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WO2025166119A1 - Protéines de liaison au récepteur de la transferrine et conjugués - Google Patents

Protéines de liaison au récepteur de la transferrine et conjugués

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Publication number
WO2025166119A1
WO2025166119A1 PCT/US2025/013966 US2025013966W WO2025166119A1 WO 2025166119 A1 WO2025166119 A1 WO 2025166119A1 US 2025013966 W US2025013966 W US 2025013966W WO 2025166119 A1 WO2025166119 A1 WO 2025166119A1
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WIPO (PCT)
Prior art keywords
antisense strand
seq
sense strand
comprises seq
conjugate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/013966
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English (en)
Inventor
Riazul ALAM
Johnny Eugene CROY
Christopher Carl Frye
Hiroaki Tani
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eli Lilly and Co
Original Assignee
Eli Lilly and Co
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Filing date
Publication date
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of WO2025166119A1 publication Critical patent/WO2025166119A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/332—Abasic residue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the BBB allows the passage of some small molecules by passive diffusion and the cells of BBB actively transport metabolic products crucial to neural function such as glucose and amino acids across the barrier using specific transport proteins.
  • the BBB has neuroprotective function by tightly controlling access to the brain; but it also impedes access of therapeutic agents to CNS.
  • BBB shuttles for improving passage of the therapeutic agents across the blood brain barrier and into the CNS have been described.
  • WO2003/009815 describes the use of antibodies directed to transferrin receptor (“TfR”) for modulating blood brain barrier transport and delivering therapeutic agents across BBB.
  • TfR transferrin receptor
  • RNA interference is a highly conserved regulatory mechanism in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA molecules (dsRNA) (Fire et al., Nature 391:806-811, 1998).
  • dsRNA double-stranded RNA molecules
  • proteins comprising one monovalent human TfR binding domain (“human TfR binding proteins”), conjugates comprising such human TfR binding proteins, e.g., human TfR binding proteins-dsRNA conjugates, pharmaceutical compositions comprising human TfR binding proteins or conjugates, and methods of treating CNS diseases (e.g., neurodegenerative disease such as neurodegenerative synucleinopathy or tauopathy) using human TfR binding proteins or conjugates.
  • CNS diseases e.g., neurodegenerative disease such as neurodegenerative synucleinopathy or tauopathy
  • the conjugates comprising human TfR binding proteins provided herein have improved yield and/or reduced microheterogeneity.
  • proteins comprising one monovalent human TfR binding domain (“human TfR binding proteins”).
  • the human TfR binding protein is any one of the human TfR binding proteins in Table 1b.
  • proteins comprising one monovalent human transferrin receptor (TfR) binding domain, wherein the protein comprises two heavy chains HC1 and HC2 and one light chain LC1, and wherein the HC1, HC2 and LC1 comprise the following sequences: (a) HC1 comprises SEQ ID NO: 11, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 12, or (b) HC1 comprises SEQ ID NO: 13, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 14.
  • the human TfR binding protein has a one arm heteromab format.
  • the human TfR binding protein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 11, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 12.
  • human TfR binding proteins comprise two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 13, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 14.
  • conjugates comprising human TfR binding proteins described herein and a therapeutic agent.
  • the therapeutic agent is selected from a double stranded RNA (e.g., siRNA, saRNA), oligonucleotide (e.g., antisense oligonucleotide), polypeptide, small molecule, nanoparticle, lipid nanoparticle, exosome, antibody or antigen binding fragment thereof, or a combination thereof.
  • the therapeutic agent is a double stranded RNA (dsRNA).
  • the dsRNA comprises a sense strand and an antisense stand, wherein the antisense strand is complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A or CACNA1B mRNA.
  • the therapeutic agent is linked to the human TfR binding protein through a linker.
  • the linker is a Mal-Tet-TCO linker, SMCC linker, or GDM linker (structures of these linkers shown in Table 3).
  • dsRNA double stranded RNA
  • L is a linker, or optionally absent
  • P is a protein comprising one monovalent human TfR binding domain, wherein P comprises two heavy chains HC1 and HC2 and one light chain LC1, and where
  • the dsRNA comprises an antisense strand complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A or CACNA1B mRNA.
  • the dsRNA comprises an antisense strand complementary to SNCA mRNA.
  • the dsRNA comprises an antisense strand complementary to MAPT mRNA.
  • Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human SNCA mRNA are provided in Table 4a.
  • the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 26, and the antisense strand comprises SEQ ID NO: 27; (b) the sense strand comprises SEQ ID NO: 28, and the antisense strand comprises SEQ ID NO: 29; (c) the sense strand comprises SEQ ID NO: 30, and the antisense strand comprises SEQ ID NO: 31; (d) the sense strand comprises SEQ ID NO: 32, and the antisense strand comprises SEQ ID NO: 33; (e) the sense strand comprises SEQ ID NO: 34, and the antisense strand comprises SEQ ID NO: 35; and (f) the sense strand comprises SEQ ID NO: 36, and the antisense strand comprises SEQ ID NO: 37; (g) the sense strand comprises SEQ ID NO: 38, and the antisense strand comprises SEQ ID NO: 27, wherein optionally one or more
  • the sense strand comprises SEQ ID NO: 26 and the antisense strand comprises SEQ ID NO: 27.
  • Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human MAPT mRNA are provided in Table 4b.
  • the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 39, and the antisense strand comprises SEQ ID NO: 40; (b) the sense strand comprises SEQ ID NO: 41, and the antisense strand comprises SEQ ID NO: 42; and (c) the sense strand comprises SEQ ID NO: 43, and the antisense strand comprises SEQ ID NO: 44, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.
  • the dsRNA can include modifications.
  • the modifications can be made to one or more nucleotides of the sense and/or antisense strand or to the internucleotide linkages.
  • one or more nucleotides of the sense strand and/or the antisense strand are independently modified nucleotides, which means the sense strand and the antisense strand can have different modified nucleotides.
  • each nucleotide of the sense strand is a modified nucleotide.
  • each nucleotide of the antisense strand is a modified nucleotide.
  • the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-C 16 alkyl) modified nucleotide.
  • each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, e.g., a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-C 16 alkyl) modified nucleotide.
  • the sense strand has four 2'-fluoro modified nucleotides, e.g., at positions 7, 9, 10, 11 from the 5’ end of the sense strand.
  • the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides.
  • the antisense strand has four 2'-fluoro modified nucleotides, e.g., at positions 2, 6, 14, 16 from the 5’ end of the antisense strand.
  • the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.
  • the sense strand has three 2'-fluoro modified nucleotides, e.g., at positions 9, 10, 11 from the 5’ end of the sense strand.
  • the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides.
  • the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 7, 14, 16 from the 5’ end of the antisense strand.
  • the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 8, 14, 16 from the 5’ end of the antisense strand.
  • the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 3, 7, 14, 16 from the 5’ end of the antisense strand.
  • the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.
  • the 5’ end of the antisense strand has a phosphate analog, e.g., 5’-vinylphosphonate (5’-VP).
  • the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety.
  • the sense strand and the antisense strand have one or more modified internucleotide linkages.
  • the modified internucleotide linkage is phosphorothioate linkage.
  • the sense strand has four or five phosphorothioate linkages.
  • the antisense strand has four or five phosphorothioate linkages.
  • the sense strand and the antisense strand each has four or five phosphorothioate linkages.
  • the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.
  • Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human SNCA mRNA are provided in Table 6a.
  • Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human MAPT mRNA are provided in Table 6b.
  • methods of treating a CNS disease e.g., a neurodegenerative disease, in a patient in need thereof, and such the method comprises administering to the patient an effective amount of the human TfR binding protein or conjugate or a pharmaceutical composition described herein.
  • a neurodegenerative synucleinopathy in a patient in need thereof, and such the method comprises administering to the patient an effective amount of the human TfR binding proteins or conjugate or a pharmaceutical composition described herein (e.g., a TBP-SNCA siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-SNCA siRNA conjugate).
  • the neurodegenerative synucleinopathy is selected from Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia.
  • the human TfR binding protein or conjugate or a pharmaceutical composition can be administered to the patient intravenously or subcutaneously.
  • a tauopathy in a patient in need thereof, and such the method comprises administering to the patient an effective amount of the human TfR binding proteins or conjugate or a pharmaceutical composition described herein (e.g., a TBP-MAPT siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-MAPT siRNA conjugate).
  • a pharmaceutical composition described herein e.g., a TBP-MAPT siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-MAPT siRNA conjugate.
  • the tauopathy is selected from Alzheimer’s disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson’s discase, Pick’s disease (PiD), primary progressive aphasia - semantic (PPA-S), primary progressive aphasia - logopenic (PPA-L), multiple system tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British type amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephal
  • the human TfR binding protein or conjugate or a pharmaceutical composition can be administered to the patient intravenously or subcutaneously.
  • provided herein are human TfR binding proteins or conjugates described herein or pharmaceutical compositions comprising such human TfR binding proteins or conjugates for use in a therapy.
  • human TfR binding proteins or conjugates described herein or pharmaceutical compositions comprising such human TfR binding proteins or conjugates e.g., a TBP-SNCA siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-SNCA siRNA conjugate
  • a neurodegenerative synucleinopathy e.g., Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia.
