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WO2021184021A1 - Constructions de vésicules extracellulaires - aso ciblant pmp22 - Google Patents

Constructions de vésicules extracellulaires - aso ciblant pmp22 Download PDF

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Publication number
WO2021184021A1
WO2021184021A1 PCT/US2021/022433 US2021022433W WO2021184021A1 WO 2021184021 A1 WO2021184021 A1 WO 2021184021A1 US 2021022433 W US2021022433 W US 2021022433W WO 2021184021 A1 WO2021184021 A1 WO 2021184021A1
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Prior art keywords
seq
aso
aspects
extracellular vesicle
sequence
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PCT/US2021/022433
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English (en)
Inventor
Wendy Broom
Ke Xu
Jing Zhou
Shailendra PATEL
Wei Zhang
Sriram SATHYANARAYAN
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Codiak Biosciences, Inc.
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Publication of WO2021184021A1 publication Critical patent/WO2021184021A1/fr

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    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • 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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present disclosure relates to extracellular vesicles (EVs), e.g., exosomes, comprising an antisense oligonucleotide (ASO), wherein the ASO comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within a PMP22 transcript.
  • the extracellular vesicle further comprises a scaffold protein.
  • Exosomes are small extracellular vesicles that are naturally produced by every eukaryotic cell. Exosomes comprise a membrane that encloses an internal space (i.e., lumen).
  • EVs As drug delivery vehicles, EVs, e.g., exosomes, offer many advantages over traditional drug delivery methods as a new treatment modality in many therapeutic areas. In particular, exosomes have intrinsically low immunogenicity, even when administered to a different species.
  • Antisense oligonucleotides have emerged as a powerful means of regulating target gene expression in vitro or in vivo. However, there remains a need to improve the stability and targeting of ASOs in vivo.
  • new and more effective engineered-EVs particularly those that can be used to deliver therapeutic agents that can reduce the expression of a gene associated with a disease (e.g., N for cancer), are necessary to better enable therapeutic use and other applications of EV-based technologies.
  • the present disclosure provides an extracellular vesicle comprising an antisense oligonucleotide (ASO) which comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within a PMP22 transcript (SEQ ID NO:164).
  • ASO antisense oligonucleotide
  • the extracellular vesicle targets a Schwann cell.
  • the ASO comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within nucleotides 1 to 1828 of a PMP22 transcript corresponding to a nucleotide sequence as set forth in SEQ ID NO: 164 (PMP22 full mRNA transcript) or nucleotides 208 to 690 of a PMP22 transcript corresponding to a nucleotide sequence as set forth in SEQ ID NO: 165 (PMP22 coding sequence).
  • the contiguous nucleotide sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% complementary to the nucleic acid sequence within the PMP22 transcript.
  • the ASO is capable of reducing PMP22 protein expression in a human cell (e.g., a Schwan cell), wherein the human cell expresses the PMP22 protein.
  • the PMP22 protein expression is reduced by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to PMP22 protein expression in a human cell that is not exposed to the ASO.
  • the ASO is capable of reducing a level of PMP22 mRNA in a human cell (e.g., an immune cell), wherein the human cell expresses the PMP22 mRNA.
  • the level of PMP22 mRNA is reduced by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to the level of the PMP22 mRNA in a human cell that is not exposed to the ASO.
  • the ASO is a gapmer, a mixmer, or a totalmer.
  • the ASO comprises one or more nucleoside analogs.
  • one or more of the nucleoside analogs comprises a 2'-O-alkyl-RNA; 2'-O-methyl RNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluro-RNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro-ANA; or bicyclic nucleoside analog.
  • one or more of the nucleoside analogs is a sugar modified nucleoside.
  • the sugar modified nucleoside is an affinity enhancing 2' sugar modified nucleoside.
  • one or more of the nucleoside analogs comprises a nucleoside comprising a bicyclic sugar. In some aspects, one or more of the nucleoside analogs comprises an LNA. In some aspects, one or more of the nucleotide analogs is selected from the group consisting of constrained ethyl nucleoside (cEt), 2',4'-constrained 2′-O-methoxyethyl (cMOE), ⁇ -L-LNA, ⁇ -D-LNA, 2'-O,4'- C-ethylene-bridged nucleic acids (ENA), amino-LNA, oxy-LNA, thio-LNA, and any combination thereof.
  • cEt constrained ethyl nucleoside
  • cMOE 2',4'-constrained 2′-O-methoxyethyl
  • ENA 2'-O,4'- C-ethylene-bridged nucleic acids
  • the ASO comprises one or more 5'-methyl-cytosine nucleobases.
  • the contiguous nucleotide sequence is complementary to a nucleic acid sequence within (i) a 5' untranslated region (UTR); (ii) a coding region; or (iii) a 3' UTR of the PMP22 transcript.
  • the contiguous nucleotide sequence is complementary to a nucleic acid sequence comprising (i) nucleotides 1 – 173 of SEQ ID NO: 164 (exon 1); (ii) nucleotides 174-285 of SEQ ID NO: 164 (exon 2); (iii) nucleotides 286 - 385 of SEQ ID NO: 164 (exon 3); (iv) nucleotides 386 – 526 of SEQ ID NO: 164 (exon 4); (v) 527 – 1828 of SEQ ID NO: 164 (exon 5); (vi) 200 – 300 of SEQ ID NO: 164, (vii) nucleotides 200 – 400 of SEQ ID NO: 164; (viii) nucleotides 500 -600 of SEQ ID NO: 164; (ix) nucleotides 600 - 700 of SEQ ID NO: 164; (x) nucleotides 600 – 800 of SEQ ID NO:
  • the contiguous nucleotide sequence is complementary to a nucleic acid sequence comprising nucleotides 152-168 (SEQ ID NO: 130), 225-244 (SEQ ID NO:131), 227-246 (SEQ ID NO:132), 235-254 (SEQ ID NO:133), 265-284 (SEQ ID NO:134), 271-290 (SEQ ID NO:135), 380-399 (SEQ ID NO:136), 383-402 (SEQ ID NO:137), 385-404 (SEQ ID NO:138), 418-437 (SEQ ID NO:139), 479-498 (SEQ ID NO:140), 583-602 (SEQ ID NO:141), 671-690 (SEQ ID NO:142), 672-691 (SEQ ID NO:143), 673-692 (SEQ ID NO:144), 674-693 (SEQ ID NO:145), 675-691 (SEQ ID NO:146), 676-691 (SEQ ID NO:147), 678-693 (SEQ ID NO:
  • the contiguous nucleotide sequence comprises a nucleotide sequence complementary to a nucleic acid sequence comprising nucleotides 152-168 (SEQ ID NO:130), 235-254 (SEQ ID NO:133), 385-404 (SEQ ID NO:138), 479-498 (SEQ ID NO:140), 672-691 (SEQ ID NO:143), 675-691 (SEQ ID NO:146), 939-958 (SEQ ID NO:149), 1130-1149 (SEQ ID NO:152), 1293-1312 (SEQ ID NO:153), 1365-1384 (SEQ ID NO:157), 1404-1423 (SEQ ID NO:158), or 1605-1624 (SEQ ID NO:160) of SEQ ID NO: 164.
  • the present disclosure also provides an extracellular vesicle wherein the continuous nucleotide sequence is fully complementary to a nucleotide sequence within the PMP22 transcript.
  • the ASO comprises a nucleotide sequence selected from SEQ ID NOs: 130-163 or 202-236, with one or two mismatches.
  • the ASO comprises at least one LNA.
  • the ASO is from 16 to 20 nucleotides in length.
  • the contiguous nucleotide sequence comprises one or more modified internucleoside linkages.
  • the one or more modified internucleoside linkages is a phosphorothioate linkage.
  • the extracellular vesicle further comprises an anchoring moiety.
  • the ASO is linked to the anchoring moiety.
  • the extracellular vesicle further comprising an exogenous targeting moiety.
  • the exogenous targeting moiety comprises a peptide, an antibody or an antigen-binding fragment thereof, a chemical compound, an RNA aptamer, or any combination thereof.
