates were purified by anion exchange chromatography using Resource Q column (equilibration buffer: 50mM Tris. pH 7.5, elution buffer: 50 mM Tris, pH 7.5 + 2 M NaCl) to remove unwanted and unconjugated products. Purity of the SNCA ASO conjugates were determined by LC / MS and SEC. The resulting conjugates are referred to as OTV or OTV SNCA.Example 3. In Vivo Methods.

[0256] Mouse handling and tissue collection. Mice were peripherally administered therapeutic treatment via intravenous (IV) tail vein injection (-200 pL total volume). For in-life plasma collection, blood was collected via submental puncture and transferred to EDTA coated tubes, then spun down at 12,700 rpm for 7 min at 4 °C before collecting the top plasma layer. For tissue collection, animals were anesthetized with tribromoethanol and whole blood was collected via cardiac puncture into EDTA coated tubes for plasma drug concentration assessment. Following transfer to EDTA coated tubes, whole blood was spun down at 12,700 rpm for 7 min at 4 °C before collecting the top plasma layer. Mice were then perfused with ice-cold PBS transcardially at a rate of 5 mL / min for 5 min. For biochemical analysis, tissues were collected, weighed, snap frozen on dry ice, and then stored at -80°C.

[0257] Intracerebroventricular Bolus (ICV) surgery. Procedures were performed as described in DeVos SL (“Direct intraventricular delivery of drugs to the rodent central nervous system” J Vis Exp 2013 May 12:(75):e50326; incorporated herein by reference) and summarized in brief as follows. In preparation for the surgery, the surgical area was sterilized with 70% ethanol. Mice were brought under anesthesia with 4% isoflurane. Hair was shaved between from the shoulder region to between the eyes prior to placing mouse on stereotax surface. With a maintenance level of 2% isoflurane, an incision was made from the base of the neck up to between the eyes. Following cleaning with hydrogen peroxide, a needle was slowly driven through the skull at a rate of 1 mm / s. After a 2-3 min period to allow for brain sealing around the needle, a dose of 10 pL ASO at 1 pL per second was administered. With a cotton swab held against the skull at the base of the needle, the needle was raised at a rate of 1 mm per second. The cotton swab was held at the site of injection for 1 min to limit drug leakage. Following ICV bolus, the incision was sutured and treated with an antibiotic ointment. The mouse was transferred to a heated recovery pad and observed for full recover}'. Mice were monitored daily after surgery to check for pain, discomfort, or infections.

[0258] Tissue homogenization for drug concentration measurement and protein assays. Weighed frozen tissue samples were processed for biochemical assays by adding 10x volume chilled 1% NP40 + PBS homogenization buffer with added complete Protease Inhibitor (Roche #04693132001) and PhosStop (Roche 04906837001) phosphatase inhibitors. Samples were homogenized using 3 mm tungsten carbide beads in 1.5 mL Eppendorf tubes, shaken using the Qiagen TIssueLyzer II (Cat No. / ID: 85300) (2x3 min at 27 Hz). For protein assays, samples were then centrifuged for 15 min at 17000xg, and the supernatant was removed and used for assays.

[0259] Tissue homogenization for RNA measurements. Weighed frozen tissue samples were processed for RNA assays by adding 10x volume Qiazol reagent. Samples were homogenized using 5 mm tungsten carbide beads in 2 mL Eppendorf tubes, shaken using the Qiagen TIssueLyzer II (Cat No. / ID: 85300) (2x3 min at 27 Hz). After lysis, samples were incubated for 5 min at room temperature, then chloroform was added. Samples were vortexed, incubated at room temperature for 3 min, then centrifuged for 15 min at 12000xg at 4 °C. The aqueous phase was then isolated. RNA was then isolated by adding isopropanol, vortexing, incubating for 10 min at room temperature, then centrifuging for 10 min at 12000xg at 4 °C. The resultingpellet was then resuspended in 75% ethanol, vortexed and centrifuged for 5 min at 7500 / g at 4 °C. The final pellet was resuspended in water.

[0260] huIgG Assay. Quantification of humanized antibodies in mouse plasma and tissue lysates were measured using a generic electrochemiluminescence immunoassay (ECLIA). Briefly, to the wells of an MSD GOLD 96-well streptavidin-coated microtiter plate (Meso Scale Discovery, Rockville, MD), a working concentration of biotinylated goat anti-human IgG polyclonal primary antibody (Southern Biotech, Birmingham, AL) prepared in assay diluent was incubated for approximately 1 hr. Following this incubation and a plate wash step, prepared test samples (with sample pre-dilution, where appropriate) and relevant standards were added to the assay plate and allowed to incubate for approximately 1 hr. Following test sample incubation and a plate wash step, secondary ruthenylated (SULFO-TAG) goat antihuman IgG antibody (Meso Scale Discovery, Rockville, MD) at a working concentration in assay diluent was added to the assay plate and incubated for approximately 1 hr. Following a plate wash, a 1 x MSD Read Buffer T (Meso Scale Discovery, Rockville, MD) was then added to generate the electrochemiluminescence (ECL) assay signal, which was then expressed in ECL units (ECLU). All of the assay reaction steps were performed at ambient temperature with shaking on a plate shaker (where appropriate); and all test samples were pre-diluted at the assay MRD of 1:20 prior to analyzing in the assay plate. Sample ECLU signals generated in the assay subsequently were processed into concentrations by back-calculating off the assay calibration (CS) curve. The assay CS curve was fitted with a weighted four-parameter nonlinear logistic regression for use in calculating concentrations for unknown / test samples.