  • human TfR binding proteins or conjugates described herein or pharmaceutical compositions comprising such human TfR binding proteins or conjugates for use in the treatment of a tauopathy, e.g., e.g., Alzheimer’s disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson’s discase, Pick’s disease (PiD), primary progressive aphasia - semantic (PPA-S), primary progressive aphasia - logopenic (PPA-L), multiple system tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia,
  • a tauopathy e.g., e.g., Alzheimer’s disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to
  • a neurodegenerative disease e.g., a neurodegenerative disease.
  • the neurodegenerative disease is a neurodegenerative synucleinopathy, e.g., Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia.
  • the neurodegenerative disease is a tauopathy, e.g., Alzheimer’s disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson’s discase, Pick’s disease (PiD), primary progressive aphasia - semantic (PPA-S), primary progressive aphasia - logopenic (PPA-L), multiple system tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British type amyloid angiopathy, cerebral amyloid angiopathy
  • Figure 1A shows an exemplary analytical anion exchange (AEX) chromatogram of DAR profile for TBP3-SMCC-dsRNA No. 26 conjugate after purification.
  • Figure 1B shows an exemplary AEX chromatogram of DAR profile for TBP3-SMCC-dsRNA No. 13 conjugate after purification.
  • Figure 1C shows an exemplary AEX chromatogram of DAR profile for TBP2-SMCC-dsRNA No. 26 conjugate after purification.
  • Figure 1D shows an exemplary AEX chromatogram of DAR profile for TBP2-SMCC-dsRNA No.13 conjugate after purification.
  • Figure 1E shows an exemplary AEX chromatogram of DAR profile for mTBP2-SMCC-dsRNA No. 26 conjugate after purification.
  • Figure 2A shows MAPT mRNA reduction in Cynomolgus monkey tissues 29 days after a single intravenous (IV) delivery of TBP2-SMCC-dsRNA No.26 conjugate at 20 mg/kg siRNA dose.
  • Figure 2B shows MAPT mRNA reduction in Cynomolgus monkey tissues 29 days after a single IV delivery of TBP3-SMCC-dsRNA No. 26 conjugate at 20 mg/kg siRNA dose.
  • Figure 3 show SNCA mRNA reduction in mouse brain 28 days following a single IV delivery of TBP2-SMCC-dsRNA No. 13 or TBP3-SMCC-dsRNA No. 13 conjugate at 0.1, 0.5 or 5 mg/kg siRNA dose.
  • the error bars in Figure 3 are Standard Deviations and statistical analysis was performed with a one-way ANOVA with Dunnett’s multiple comparison test against PBS control group.
  • Figure 4 show MAPT mRNA reduction in human tau transgenic mouse brain 28 days following a single IV delivery of mTBP2-SMCC-dsRNA No. 26 conjugate at 1, 10 or 100 mg/kg siRNA dose.
  • the error bars in Figure 4 are Standard Deviations and statistical analysis was performed with a one-way ANOVA with Dunnett’s multiple comparison test against PBS control group.
  • proteins comprising one monovalent human TfR binding domain (“human TfR binding proteins”), conjugates comprising such human TfR binding proteins, e.g., human TfR binding proteins-dsRNA conjugates, pharmaceutical compositions comprising human TfR binding proteins or conjugates, and methods of treating CNS diseases (e.g., neurodegenerative disease such as neurodegenerative synucleinopathy or tauopathy) using human TfR binding proteins or conjugates.
  • CNS diseases e.g., neurodegenerative disease such as neurodegenerative synucleinopathy or tauopathy
  • the conjugates comprising human TfR binding proteins provided herein have improved yield and/or reduced microheterogeneity.
  • Human TfR binding proteins [00034]
  • proteins comprising one monovalent human TfR binding domain (“human TfR binding proteins”).
  • the monovalent human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3.
  • the monovalent human TfR binding domain comprises a VH comprising HCDR1, HCDR2, and HCDR3 selected from Table 1a.
  • the monovalent human TfR binding domain comprises a VL comprising LCDR1, LCDR2, and LCDR3 selected from Table 1a.
  • the monovalent human TfR binding domain comprises a VH comprising HCDR1, HCDR2, and HCDR3 selected from Table 1a, and/or a VL comprising LCDR1, LCDR2, and LCDR3 selected from Table 1a. In some embodiments, the monovalent human TfR binding domain comprises a VH and/or a VL selected from Table 1a. In some embodiments, the human TfR binding proteins described herein also bind cynomolgus monkey TfR. Table 1a.
  • the monovalent human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3.
  • HCDR1 comprises SEQ ID NO: 1
  • HCDR2 comprises SEQ ID NO: 2
  • HCDR3 comprises SEQ ID NO: 3
  • LCDR1 comprises SEQ ID NO: 4
  • LCDR2 comprises SEQ ID NO: 5
  • LCDR3 comprises SEQ ID NO: 6.
  • VH comprises SEQ ID NO: 7, and VL comprises SEQ ID NO: 8. In some embodiments, VH comprises a sequence having at least 95% sequence identity to SEQ ID NO: 7, and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 8.
  • the monovalent human TfR binding domain is an antibody fragment, e.g., Fab, scFv, Fv, or scFab (single chain Fab). In some embodiments, the monovalent human TfR binding domain is Fab. In some embodiments, the human TfR binding domain further comprises a heavy chain constant region and/or a light chain constant region.
  • the human TfR binding protein further comprises a half- life extender, e.g., an immunoglobulin Fc region or a VHH that binds human serum albumin (HSA).
  • the human TfR binding protein further comprises an immunoglobulin Fc region, e.g., a modified human IgG4 Fc region, or a modified human IgG1 Fc region.
  • the human TfR binding protein further comprises a modified human IgG4 Fc region comprising proline at residue 228, and alanine at residues 234 and 235 (all residues are numbered according to the EU Index numbering, also called hIgG4PAA Fc region).
  • the human TfR binding protein further comprises a modified human IgG1 Fc region comprising alanine at residues 234, 235, and 329, serine at position 265, aspartic acid at position 436 (all residues are numbered according to the EU Index numbering, also called hIgG1 effector null or hIgG1EN Fc region).
  • the human TfR binding protein comprises heterodimeric mutations.
  • the human TfR binding protein comprises a modified Fc region comprising a first Fc CH3 domain comprising serine at residue 349, methionine at residue 366, tyrosine at residue 370, and valine at residue 409, and a second Fc CH3 domain comprising glycine at residue 356, aspartic acid at residue 357, glutamine at residue 364 and alanine at residue 407 (all residues are numbered according to the EU Index numbering).
  • the human TfR binding protein comprises a modified Fc region comprising a first Fc CH3 domain comprising leucine at residue 405, and a second Fc CH3 domain comprising arginine at residue 409 (all residues are numbered according to the EU Index numbering).
  • the human TfR binding protein comprises one or more native cysteine residues, which can be used for conjugation.
  • the human TfR binding protein comprises a native cysteine at position 220 of the light chain and/or a native cysteine at position 226 of the heavy chain, which can be used for conjugation (all residues according to the EU Index numbering).
  • the human TfR binding protein comprises engineered cysteine residues for conjugation.
  • the approach of including engineered cysteines as a means for conjugation has been described in WO 2018/232088.
  • the human TfR binding protein comprises a heavy chain comprising one or more cysteines at the following residues: 124, 157, 162, 262, 373, 375, 378, 397, 415 (all residues according to the EU Index numbering).
  • the human TfR binding protein comprises a light chain (e.g., a kappa light chain) comprising one or more cysteines at the following residues: 156, 171, 191, 193, 202, 208 (all residues according to the EU Index numbering).
  • the human TfR binding protein comprises a heavy chain constant region comprising cysteine at residue 124 (according to the EU Index numbering).
  • the human TfR binding protein comprises a light chain constant region comprising cysteine at residue 156 (according to the EU Index numbering).
  • the human TfR binding protein comprises an immunoglobulin Fc region comprising cysteine at residue 378 (according to the EU Index numbering).
  • the human TfR binding protein is any one of the human TfR binding proteins in Table 1b.
  • proteins comprising one monovalent human transferrin receptor (TfR) binding domain, wherein the protein comprises two heavy chains HC1 and HC2 and one light chain LC1, and wherein the HC1, HC2 and LC1 comprise the following sequences: (c) HC1 comprises SEQ ID NO: 11, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 12, or (d) HC1 comprises SEQ ID NO: 13, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 14.
  • the human TfR binding protein has a one arm heteromab format.
  • the human TfR binding protein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 11, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 12.
  • HC1 comprises SEQ ID NO: 13
  • LC1 comprises SEQ ID NO: 10
  • HC2 comprises SEQ ID NO: 14.
  • the human TfR binding proteins described herein can be recombinantly produced in a host cell, for example, using an expression vector.
  • an expression vector may include a sequence that encodes one or more signal peptides that facilitate secretion of the polypeptide(s) from a host cell.