  • the exogenous targeting moiety comprises a peptide.
  • the exogenous targeting moiety comprises a microprotein, a designed ankyrin repeat protein (darpin), an anticalin, an adnectin, an aptamer, a peptide mimetic molecule, a natural ligand for a receptor, a camelid nanobody, or any combination thereof.
  • the exogenous targeting moiety comprises a full-length antibody, a single domain antibody, a heavy chain only antibody (VHH), a single chain antibody, a shark heavy chain only antibody (VNAR), an scFv, a Fv, a Fab, a Fab', a F(ab')2, or any combination thereof.
  • the antibody is a single chain antibody.
  • the exogenous targeting moiety targets the exosome to peripheral myelinated nerve fibers.
  • the exogenous targeting moiety targets the exosome to a Schwan cells.
  • the EV comprises a scaffold moiety linking the exogenous targeting moiety to the EV.
  • the anchoring moiety and/or the scaffold moiety is a Scaffold X.
  • the anchoring moiety and/or the scaffold moiety is a Scaffold Y.
  • the Scaffold X is a scaffold protein that is capable of anchoring the ASO on the luminal surface of the EV and/or on the exterior surface of the EV.
  • the Scaffold X is selected from the group consisting of prostaglandin F2 receptor negative regulator (the PTGFRN protein); basigin (the BSG protein); immunoglobulin superfamily member 2 (the IGSF2 protein); immunoglobulin superfamily member 3 (the IGSF3 protein); immunoglobulin superfamily member 8 (the IGSF8 protein); integrin beta-1 (the ITGB1 protein); integrin alpha-4 (the ITGA4 protein); 4F2 cell-surface antigen heavy chain (the SLC3A2 protein); a class of ATP transporter proteins (the ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4 proteins); a functional fragment thereof; and any combination thereof.
  • the PTGFRN protein prostaglandin F2 receptor negative regulator
  • basigin the BSG protein
  • immunoglobulin superfamily member 2 the IGSF2 protein
  • immunoglobulin superfamily member 3 the
  • the anchoring moiety and/or the scaffold moiety is PTGFRN protein or a functional fragment thereof.
  • the anchoring moiety and/or the scaffold moiety comprises an amino acid sequence as set forth in SEQ ID NO: 1 (PTGFRN).
  • the anchoring moiety and/or the scaffold moiety comprises an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identical to SEQ ID NO: 1 (PTGFRN).
  • the Scaffold Y is a scaffold protein that is capable of anchoring the ASO on the luminal surface of the EV and/or on the exterior surface of the EV.
  • the Scaffold Y is selected from the group consisting of myristoylated alanine rich Protein Kinase C substrate (the MARCKS protein), myristoylated alanine rich Protein Kinase C substrate like 1 (the MARCKSL1 protein), brain acid soluble protein 1 (the BASP1 protein), a functional fragment thereof, and any combination thereof.
  • the Scaffold Y is a BASP1 protein or a functional fragment thereof.
  • the ASO is linked to the anchoring moiety and/or the scaffold moiety on the exterior surface of the EV. In some aspects, the ASO is linked to the anchoring moiety and/or the scaffold moiety on the luminal surface of the EV. In some aspects, the anchoring moiety comprises sterol, GM1, a lipid, a vitamin, a small molecule, a peptide, or a combination thereof. In some aspects, the anchoring moiety comprises cholesterol. In some aspects, the anchoring moiety comprises a phospholipid, a lysophospholipid, a fatty acid, a vitamin (e.g., vitamin D and/or vitamin E), or any combination thereof.
  • the ASO is linked to the anchoring moiety and/or the scaffold moiety by a linker.
  • the ASO is linked to the EV by a linker.
  • the linker is a polypeptide.
  • the linker is a non-polypeptide moiety.
  • the linker comprises ethylene glycol.
  • the linker comprises HEG, TEG, PEG, or any combination thereof.
  • the linker comprises acrylic phosphoramidite (e.g., ACRYDITETM), adenylation, azide (NHS Ester), digoxigenin (NHS Ester), cholesterol-TEG, I-LINKERTM, an amino modifier (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, or UNI-LINKTM amino modifier), alkyne, 5' Hexynyl, 5-Octadiynyl dU, biotinylation (e.g., biotin, biotin (Azide), biotin dT, biotin-TEG, dual biotin, PC biotin, or desthiobiotin), thiol modification (thiol modifier C3 S-S, dithiol or thiol modifier C6 S-S), or any combination thereof.
  • acrylic phosphoramidite e.g., ACRYDITETM
  • adenylation azide
  • NHS Ester digoxigenin
  • the linker is a cleavable linker.
  • the linker comprises valine-alanine-p-aminobenzylcarbamate or valine-citrulline-p- aminobenzylcarbamate.
  • the linker comprises (i) a maleimide moiety and (ii) valine-alanine-p-aminobenzylcarbamate or valine-citrulline-p-aminobenzylcarbamate.
  • the EV is an exosome.
  • the present disclosure also provides an antisense oligonucleotide (ASO) comprising comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within a PMP22 transcript (SEQ ID NO:164). In some aspects, the ASO is not (SEQ ID NO: 237).
  • the ASO comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within nucleotides 1 to 1828 of a PMP22 transcript corresponding to a nucleotide sequence as set forth in SEQ ID NO: 164 (PMP22 full mRNA transcript) or nucleotides 208 to 690 of a PMP22 transcript corresponding to a nucleotide sequence as set forth in SEQ ID NO: 165 (PMP22 coding sequence).
  • the contiguous nucleotide sequence thereof is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% complementary to the nucleic acid sequence within the PMP22 transcript.
  • the ASO is capable of reducing PMP22 protein expression in a human cell (e.g., aa Schwann cell), wherein the human cell expresses the PMP22 protein (e.g., a protein of SEQ ID NO:166 or a fragment or variant thereof, e.g., a variant comprising one or more mutations).
  • the PMP22 protein expression is reduced by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to PMP22 protein expression in a human cell that is not exposed to the ASO.
  • the ASO is capable of reducing a level of PMP22 mRNA in a human cell (e.g., a Schwann cell), wherein the human cell expresses the PMP22 mRNA.
  • the level of PMP22 mRNA is reduced by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to the level of the PMP22 mRNA in a human cell that is not exposed to the ASO.
  • the ASO is a gapmer, a mixmer, or a totalmer.
  • the ASO comprises one or more nucleoside analogs.
  • one or more of the nucleoside analogs comprises a 2'-O-alkyl-RNA; 2'-O-methyl RNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluro-RNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro-ANA; or bicyclic nucleoside analog (LNA).
  • one or more of the nucleoside analogs is a sugar modified nucleoside.
  • the sugar modified nucleoside is an affinity enhancing 2' sugar modified nucleoside.
  • one or more of the nucleoside analogs comprises a nucleoside comprising a bicyclic sugar.
  • one or more of the nucleoside analogs comprises an LNA.
  • the LNA is selected from the group consisting of constrained ethyl nucleoside (cEt), 2',4'-constrained 2′-O-methoxyethyl (cMOE), ⁇ -L-LNA, ⁇ -D-LNA, 2'-O,4'- C-ethylene-bridged nucleic acids (ENA), amino-LNA, oxy-LNA, thio-LNA, and any combination thereof.
  • the ASO comprises one or more 5'-methyl-cytosine nucleobases.
  • the ASO comprises any one of SEQ ID NO: 130 to SEQ ID NO: 163, or SEQ ID NO: 202 to SEQ ID NO: 236.
  • the ASO comprises at least one LNA.
  • the ASO is from 14 to 20 nucleotides in length.
  • the contiguous nucleotide sequence comprises one or more modified internucleoside linkages.
  • the one or more modified internucleoside linkages is a phosphorothioate linkage.
  • the present disclosure also provides a conjugate comprising an ASO of the present disclosure, wherein the ASO is covalently attached to at least one non-nucleotide or non-polynucleotide moiety.
  • the non-nucleotide or non-polynucleotide moiety comprises a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or any combinations thereof.
  • the disclosure also provides an extracellular vesicle comprising an ASO or conjugate disclosed herein.