[0261] Total ASO Assay. Quantification of total ASO (in conjugated and free forms) in mouse plasma and tissue homogenates were measured using a hybridization-based electrochemiluminescence immunoassay (ECLIA). Briefly, custom biotinylated and digoxigenin-conjugated antisense probes (synthesized by Integrated DNA Technologies, Coralville, IA) at working concentrations were combined with prepared test samples (with sample pre-dilution, where appropriate) and relevant standards in TE Buffer (10 mM Tris-HCL containing 1 mM EDTA). Prepared samples in TE buffer were added, in a 1: 1 mix, into 1 x SSC Buffer (Sigma-Aldrich. St. Louis, MO) containing a working concentration of recombinant proteinase K enzyme (ThermoFisher, Waltham, MA). Hybridization / Enzyme mixture was then digested, denatured, annealed, and cooled in a thermal cycler instrument. Following hy brid product incubation, samples were added to the wells of an MSD GOLD 96-well streptavidin-coated microtiter plate (Meso Scale Discovery, Rockville, MD) and incubated for approximately 30 min. Following incubation and a plate wash step, secondaryruthenylated (SULFO-TAG) sheep anti-digoxigenin antibody (Novus Biologicals, Littleton, CO) at a working concentration in assay diluent was added to the plate and incubated for approximately 30 min. Following a plate wash, a lx MSD Read Buffer T (Meso Scale Discovery, Rockville, MD) was then added to generate the electrochemiluminescence (ECL) assay signal, which was then expressed in ECL units (ECLU). All of the assay reaction steps were performed at ambient temperature with shaking on a plate shaker (where appropriate); and all test samples were pre-diluted at the assay MRD of 1:20 prior to analyzing in the assay plate. Sample ECLU signals generated in the assay subsequently were processed into concentrations by back-calculating off the assay calibration (CS) curve. The assay CS curve was fitted with a weighted four-parameter nonlinear logistic regression for use in calculating concentrations for unknown / test samples.

[0262] qPCR analysis for hSNCA. To evaluate target mRNA levels, qRT-PCR was run on RNA extracted from tissue lysates. Target mRNA levels were evaluated using Taqman probes (hSNCA, mGapdh) and the Express One-Step Kit. For each sample, hSNCA mRNA levels were normalized to the housekeeping gene Gapdh. qRT-PCR was performed using a QuantStudio 6 Flex system (Applied Biosystems) and average CT values were measured for each probe using technical duplicates. Next, the delta delta CT (AACT) values were calculated relative to the non-ASO treated group and plotted as relative expression levels.

[0263] MSD analysis for a-synuclein protein. To evaluate target a-synuclein protein levels in tissue homogenates, samples were evaluated using the MSD U-PLEX Plus human a-synuclein kit. Brain samples were diluted 1:100,000 for the assay. Protein concentrations following treatment were normalized to age-matched saline treated animals to evaluate relative expression levels.Example 4. Modified ASO ICV bolus screening in hSNCA: TfR KI mice.

[0264] 43 backbone modified versions of two previously identified parent ASOs (referred to as “Parent E’ and “Parent_2”) were generated as described. The 43 modified ASOs are found in Table 1. To confirm modified ASO potency, the ASOs were diluted in sterile saline and administered to hSNCA (mSNCA⁻ / ⁻); TfR KI mice via ICV injection at a dose of 25 pg in a volume of 10 pL. Unmodified ASOs Parent l and Parent_1 were also tested as a control. 10 days after dosing, tissues were harvested and SNCA expression was measured in the brain via bulk RNA isolation followed by qPCR of SNCA and Gapdh. SNCA knockdown in brain isshown in Table 7. These data suggest that many of the modified ASOs maintain comparable potency relative to the unmodified parent ASOs.Table 7. ICV bolus assessment of modified SCNA ASO potency in hSNCA: TfR KI mice (RNA expression (Normalized to Saline and Gapdh, Mean±S. E. M.)ASO ID SNCA expression ASO ID SNCA expressionSaline l±0.04 Parent_2 0.6±0.03Parent l 0.53±0.04 ASO 4 0.58±0.08ASO l 0.44±0.12 ASO 6 0.69±0.03ASO 2 0.52±0.02 ASO 7 0.69±0.02ASO 3 0.54±0.08 ASO 8 0.71±0.13ASO_5 0.68±0.02 ASO 10 0.71±0.07ASO 9 0.57±0.02 ASO ll 0.54±0.04ASO 13 0.6±0.03 ASO 12 0.55±0.01ASO 16 0.62±0.07 ASO 14 0.61±0.04ASO 18 0.64±0.06 ASO 15 0.62±0.05ASO 20 0.65±0.06 ASO 17 0.63±0.02ASO 22 0.66±0.05 ASO 19 0.64±0.05ASO 24 0.67±0.06 ASO 21 0.66±0.05ASO 26 0.69±0.06 ASO 23 0.66±0.08ASO 28 0.75±0.05 ASO 25 0.69±0.02ASO 32 0.74±0.05 ASO 27 0.7±0.11ASO 33 0.74±0.06 ASO 29 0.79±0.04ASO 34 0.67±0.07 ASO 30 0.81±0.05ASO 38 0.79±0.07 ASO 31 0.6±0.01ASO 35 0.71±0.05ASO 36 0.72±0.09ASO 37 0.73±0.05ASO 39 0.79±0.04Example 5. Rodent Plasma Pharmacokinetics of modified OTV molecules.