  • Expression vectors containing a polynucleotide of interest e.g., a polynucleotide encoding a heavy chain or light chain of the TfR binding proteins
  • expression vectors may contain one or more selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to aide in detection of host cells transformed with the desired polynucleotide sequences.
  • a host cell includes cells stably or transiently transfected, transformed, transduced or infected with one or more expression vectors expressing all or a portion of the TfR binding proteins described herein.
  • a host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing HC polypeptides and an expression vector expressing LC polypeptides of the TfR binding proteins described herein.
  • a host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing HC and LC polypeptides of the TfR binding proteins described herein.
  • the TfR binding proteins may be produced in mammalian cells such as CHO, NS0, HEK293 or COS cells according to techniques well known in the art.
  • Medium, into which the TfR binding proteins has been secreted may be purified by conventional techniques, such as mixed-mode methods of ion-exchange and hydrophobic interaction chromatography.
  • the medium may be applied to and eluted from a Protein A or G column using conventional methods; mixed-mode methods of ion-exchange and hydrophobic interaction chromatography may also be used.
  • Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography.
  • Mouse TfR binding proteins [00048] Some conjugates used in the Examples below comprise a protein comprising one monovalent mouse TfR binding domain (“mouse TfR binding proteins” or mTBP). Exemplary sequences of mouse TfR binding proteins are provided in Tables 2A and 2B. Such conjugates comprising a mouse TfR binding protein can serve as surrogate molecules in mouse models. Table 2A.
  • Exemplary sequences of mouse TfR binding proteins Region Sequence SEQ ID NO HCDR1 GSYWIC 15 HC2 ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLM 25 ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG Table 2B.
  • Exemplary sequences of mouse TfR binding proteins Mouse TfR binding HC1 LC1 HC2 protein (mTBP) Conjugates comprising human TfR binding protein [00049]
  • conjugates comprising human TfR binding proteins or antibodies described herein and a therapeutic agent.
  • the therapeutic agent is selected from a double stranded RNA (e.g., siRNA, saRNA), oligonucleotide (e.g., antisense oligonucleotide), polypeptide, small molecule, nanoparticle, lipid nanoparticle, exosome, antibody or antigen binding fragment thereof, or a combination thereof.
  • the therapeutic agent is a double stranded RNA (dsRNA).
  • the dsRNA comprises a sense strand and an antisense stand, wherein the antisense strand is complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A or CACNA1B mRNA.
  • the dsRNA comprises a sense strand and an antisense stand, wherein the antisense strand is complementary to SNCA mRNA.
  • the dsRNA comprises a sense strand and an antisense stand, wherein the antisense strand is complementary to MAPT mRNA.
  • the human TfR binding proteins described herein comprise one or more native cysteine residues, which can be used for conjugation.
  • the human TfR binding protein described herein comprises a native cysteine at position 220 of the light chain and/or a native cysteine at position 226 of the heavy chain, which can be used for conjugation (all residues according to the EU Index numbering).
  • the human TfR binding proteins described herein comprise one or more engineered cysteine residues for conjugation. The approach of including engineered cysteines as a means for conjugation has been described in WO 2018/232088.
  • the human TfR binding proteins described herein comprise a heavy chain comprising one or more cysteines at the following residues: 124, 157, 162, 262, 373, 375, 378, 397, 415 (all residues according to the EU Index numbering).
  • the human TfR binding proteins described herein comprise a light chain (e.g., a kappa light chain) comprising one or more cysteines at the following residues: 156, 171, 191, 193, 202, 208 (all residues according to the EU Index numbering).
  • the human TfR binding proteins described herein comprise a heavy chain constant region comprising cysteine at residue 124 (according to the EU Index numbering).
  • the human TfR binding proteins described herein comprise a light chain constant region comprising cysteine at residue 156 (according to the EU Index numbering). In some embodiments, the human TfR binding proteins described herein comprise an immunoglobulin Fc region comprising cysteine at residue 378 (according to the EU Index numbering).
  • the therapeutic agent is linked to the human TfR binding protein through a linker.
  • the linker is a Mal-Tet-TCO linker, SMCC linker, or GDM linker (structures of these linkers shown in Table 3). Table 3.
  • exemplary linker structures Linker Structure 10 Hydrolyzed ring open form of SMCC linker 2
  • conjugates of Formula (I): R-L-P wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand; wherein L is a linker, or optionally absent, wherein P is a protein comprising one monovalent human TfR binding domain, wherein P comprises two heavy chains HC1 and HC2 and one light chain LC1, and wherein the HC1, HC2 and LC1 comprise the following sequences: (a) HC1 comprises SEQ ID NO: 11, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 12, or (b) HC1 comprises SEQ ID NO: 13, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 14.
  • dsRNA double stranded RNA
  • L is a linker, or optionally absent
  • P is a protein comprising one monovalent human TfR
  • P comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 11, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 12.
  • P comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 13, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 14.
  • L is present and selected from: a Mal-Tet-TCO linker, SMCC linker, or GDM linker (see Table 3).
  • L is a SMCC linker.
  • L is absent.
  • P is linked to the 3’ end of the sense strand of the dsRNA. In some embodiments, P is linked to the 5’ end of the sense strand of the dsRNA. In some embodiments, P is linked to an internal position of the sense strand of the dsRNA. In some embodiments, P is linked to the 3’ end of the antisense strand of the dsRNA. In some embodiments, P is linked to an internal position of the antisense strand of the dsRNA. [00058]
  • the conjugates described herein can be made by a variety of procedures known to one of ordinary skill in the art, some of which are illustrated in the preparations and examples below, e.g., in Example 3.
  • the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare conjugates.
  • the product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization.
  • the reagents and starting materials are readily available to one of ordinary skill in the art.
  • the TfR binding proteins with native or engineered cysteines described herein can be first treated with a reducing agent, e.g., DTT, and then re- oxidized with an oxidizing agent, e.g., DHAA.
  • the dsRNA comprises an antisense strand complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A or CACNA1B mRNA.
  • the dsRNA comprises an antisense strand complementary to SNCA mRNA.
  • the dsRNA comprises an antisense strand complementary to MAPT mRNA.
  • the sense strand and the antisense strand of the dsRNA are each 15-30 nucleotides in length, e.g., 20-25 nucleotides in length.
  • the dsRNA has a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides.
  • the sense strand and antisense strand of the dsRNA may have overhangs at either the 5’ end or the 3’ end (i.e., 5’ overhang or 3’ overhang).
  • the sense strand and the antisense strand may have 5’ or 3’ overhangs of 1 to 5 nucleotides or 1 to 3 nucleotides.
  • the antisense strand comprises a 3’ overhang of two nucleotides.
  • Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human SNCA mRNA are provided in Table 4a.
  • Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human MAPT mRNA are provided in Table 4b.
  • Table 4a. Unmodified Nucleic Acid Sequences of dsRNA targeting human SNCA mRNA (SNCA siRNA) dsRNA Sense Strand (5' to 3') SEQ Antisense Strand (5' to 3') SEQ Start No. ID ID position NO NO of target n pt 03 Table 4b.
  • dsRNA targeting human MAPT mRNA MAPT siRNA
  • dsRNA Sense Strand 5' to 3'
  • SEQ Antisense Strand 5' to 3'
  • SEQ Start No. ID ID position t n pt 11 the dsRNA targets SNCA mRNA.
  • the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 26, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 27; (b) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 28, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 29; (c) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 30, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 31; (d) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 32, and the antisense strand
  • the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 26, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 27; (b) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 28, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 29; (c) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 30, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 31; (d) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:
  • the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 26, and the antisense strand comprises SEQ ID NO: 27; (b) the sense strand comprises SEQ ID NO: 28, and the antisense strand comprises SEQ ID NO: 29; (c) the sense strand comprises SEQ ID NO: 30, and the antisense strand comprises SEQ ID NO: 31; (d) the sense strand comprises SEQ ID NO: 32, and the antisense strand comprises SEQ ID NO: 33; (e) the sense strand comprises SEQ ID NO: 34, and the antisense strand comprises SEQ ID NO: 35; (f) the sense strand comprises SEQ ID NO: 36, and the antisense strand comprises SEQ ID NO: 37; and (g) the sense strand comprises SEQ ID NO: 38, and the antisense strand comprises SEQ ID NO: 27, wherein optional
  • the dsRNA targets MAPT mRNA.
  • the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 39, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 40; (b) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 41, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 42; and (c) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 43, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 44, wherein optionally one or more nucleotides
  • the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 39, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 40; (b) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 41, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 42; and (c) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 43, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 44, wherein optionally one or more nucleotides of the sense strand and the antisense strand comprises a pair of nucle
  • the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 39, and the antisense strand comprises SEQ ID NO: 40; (b) the sense strand comprises SEQ ID NO: 41, and the antisense strand comprises SEQ ID NO: 42; and (c) the sense strand comprises SEQ ID NO: 43, and the antisense strand comprises SEQ ID NO: 44, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.
  • the dsRNA can include modifications.
  • the modifications can be made to one or more nucleotides of the sense and/or antisense strand or to the internucleotide linkages, which are the bonds between two nucleotides in the sense or antisense strand.