  • a pharmaceutical composition comprising (i) an extracellular vesicle (e.g., an exosome) disclosed herein, an ASO disclosed herein, or a conjugate disclosed herein, and (ii) a pharmaceutically acceptable diluent, carrier, salt, or adjuvant.
  • the pharmaceutically acceptable salt comprises a sodium salt, a potassium salt, an ammonium salt, or any combination thereof.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the additional therapeutic agent is a PMP22 antagonist.
  • the PMP22 antagonist is a chemical compound, an siRNA, an shRNA, an antisense oligonucleotide, a protein, or any combination thereof.
  • the PMP22 antagonist is a progesterone antagonist.
  • the progesterone antagonist is onapristone.
  • the disclosure also provides a kit comprising an extracellular vesicle, ASO, conjugate, or pharmaceutical composition disclosed herein, and instructions for use. Also provided is a diagnostic kit comprising an extracellular vesicle, ASO, conjugate, or pharmaceutical composition disclosed herein, and instructions for use.
  • Also provided is a method of inhibiting or reducing PMP22 protein expression in a cell comprising administering an extracellular vesicle, ASO, conjugate, or pharmaceutical composition disclosed herein to the cell expressing PMP22 protein, wherein the PMP22 protein expression in the cell is inhibited or reduced after the administration.
  • method of treating a neuropathy in a subject in need thereof comprising administering an effective amount of extracellular vesicle, ASO, conjugate, or pharmaceutical composition disclosed herein to the subject.
  • the disclosure also provides the use of an extracellular vesicle, ASO, conjugate, or pharmaceutical composition disclosed herein in the manufacture of a medicament for the treatment of a neuropathy in a subject in need thereof.
  • an extracellular vesicle, ASO, conjugate, or pharmaceutical composition disclosed herein for use in the treatment of a neuropathy in a subject in need thereof.
  • the disclosure also provides a method of treating a disease or disorder in a subject in need thereof, comprising administering an effective amount of an extracellular vesicle, ASO, conjugate, or pharmaceutical composition disclosed herein to the subject, wherein the disease or disorder is Charcot-Marie-Tooth disease 1A (CMT1A).
  • CMT1A Charcot-Marie-Tooth disease 1A
  • the disclosure also provides the use of an extracellular vesicle, ASO, conjugate, or pharmaceutical composition disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder in a subject in need thereof, wherein the disease or disorder is Charcot-Marie-Tooth disease 1A (CMT1A).
  • CMT1A Charcot-Marie-Tooth disease 1A
  • the ASO inhibits or reduces expression of PMP22 mRNA in the cell after the administration.
  • a level of PMP22 mRNA is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% after the administration compared to the level of PMP22 mRNA in a cell not exposed to the ASO.
  • the expression of PMP22 protein is reduced by at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% after the administration compared to the expression of PMP22 protein in a cell not exposed to the ASO.
  • the administration of the extracellular vesicle, the ASO, the conjugate, or the pharmaceutical composition of the present disclosure is intrathecal.
  • the intrathecal administration is into the spinal canal and/or into the subarachnoid space.
  • the extracellular vesicle is targeted to Schwann cells.
  • the extracellular vesicle comprises a surface anchored anti-phagocytic signal.
  • the anti-phagocytic signal is CD47, CD24, a fragment or variant thereof, or a combination thereof.
  • the extracellular vesicle comprises a tissue or cell-specific target ligand which increases extracellular vesicle tropism to specific peripheral nerves.
  • the extracellular vesicle comprises a tissue or cell-specific target ligand which increases extracellular vesicle tropism to Schwann cells.
  • the cell-specific target ligand which increases extracellular vesicle tropism to a Schwann cells binds to a Schwann cell surface marker.
  • the Schwann cell surface marker is selected from Myelin Basic Protein (MBP) and isoforms thereof, Myelin Protein Zero (P0), P75NTR, NCAM, and PMP22.
  • the cell-specific target ligand comprises an antibody or an antigen-binding portion thereof, an aptamer, or an agonist or antagonist of a receptor expressed on the surface of the Schwann cell.
  • the present disclosure provides an extracellular vesicle comprising an antisense oligonucleotide (ASO) which comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within a PMP22 transcript (SEQ ID NO: 164), wherein the extracellular vesicle comprises a EV comprises a surface anchored anti-phagocytic signal and a tissue or cell-specific target ligand which increases EV tropism to Schwann cells.
  • the anti-phagocytic signal is CD47, CD24, a fragment or variant thereof, or a combination thereof.
  • the extracellular vesicle further comprises a targeting moiety that targets a CNS specific peripheral nerve.
  • the targeting moiety comprises a ligand that binds to a transferrin receptor (TfR), apolipoprotein D (ApoD), Galectin 1 (LGALS1), Myelin proteolipid protein (PLP), Glypican 1, or Syndecan 3.
  • TfR is TfR1.
  • the ligand that binds to TfR1 is an antibody against TfR1 or transferrin.
  • the transferrin is a serum transferrin, lacto transferrin (lactoferrin) ovotransferrin, or melanotransferrin.
  • the transferrin is an asialo transferrin, a monosialo transferrin, a disialo transferrin, a trisialo transferrin, a tetrasialo transferrin, a pentasialo transferrin, an hexasialo transferrin, or a combination thereof.
  • the targeting moiety comprises an antibody or an antigen-binding portion thereof, a vNAR, an aptamer, or an agonist or antagonist of a receptor expressed on the surface of the Schwann cell.
  • the extracellular vesicle further comprises a targeting moiety that targets a sensory neuron.
  • the targeting moiety comprises a neurotrophin that binds to a tropomyosin receptor kinase (Trk) receptor.
  • Trk receptor is TrkA, TrB, TrkC, or a combination thereof.
  • the neurotrophin is Nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4 (NT-4), or a combination thereof.
  • the extracellular vesicle further comprises a targeting moiety that targets a motor neuron.
  • the targeting moiety comprises a Rabies Virus Glycoprotein (RVG) peptide, a Targeted Axonal Import (TAxI) peptide, a P75R peptide, or a Tet-C peptide.
  • RVG Rabies Virus Glycoprotein
  • TxI Targeted Axonal Import
  • P75R P75R
  • Tet-C peptide Tet-C peptide.
  • FIG. 1 shows a preliminary selection of targeting areas and antisense oligonucleotides (ASOs) in the PMP22 coding region (denoted by dark grey) and 3’ UTR. Selection was conducted using the Selection method #1 described in Example 1. The final selection included 2115-mers, 2216-mers, 1917-mers and 3820-mers.
  • FIG. 2 shows the starting nucleotide location of the ASOs of FIG.
  • FIG. 3 shows the results of a second round to ASO selection conducted according to the criteria described in Selection method #2 in Example 1.
  • the graph shows the starting nucleotide location of the ASOs of FIG.1 versus number of 4 mismatch hits in spliced transcriptome.
  • FIG. 4 illustrates the multiple transcripts of the PMP22 gene. The protein coding sequences are identical, but there are differences in the 5’ and 3’ UTRs.
  • FIG. 5 shows additional ASOs identified according to Selection method #3 described in Example 1.
  • FIG. 6 shows 2 dose (2nM and 20nM ASO) KD activity in HEK293 cells as described in Example 2.
  • FIG. 7 shows a comparison between the reduction in PMP22 mRNA levels remaining after administration of either 2nM anti-PMP22 ASO or 20mM anti-PMP22 ASO.
  • FIG.8 shows the percentage of PMP mRNA remaining after administration of different anti-PMP22 ASOs at two concentrations (2 nM and 20 nM) with respect to their respective starting nucleotide location on the PMP22 mRNA transcript for the best 34 ASOs screened.
  • FIG.9 shows the species cross-reactivity for the 34 top screened ASOs.
  • FIG.10 shows the KD activity for the 34 top screened ASOs.
  • FIG.11 shows the result of a dose response analysis for the ASOs of SEQ ID NOS: 130, 133, 138, 140, 143, 146, 149, 152, 153, 157, 158 and 160. The lowest IC50 corresponded to SEQ ID NOs: 146 and 157.