[0265] To assess pharmacokinetics of modified ASOs when conjugated to the TV platform, ASO_1-ASO_43 were conjugated to an OTV (TfR-targeting Fab-Fc fusion polypeptide dimer comprising SEQ ID NOs: 132, 133, and 134) (except ASO_28, which was excluded from the OTV analysis) to form SNCA ASO conjugates. The SNCA ASO conjugates were administered to Sprague Dawley rats at a concentration of 10 mg / kg via intravenous injection. For a control, SNCA ASO conjugates comprising with “Parent_2” orc'Parent_4” ASOs were also administered. All drugs w ere administered at a dose volume of 2 mL / kg. Plasma w as collected at 0.25, 4, 24, 48, 72, and 168 hr after injection. hlgG and ASO concentrations were measuredin plasma using the methods described above. Pharmacokinetics parameters for the systemic exposure of the SNCA ASO conjugates are shown in Table 8. These results demonstrate slower systemic clearance profiles with the SNCA ASO conjugates containing the modified SNCA ASOs compared to the parent SNCA ASOs. The modified ASOs persisted longer, which can enable greater uptake into relevant tissues and enhance the SNCA ASO conjugates’ therapeutic indices.Table 8. Rodent plasma PK assessment of SNCA ASO conjugatesConjugate huIgG ASOASO Cmax AUClast CL Cmax AUClast CL Parent_2 1.78 31.4 46.6 1.32 16.5 83.1 Parent_4 1.86 28.2 56.2 1.29 17.2 79.5 ASO l 1.68 60.7 24.7 1.14 33.1 46.7 ASO 2 1.58 56.1 27.9 1.17 31.8 49.3 ASO 3 1.93 52.2 30.0 1.34 25.2 62.2 ASO 4 1.73 51.1 30.6 0.913 17.8 88.7 ASO 5 2.35 100 15.3 1.48 48.2 32.0 ASO 6 2.16 75.0 16.8 1.10 24.0 63.4 ASO 7 1.81 57.3 27.4 1.09 17.9 87.9 ASO 8 1.89 58.8 26.9 0.787 13.9 117ASO 9 2.69 78.4 19.9 0.588 7.89 206 ASO lO 2.62 101 15.2 0.611 10.9 140 ASO 11 1.66 46.3 32.0 1.02 18.1 86.7 ASO 12 2.46 116 10.3 1.27 28.3 53.6 ASO 13 2.23 58.3 25.7 0.778 12.0 130ASO 14 1.90 61.9 23.0 1.05 17.2 91.4 ASO 15 1.70 79.9 14.7 1.31 24.3 63.4 ASO 16 1.84 53.7 29.5 1.45 23.5 66.7 ASO 17 2.42 52.7 30.1 0.485 5.97 285 ASO 18 2.49 102 13.6 1.63 37.9 40.5 ASO 19 2.64 62.2 24.3 0.655 9.10 173 ASO 20 2.08 49.9 28.5 0.826 10.4 152 ASO 21 2.58 73.5 20.2 1.22 19.6 80.6 ASO 22 2.40 40.0 37.9 N / A N / A N / A ASO 23 1.78 51.9 29.0 1.10 19.0 86.2 ASO 24 1.81 46.1 34.3 N / A N / A N / A ASO 25 1.91 49.6 31.7 N / A N / A N / A ASO 26 2.81 55.6 25.7 N / A N / A N / A ASO 27 2.41 37.6 41.6 N / A N / A N / A ASO 29 2.48 111 8.96 N / A N / A N / AASO 30 2.78 35.3 42.2 N / A N / A N / A ASO 31 3.15 171 7.76 0.844 20.6 75.5ASO 32 3.02 118 13.3 0.876 19.2 81.9ASO 33 3.85 148 10.3 1.12 24.4 65.5 ASO 34 1.95 56.5 27.9 1.10 18.0 87.7ASO 35 2.26 60.2 23.7 N / A N / A N / A ASO 36 2.61 74.8 21.0 N / A N / A N / A ASO 37 2.39 61.6 25.3 0.899 12.8 123 ASO 38 2.72 37.6 42.3 N / A N / A N / A ASO 39 3.32 62.7 23.2 N / A N / A N / AASO 40 1.74 18.4 77.8 N / A N / A N / A ASO 41 2.69 63.6 23.7 N / A N / A N / A ASO 42 2.24 67.3 22.7 N / A N / A N / AASO 43 1.86 31.3 41.4 N / A N / A N / AExample 6. hSNCA knockdown duration of action in hSNCA. TfR KI mice.

[0266] To further assess the pharmacokinetic and pharmacodynamic profdes of SNCA conjugates, two SNCA conjugates (containing ASO 5 and ASO_6: OTV_5 and OTV 6. respectively) were dosed in hSNCA: TfR KI mice. Mice received 4, 25 mg / kg doses of SNCA conjugate over a 10 day period, at a dose concentration of 5 mg / mL. 1 day prior to the first dose of SNCA conjugate, mice received a single 5 mg dose of anti-CD4 antibodies. Brain and plasma were harvested either 7 or 40 days after the last SNCA conjugate dose was administered. huIgG and ASO concentrations were measured in plasma and brain using the methods described above. Plasma exposures of hlgG and ASO are show n in FIGs. 1A-B. Brain exposures of hlgG and ASO are shown in FIGs. 1C-D. SNCA RNA expression was measured in the brain via bulk RNA isolation follow ed by qPCR of SNCA and Gapdh according to the protocol described above. SNCA RNA knockdown relative to saline treated controls is shown in FIG. IE. Brain a-synuclein protein levels were also measured in the brain via bulk brain homogenization and MSD assay quantification. Reduction of brain a-synuclein levels in the brain are shown in FIG. IF. These findings demonstrate that the SNCA conjugates targeting SNCA can achieve robust exposure in the brain with systemic delivery and reduced SNCA and a-synuclein expression.