  • some 2’- modifications of ribose or deoxyribose can increase RNA or DNA stability and half-life.
  • Such 2’-modifications can be 2’-fluoro, 2’-O-methyl (i.e., 2’-methoxy), or 2'-O-alkyl (e.g., 2’-O-C16 alkyl).
  • one or more nucleotides of the sense strand and/or the antisense strand are independently modified nucleotides, which means the sense strand and the antisense strand can have different modified nucleotides.
  • each nucleotide of the sense strand is a modified nucleotide.
  • each nucleotide of the antisense strand is a modified nucleotide.
  • the modified nucleotide is a 2'- fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-C16 alkyl) modified nucleotide.
  • each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, e.g., a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-C16 alkyl) modified nucleotide.
  • the sense strand has four 2'-fluoro modified nucleotides, e.g., at positions 7, 9, 10, 11 from the 5’ end of the sense strand.
  • the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides.
  • the antisense strand has four 2'-fluoro modified nucleotides, e.g., at positions 2, 6, 14, 16 from the 5’ end of the antisense strand.
  • the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.
  • the sense strand has three 2'-fluoro modified nucleotides, e.g., at positions 9, 10, 11 from the 5’ end of the sense strand.
  • the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides.
  • the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 7, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 8, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 3, 7, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.
  • the 5’ end of the antisense strand has a phosphate analog, e.g., 5’-vinylphosphonate (5’-VP).
  • the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety, e.g., a moiety shown in Table 5.
  • the sense strand and the antisense strand have one or more modified internucleotide linkages.
  • the modified internucleotide linkage is phosphorothioate linkage.
  • the sense strand has four or five phosphorothioate linkages.
  • the antisense strand has four or five phosphorothioate linkages.
  • the sense strand and the antisense strand each has four or five phosphorothioate linkages.
  • the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.
  • Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human SNCA mRNA are provided in Table 6a.
  • Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human MAPT mRNA are provided in Table 6b.
  • the dsRNA comprises a sense strand that comprises a sequence that has 1, 2, or 3 differences from a sense stand sequence in Table 4a or 6a.
  • the dsRNA comprises an antisense strand that comprises a sequence that has 1, 2, or 3 differences from an antisense stand sequence in Table 4a or 6a.
  • the dsRNA comprises a sense strand that comprises a sequence that has 1, 2, or 3 differences from a sense stand sequence in Table 4b or 6b.
  • the dsRNA comprises an antisense strand that comprises a sequence that has 1, 2, or 3 differences from an antisense stand sequence in Table 4b or 6b.
  • Table 6a Modified Nucleic Acid Sequences of dsRNA targeting human SNCA mRNA (SNCA siRNA) dsRNA SEQ ID Strand Oligo Sequence 5' to 3' No.
  • n is the 61 S 21 abasic moiety in Table 10.
  • 9 2 9 “iAb” indicates inverted abasic moiety in Table 10; “S” means the sense strand; “AS” means the antisense strand.
  • Table 6b Modified Nucleic Acid Sequences of dsRNA targeting human MAPT mRNA (MAPT siRNA) dsRNA SEQ ID Strand Oligo Sequence 5' to 3' No.
  • the 5’ end of the AS may be substituted with 5’-vinylphosphonate.
  • Abbreviations – “m” indicates 2’-OMe; “f” indicated 2’-fluoro; “*” indicates phosphorothioate linkage; “iAb” indicates inverted abasic moiety in Table 10; “S” means the sense strand; “AS” means the antisense strand.
  • the dsRNA targets SNCA mRNA.
  • the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 45, and the antisense strand comprises SEQ ID NO: 46; (b) the sense strand comprises SEQ ID NO: 47, and the antisense strand comprises SEQ ID NO: 48; (c) the sense strand comprises SEQ ID NO: 47, and the antisense strand comprises SEQ ID NO: 49; (d) the sense strand comprises SEQ ID NO: 47, and the antisense strand comprises SEQ ID NO: 50; (e) the sense strand comprises SEQ ID NO: 51, and the antisense strand comprises SEQ ID NO: 46; (f) the sense strand comprises SEQ ID NO: 52, and the antisense strand comprises SEQ ID NO: 53; (g) the sense strand comprises SEQ ID NO: 54, and the antisense strand comprises SEQ ID NO: 55; (h) the group consisting of
  • the sense strand and the antisense strand of the dsRNA have a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand consists of SEQ ID NO: 45, and the antisense strand consists of SEQ ID NO: 46; (b) the sense strand consists of SEQ ID NO: 47, and the antisense strand consists of SEQ ID NO: 48; (c) the sense strand consists of SEQ ID NO: 47, and the antisense strand consists of SEQ ID NO: 49; (d) the sense strand consists of SEQ ID NO: 47, and the antisense strand consists of SEQ ID NO: 50; (e) the sense strand consists of SEQ ID NO: 51, and the antisense strand consists of SEQ ID NO: 46; (f) the sense strand consists of SEQ ID NO: 52, and the antisense strand consists of SEQ ID NO: 53; (g) the sense strand consists of SEQ ID NO
  • the dsRNA targets MAPT mRNA.
  • the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 63, and the antisense strand comprises SEQ ID NO: 64; (b) the sense strand comprises SEQ ID NO: 65, and the antisense strand comprises SEQ ID NO: 66; (c) the sense strand comprises SEQ ID NO: 67, and the antisense strand comprises SEQ ID NO: 68; (d) the sense strand comprises SEQ ID NO: 69, and the antisense strand comprises SEQ ID NO: 70; (e) the sense strand comprises SEQ ID NO: 71, and the antisense strand comprises SEQ ID NO: 72; and (f) the sense strand comprises SEQ ID NO: 73, and the antisense strand comprises SEQ ID NO: 74.
  • the sense strand and the antisense strand of the dsRNA have a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand consists of SEQ ID NO: 63, and the antisense strand consists of SEQ ID NO: 64; (b) the sense strand consists of SEQ ID NO: 65, and the antisense strand consists of SEQ ID NO: 66; (c) the sense strand consists of SEQ ID NO: 67, and the antisense strand consists of SEQ ID NO: 68; (d) the sense strand consists of SEQ ID NO: 69, and the antisense strand consists of SEQ ID NO: 70; (e) the sense strand consists of SEQ ID NO: 71, and the antisense strand consists of SEQ ID NO: 72; and (f) the sense strand consists of SEQ ID NO: 73, and the antisense strand consists of SEQ ID NO:
  • the sense strand and antisense strand of dsRNA can be synthesized using any nucleic acid polymerization methods known in the art, for example, solid-phase synthesis by employing phosphoramidite chemistry methodology (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), H- phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, for example, MerMadeTM 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems.
  • phosphoramidite chemistry methodology e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA
  • H- phosphonate e.g
  • Phosphorothioate linkages can be introduced using a sulfurizing reagent such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene) amino)-3H-l,2,4-dithiazaoline-3-thione). It is well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products to synthesize modified oligonucleotides or conjugated oligonucleotides. [00084] Purification methods can be used to exclude the unwanted impurities from the final oligonucleotide product.
  • oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm. The sense strand and antisense strand can then be annealed to form a dsRNA.
  • Pharmaceutical Composition [00085] In another aspect, provided herein are pharmaceutical compositions comprising any of the human TfR binding proteins or conjugates described herein and a pharmaceutically acceptable carrier.
  • compositions can also comprise one or more pharmaceutically acceptable excipient, diluent, or carrier.
  • Pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A. Loyd et al., Academic Press). Method of Treatment and Therapeutic Use [00086]
  • a CNS disease e.g., a neurodegenerative disease
  • the method comprises administering to the patient an effective amount of the human TfR binding protein or conjugate or a pharmaceutical composition described herein.
  • a neurodegenerative synucleinopathy in a patient in need thereof, and such the method comprises administering to the patient an effective amount of the human TfR binding proteins or conjugate or a pharmaceutical composition described herein, e.g., a TBP-SNCA siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-SNCA siRNA conjugate.
  • exemplary neurodegenerative synucleinopathy includes, but are not limited to, Parkinson’s disease; multiple system atrophy; Lewy body dementia or dementia with Lewy bodies; pure autonomic failure; Alzheimer’s disease; Lewy body dysphagia; and incidental Lewy body disease.
  • the neurodegenerative synucleinopathy is selected from Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia.
  • the human TfR binding protein or conjugate or a pharmaceutical composition can be administered to the patient intravenously or subcutaneously.
  • the method comprises administering to the patient an effective amount of the human TfR binding proteins or conjugate or a pharmaceutical composition described herein, e.g., a TBP-MAPT siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-MAPT siRNA conjugate.
  • Exemplary tauopathy includes, but are not limited to, Alzheimer’s disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson’s discase, Pick’s disease (PiD), primary progressive aphasia - semantic (PPA-S), primary progressive aphasia - logopenic (PPA-L), multiple system tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British type amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic
  • the human TfR binding protein or conjugate or a pharmaceutical composition can be administered to the patient intravenously or subcutaneously.
  • Human TfR binding protein or conjugate dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Dosage values may vary with the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
  • human TfR binding proteins or conjugates described herein or pharmaceutical compositions comprising such human TfR binding proteins or conjugates for use in a therapy.