  • FIG.12 shows a dose-response for the ASO is SEQ ID: 146.
  • FIG.13 shows a dose-response for the ASO is SEQ ID: 157.
  • FIG. 14 shows PMP22 expression in Panc-1 cells. Accordingly, Panc-1 cells can be used as an in vitro model for ASO screening.
  • FIGs.15A-15D are schematic drawings of exemplary CD47-Scaffold X fusion constructs that can be delivered on the extracellular vesicles described herein, along with an ASO targeting a PMP22 transcript.
  • FIG.15A-15D are schematic drawings of exemplary CD47-Scaffold X fusion constructs that can be delivered on the extracellular vesicles described herein, along with an ASO targeting a PMP22 transcript.
  • FIG. 15A shows constructs comprising the extracellular domain of wild-type CD47 (with a C15S substitution) fused to either a flag-tagged (1083 and 1084) or non-flag-tagged (1085 and 1086) full length Scaffold X (1083 and 1086) or a truncated Scaffold X (1084 and 1085).
  • FIG.15B shows constructs comprising the extracellular domain of Velcro-CD47 fused to either a flag-tagged (1087 and 1088) or non-flag-tagged (1089 and 1090) full length Scaffold X (1087 and 1090) or a truncated Scaffold X (1088 and 1089).
  • FIG. 15C shows constructs wherein the first transmembrane domain of wild-type CD47 (with a C15S substitution; 1127 and 1128) or Velcro-CD47 (1129 and 1130) is replaced with a fragment of Scaffold X, comprising the transmembrane domain and the first extracellular motif of Scaffold X.
  • FIG. 15D shows various constructs comprising a minimal "self" peptide SEQ ID NO: 102) fused to either a flag-tagged (1158 and 1159) or non-flag-tagged (1160 and 1161) full length Scaffold X (1158 and 1161) or a truncated Scaffold X (1159 and 1160). [0061] FIG.
  • FIG. 16 shows the expression of exemplary mouse CD47-Scaffold X fusion constructs that can be delivered on the surface of modified exosomes, along with an ASO targeting a PMP transcript.
  • the construct comprises the extracellular domain of wild-type murine CD47 (with a C15S substitution) fused to either a flag-tagged (1923 and 1925) or non- flag-tagged (1924 and 1922) full length Scaffold X (1923 and 1922) or a truncated Scaffold X (1925 and 1924).
  • FIG.17A shows a schematic diagram of exemplary extracellular vesicle (e.g., exosome) targeting Trks using neurotrophin-Scaffold X fusion construct that can be delivered along with a PMP22 ASO.
  • Neurotrophins bind to Trk receptors as a homo dimer and allow the EV to target a sensory neuron.
  • FIG.17B shows a schematic diagram of exemplary extracellular vesicle (e.g., exosome) having (i) neuro-tropism as well as (ii) an anti-phagocytic signal, e.g., CD47 and/or CD24, on the exterior surface of the EV that can be delivered along with (iii) a PMP22 ASO.
  • FIG. 18 shows transferrin receptor expression levels in cell lines Panc-1, HepG2, Hep3B2.1-7, Neuro-2A, JeLa, and HEK293. [0065] FIG.
  • FIG. 19 shows transferrin receptor expression levels in cell lines HEK293, Neuro2A, Sol8, and C2C12, and in humau (human SC), and mouse (mouse SC) Schwann cells.
  • the left side panel shows a SDS-PAGE gel under reducing conditions, and the right side panel shows a Western blot conducted using an anti-TfR monoclonal antibody.
  • exosome 20 shows the differental uptake of exosomes by mouse Schwann cells 2 hours or 22 hours after incubation of the cells with the exosomes depending on whether the exosomes express transferrin on their surfaces, e.g., exoTransferrin exosomes comprising human transferrin (1597 construct), or exomTransferrin exosomes comprising mouse transferrin (1598 construct), or transferrin is not present on the exosome surface (exoLinker exosomes) (2022 construct).
  • FIG.21 shows the differental uptake of exosomes by human Schwann cells 2 hours or 22 hours after incubation of the cells with exosomes depending expressing transferrin on their surfaces (exoTransferrin exosomes comprising human transferrin, or exomTransferrin exosomes comprising mouse transferrin) or not expressing transferrin on their surfaces (exoLinker exosomes).
  • FIG. 22 present high magnification microscopy images with cytoskeleton counter stain of mouse (left) and human (right) Schwann cells after incubation with exotransferrin exosomes.
  • FIG. 23 shows the sequences of the PMP22 ASOs used in the experiments presented in FIGS.24-30.
  • FIG.24 shows a PMP22 knockdown dose-response for the ASOs presented in FIG .23 in human Schwann cells (HSC).
  • HSC human Schwann cells
  • the ASOs were transfected into the Schwann cells usng Lipofectamine RNAiMAX. RNA levels were quantitated using qPCR. ASO concentrations up to 200 nM were used.
  • FIG.25 shows a PMP22 knockdown dose-response for the ASOs presented in FIG.23 in mouse Schwann cells (MSC).
  • FIG. 26 shows a PMP22 knockdown dose-response for the “control” ASO presented in FIG.23 and an ASO specifically targeting PMP22 in mouse Schwann cells (MSC).
  • the ASOs were transfected into the Schwann cells using Lipofectamine RNAiMAX. RNA levels were quantitated using qPCR. ASO concentrations up to 500 nM were used.
  • the ASO targeting PMP22 used was X61832, i.e., a PMP22 ASO of sequence CTCATTCGCGTTTCCGC (SEQ ID NO: 146).
  • FIG. 27 shows a PMP22 knockdown dose-response for the “control” ASO presented in FIG.23 and an ASO specifically targeting PMP22 in mouse Schwann cells (MSC).
  • the ASOs were transfected into the Schwann cells usng Lipofectamine RNAiMAX. RNA levels were quantitated using qPCR. ASO concentrations up to 500 nM were used.
  • the ASO targeting PMP22 used was X61832, i.e., a PMP22 ASO of sequence (SEQ ID NO: 146).
  • FIG.28 shows a PMP22 knockdown assay in mouse Schwann cells comprising the administration of ExomTF-PMP22 ASO contructs, i.e., exosomes expressing mouse transferrin on their surfaces and also comprising an ASO against PMP22.
  • ASOs were administered at 125 nM, 250 nM, and 500 nM concentrations.
  • Exosomes comprising the scrambled sequence of the PMP22 ASO were used as controls.
  • RNAiMAX RNAimax only
  • PMP22 ASO alone Free PMP22 ASO
  • exomTransferrin mouse Transferrin
  • samples transfected with PMP22 ASO with RNAiMAX PMP22trans
  • samples transfected with control ASO with RNAiMAX controltrans.
  • the ASO used was X61832, i.e., a PMP22 ASO of sequence CTCATTCGCGTTTCCGC (SEQ ID NO: 146).
  • FIG. 29 shows that PMP22 is expressed in Panc-1 cells, a human pancreatic cancer cell line isolated from a pancreatic carcinoma of ductal cell origin.
  • Panc-1 cells can be used as an in vitro screening model for PMP22 ASOs instead of using human or mouse Schwann cells.
  • FIG.30 shows the mRNA knock-down effect of a PMP22 ASO of the present disclosure compared to four ASO controls using Panc-1 cells instead of human or mouse Schwann cells.
  • Certain aspects of the present disclosure are directed to an extracellular vesicle (EV), e.g., an exosome, comprising an antisense oligonucleotide (ASO), wherein the ASO comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within a PMP22 transcript.
  • EV extracellular vesicle
  • ASO antisense oligonucleotide
  • a or “an” entity refers to one or more of that entity; for example, "a nucleotide sequence,” is understood to represent one or more nucleotide sequences.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
  • ASO antisense oligonucleotide
  • the term “antisense oligonucleotide” refers to an oligomer or polymer of nucleosides, such as naturally-occurring nucleosides or modified forms thereof, that are covalently linked to each other through internucleotide linkages.
  • the ASO useful for the disclosure includes at least one non-naturally occurring nucleoside.