Claims

1. Claims:

1. A synuclein (SNCA) antisense oligonucleotides (ASO) conjugate comprising a transferrin receptor (TfR)-targeting Fc polypeptide dimer and a SNCA ASO, wherein:3.(a) the TfR-targeting Fc polypeptide dimer comprises:4.(i) a first Fc polypeptide, and5.(ii) a second Fc polypeptide, wherein the second Fc polypeptide comprises a modified constant domain that specifically binds to human transferrin receptor 1 (TfR.) and comprises a sequence having at least 90% sequence identity to SEQ ID NO: 12, wherein second Fc polypeptide forms an Fc dimer with the first Fc polypeptide6.(b) the SNCA ASO comprises7.(i) +[%C]$+A$+[%C]$dA*dT*dT$dG$dG$dA$dA*dC*dT*dG*dA*dG$dC$+A$+[%C]$+T (SEQ ID NO: 111)8.(ii) +GS+TS+TSdA*dA*dA*dT*dCSdT$dASdGSdT*dT*dG$dT$+[%C]$+[%C]S+A (SEQ ID NO: 112);9.(iii) +T$+[%C]$+T$dC*dT$dA$dT$dA*dT*dA*dA*dC*dA*dT$dC$+A$+[%C]S+T (SEQ ID NO: 113);10.(iv) +AS+AS+[%C]SdT*dG$dC$dT$dT$dA$dG*dT*dG*dA*dT$dT$+[%C]S+[%C]S+A (SEQ ID NO: 114); or11.(v) +GS+GS+TSdASdASdC*dT*dT*dA*dG*dG*dA*dC*dASdAS+GS+G$+T12.(SEQ ID NO: 115);13.wherein14.+AL, +[%C], +G, and +T are adenosine, 5-methylcytosine, guanosine, and thymine locked nucleosides, respectively,15.dA, dC, dG, and dT are deoxyadenosine, deoxy cytidine, deoxyguanidine, and deoxythymidine nucleosides, respectively,16.each * is a phosphorothioate (PS) internucleoside linkage, and each $ is independently a PS internucleoside linkage or a stabilizing internucleoside linkage,17.wherein the Fc polypeptide dimer is linked to the SNCA.

2. The SNCA ASO conjugate of claim 1, wherein each $ is a PS internucleoside linkage.

3. The SNCA ASO conjugate of claim 1, wherein each $ is a stabilizing internucleoside linkage.

4. The SNCA ASO conjugate of claim 1, wherein each $ is phosphorodithioate (PS2) internucleoside linkage.

5. The SNCA ASO conjugate of claim 1, wherein each $ is a phosphorylguanidine (PN) internucleoside linkage.

6. The SNCA ASO conjugate of claim 1, wherein each $ is a mesylphosphoramidate (MsPA) internucleoside linkages.

7. The SNCA ASO conjugate of claim 1. wherein the SNCA ASO comprises the oligonucleotide:23.5 ' +[%C] $+ A$+[%C] $d A*dT*dT$dG$dG$d A$dA*dC*dT*dG*dA*dG$dC$+ A$+ [%C] $+T 3 ' (SEQ ID NO: 111)24.wherein25.+[%C] is a 5-methylcytosine locked nucleoside;26.+A is an adenosine locked nucleoside;27.+T is a thymine locked nucleoside;28.dA is a deoxy adenosine;29.dC is a deoxy cytidine;30.dT is a deoxythymidine;31.dG is a deoxy guanosine;32.* is a PS internucleoside linkage; and33.each $ is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkage.

8. The SNCA ASO conjugate of claim 1, wherein the SNCA ASO comprises the oligonucleotide:34.5' +G$+T$+T$dA*dA*dA*dT*dC$dT$dA$dG$dT*dT*dG$dT$+[%C]$+[%C]$+A 3' (SEQ ID NO: 112)35.wherein36.+G is a guanosine locked nucleoside;37.+[%C] is a 5-methylcytosine locked nucleoside;38.+A is an adenosine locked nucleoside;39.+T is a thymine locked nucleoside;40.dA is a deoxy adenosine; dC is a deoxy cytidine;41.dT is a deoxythymidine;42.dG is a deoxy guanosine;43.* is a PS internucleoside linkage; and44.each $ is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkage.

9. The SNCA ASO conjugate of claim 8, wherein the SNCA ASO comprises the oligonucleotide:46.(a) +G*+T*+T*dA*dA*dA*dT*dC*dTtdAtdG*dT*dT*dGndTn+[%C]n+[%C]n+A; (b) +Gn+Tn+T*dA*dA*dA*dT*dC*dTtdAtdG*dT*dT*dG*dT*+[%C]n+[%C]n+A; (c) +G*+T*+T*dA*dA*dA*dT*dC*dT*dAtdG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (d) +Gn+Tn+TndA*dA*dA*dT*dC*dTtdAtdG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (e) +G*+T*+T*dA*dA*dA*dT*dC*dT*dAtdG*dT*dT*dG*dT*+[%C]*+[%C]t+A; (f) +G*+T*+T*dA*dA*dA*dT*dCndTndAndG*dT*dT*dG*dTu+[%C]u+[%C]u+A; (g) +G*+T*+T*dA*dA*dA*dT*dC*dTudAudGudT*dT*dG*dTn+[%C]n+[%C]n+A; (h) +Gn+Tn+TndA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (i) +G*+T*+T*dA*dA*dA*dT*dCndTndAndGsicdT*dT*dG*dT*+[%C]*+[%C]u+A; (j) +G*+T*+T*dA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (k) +G*+T*+T*dA*dA*dA*dT*dCudTudAudG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (l) +G*+T*+T*dA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dTn+[%C]n+[%C]u+A; (m)+G*+T*+T*dA*dA*dA*dT*dC*dTndAndGndT*dT*dG*dT*+[%C]*+[%C]u+A; (n) +G*+T*+T*dA*dA*dA*dT*dC*dT*dAudG*dT*dT*dG*dT*+[%C]*+[%C]u+A; (o) +G*+T*+T*dA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dT*+[%C]*+[%C]u+A; (p) +G*+T*+T*dA*dA*dA*dT*dC*dTndAndG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (q) +G*+T*+T*dA*dA*dA*dT*dC*dTndAndG*dT*dT*dG*dT*+[%C]n+[%C]n+A; wherein47.+G is a guanosine locked nucleoside;48.+[%C] is a 5-methylcytosine locked nucleoside;49.+A is an adenosine locked nucleoside;50.+T is a thymine locked nucleoside;51.dA is a deoxy adenosine;52.dC is a deoxy cytidine;53.dT is a deoxythymidine; dG is a deoxy guanosine;54.* is a PS internucleoside linkage;55.t is a PS2 internucleoside linkage;56.n is a PN internucleoside linkage; and57.u is a MsPA internucleoside linkage.