  • human TfR binding proteins or conjugates described herein or pharmaceutical compositions comprising such human TfR binding proteins or conjugates e.g., a TBP-SNCA siRNA conjugate described herein or a pharmaceutical composition comprising such a TBP-SNCA siRNA conjugate
  • a neurodegenerative synucleinopathy e.g., Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia.
  • human TfR binding proteins or conjugates described herein or pharmaceutical compositions comprising such human TfR binding proteins or conjugates for use in the treatment of a tauopathy, e.g., Alzheimer’s disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson’s discase, Pick’s disease (PiD), primary progressive aphasia - semantic (PPA-S), primary progressive aphasia - logopenic (PPA-L), multiple system tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD
  • a tauopathy e.g., Alzheimer’s disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17),
  • a neurodegenerative disease e.g., a neurodegenerative disease.
  • the neurodegenerative disease is a neurodegenerative synucleinopathy, e.g., Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, or Lewy body dementia.
  • the neurodegenerative disease is a tauopathy, e.g., Alzheimer’s disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson’s discase, Pick’s disease (PiD), primary progressive aphasia - semantic (PPA-S), primary progressive aphasia - logopenic (PPA-L), multiple system tauopathy with presenile dementia (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British type amyloid angiopathy, cerebral amyloid angiopathy
  • alkyl means saturated linear or branched-chain monovalent hydrocarbon radical, containing the indicated number of carbon atoms.
  • C 1 -C 20 alkyl means a radical having 1-20 carbon atoms in a linear or branched arrangement.
  • antibody refers to a molecule that binds an antigen.
  • Embodiments of an antibody include a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, heterodimeric antibody, bispecific or multispecific antibody, or conjugated antibody .
  • the antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA), and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).
  • An immunoglobulin G (IgG) type antibody comprised of four polypeptide chains: two heavy chains (HC) and two light chains (LC) that are cross-linked via inter-chain disulfide bonds.
  • the amino-terminal portion of each of the four polypeptide chains includes a variable region of about 100-125 or more amino acids primarily responsible for antigen recognition.
  • the carboxyl-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function.
  • Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain is comprised of a light chain variable region (VL) and a light chain constant region.
  • the IgG isotype may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).
  • VH and VL regions can be further subdivided into regions of hyper- variability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen binding specificity.
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3” and the three CDRs of the light chain are referred to as “LCDR1, LCDR2 and LCDR3”.
  • the CDRs contain most of the residues that form specific interactions with the antigen. Assignment of amino acid residues to the CDRs may be done according to the well-known schemes, including those described in Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md.
  • Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments that, as used herein, comprise at least a portion of an antibody retaining the ability to specifically interact with an antigen or an epitope of the antigen, such as Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fvs (sdFv), a Fd fragment.
  • TfR binding domain refers to a portion of an antibody or antibody fragment that binds TfR or an epitope of TfR.
  • heterodimeric antibody refers to an antibody that comprises two distinct antigen-binding domains.
  • antisense strand means a single-stranded oligonucleotide that is complementary to a region of a target sequence.
  • sense strand means a single-stranded oligonucleotide that is complementary to a region of an antisense strand.
  • bind and “binds” as used herein are intended to mean, unless indicated otherwise, the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in proximity of the two proteins or molecules as determined by common methods known in the art.
  • “complementary” means a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand, e.g., a hairpin) that permits the two nucleotides to form base pairs with one another.
  • a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another.
  • Complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes.
  • two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity, as described herein.
  • duplex in reference to nucleic acids or oligonucleotides, means a structure formed through complementary base pairing of two antiparallel sequences of nucleotides (i.e., in opposite directions), whether formed by two separate nucleic acid strands or by a single, folded strand (e.g., via a hairpin).
  • An “effective amount” refers to an amount necessary (for periods of time and for the means of administration) to achieve the desired therapeutic result.
  • An effective amount of a protein or conjugate may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein or conjugate to elicit a desired response in the individual.
  • epitope refers to the amino acid residues, of an antigen, that are bound by an antibody.
  • An epitope can be a linear epitope, a conformational epitope, or a hybrid epitope.
  • epitope may be used in reference to a structural epitope.
  • a structural epitope may be used to describe the region of an antigen which is covered by an antibody or antigen binding protein.
  • a structural epitope may describe the amino acid residues of the antigen that are within a specified proximity (e.g., within a specified number of Angstroms) of an amino acid residue of the antibody or antigen binding protein.
  • the term “epitope” may also be used in reference to a functional epitope.
  • a functional epitope may be used to describe amino acid residues of the antigen that interact with amino acid residues of the antibody or antigen binding protein in a manner contributing to the binding energy between the antigen and the antibody or antigen binding protein.
  • An epitope can be determined according to different experimental techniques, also called “epitope mapping techniques.” It is understood that the determination of an epitope may vary based on the different epitope mapping techniques used and may also vary with the different experimental conditions used, e.g., due to the conformational changes or cleavages of the antigen induced by specific experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3 rd ed.
  • Fc region refers to a polypeptide comprising the CH2 and CH3 domains of a constant region of an immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4.
  • the Fc region may include a portion of the hinge region or the entire hinge region of an immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4.
  • the Fc region is a human IgG Fc region, e.g., a human IgG1 Fc region, human IgG2 Fc region, human IgG3 Fc region or human IgG4 Fc region.
  • the Fc region is a modified IgG Fc region with reduced or eliminated effector functions compared to the corresponding wild type IgG Fc region.
  • the numbering of the residues in the Fc region is based on the EU index as described in Kabat (Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991).
  • the boundaries of the Fc region of an immunoglobulin heavy chain might vary, and the human IgG heavy chain Fc region is usually defined as the stretch from the N-terminus of the CH2 domain (e.g., the amino acid residue at position 231 according to the EU index numbering) to the C-terminus of the CH3 domain (or the C-terminus of the immunoglobulin).
  • modified internucleotide linkage means an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage having a phosphodiester bond.
  • a modified internucleotide linkage can be a non-naturally occurring linkage.
  • the modified internucleotide linkage is phosphorothioate linkage.
  • modified nucleotide refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide.
  • a modified nucleotide can have, for example, one or more chemical modification in its sugar, nucleobase, and/or phosphate group. Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide.
  • the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-C 16 alkyl) modified nucleotide.
  • the modified nucleotide has a phosphate analog, e.g., 5’-vinylphosphonate.
  • the modified nucleotide has an abasic moiety or inverted abasic moiety, e.g., a moiety shown in Table 10.
  • the term “neurodegenerative synucleinopathy” refers to a neurodegenerative disorder characterized by fibrillary aggregates of alpha-synuclein protein in the cytoplasm of selective populations of neurons and glia in the central and/or peripheral nervous systems.
  • nucleotide means an organic compound having a nucleoside (a nucleobase, e.g., adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar, e.g., ribose or 2'-deoxyribose) linked to a phosphate group.
  • a “nucleotide” can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • a “null arm” means an antibody arm that does not bind any known human target.
  • oligonucleotide means a polymer of linked nucleotides, each of which can be modified or unmodified. An oligonucleotide is typically less than about 100 nucleotides in length.
  • overhang means the unpaired nucleotide or nucleotides that protrude from the duplex structure of a double stranded oligonucleotide. An overhang may include one or more unpaired nucleotides extending from a duplex region at the 5’ terminus or 3’ terminus of a double stranded oligonucleotide.
  • the overhang can be a 3’ or 5’ overhang on the antisense strand or sense strand of a double stranded oligonucleotide.
  • patient refers to a human patient.
  • phosphate analog means a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group.
  • a phosphate analog is positioned at the 5’ terminal nucleotide of an oligonucleotide in place of a 5’- phosphate, which is often susceptible to enzymatic removal.
  • a 5’ phosphate analog can include a phosphatase-resistant linkage.
  • phosphate analogs include 5’ methylene phosphonate (5’-MP) and 5’-(E)-vinylphosphonate (5’-VP). In some embodiments, the phosphate analog is 5’-VP.
  • % sequence identity or “percentage sequence identity” with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical with the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp. 30, section 7.7.18, Table 7.7.1), and including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Clustal W2.0 or Clustal X2.0 software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Percentage of “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the nucleic acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage can be calculated by determining the number of positions at which the identical nucleotide, nucleoside, or nucleobase occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • the output is the percent identity of the subject sequence with respect to the query sequence.
  • polypeptide or “protein”, as used herein, refers to a polymer of amino acid residues. The term applies to polymers comprising naturally occurring amino acids and polymers comprising one or more non-naturally occurring amino acids.
  • strand refers to a single, contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have two free ends (e.g., a 5’ end and a 3’ end).
  • SNCA refers to an alpha-synuclein (SNCA) mRNA, protein, or polypeptide.