  • An ASO is at least partially complementary to a target nucleic acid, such that the ASO hybridizes to the target nucleic acid sequence.
  • nucleic acids or “nucleotides” is intended to encompass plural nucleic acids.
  • the term “nucleic acids” or “nucleotides” refers to a target sequence, e.g., pre-mRNAs, mRNAs, or DNAs in vivo or in vitro.
  • the nucleic acids or nucleotides can be naturally occurring sequences within a cell.
  • nucleic acids or nucleotides refer to a sequence in the ASOs of the disclosure.
  • the nucleic acids or nucleotides can be non-naturally occurring, i.e., chemically synthesized, enzymatically produced, recombinantly produced, or any combination thereof.
  • the nucleic acids or nucleotides in the ASOs are produced synthetically or recombinantly, but are not a naturally occurring sequence or a fragment thereof.
  • the nucleic acids or nucleotides in the ASOs are not naturally occurring because they contain at least one nucleoside analog that is not naturally occurring in nature.
  • nucleotide refers to a glycoside comprising a sugar moiety, a base moiety and a covalently linked group (linkage group), such as a phosphate or phosphorothioate internucleotide linkage group, and covers both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to as "nucleotide analogs" herein.
  • a single nucleotide can be referred to as a monomer or unit.
  • nucleotide analogs refers to nucleotides having modified sugar moieties.
  • nucleotides having modified sugar moieties e.g., LNA
  • nucleotide analogs refers to nucleotides having modified nucleobase moieties.
  • nucleotides having modified nucleobase moieties include, but are not limited to, 5-methyl-cytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5- propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6- aminopurine.
  • nucleotide “unit” and “monomer” are used interchangeably. It will be recognized that when referring to a sequence of nucleotides or monomers, what is referred to is the sequence of bases, such as A, T, G, C or U, and analogs thereof.
  • nucleoside as used herein is used to refer to a glycoside comprising a sugar moiety and a base moiety, and can therefore be used when referring to the nucleotide units, which are covalently linked by the internucleotide linkages between the nucleotides of the ASO.
  • nucleotide is often used to refer to a nucleic acid monomer or unit.
  • nucleotide can refer to the base alone, i.e., a nucleobase sequence comprising cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA) and uracil (RNA), in which the presence of the sugar backbone and internucleotide linkages are implicit.
  • nucleotide can refer to a "nucleoside.”
  • nucleotide can be used, even when specifying the presence or nature of the linkages between the nucleosides.
  • nucleotide length means the total number of the nucleotides (monomers) in a given sequence. For example, the sequence of the PMP22 ASO of SEQ ID NO:146 has 17 nucleotides; thus the nucleotide length of the sequence is 17.
  • nucleotide length is therefore used herein interchangeably with “nucleotide number.”
  • nucleotide number is therefore used herein interchangeably with “nucleotide number.”
  • the 5' terminal nucleotide of an oligonucleotide does not comprise a 5' internucleotide linkage group, although it can comprise a 5' terminal group.
  • the compounds described herein can contain several asymmetric centers and can be present in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
  • the asymmetric center can be an asymmetric carbon atom.
  • asymmetric carbon atom means a carbon atom with four different substituents. According to the Cahn-Ingold-Prelog Convention an asymmetric carbon atom can be of the "R” or "S” configuration.
  • bicyclic sugar refers to a modified sugar moiety comprising a 4 to 7 membered ring comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure.
  • the bridge connects the C2' and C4' of the ribose sugar ring of a nucleoside (i.e., 2'-4' bridge), as observed in LNA nucleosides.
  • a "coding region” or “coding sequence” is a portion of polynucleotide which consists of codons translatable into amino acids.
  • a "stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, untranslated regions ("UTRs"), and the like, are not part of a coding region.
  • the boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide.
  • non-coding region means a nucleotide sequence that is not a coding region.
  • non-coding regions include, but are not limited to, promoters, ribosome binding sites, transcriptional terminators, introns, untranslated regions ("UTRs"), non-coding exons and the like. Some of the exons can be wholly or part of the 5' untranslated region (5' UTR) or the 3' untranslated region (3' UTR) of each transcript. The untranslated regions are important for efficient translation of the transcript and for controlling the rate of translation and half-life of the transcript.
  • region when used in the context of a nucleotide sequence refers to a section of that sequence.
  • region within a nucleotide sequence or “region within the complement of a nucleotide sequence” refers to a sequence shorter than the nucleotide sequence, but longer than at least 10 nucleotides located within the particular nucleotide sequence or the complement of the nucleotides sequence, respectively.
  • sequence or “subsequence” can also refer to a region of a nucleotide sequence.
  • downstream when referring to a nucleotide sequence, means that a nucleic acid or a nucleotide sequence is located 3' to a reference nucleotide sequence.
  • downstream nucleotide sequences relate to sequences that follow the starting point of transcription.
  • the translation initiation codon of a gene is located downstream of the start site of transcription.
  • upstream refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence.
  • regulatory region refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region.
  • Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, UTRs, and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • mRNA messenger RNA
  • pre-mRNA precursor messenger RNA
  • transcript can be interchangeably used with "pre-mRNA” and "mRNA.” After DNA strands are transcribed to primary transcripts, the newly synthesized primary transcripts are modified in several ways to be converted to their mature, functional forms to produce different proteins and RNAs, such as mRNA, tRNA, rRNA, lncRNA, miRNA and others. Thus, the term “transcript” can include exons, introns, 5' UTRs, and 3' UTRs. [0100]
  • expression refers to a process by which a polynucleotide produces a gene product, for example, a RNA or a polypeptide.
  • RNA messenger RNA
  • expression produces a "gene product.”
  • a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • nucleic acids refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity can be measured using sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.
  • sequence alignment algorithm is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad.
  • BLAST-2 Altschul et al., 1996, Methods in Enzymology, 266:460-480
  • ALIGN ALIGN-2
  • Megalign Megalign
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6).
  • the GAP program in the GCG software package which incorporates the algorithm of Needleman and Wunsch (J.
  • Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5).
  • the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)).
  • the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4.
  • One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software.
  • the default parameters of the alignment software are used.
  • the percentage identity "X" of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.
  • Different regions within a single polynucleotide target sequence that align with a polynucleotide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
  • the terms “homologous” and “homology” are interchangeable with the terms “identity” and “identical.”
  • naturally occurring variant thereof refers to variants of the PMP22 polypeptide sequence or PMP22 nucleic acid sequence (e.g., transcript) which exist naturally within the defined taxonomic group, such as mammalian, such as mouse, monkey, and human.
  • Naturally occurring variants of a polynucleotide the term also can encompass any allelic variant of the PMP22-encoding genomic DNA which is found at Chromosomal position 17p12 at 15,229,777-15,265,326 (i.e., nucleotides 15,229,777- 15,265,326 of GenBank Accession No. NC_000017.11) by chromosomal translocation or duplication, and the RNA, such as mRNA derived therefrom.
  • “Naturally occurring variants” can also include variants derived from alternative splicing of the PMP22 mRNA.
  • the term when referenced to a specific polypeptide sequence, e.g., the term also includes naturally occurring forms of the protein, which can therefore be processed, e.g., by co- or post-translational modifications, such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc.
  • the degree of "complementarity” is expressed as the percentage identity (or percentage homology) between the sequence of the ASO (or region thereof) and the sequence of the target region (or the reverse complement of the target region) that best aligns therewith. The percentage is calculated by counting the number of aligned bases that are identical between the two sequences, dividing by the total number of contiguous monomers in the ASO, and multiplying by 100.
  • complement indicates a sequence that is complementary to a reference sequence. It is well known that complementarity is the base principle of DNA replication and transcription as it is a property shared between two DNA or RNA sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary, much like looking in the mirror and seeing the reverse of things.
  • the complement of a sequence of 5'"ATGC"3' can be written as 3'"TACG"5' or 5'"GCAT"3'.
  • the terms “reverse complement”, “reverse complementary”, and “reverse complementarity” as used herein are interchangeable with the terms “complement”, “complementary”, and “complementarity.”