10. The SNCA ASO conjugate of claim 1, wherein the SNCA ASO comprises the oligonucleotide:59.5' +T$+[%C]$+T$dC*dT$dA$dT$dA*dT*dA*dA*dC*dA*dT$dC$+A$+[%C]$+T 3' (SEQ ID NO: 113)60.wherein61.+T is a thymine locked nucleoside;62.+[%C] is a 5-methylcytisine locked nucleoside;63.+A is an adenosine locked nucleoside;64.dA is a deoxy adenosine;65.dC is a deoxy cytidine;66.dT is a deoxythymidine;67.dG is a deoxy guanosine;68.* is a PS internucleoside linkage; and69.each $ is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkage.

11. The SNCA ASO conjugate of claim 1, wherein the SNCA ASO comprises the oligonucleotide:70.5' +A$+A$+[%C]$dT*dG$dC$dT$dT$dA$dG*dT*dG*dA*dT$dT$+[%C]$+[%C]$+A 3' (SEQ ID NO: 114)71.wherein72.+A is an adenosine locked nucleoside;73.+[%C] is a 5-methylcytosine locked nucleoside;74.dA is a deoxy adenosine;75.dC is a deoxy cytidine;76.dT is a deoxythymidine;77.dG is a deoxy guanosine;78.* is a PS internucleoside linkage; and each $ is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkage.

12. The SNCA ASO conjugate of claim 11, wherein the SNCA ASO comprises the oligonucleotide:80.(a) +A*+A*+[%C]*dT*dG*dC*dTtdT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (b) +An+An+[%C]ndT*dG*dC*dTtdTtdA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (c) +An+An+[%C]*dT*dG*dC*dTtdTtdA*dG*dT*dG*dA*dT*dT*+[%C]n+[%C]n+A (d) +A*+A*+[%C]*dT*dG*dC*dTtdTtdA*dG*dT*dG*dA*dTndTn+[%C]n+[%C]n+A (e) +A*+A*+[%C]*dT*dG*dC*dTtdT*dA*dG*dT*dG*dA*dT*dT*+[%C]*+[%C]t+A (f) +A*+A*+[%C]*dT*dG*dCudTudTudA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (g) +An+An+[%C]ndT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (h) +A*+A*+[%C]*dT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (i) +A*+A*+[%C]*dT*dGudCudTudT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (j) +A*+A*+[%C]*dT*dG*dC*dTndTndAndG*dT*dG*dA*dT*dT*+[%C]*+[%C]u+A (k) +A*+A*+[%C]*dT*dG*dCndTndTndA*dG*dT*dG*dA*dT*dT*+[%C]*+[%C]u+A (l) +A*+A*+[%C]*dT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]u+A (m)+A*+A*+[%C]*dT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]u+A (n) +A*+A*+[%C]*dT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (o) +A*+A*+[%C]*dT*dG*dC*dTudTudAudG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (p) +An+An+[%C]ndT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (q) +A*+A*+[%C]*dT*dG*dC*dTndTndAndG*dT*dG*dA*dT*dTu+[%C]u+[%C]u+A (r) +A*+A*+[%C]*dT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dT*+[%C]*+[%C]u+A (s) +A*+A*+[%C]*dT*dG*dCndTndT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (t) +A*+A*+[%C]*dT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]u+A (u) +A*+A*+[%C]*dT*dG*dC*dTndTndA*dG*dT*dG*dA*dT*dT*+[%C]n+[%C]n+A (v) +A*+A*+[%C]*dT*dG*dCndTndT*dA*dG*dT*dG*dA*dT*dT*+[%C]n+[%C]n+A wherein81.+A is an adenosine locked nucleoside;82.+[%C] is a 5-methylcytosine locked nucleoside;83.dA is a deoxy adenosine;84.dC is a deoxy cytidine;85.dT is a deoxythymidine;86.dG is a deoxy guanosine; * is a PS internucleoside linkage;87.t is a PS2 internucleoside linkage;88.n is a PN internucleoside linkage; and89.u is a MsPA internucleoside linkage.

13. The SNCA ASO conjugate of claim 1. wherein the SNCA ASO comprises the oligonucleotide:91.5' +G$+G$+T$dA$dA$dC*dT*dT*dA*dG*dG*dA*dC*dA$dA$+G$+G$+T 3' (SEQ ID NO: 115)92.wherein93.+G is a guanosine locked nucleoside;94.+T is a thymine locked nucleoside;95.dA is a deoxy adenosine;96.dC is a deoxy cytidine;97.dT is a deoxythymidine;98.dG is a deoxy guanosine;99.* is a PS internucleoside linkage; and100.each $ is independently a PS internucleoside linkage, a PS2 internucleoside linkage, a MsPA internucleoside linkage, or a PN internucleoside linkage.

14. The SNCA ASO conjugate of any one of claims 1-13, wherein the modified constant domain that specifically binds to TfR. comprises a glutamate at position 380, a tyrosine at position 384, a threonine at position 386, a glutamate at position 387, a tryptophan at position 388, an alanine at position 389, an asparagine at position 390. a threonine at position 413, a glutamate at position 415, a glutamate at position 416, and a phenylalanine at position 421, according to EU numbering.

15. The SNCA ASO conjugate of any one of claims 1-14. wherein the first Fc polypeptide and / or the second Fc polypeptide comprises at least one cysteine substitution.

16. The SNCA ASO conjugate of claim 15, wherein at least one cysteine substitution is selected from the group consisting of a S239C substitution, a S442C substitution, a A330C substitution, a K149C substitution, or aT289C substitution.

17. The SNCA ASO conjugate of claim 16, wherein the first Fc polypeptide comprises the at least one cysteine substitution.

18. The SNCA ASO conjugate of any one of claims 15-17, wherein Fc dimer is linked to the SNCA ASO via a linking group attached to the at least one cysteine substitution and the 5' end of the SNCA ASO, wherein the linking group is105.

19. The SNCA ASO conjugate of any one of claims 1-18, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprises one or more mutations that modulate effector function.

20. The SNCA ASO conjugate of claim 19, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises an alanine at position 234, an alanine at position 235, and a serine or glycine at position 329, each according to EU numbering.