  • the nucleic acid sequence of a human SNCA mRNA transcript can be found at NM_000345.4: 1 GGCGACGACC AGAAGGGGCC CAAGAGAGGG GGCGAGCGAC CGAGCGCCGC GACGCGGAAG 61 TGAGGTGCGT GCGGGCTGCA GCGCAGACCC CGGCCCGGCC CCTCCGAGAG CGTCCTGGGC 121 GCTCCCTCAC GCCTTGCCTT CAAGCCTTCT GCCTTTCCAC CCTCGTGAGC GGAGAACTGG 181 GAGTGGCCAT TCGACGACAG TGTGGTGTAA AGGAATTCAT TAGCCATGGA TGTATTCATG 241 AAAGGACTTT CAAAGGCCAA GGAGGGAGTT GTGGCTGCTG CTGAGAAAAC CAAACAGGGT 301 GTGGCAGAAG CAGCAGGAAA GACAAAAGAGGGTGTTCTCT ATGTAGGCTC CAAAACCAAG 361 GAGGGAGTGG TGCATGGTGT GGCAACAGTG GC
  • the amino acid sequence of a human SNCA protein can be found at NP_000336.1: 1 MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK 61 EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP 121 DNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 76).
  • the nucleic acid sequence of a mouse SNCA mRNA transcript can be found at NM_001042451.2; and the amino acid sequence of a mouse SNCA protein can be found at NP_001035916.1.
  • the nucleic acid sequence of a rat SNCA mRNA transcript can be found at NM_019169.3; and the amino acid sequence of a rat SNCA protein can be found at NP_062042.1.
  • the nucleic acid sequence of a monkey SNCA mRNA transcript can be found at XM_005555422.2; and the amino acid sequence of a monkey SNCA protein can be found at XP_005555479.1.
  • “MAPT” refers to a human MAPT mRNA transcript, encoding a microtubule associated protein Tau.
  • the nucleotide sequences of human MAPT transcript variants and amino acid sequences of human Tau protein isoforms can be found at: i.
  • MAPT transcript variant 4 Tau protein isoform 4: NM_016841.5 (nucleotide sequence) ⁇ NP_058525.1 (amino acid sequence); v. MAPT transcript variant 5 ⁇ Tau protein isoform 5: NM_001123067.4 (nucleotide sequence) ⁇ NP_001116539.1 (amino acid sequence); vi. MAPT transcript variant 6 ⁇ Tau protein isoform 6: NM_001123066.4 (nucleotide sequence) ⁇ NP_001116538.2 (amino acid sequence); vii.
  • MAPT transcript variant 7 Tau protein isoform 7: NM_001203251.2 (nucleotide sequence) ⁇ NP_001190180.1 (amino acid sequence); viii.
  • MAPT transcript variant 8 Tau protein isoform 8: NM_001203252.2 (nucleotide sequence) ⁇ NP_001190181.1 (amino acid sequence); ix.
  • MAPT transcript variant 9 Tau protein isoform 9: NM_001377265.1 (nucleotide sequence) ⁇ NP_001364194.1 (amino acid sequence); x.
  • MAPT transcript variant 10 Tau protein isoform 10: NM_001377266.1 (nucleotide sequence) ⁇ NP_001364195.1 (amino acid sequence); xi.
  • MAPT transcript variant 11 Tau protein isoform 11: NM_001377267.1 (nucleotide sequence) ⁇ NP_001364196.1 (amino acid sequence); xii.
  • MAPT transcript variant 12 ⁇ Tau protein isoform 4: NM_001377268.1 (nucleotide sequence) ⁇ NP_001364197.1 (amino acid sequence).
  • the nucleotide sequence of the human MAPT transcript variant 6 (encoding 2N4R Tau) can be found at NM_001123066.4: 1 GCAGTCACCG CCACCCACCA GCTCCGGCAC CAACAGCAGC GCCGCTGCCA CCGCCCACCT 61 TCTGCCGCCG CCACCACAGC CACCTTCTCC TCCTCCGCTG TCCTCTCCCG TCCTCGCCTC 121 TGTCGACTAT CAGGTGAACT TTGAACCAGG ATGGCTGAGC CCCGCCAGGA GTTCGAAGTG 181 ATGGAAGATC ACGCTGGGAC GTACGGGTTG GGGGACAGGA AAGATCAGGG GGGCTACACC 241 ATGCACCAAG ACCAAGGG TGACACGGAC GCTGGCCTGA AAGAATCTCC CCTGCAGACC 301 CCCACTGAGG ACGGATCTGA GGAACCGGGC TCTGAAACCT CTGATGCTAA GAGCACTCCA 361 ACAGCGGA
  • the corresponding amino acid sequence of human Tau protein isoform 6 can be found at NP_001116538.2: 1 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 61 SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 121 HVTQEPESGK VVQEGFLREP GPPGLSHQLM SGMPGAPLLP EGPREATRQP SGTGPEDTEG 181 GRHAPELLKH QLLGDLHQEG PPLKGAGGKE RPGSKEEVDE DRDVDESSPQ DSPPSKASPA 241 QDGRPPQTAA REATSIPGFP AEGAIPLPVD FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE 301 FTFHVEITPN VQKEQAHSEE HLGRAAFPGA PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
  • the nucleotide sequence of a human MAPT transcript variant 5 (encoding 1N4R Tau) can be found at NM_001123067.4: 1 GCAGTCACCG CCACCCACCA GCTCCGGCAC CAACAGCAGC GCCGCTGCCA CCGCCCACCT 61 TCTGCCGCCG CCACCACAGC CACCTTCTCC TCCTCCGCTG TCCTCTCCCG TCCTCGCCTC 121 TGTCGACTAT CAGGTGAACT TTGAACCAGG ATGGCTGAGC CCCGCCAGGA GTTCGAAGTG 181 ATGGAAGATC ACGCTGGGAC GTACGGGTTG GGGGACAGGA AAGATCAGGG GGGCTACACC 241 ATGCACCAAG ACCAAGGG TGACACGGAC GCTGGCCTGA AAGAATCTCC CCTGCAGACC 301 CCCACTGAGG ACGGATCTGA GGAACCGGGC TCTGAAACCT CTGATGCTAA GAGCACTCCA 361 ACAGCGGAAG
  • the corresponding amino acid sequence of human Tau protein isoform 5 can be found at NP_001116539.1: 1 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 61 SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKT 121 KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 181 SRTPSLPTPP TREPKKVAVV RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 241 PGGGKVQIIN KKLDLSNVQS KCGSKDNIKH VPGGGSVQIV YKPVDLSKVT SKCGSLGNIH 301 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETH
  • the nucleotide sequence of the human MAPT transcript variant 4 (encoding 0N3R Tau) can be found at NM_016841.5: 1 GCAGTCACCG CCACCCACCA GCTCCGGCAC CAACAGCAGC GCCGCTGCCA CCGCCCACCT 61 TCTGCCGCCG CCACCACAGC CACCTTCTCC TCCTCCGCTG TCCTCTCCCG TCCTCGCCTC 121 TGTCGACTAT CAGGTGAACT TTGAACCAGG ATGGCTGAGC CCCGCCAGGA GTTCGAAGTG 181 ATGGAAGATC ACGCTGGGAC GTACGGGTTG GGGGACAGGA AAGATCAGGG GGGCTACACC 241 ATGCACCAAG ACCAAGGG TGACACGGAC GCTGGCCTGA AAGCTGAAGA AGCAGGCATT 301 GGAGACACCC CCAGCCTGGA AGACGAAGCT GCTGGTCACG TGACCCAAGC TCGCATGGTC 361 AGTAAAAG
  • the corresponding amino acid sequence of human Tau protein isoform 4 can be found at NP_058525.1: 1 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 61 AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA 121 PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS 181 AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH 241 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG 301 AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ
  • tauopathy refers to a disease associated with abnormal tau protein expression, secretion, phosphorylation, cleavage, and/or aggregation.
  • TfR refers to a transferrin receptor protein or polypeptide, e.g., a human transferrin receptor protein or polypeptide.
  • the amino acid sequence of the human transferrin receptor protein can be found at NP_001121620.1: 1 MMDQARSAFS NLFGGEPLSY TRFSLARQVD GDNSHVEMKL AVDEEENADN NTKANVTKPK 61 RCSGSICYGT IAVIVFFLIG FMIGYLGYCK GVEPKTECER LAGTESPVRE EPGEDFPAAR 121 RLYWDDLKRK LSEKLDSTDF TGTIKLLNEN SYVPREAGSQ KDENLALYVE NQFREFKLSK 181 VWRDQHFVKI QVKDSAQNSV IIVDKNGRLV YLVENPGGYV AYSKAATVTG KLVHANFGTK 241 KDFEDLYTPV NGSIVIVRAG KITFAEKVAN AESLNAIGVL IYMDQTKFPI VNAELSFFGH 301 AHLGTGDPYT PGFPSFNHTQ FPPSRSSGLP NIPVQTISRA AAEKLF
  • treatment refers to all processes wherein there may be a slowing, controlling, delaying, or stopping of the progression of the disorders or disease disclosed herein, or ameliorating disorder or disease symptoms, but does not necessarily indicate a total elimination of all disorder or disease symptoms.
  • Treatment includes administration of a protein or nucleic acid or vector or composition for treatment of a disease or condition in a patient, particularly in a human.