  • the term “complementary” refers to 100% match or complementarity (i.e., fully complementary) to a contiguous nucleic acid sequence within a PMP22 transcript.
  • the term "complementary" refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% match or complementarity to a contiguous nucleic acid sequence within a PMP22 transcript.
  • the terms "corresponding to” and “corresponds to,” when referencing two separate nucleic acid or nucleotide sequences can be used to clarify regions of the sequences that correspond or are similar to each other based on homology and/or functionality, although the nucleotides of the specific sequences can be numbered differently.
  • different isoforms of a gene transcript can have similar or conserved portions of nucleotide sequences whose numbering can differ in the respective isoforms based on alternative splicing and/or other modifications.
  • different numbering systems can be employed when characterizing a nucleic acid or nucleotide sequence (e.g., a gene transcript and whether to begin numbering the sequence from the translation start codon or to include the 5'UTR).
  • nucleic acid or nucleotide sequence of different variants of a gene or gene transcript can vary.
  • nucleotide sequence of a PMP22 transcript corresponding to nucleotides X to Y of SEQ ID NO: 164 refers to an PMP22 transcript sequence (e.g., PMP22 pre-mRNA or mRNA) that has an identical sequence or a similar sequence to nucleotides X to Y of SEQ ID NO: 164, wherein X is the start site and Y is the end site.
  • corresponding nucleotide analog and “corresponding nucleotide” are intended to indicate that the nucleobase in the nucleotide analog and the naturally occurring nucleotide have the same pairing, or hybridizing, ability.
  • the 2-deoxyribose unit of the nucleotide is linked to an adenine
  • the "corresponding nucleotide analog” contains a pentose unit (different from 2-deoxyribose) linked to an adenine.
  • Beta-D-oxy LNA nucleotides are designated by OxyB where B designates a nucleotide base such as thymine (T), uridine (U), cytosine (C), 5-methylcytosine (MC), adenine (A) or guanine (G), and thus include OxyA, OxyT, OxyMC, OxyC and OxyG.
  • DNA nucleotides are designated by DNAb, where the lower case b designates a nucleotide base such as thymine (T), uridine (U), cytosine (C), 5- methylcytosine (Mc), adenine (A) or guanine (G), and thus include DNAa, DNAt, DNA and DNAg.
  • T thymine
  • U uridine
  • U cytosine
  • Mc 5- methylcytosine
  • A guanine
  • G guanine
  • DNAa DNAt, DNA and DNAg.
  • the letter M before C or c indicates 5-methylcytosine.
  • the letter “s” indicates a phosphorothioate internucleotide linkage.
  • “Potency” is normally expressed as an IC50 or EC50 value, in ⁇ M, nM or pM unless otherwise stated. Potency can also be expressed in terms of percent inhibition.
  • IC50 is the median inhibitory concentration of a therapeutic molecule.
  • EC 50 is the median effective concentration of a therapeutic molecule relative to a vehicle or control (e.g., saline).
  • IC50 is the concentration of a therapeutic molecule that reduces a biological response, e.g., transcription of mRNA or protein expression, by 50% of the biological response that is achieved by the therapeutic molecule.
  • EC 50 is the concentration of a therapeutic molecule that produces 50% of the biological response, e.g., transcription of mRNA or protein expression.
  • IC50 or EC50 can be calculated by any number of means known in the art.
  • the term “inhibiting,” e.g., the expression of PMP22 gene transcript and/or PMP22 protein refers to the ASO reducing the expression of the PMP22 gene transcript and/or PMP22 protein in a cell or a tissue. In some aspects, the term “inhibiting” refers to complete inhibition (100% inhibition or non-detectable level) of PMP22 gene transcript or PMP22 protein.
  • the term “inhibiting” refers to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% inhibition of PMP22 gene transcript and/or PMP22 protein expression in a cell or a tissue.
  • extracellular vesicle or "EV” refers to a cell-derived vesicle comprising a membrane that encloses an internal space.
  • Extracellular vesicles comprise all membrane-bound vesicles (e.g., exosomes, nanovesicles) that have a smaller diameter than the cell from which they are derived.
  • extracellular vesicles range in diameter from 20 nm to 1000 nm, and can comprise various macromolecular payload either within the internal space (i.e., lumen), displayed on the external surface of the extracellular vesicle, and/or spanning the membrane.
  • the payload can comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and/or combinations thereof.
  • an extracellular vehicle comprises a scaffold moiety.
  • extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane).
  • Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, prokaryotic or eukaryotic cells, and/or cultured cells. In some aspects, the extracellular vesicles are produced by cells that express one or more transgene products.
  • extracellular vesicle or EV refers to a population of extracellular vesicles (EVs).
  • the term "exosome” refers to an extracellular vesicle with a diameter between 20-300 nm (e.g., between 40-200 nm). Exosomes comprise a membrane that encloses an internal space (i.e., lumen), and, in some aspects, can be generated from a cell (e.g., producer cell) by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. In certain aspects, an exosome comprises a scaffold moiety.
  • exosome can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.
  • the EVs, e.g., exosomes, of the present disclosure are produced by cells that express one or more transgene products.
  • the term exosome refers to a population of exosomes.
  • the term "nanovesicle” refers to an extracellular vesicle with a diameter between 20-250 nm (e.g., between 30-150 nm) and is generated from a cell (e.g., producer cell) by direct or indirect manipulation such that the nanovesicle would not be produced by the cell without the manipulation.
  • Appropriate manipulations of the cell to produce the nanovesicles include but are not limited to serial extrusion, treatment with alkaline solutions, sonication, or combinations thereof. In some aspects, production of nanovesicles can result in the destruction of the producer cell.
  • population of nanovesicles described herein are substantially free of vesicles that are derived from cells by way of direct budding from the plasma membrane or fusion of the late endosome with the plasma membrane.
  • a nanovesicle comprises a scaffold moiety. Nanovesicles, once derived from a producer cell, can be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.
  • the term "surface-engineered EVs" refers to an EV with the membrane or the surface of the EV modified in its composition so that the surface of the engineered EV is different from that of the EV prior to the modification or of the naturally occurring EV.
  • the engineering can be on the surface of the EV or in the membrane of the EV so that the surface of the EV is changed.
  • the membrane is modified in its composition of a protein, a lipid, a small molecule, a carbohydrate, etc.
  • the composition can be changed by a chemical, a physical, or a biological method or by being produced from a cell previously or concurrently modified by a chemical, a physical, or a biological method.
  • the composition can be changed by a genetic engineering or by being produced from a cell previously modified by genetic engineering.
  • a surface-engineered EV comprises an exogenous protein (i.e., a protein that the EV does not naturally express) or a fragment or variant thereof that can be exposed to the surface of the EV or can be an anchoring point (attachment) for a moiety exposed on the surface of the EV.
  • a surface-engineered EV comprises a higher expression (e.g., higher number) of a natural exosome protein (e.g., Scaffold X) or a fragment or variant thereof that can be exposed to the surface of the EV or can be an anchoring point (attachment) for a moiety exposed on the surface of the EV.
  • a natural exosome protein e.g., Scaffold X
  • a fragment or variant thereof that can be exposed to the surface of the EV or can be an anchoring point (attachment) for a moiety exposed on the surface of the EV.
  • the term "lumen-engineered exosome” refers to an EV with the membrane or the lumen of the EV modified in its composition so that the lumen of the engineered EV is different from that of the EV prior to the modification or of the naturally occurring EV.
  • the engineering can be directly in the lumen or in the membrane of the EV so that the lumen of the EV is changed.
  • the membrane is modified in its composition of a protein, a lipid, a small molecule, a carbohydrate, etc. so that the lumen of the EV is modified.
  • the composition can be changed by a chemical, a physical, or a biological method or by being produced from a cell previously modified by a chemical, a physical, or a biological method.
  • the composition can be changed by a genetic engineering or by being produced from a cell previously modified by genetic engineering.
  • a lumen-engineered exosome comprises an exogenous protein (i.e., a protein that the EV does not naturally express) or a fragment or variant thereof that can be exposed in the lumen of the EV or can be an anchoring point (attachment) for a moiety exposed on the inner layer of the EV.