21. The SNCA ASO conjugate of any one of claims 1-20, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises one or more mutations that increase serum stability.

22. The SNCA ASO conjugate of any one of claims 1-21, wherein the second Fc polypeptide further comprises a try ptophan at position 366, according to EU numbering.

23. The SNCA ASO conjugate of claim 22, wherein the first Fc polypeptide further comprises a serine a position 366, an alanine at position 368, and a valine at position 407, according to EU numbering.

24. The SNCA ASO conjugate of any one of claims 1-21, wherein the second Fc polypeptide further comprises a serine a position 366, an alanine at position 368, and a valine at position 407, according to EU numbering.

25. The SNCA ASO conjugate of claim 24, wherein the first Fc polypeptide further comprises a try ptophan at position 366, according to EU numbering.

26. The SNCA ASO conjugate of any one of claims 1-14. wherein the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 47-48, 51-54, 61-63, 66,73-78, 80, and 90; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 4-29.

36. 43-44, 49, and 50.

27. The SNCA ASO conjugate of claim 26, wherein the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO:63 or 80; and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12.

28. The SNCA ASO conjugate of any one of claims 1-27, wherein the first Fc polypeptide is fused to a first non-targeting Fab (NTF) via a first hinge region to form a first Fab-Fc fusion polypeptide29. The SNCA ASO conjugate of any one of claims 1-27, wherein the second Fc polypeptide is fused to second NTF via a second hinge region to form a second Fab-Fc fusion polypeptide.

30. The SNCA ASO conjugate of any one of claims 1-27, wherein the first Fc polypeptide is fused to a NTF via a first hinge region to form a first Fab-Fc fusion polypeptide and the second Fc polypeptide is fused to second NTF via a second hinge region to form a second Fab-Fc fusion polypeptide.

31. The SNCA ASO conjugate of any one of claims 27-30, wherein the first Fab-Fc fusion polypeptide and / or the second Fab-Fc fusion polypeptide comprises at least one cysteine substitution, optionally wherein the cysteine substitution is selected form the group consisting of position 114 of the (according to Kabat numbering) of the heavy chain, position 124 (according to EU numbering) of the heavy chain; position 149 (according to EU numbering) of the light chain, or position 156 (according to EU numbering) of the light chain.

32. The SNCA ASO conjugate of any one of claims 27-31, wherein the first and second NTFs each comprise a heavy chain comprising SEQ ID NO: 109 or 130 and a light chain comprising SEQ ID NO: 108 or 129. optionally wherein the heavy chain further comprises a hinge region comprising SEQ ID NO:91, 92, 93, or 121.

33. The SNCA ASO conjugate of any one of claims 27-32, wherein the first Fab-Fc fusion polypeptide comprises SEQ ID NO: 100 or 101 and the second Fab-Fc fusion polypeptide comprises SEQ ID NO:98 or 99.

34. A SNCA ASO conjugate comprising a TfR-targeting Fc polypeptide dimer and at least one SNCA ASO, wherein:121.(a) the TfR-targeting Fc polypeptide dimer comprises122.(i) a first Fc polypeptide,123.(ii) a second Fc polypeptide, wherein the second Fc polypeptide comprises an alanine at position 234, an alanine at position 235, and serine at position 329, each according to EU numbering, and a modified constant domain that specifically binds to human transferrin receptor 1 (TfR) and comprises a sequence having at least 90% sequence identity to SEQ ID NO: 12, wherein second Fc polypeptide forms an Fc dimer with the first Fc polypeptide,124.(iii) a first non-targeting Fab (NTF) fused to the first Fc polypeptide via a first hinge region to form a first Fab-Fc fusion polypeptide and a second NTF fused to the second Fc polypeptide via a second hinge region to form a second Fab-Fc fusion polypeptide, (b) the at least one SNCA ASO comprises125.(i) +[%C]$+A$+[%C]$dA*dT*dT$dG$dG$dA$dA*dC*dT*dG*dA*dG$dC$+A$+[%C]$+T (SEQ ID NO: 111)126.(ii) +GS+TS+TSdA*dA*dA*dT*dCSdT$dASdGSdT*dT*dG$dT$+[%C]$+[%C]$+A127.(SEQ ID NO: 112);128.(iii)+T$+[%C]$+T$dC*dT$dA$dT$dA*dT*dA*dA*dC*dA*dT$dC$+A$+[%C]$+T129.(SEQ ID NO: 113);130.(iv)+AS+AS+[%C]SdT GSdC$dT$dT$dA$dG*dT*dG*dA*dT$dT$+[%C]$+[%C]S+A (SEQ ID NO: 114); or131.(v) +GS+GS+TSdASdASdC*dT*dT*dA*dG*dG*dA*dC*dASdAS+GS+G$+T132.(SEQ ID NO: 115);133.wherein134.+AL, +[%C], +G, and +T are adenosine, 5-methylcytosine, guanosine, and thymine locked nucleosides, respectively,135.dA, dC, dG, and dT are deoxyadenosine, deoxycytidine,136.deoxy guanidine, and deoxythymidine nucleosides, respectively,137.each * is a phosphorothioate (PS) internucleoside linkage, and each $ is independently a PS internucleoside linkage or a stabilizing internucleoside linkage,138.wherein the first Fab-Fc fusion polypeptide and / or the second Fab-Fc fusion polypeptide comprises at least one cysteine substitution, and wherein the at least one SNCA ASO is conjugated to the first Fab-Fc fusion polypeptide and / or the second Fab-Fc fusion polypeptide to the at least one cysteine substitution via a linker.