  • TfR binding proteins Generation of human TfR binding proteins
  • Antibody against human TfR was generated by immunizing AlivaMab® transgenic mice with the extracellular domains of human Transferrin Receptor 1 protein with a His tag (hTfR-ECD-6His, SEQ ID NO: 85, see Table 7) and mouse Transferrin Receptor protein (mTfR, SEQ ID NO:84).
  • Antigen positive B-cells were sorted from pooled spleens. Binding of individual antibodies cloned from those B-cells to his-tagged hTfR-ECD was verified.
  • Human TfR binding proteins were generated by recombinant DNA technology. Such human TfR binding proteins can be expressed in a mammalian cell line such as HEK293 or CHO, either transiently or stably transfected with an expression system using an optimal predetermined HC:LC vector ratio or a single vector system encoding both HC and LC.
  • Binding affinity and binding stoichiometry of the exemplified human TfR binding proteins to human and cynomolgus TfR was characterized using a surface plasmon resonance assay on a Biacore 8K instrument primed with HBS-EP+ (10mM Hepes pH7.4 + 150mM NaCl + 3mM EDTA + 0.05% (w/v) surfactant P20) running buffer and analysis temperature set at 37 °C.
  • TfR ECD Target human and cynomologus TfR ECD’s were immobilized on a CM4 chip (Cytiva P/N 29104989) using standard NHS-EDC amine coupling.
  • the TfR binding proteins were prepared at a final concentration of 0.3, 0.1, 0.033, 0.01, 0.0033, 0.001, 0.00033, 0.0001 ⁇ M respectively by dilution of stock solution into running buffer.
  • Binding analysis was performed in a multi-cycle kinetics manner.
  • Each analysis cycle consists of (1) injection of the lowest to highest concentration proteins over all Fc at 50 ⁇ L/min for 140 seconds followed by return to buffer flow for 400 seconds to monitor dissociation phase; (2) regeneration of chip surfaces with injection of 3M magnesium chloride, for 30 seconds at 100 ⁇ L/min over all cells; and (3) equilibration of chip surfaces with a 50 ⁇ L (30-sec) injection of HBS-EP+.
  • Data were processed using standard double-referencing and fit to a 2-state binding model using Biacore 8K Evaluation software, to determine the association rate (kon, M -1 s -1 units), dissociation rate (koff, s -1 units), and Rmax (RU units).
  • MSD Meso Scale Discovery
  • Target human and cynomolgus TfR ECD were diluted to 1 ⁇ g/mL in 1X PBS buffer and 80 ⁇ L dispensed in 96- well MSD plate and agitated on plater shaker for ⁇ 1 min and stored at 4 °C overnight to coat the plate surface. Coated plates were washed with three times with PBS containing 0.02% Tween-20 (PBS-T), followed by addition of 200 ⁇ L of 3% blocker A to each well and agitated for 1 hour at ambient temperature. TBPs were prepared by diluting into PBS-T, starting at 2 ⁇ M top concentration and serially diluting down 1 to 3 fold.
  • MSD plate was washed three times with 200 ⁇ L PBS-T and 80 ⁇ L serially diluted TBPs were added to each well, followed by agitation on plate shaker for 1 hour at ambient temperature.
  • Sulfo-tag anti-human kappa chain secondary antibody (MSD, Cat. D20TF) was prepared by diluting at 1 ⁇ g/mL into PBS-T buffer and 80 ⁇ L added to each well and agitated on plate shaker for 1 hour at ambient temperature.
  • Blocked plate was washed for three times with 200 ⁇ L PBS-T.
  • 200 ⁇ L MSD Rad Buffer T (MSD, Cat. R92TC) was added to each well and read on MSD plate reader immediately.
  • the sense strands were synthesized using phthalamido amino C6 lcaa CPG 500 ⁇ (Chemgenes) whereas the antisense strands used standard support (LGC Biosearch Technologies).
  • the oligonucleotides were synthesized via phosphoramidite chemistry at either 5, 10, or 50 ⁇ mol scales.
  • Standard reagents were used in the oligo synthesis (Table 10), where 0.1M xanthane hydride in pyridine was used as the sulfurization reagent and 20% DEA in ACN was used as an auxiliary wash post synthesis. All monomers (Table 11) were made at 0.1M in ACN and contained a molecular sieves trap bag.
  • the oligonucleotides were cleaved and deprotected (C/D) at 45 °C for 20 hours.
  • the sense strands were C/D from the CPG using cold 50% (methylamine/ammonia hydroxide 28-30%) at ambient temperature for 3 hrs, whereas 3% DEA in ammonia hydroxide (28-30%, cold) was used for the antisense strands.
  • C/D was determined complete by IP-RP LCMS when the resulting mass data confirmed the identity of sequence.
  • the CPG was filtered via 0.45 um PVDF syringeless filter, 0.22 um PVDF Steriflip® vacuum filtration or 0.22 um PVDF Stericup® Quick release.
  • the CPG was back washed/rinsed with either 30% EtOH/RNAse free water then filtered through the same filtering device and combined with the first filtrate. This was repeated twice. The material was then divided evenly into 50 mL falcon tubes to remove organics via GenevacTM. After concentration, the crude oligonucleotides were diluted back to synthesized scale with RNAse free water and filtered either by 0.45 ⁇ m PVDF syringeless filter, 0.22 ⁇ m PVDF Steriflip® vacuum filtration or 0.22 ⁇ m PVDF Stericup® Quick release. [000140] The crude oligonucleotides were purified via AKTATM Pure purification system using anion-exchange (AEX).
  • AEX anion-exchange
  • oligonucleotides were desalted using 15 mL 3K MWCO centrifugal spin tubes at 3500xg for ⁇ 30 min. The oligonucleotides were rinsed with RNAse free water until the eluent conductivity reached ⁇ 100 usemi/cm.
  • RNAse free water was added then aspirated 10x, the retainment was transferred to a 50 mL falcon tube, this was repeated until complete transfer of oligo by measuring concentration of compound on filter via nanodrop.
  • the final oligonucleotide was then nano filtered 2x via 15 mL 100K MWCO centrifugal spin tubes at 3500xg for 2 min.
  • the final desalted oligonucleotides were analyzed for concentration (nano drop at A260), characterized by IP-RP LC/MS for mass purity (Table 9) and UPLC for UV-purity.
  • Step B shows the acidic deprotection of compound (2) with an acid such as TFA in a suitable solvent such as DCM followed by an amide coupling with methyltetrazine-PEG4-acid using an amide coupling reagent such as HATU with an appropriate base such as N,N-diisopropyl amine in a solvent system such as DMF and THF to give compound (3).
  • an amide coupling reagent such as HATU with an appropriate base such as N,N-diisopropyl amine in a solvent system such as DMF and THF
  • step A depicts the transformation of a cis-olefin compound (4) to the trans olefin compounds (5) and (6) through using a closed-loop flow apparatus using irradiation and capture on a column of silver nitrate absorbed onto silica gel.
  • Step B shows the reaction of compound (5) with N,N’-disuccinimidyl carbonate using a suitable base such as TEA in a solvent such as ACN to give compound (7).
  • Scheme 3 Step A [000145] a one anhydride using an appropriate base such as DIEA in a solvent such as THF followed by an amide coupling with N-hydroxysuccinimide using an appropriate coupling reagent such as 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with an appropriate base such as 4- dimethylaminopyridine to give compound (9).
  • an appropriate coupling reagent such as 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with an appropriate base such as 4- dimethylaminopyridine
  • Scheme 4 a solvent such as acetic acid followed by treatment with TEA in a solvent such as toluene to give compound (11).
  • Step B depicts the conversion of compound (11) to compound (12) in a manner essentially analogous to scheme 1, step B.
  • Preparation 1 tert-Butyl 4-[2-(2,5-dioxopyrrol-1-yl)ethyl]piperazine-1-carboxylate [000147] tert-Butyl 4-(2-aminoethyl)piperazine-1-carboxylate (3.00 g, 13.1 mmol) was dissolved in acetic acid (6 mL). Added furan-2,5-dione (1.28 g, 13.1 mmol) and stirred at ambient temperature for 7 h. The mixture was then stored in a refrigerator for 18 h. Removed most of the acetic acid under vacuum at 50 °C.
  • this lamp produces between 4400 and 4750 microwatts/cm ⁇ 2 intensity at 0.75" for 254 nM light.
  • FMI pump set to 10 mL/min that draws the reaction mixture from a Pyrex® round bottom flask (250 mL). This was connected to FEP 1/16" tubing that was wrapped around a cold finger (total 7 mL loop, air cooling). The UV lamp was placed in the center of the cold finger to irradiate the sample with air cooling.
  • the sample tubing continued into an ISCO SLM that contained 25 g of silver nitrate impregnated silica gel (See Fox, et.al., Angewandte Chemie, International Edition Engl 2009, 48(38), 7013-7016; Synthesis 2018, 50, 4875).
  • the following steps were performed. Loaded a 50 g silica gel cartridge with 25 g of silver nitrate absorbed onto silica gel on top, covered in aluminum foil, and conditioned by pumping the 1:1 hexanes/diethyl ether solvent mixture for 1 h.