  • a lumen-engineered EV comprises a higher expression of a natural exosome protein (e.g., Scaffold X or Scaffold Y) or a fragment or variant thereof that can be exposed to the lumen of the exosome or can be an anchoring point (attachment) for a moiety exposed in the lumen of the exosome.
  • a modified EV described herein refers to an alteration or engineering of an EV and/or its producer cell, such that the modified EV is different from a naturally-occurring EV.
  • a modified EV described herein comprises a membrane that differs in composition of a protein, a lipid, a small molecular, a carbohydrate, etc. compared to the membrane of a naturally-occurring EV (e.g., membrane comprises higher density or number of natural exosome proteins and/or membrane comprises proteins that are not naturally found in exosomes (e.g. ⁇ an ASO).
  • scaffold moiety refers to a molecule that can be used to anchor a payload or any other compound of interest (e.g., an ASO) to the EV either on the luminal surface or on the exterior surface of the EV.
  • a scaffold moiety comprises a synthetic molecule.
  • a scaffold moiety comprises a non- polypeptide moiety.
  • a scaffold moiety comprises a lipid, carbohydrate, or protein that naturally exists in the EV. In some aspects, a scaffold moiety comprises a lipid, carbohydrate, or protein that does not naturally exist in the EV. In certain aspects, a scaffold moiety is Scaffold X. In some aspects, a scaffold moiety is Scaffold Y. In further aspects, a scaffold moiety comprises both Scaffold X and Scaffold Y.
  • Non-limiting examples of other scaffold moieties that can be used with the present disclosure include: aminopeptidase N (CD13); Neprilysin, AKA membrane metalloendopeptidase (MME); ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1); Neuropilin-1 (NRP1); CD9, CD63, CD81, PDGFR, GPI anchor proteins, lactadherin (MFGE8), LAMP2, and LAMP2B.
  • the term "Scaffold X" refers to exosome proteins that have recently been identified on the surface of exosomes. See, e.g., U.S. Pat.
  • Non-limiting examples of Scaffold X proteins include: prostaglandin F2 receptor negative regulator ("the PTGFRN protein”); basigin (“the BSG protein”); immunoglobulin superfamily member 2 (“the IGSF2 protein”); immunoglobulin superfamily member 3 (“the IGSF3 protein”); immunoglobulin superfamily member 8 (“the IGSF8 protein”); integrin beta-1 ("the ITGB1 protein); integrin alpha-4 (“the ITGA4 protein”); 4F2 cell-surface antigen heavy chain (“the SLC3A2 protein”); a class of ATP transporter proteins ("the ATP1A1 protein,” “the ATP1A2 protein,” “the ATP1A3 protein,” “the ATP1A4 protein,” “the ATP1B3 protein,” “the ATP2B1 protein,” “the ATP2B2 protein,” “the ATP2B3 protein,” “the ATP2B protein”); and a functional fragment thereof.
  • the PTGFRN protein prostaglandin F2 receptor negative regulator
  • a Scaffold X protein can be a whole protein or a fragment thereof (e.g., functional fragment, e.g., the smallest fragment that is capable of anchoring another moiety on the exterior surface or on the luminal surface of the EV).
  • a Scaffold X can anchor a moiety (e.g., an ASO) to the external surface or the luminal surface of the EV.
  • the term "Scaffold Y" refers to exosome proteins that were newly identified within the lumen of exosomes. See, e.g., International Publ. No. WO/2019/099942, which is incorporated herein by reference in its entirety.
  • Non-limiting examples of Scaffold Y proteins include: myristoylated alanine rich Protein Kinase C substrate ("the MARCKS protein”); myristoylated alanine rich Protein Kinase C substrate like 1 (“the MARCKSL1 protein”); and brain acid soluble protein 1 (“the BASP1 protein”).
  • a Scaffold Y protein can be a whole protein or a fragment thereof (e.g., functional fragment, e.g., the smallest fragment that is capable of anchoring a moiety to the luminal surface of the exosome).
  • a Scaffold Y can anchor a moiety (e.g., an ASO) to the luminal surface of the EV.
  • a Scaffold Y can anchor a moiety (e.g., an ASO) to the exterior surface of the EV.
  • a moiety e.g., an ASO
  • fragment refers to an amino acid sequence of a protein that is shorter than the naturally-occurring sequence, N- and/or C-terminally deleted or any part of the protein deleted in comparison to the naturally occurring protein.
  • functional fragment refers to a protein fragment that retains protein function.
  • a functional fragment of a Scaffold X protein retains the ability to anchor a moiety on the luminal surface or on the exterior surface of the EV.
  • a functional fragment of a Scaffold Y protein retains the ability to anchor a moiety on the luminal surface or exterior surface of the EV.
  • Whether a fragment is a functional fragment can be assessed by any art known methods to determine the protein content of EVs including Western Blots, FACS analysis and fusions of the fragments with autofluorescent proteins like, e.g., GFP.
  • a functional fragment of a Scaffold X protein retains at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% of the ability, e.g., an ability to anchor a moiety, of the naturally occurring Scaffold X protein.
  • a functional fragment of a Scaffold Y protein retains at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% of the ability, e.g., an ability to anchor another molecule, of the naturally occurring Scaffold Y protein.
  • variant of a molecule refers to a molecule that shares certain structural and functional identities with another molecule upon comparison by a method known in the art.
  • a variant of a protein can include a substitution, insertion, deletion, frameshift or rearrangement in another protein.
  • a variant of a Scaffold X comprises a variant having at least about 70% identity to the full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporter proteins or a fragment (e.g., functional fragment) of the PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporter proteins.
  • variants or variants of fragments of PTGFRN share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with PTGFRN according to SEQ ID NO: 1 or with a functional fragment thereof (e.g., SEQ ID NO:2, 103, 104, 105, 106, 107 or 108).
  • the variant or variant of a fragment of Scaffold X protein disclosed herein retains the ability to be specifically targeted to EVs.
  • the Scaffold X includes one or more mutations, for example, conservative amino acid substitutions.
  • a variant of a Scaffold Y comprises a variant having at least 70% identity to MARCKS, MARCKSL1, BASP1, or a fragment of MARCKS, MARCKSL1, or BASP1.
  • variants or variants of fragments of MARCKS share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with MARCKS according to SEQ ID NO: 8 or with a functional fragment thereof.
  • variants or variants of fragments of BASP1 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with BASP1 according to SEQ ID NO: 10 or with a functional fragment thereof.
  • the variant or variant of a fragment of Scaffold Y protein retains the ability to be specifically targeted to the luminal surface of EVs.
  • the Scaffold Y includes one or more mutations, e.g., conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e
  • a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • the term "percent sequence identity" or “percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences.
  • a matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence.
  • Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids.
  • gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence.
  • the percentage of sequence identity is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base 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 comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using readily available software both for online use and for download.
  • Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences.
  • One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov).
  • Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • sequence alignments are not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments.
  • One suitable program to generate multiple sequence alignments is ClustalW2, available from www.clustal.org.
  • Another suitable program is MUSCLE, available from www.drive5.com/muscle/.
  • sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data.
  • a suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively available, e.g., from the EBI.
  • T-Coffee available at www.tcoffee.org
  • alternatively available e.g., from the EBI.
  • the final alignment used to calculate percent sequence identity can be curated either automatically or manually.
  • the polynucleotide variants can contain alterations in the coding regions, non- coding regions, or both.
  • the polynucleotide variants contain alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
  • nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code.
  • variants in which 5- 10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.
  • Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to others, e.g., a bacterial host such as E. coli).
  • Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present disclosure.
  • non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.
  • variants can be generated to improve or alter the characteristics of the polypeptides. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. Ron et al., J. Biol. Chem.268: 2984-2988 (1993), incorporated herein by reference in its entirety, reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues.
  • interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein.
  • Gayle and coworkers conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule.
  • polypeptide variants include, e.g., modified polypeptides.
  • Modifications include, e.g., acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation,
  • Scaffold X and/or Scaffold Y is modified at any convenient location.