35. An SNCA ASO comprising:140.(i) +[%C]$+A$+[%C]$dA*dT*dT$dG$dG$dA$dA*dC*dT*dG*dA*dG$dC$+A$+[%C]$+T (SEQ ID NO: 111)141.(ii) +GS+TS+TSdA*dA*dA*dT*dCSdT$dASdGSdT*dT*dG$dT$+[%C]$+[%C]$+A142.(SEQ ID NO: 112);143.(iii) +TS+[%C]S+TSdC*dTSdASdT$dA*dT*dA*dA*dC*dA*dT$dC$+A$+[%C]$+T (SEQ ID NO: 113);144.(iv)+AS+AS+[%C]SdT*dGSdC$dT$dT$dA$dG*dT*dG*dA*dT$dT$+[%C]$+[%C]S+A (SEQ ID NO: 114); or145.(v) +G$+GS+TSdASdASdC*dT*dT*dA*dG*dG*dA*dC*dASdA$+GS+G$+T146.(SEQ ID NO: 115);147.wherein148.+AL, +[%C], +G, and +T are adenosine, 5-methylcytosine, guanosine, and thymine locked nucleosides, respectively,149.dA, dC, dG, and dT are deoxyadenosine, deoxycytidine,150.deoxy guanidine, and deoxythymidine nucleosides, respectively,151.each * is a phosphorothioate (PS) internucleoside linkage, and each $ is independently a PS internucleoside linkage or a stabilizing internucleoside linkage.

36. The SNCA ASO of claim 35, wherein each $ is independently a PS2 internucleoside linkage, a PN internucleoside linkage, or a MsPA internucleoside linkage.

37. The SNCA ASO of claim 36, wherein the SNCA ASO comprises the oligonucleotide:154.(a) +G*+T*+T*dA*dA*dA*dT*dC*dTtdAtdG*dT*dT*dGndTn+[%C]n+[%C]n+A; (b) +Gn+Tn+T*dA*dA*dA*dT*dC*dTtdAtdG*dT*dT*dG*dT*+[%C]n+[%C]n+A; (c) +G*+T*+T*dA*dA*dA*dT*dC*dT*dAtdG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (d) +Gn+Tn+TndA*dA*dA*dT*dC*dTtdAtdG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (e) +G*+T*+T*dA*dA*dA*dT*dC*dT*dAtdG*dT*dT*dG*dT*+[%C]*+[%C]t+A; (f) +G*+T*+T*dA*dA*dA*dT*dCndTndAndG*dT*dT*dG*dTu+[%C]u+[%C]u+A; (g) +G*+T*+T*dA*dA*dA*dT*dC*dTudAudGudT*dT*dG*dTn+[%C]n+[%C]n+A; (h) +Gn+Tn+TndA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (i) +G*+T*+T*dA*dA*dA*dT*dCndTndAndG*dT*dT*dG*dT*+[%C]*+[%C]u+A; (j) +G*+T*+T*dA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (k) +G*+T*+T*dA*dA*dA*dT*dCudTudAudG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (l) +G*+T*+T*dA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dTn+[%C]n+[%C]u+A; (m)+G*+T*+T*dA*dA*dA*dT*dC*dTndAndGndT*dT*dG*dT*+[%C]*+[%C]u+A; (n) +G*+T*+T*dA*dA*dA*dT*dC*dT*dAudG*dT*dT*dG*dT*+[%C]*+[%C]u+A; (o) +G*+T*+T*dA*dA*dA*dT*dC*dTudAudG*dT*dT*dG*dT*+[%C]*+[%C]u+A; (p) +G*+T*+T*dA*dA*dA*dT*dC*dTndAndG*dT*dT*dG*dTn+[%C]n+[%C]n+A; (q) +G*+T*+T*dA*dA*dA*dT*dC*dTndAndG*dT*dT*dG*dT*+[%C]n+[%C]n+A; wherein155.+G is a guanosine locked nucleoside;156.+[%C] is a 5-methylcytosine locked nucleoside;157.+A is an adenosine locked nucleoside;158.+T is a thymine locked nucleoside;159.dA is a deoxy adenosine;160.dC is a deoxy cytidine;161.dT is a deoxythymidine;162.dG is a deoxy guanosine;163.* is a PS internucleoside linkage;164.t is a PS2 internucleoside linkage;165.n is a PN internucleoside linkage; and166.u is a MsPA internucleoside linkage.

38. The SNCA ASO of claim 36, wherein the SNCA ASO comprises the oligonucleotide:168.(a) +A*+A*+[%C]*dT*dG*dC*dTtdT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (b) +An+An+[%C]ndT*dG*dC*dTtdTtdA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (c) +An+An+[%C]*dT*dG*dC*dTtdTtdA*dG*dT*dG*dA*dT*dT*+[%C]n+[%C]n+A (d) +A*+A*+[%C]*dT*dG*dC*dTtdTtdA*dG*dT*dG*dA*dTndTn+[%C]n+[%C]n+A (e) +A*+A*+[%C]*dT*dG*dC*dTtdT*dA*dG*dT*dG*dA*dT*dT*+[%C]*+[%C]t+A (f) +A*+A*+[%C]*dT*dG*dCudTudTudA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (g) +An+An+[%C]ndT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (h) +A*+A*+[%C]*dT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (i) +A*+A*+[%C]*dT*dGudCudTudT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (j) +A*+A*+[%C]*dT*dG*dC*dTndTndAndG*dT*dG*dA*dT*dT*+[%C]*+[%C]u+A (k) +A*+A*+[%C]*dT*dG*dCndTndTndA*dG*dT*dG*dA*dT*dT*+[%C]*+[%C]u+A (l) +A*+A*+[%C]*dT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]u+A (m)+A*+A*+[%C]*dT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]u+A (n) +A*+A*+[%C]*dT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (o) +A*+A*+[%C]*dT*dG*dC*dTudTudAudG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (p) +An+An+[%C]ndT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (q) +A*+A*+[%C]*dT*dG*dC*dTndTndAndG*dT*dG*dA*dT*dTu+[%C]u+[%C]u+A (r) +A*+A*+[%C]*dT*dG*dC*dTudTudA*dG*dT*dG*dA*dT*dT*+[%C]*+[%C]u+A (s) +A*+A*+[%C]*dT*dG*dCndTndT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]n+A (t) +A*+A*+[%C]*dT*dG*dCudTudT*dA*dG*dT*dG*dA*dT*dTn+[%C]n+[%C]u+A (u) +A*+A*+[%C]*dT*dG*dC*dTndTndA*dG*dT*dG*dA*dT*dT*+[%C]n+[%C]n+A (v) +A*+A*+[%C]*dT*dG*dCndTndT*dA*dG*dT*dG*dA*dT*dT*+[%C]n+[%C]n+A wherein169.+A is an adenosine locked nucleoside;170.+1%C] is a 5-methylcytosine locked nucleoside;171.dA is a deoxy adenosine;172.dC is a deoxy cytidine;173.dT is a deoxythymidine;174.dG is a deoxy guanosine;175.* is a PS internucleoside linkage;176.t is a PS2 internucleoside linkage;177.n is a PN internucleoside linkage; and178.u is a MsPA internucleoside linkage.