  • MAPT dsRNA functionalization and anneal can be performed in the same way as SNCA dsRNA described above.
  • Conjugation of dsRNA to TfR binding proteins [000163] Site-specific native or engineered cysteine amino acid residues in the TfR binding proteins were used to conjugate dsRNA. Cysteines can be engineered into the primary amino acid sequence of the TfR binding proteins. The approach of introducing cysteines as a means for conjugation has been described in WO 2018/232088, which is both incorporated by reference in its entirety and incorporated specifically in relation to conjugation via cysteine residues.
  • TfR binding proteins were first reduced with 40 molar equivalents reducing agent dithiothreitol (DTT) at 37 °C for two hours, followed by desalting to remove reducing agent via dialysis or desalting columns. This is followed by re-oxidation of the TfR binding protein to reform the structural disulfides with 10 molar equivalent dehydroascorbic acid (DHAA) incubation at ambient temperature for two hours. A follow up desalting was performed to remove oxidizing agent.
  • DTT dithiothreitol
  • DHAA dehydroascorbic acid
  • the second conjugation method utilized the SMCC-functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding proteins.
  • TfR binding protein was prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding proteins. This is followed by incubating the SMCC-dsRNA with the TfR binding proteins at 4 molar equivalents for overnight conjugation at 4 °C.
  • a maleimide hydrolysis step can be done to secure the linker-payload in terminal stage and avoid deconjugation during human body circulation via retro-Michael addition.
  • the third conjugation method utilized GDM-functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding protein via disulfide bond.
  • TfR binding protein was prepared similarly as above to make the engineered thiol available for conjugation by undergoing reduction and oxidation process of the TfR binding protein.
  • dithiobis(5-nitropyridine) was added in as 20 molar equivalents to the protein to generate the intermediate prior to dsRNA conjugation. Excess dithiobis(5-nitropyridine) was removed by desalting.
  • GDM-functionalized dsRNA was added to the protein intermediate in a 4 molar equivalents.
  • Step 1 TfR binding protein conjugation with dithiobis(5-nitropyridine) for intermediate generation
  • Conjugation was monitored using analytical anion exchange chromatography.
  • a ProPacTM SAX-10 HPLC Column, 10 ⁇ m particle, 4mm diameter, 250mm length was utilized with the following method. Flow rate of 1 mL/min, Buffer A: 20mM TRIS pH 7.0, Buffer B: 20 mM TRIS pH 7.0 + 1.5M NaCl, at 30 oC.
  • Table 12 HPLC gradient used to assess dsRNA conjugation to TfR binding protein Time [min] A B [%] [000170] Drug/siRNA to antibody/protein ratio (DAR) was calculated based on peak area % from the analytical anion exchange (aAEX) chromatogram. [000171] Post conjugation of dsRNA to the TfR binding protein, excess dsRNA and unconjugated protein was removed by further purification. Either preparative size exclusion chromatography (SEC) or preparative anion exchange chromatography was utilized for purification of the final conjugate. Preparative SEC was performed using Cytiva Superdex® 200 in 1X PBS pH 7.2 under an isocratic condition.
  • anion exchange e.g., ThermoFisher POROS TM XQ
  • starting buffer 20mM TRIS pH 7.0
  • eluting 20 column volume gradient with a buffer containing 20mM TRIS pH 7.0 and 1M NaCl.
  • ThermoFisher POROS TM XQ was used with starting buffer of 20mM TRIS pH 7.0 and eluting with 20 column volume gradient with a buffer containing 20mM TRIS pH 7.0 and 1M NaCl.
  • siRNA/drug to TBP/antibody ratio Average DAR % of DAR0 % of DAR1 % of DAR2 TBP3- dsRNA 1.03 0.90 95.19 3.64
  • RNA from NHP tissues were isolated using the RNadvance Tissue kit (Beckman Coulter, Indianapolis, IN) manually or on a Biomek i7 liquid handler (Beckman Coulter), following the manufacturer’s procedure with some modifications.
  • the frozen tissue sections were mixed with one 5mm stainless steel ball, lysis buffer and proteinase K, homogenized for 5 cycles of 30s at 1200rpm, with an interval of 20s between cycles, on a 2010 GenoGrinder (SPEX SamplePrep, Metuchen, NJ). Tissues from some regions were shaved on dry ice, prior to homogenization. The homogenates were incubated at 37 °C for 1 hour, then extracted with an equal volume of phenol- chloroform. The RNA in the supernatant were purified with the RNadvance tissue kit, where a 30 minute digestion with DNase was included. The concentration and the purity (A260/A280) of the RNA elute were determined by spectrophotometry.
  • the expression of the respective gene targets in the cDNA was determined using TaqMan qPCR assays on the QuantStudio 7 Pro platform (Thermo Fisher Scientific).
  • Gene expression levels of the MAPT were normalized by GAPDH using respective probes (ThermoFisher).
  • TBP3- dsRNA No. 26 at 20 mg/kg dose in NHPs also led to reduction of MAPT mRNA in key brain regions compared to PBS treatment group at 29 days following dosing.
  • MAPT mRNA reductions were demonstrated in the hippocampus (42%), temporal cortex (41%), parietal cortex (37%), motor cortex (32%) and prefrontal cortex (36%).
  • Example 5
  • TfR binding proteins-dsRNA conjugates were evaluated in human transferrin transgenic mice at various doses of siRNA concentrations.
  • TBP2-SNCA dsRNA No. 13 and TBP3-SNCA dsRNA No. 13 conjugate demonstrated significant reduction of SNCA mRNA in brain compared to the PBS treated group.
  • the SNCA mRNA reduction level was similar in each dose group treated by either TBP2-SNCA dsRNA No. 13 or TBP3-SNCA dsRNA No. 13 conjugate ( Figure 3). Specifically, for TBP2-SNCA dsRNA No.
  • mice Further characterization of the mouse TfR binding proteins-dsRNA conjugates in human Tau (hTau) transgenic mice [000177] The pharmacodynamic efficacy of the mouse TfR binding proteins-dsRNA conjugates were evaluated in human tau (hTau) transgenic mice at various doses of siRNA concentrations.
  • mTBP2-MAPT dsRNA No. 26 demonstrated significant reduction of MAPT mRNA in brain compared to the PBS treated group. Specifically, for mTBP2-MAPT dsRNA No. 26 conjugate, 100 mg/kg siRNA dose treatment resulted in 39% MAPT mRNA remaining (61% knock down), 10 mg/kg siRNA dose treatment resulted in 60% MAPT mRNA remaining (40% knock down), 1 mg/kg siRNA dose treatment resulted in 85% MAPT mRNA remaining (15% knock down).

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Abstract

L'invention concerne des protéines comprenant un domaine de liaison au TfR humain monovalent (« protéines de liaison au TfR humain »), des conjugués comprenant de telles protéines de liaison au TfR humain, par exemple, des conjugués ARNdb-protéines de liaison au TfR humain, des compositions pharmaceutiques comprenant des protéines ou des conjugués de liaison au TfR humain, et des méthodes de traitement de maladies du SNC (par exemple, une maladie neurodégénérative telle qu'une synucléinopathie neurodégénérative ou une tauopathie) à l'aide de protéines de liaison au TfR humain ou de conjugués.
PCT/US2025/013966 2024-02-02 2025-01-31 Protéines de liaison au récepteur de la transferrine et conjugués Pending WO2025166119A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003009815A2 (fr) 2001-07-25 2003-02-06 Biomarin Pharmaceutical Inc. Compositions et procedes de modulation du transport a travers la barriere hematho-encephalique
WO2018232088A1 (fr) 2017-06-16 2018-12-20 Eli Lilly And Company Composés d'anticorps modifiés et conjugués de ceux-ci
WO2024006976A2 (fr) * 2022-07-01 2024-01-04 Denali Therapeutics Inc. Conjugués de molécules de liaison au récepteur de la transferrine pour l'administration d'oligonucléotides à des cellules
WO2024026474A1 (fr) * 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions et méthodes d'administration médiée par le récepteur de la transferrine (tfr) au cerveau et au muscle
WO2024036096A2 (fr) * 2022-08-08 2024-02-15 Eli Lilly And Company Protéines de liaison au récepteur de la transferrine et conjugués

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Publication number Priority date Publication date Assignee Title
WO2003009815A2 (fr) 2001-07-25 2003-02-06 Biomarin Pharmaceutical Inc. Compositions et procedes de modulation du transport a travers la barriere hematho-encephalique
WO2018232088A1 (fr) 2017-06-16 2018-12-20 Eli Lilly And Company Composés d'anticorps modifiés et conjugués de ceux-ci
WO2024006976A2 (fr) * 2022-07-01 2024-01-04 Denali Therapeutics Inc. Conjugués de molécules de liaison au récepteur de la transferrine pour l'administration d'oligonucléotides à des cellules
WO2024026474A1 (fr) * 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions et méthodes d'administration médiée par le récepteur de la transferrine (tfr) au cerveau et au muscle
WO2024036096A2 (fr) * 2022-08-08 2024-02-15 Eli Lilly And Company Protéines de liaison au récepteur de la transferrine et conjugués

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