39. A pharmaceutical composition comprising the SNCA ASO conjugate of any one of claims 1-34 or the SNCA ASO of any one of claims 35-38 and a pharmaceutically acceptable carrier or excipient.

40. A method of generating a neuron cell with decreased synuclein expression, the method comprising delivering to the neuron cell the SNCA ASO conjugate of any one of claims 1-34, the SNCA ASO of any one of claims 35-38, or the pharmaceutical composition of claim 39, wherein the SNCA ASO decreases the expression level of an endogenous SNCA gene in the neuron cell.

41. A method of modifying a neuron cell to decrease synuclein expression, the method comprising delivering to the neuron cell the SNCA ASO conjugate of any one of claims 1-34, the SNCA ASO of any one of claims 35-38, or the pharmaceutical composition of claim 39, thereby decreasing the expression level of an endogenous SNCA gene.

42. The method of claim 41, wherein decreasing the expression level of an endogenous SNCA comprises specifically reducing the expression level of a SNCA transcript in the cell.

43. A method of reducing expression of synuclein in a cell of the spinal cord of a subject comprising administering the SNCA ASO conjugate of any one of claims 1-34, the SNCA ASO of any one of claims 35-38, or the pharmaceutical composition of claim 39 by intrathecal administration.

44. A method of reducing synuclein expression in a subject comprising administering the SNCA ASO conjugate of any one of claims 1-34, the SNCA ASO of any one of claims 35-38, or the pharmaceutical composition of claim 39 to the subject.

45. The method of claim 44, wherein synuclein expression is reduced in the CNS of the subject.

46. The method of claim 44, wherein the SNCA ASO conjugate, the SNCA ASO, or the pharmaceutical composition is administered to the subj ect by intrathecal administration, intravenous injection, or intravenous infusion.

47. The method of any one of claims 40-46, wherein the SNCA ASO decreases the expression level of an endogenous SNCA gene or reduces the level of a SNCA mRNA transcript by at least about 10%. at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% as compared to the level prior to or without administering the pharmaceutical composition.

48. The method of claim 47, wherein the expression of the endogenous SNCA gene or the level of the SNCA transcript is reduced by at least about 50%.

49. The method of claim 47, wherein the expression of the endogenous SNCA gene or the level of the SNCA transcript is reduced by at least about 70%.

50. A method of treating a synuclein-associated neurodegenerative disorder in a human subject in need thereof, the method comprising administering to the human subject the SNCA ASO conjugate of any one of claims 1-34, the SNCA ASO of any one of claims 35-38, or the pharmaceutical composition of claim 39.

51. The method of claim 50, wherein the synuclein-associated neurodegenerative syndrome is Parkinson’s disease, multiple systems atrophy, or Lewy body dementia.

52. A method of treating Parkinson’s disease, the method comprising administering to a human subject in need thereof, the SNCA ASO conjugate of any one of claims 1-34, the SNCA ASO of any one of claims 35-38, or the pharmaceutical composition of claim 39.

53. A method of reducing SNCA messenger ribonucleic acid (mRNA) expression in a human subject in need thereof, the method comprising administering to the human subject the SNCA ASO conjugate of any one of claims 1-34, the SNCA ASO of any one of claims 35-38, or the pharmaceutical composition of claim 39.

54. The pharmaceutical composition as described in claim 39, for use in delivering a SNCA ASO to the CNS of a human subject in need thereof, wherein said SNCA ASO decreases the expression level of an endogenous SNCA gene.

55. The pharmaceutical composition as described in claim 39 for use in treating a synuclein-associated neurodegenerative disorder in a human subject in need thereof.

56. A pharmaceutical composition for use as in claim 55, wherein the synuclein-associated neurodegenerative disorder is Parkinson’s disease.

57. The pharmaceutical composition of claim 39 for use in reducing SNCA mRNA expression in a human subject in need thereof.

58. Nucleic acid sequence encoding an Fc polypeptide dimer comprising a first nucleic acid encoding any of SEQ ID NOs:l, 47-48, 51-54, 61-63, 66, 73-78, 80, and 90; and a second nucleic acid sequence encoding any one of SEQ ID NOs:4-29, 36, 43-44, and 49-50.

59. The nucleic acid sequences of claim 58, wherein the first nucleic acid sequence encodes a first Fab-Fc fusion polypeptide and the second nucleic acid sequence encoding a second Fab-Fc fusion polypeptide.

60. The nucleic acid sequences of claim 59, wherein the first nucleic acid sequence encodes any one of SEQ ID NOs: 100-101 and 130-131; the second nucleic acid sequence encodes any one of SEQ ID NOs:98-99; and wherein the nucleic acid sequence sequences further comprise a third nucleic acid sequence encoding SEQ ID NOs: 108 or 129.

61. The nucleic acid sequences of claim 60, wherein the first nucleic acid sequence comprises SEQ ID NO: 133 or a sequence having at least 75% identity to SEQ ID NO: 133; the second nucleic acid sequence comprises SEQ ID NO: 132 or a sequence having at least 75% identity to SEQ ID NO: 132; and the third nucleic acid sequence comprises SEQ ID NO: 134 or a sequence having at least 75% identity to SEQ ID NO: 134.

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