  • the term "linked to” or “conjugated to” are used interchangeably and refer to a covalent or non-covalent bond formed between a first moiety and a second moiety, e.g., Scaffold X and an ASO, respectively, e.g., a scaffold moiety expressed in or on the extracellular vesicle and an ASO, e.g., Scaffold X (e.g., a PTGFRN protein), respectively, in the luminal surface of or on the external surface of the extracellular vesicle.
  • encapsulated refers to a status or process of having a first moiety (e.g., an ASO) inside a second moiety (e.g., an EV) without chemically or physically linking the two moieties.
  • a first moiety e.g., an ASO
  • a second moiety e.g., an EV
  • the term “encapsulated” can be used interchangeably with "in the lumen of.”
  • Non-limiting examples of encapsulating a first moiety (e.g., an ASO) into a second moiety are disclosed elsewhere herein.
  • the term "producer cell” refers to a cell used for generating an EV.
  • a producer cell can be a cell cultured in vitro, or a cell in vivo.
  • a producer cell includes, but not limited to, a cell known to be effective in generating EVs, e.g., HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblast cells, fHDF fibroblast cells, AGE.HN ® neuronal precursor cells, CAP ® amniocyte cells, adipose mesenchymal stem cells, RPTEC/TERT1 cells.
  • a producer cell is not an antigen-presenting cell.
  • a producer cell is not a dendritic cell, a B cell, a mast cell, a macrophage, a neutrophil, Kupffer-Browicz cell, cell derived from any of these cells, or any combination thereof.
  • the EVs useful in the present disclosure do not carry an antigen on MHC class I or class II molecule exposed on the surface of the EV but instead can carry an antigen in the lumen of the EV or on the surface of the EV by attachment to Scaffold X and/or Scaffold Y.
  • isolating or purifying is the process of removing, partially removing (e.g., a fraction) of the EVs from a sample containing producer cells.
  • an isolated EV composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In other aspects, an isolated EV composition has an amount and/or concentration of desired EVs at or above an acceptable amount and/or concentration. In other aspects, the isolated EV composition is enriched as compared to the starting material (e.g., producer cell preparations) from which the composition is obtained. This enrichment can be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to the starting material.
  • the starting material e.g., producer cell preparations
  • isolated EV preparations are substantially free of residual biological products.
  • the isolated EV preparations are 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free, or 90% free of any contaminating biological matter.
  • Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.
  • Substantially free of residual biological products can also mean that the EV composition contains no detectable producer cells and that only EVs are detectable.
  • the term "payload" refers to an agent that acts on a target (e.g., a target cell) that is contacted with the EV.
  • Payloads that can be introduced into an EV and/or a producer cell include agents such as, nucleotides (e.g., nucleotides comprising a detectable moiety or a toxin or that disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules that encode a polypeptide such as an enzyme, or RNA molecules that have regulatory function such as miRNA, dsDNA, lncRNA, and siRNA), amino acids (e.g., amino acids comprising a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins).
  • nucleotides e.g., nucleotides comprising a detectable moiety or a toxin or that disrupt transcription
  • nucleic acids e.g., DNA or mRNA molecules that encode a polypeptide such as an enzyme, or RNA molecules that have regulatory function such
  • a payload comprises an ASO.
  • the term “antibody” encompasses an immunoglobulin whether natural or partly or wholly synthetically produced, and fragments thereof. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain. "Antibody” further includes a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the term “antigen” refers to any agent that when introduced into a subject elicits an immune response (cellular or humoral) to itself.
  • antibody is meant to include whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate- human monoclonal antibodies, anti-idiotype antibodies, antibody fragments, such as, e.g., scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides.
  • Antibody includes bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function.
  • the terms "individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • the compositions and methods described herein are applicable to both human therapy and veterinary applications.
  • the subject is a mammal, and in other aspects the subject is a human.
  • a “mammalian subject” includes all mammals, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like).
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile.
  • composition can be sterile.
  • conventional exosome protein means a protein previously known to be enriched in exosomes, including but is not limited to CD9, CD63, CD81, PDGFR, GPI anchor proteins, lactadherin (MFGE8), LAMP2, and LAMP2B, a fragment thereof, or a peptide that binds thereto.
  • administering means to give a composition comprising an EV disclosed herein to a subject via a pharmaceutically acceptable route.
  • Routes of administration can be intravenous, e.g., intravenous injection and intravenous infusion. Additional routes of administration include, e.g., subcutaneous, intramuscular, oral, nasal, and pulmonary administration.
  • EVs can be administered as part of a pharmaceutical composition comprising at least one excipient.
  • An "effective amount" of, e.g., an ASO or an extracellular vesicle as disclosed herein, is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
  • Treating refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition.
  • the term also includes prophylaxis or prevention of a disease or condition or its symptoms thereof.
  • the "treating” or “treatment” includes inducing hematopoiesis in a subject in need thereof.
  • the disease or condition is associated with a hematopoiesis or a deficiency thereof.
  • the disease or condition is a cancer.
  • the treating enhances hematopoiesis in a subject having a cancer, wherein the enhanced hematopoiesis comprises increased proliferation and/or differentiation of one or more immune cell in the subject
  • "Prevent” or "preventing,” as used herein refers to decreasing or reducing the occurrence or severity of a particular outcome. In some aspects, preventing an outcome is achieved through prophylactic treatment.
  • an EV comprising an ASO, described herein is administered to a subject prophylactically.
  • the subject is at risk of developing cancer.
  • the subject is at risk of developing a hematopoietic disorder. II.
  • ASOs Antisense Oligonucleotides
  • the present disclosure employs antisense oligonucleotides (ASOs) for use in modulating the function of nucleic acid molecules encoding mammalian PMP22, such as the PMP22 nucleic acid, e.g., PMP22 transcript, including PMP22 pre-mRNA, and PMP22 mRNA, or naturally occurring variants of such nucleic acid molecules encoding mammalian PMP22.
  • ASO in the context of the present disclosure, refers to a molecule formed by covalent linkage of two or more nucleotides (i.e., an oligonucleotide).
  • the EV comprises at least one ASO.
  • the EV e.g., the exosome, comprises at least two ASOs, e.g., a first ASO comprising a first nucleotide sequence and a second ASO comprising a second nucleotide sequence.
  • the EV comprises at least three ASOs, at least four ASOs, at least five ASOs, at least six ASOs, or more than six ASOs.
  • each of the first ASO, the second ASO, the third ASO, the fourth ASO, the fifth ASO, the sixth ASO, and/or the ninth ASO is different.
  • the EV e.g.
  • the exosome comprises a first ASO and a second ASO, wherein the first ASO comprises a first nucleotide sequence that is complimentary to a first target sequence in a first transcript, and wherein the second ASO comprises a second nucleotide sequence that is complimentary to a second target sequence in the first transcript.
  • the first target sequence does not overlap with the second target sequence.
  • the first target sequence comprises at least one nucleotide that is within the 5'UTR of the transcript, and the second target sequence does not comprise a nucleotide that is within the 5'UTR.
  • the first target sequence comprises at least one nucleotide that is within the 3'UTR of the transcript, and the second target sequence does not comprise a nucleotide that is within the 3'UTR. In some aspects, the first target sequence comprises at least one nucleotide that is within the 5'UTR of the transcript, and the second target sequence comprises at least one nucleotide that is within the 3'UTR. [0156] In some aspects, the first ASO targets a sequence within an exon-intron junction, and the second ASO targets a sequence within an exon-intron junction. In some aspects, the first ASO targets a sequence within an exon-intron junction, and the second ASO targets a sequence within an exon.
  • the first ASO targets a sequence within an exon-intron junction
  • the second ASO targets a sequence within an intron.
  • the first ASO targets a sequence within an exon
  • the second ASO targets a sequence within an exon.
  • the first ASO targets a sequence within an intron
  • the second ASO targets a sequence within an exon.
  • the first ASO targets a sequence within an intron
  • the second ASO targets a sequence within an intron.
  • the first ASO targets a sequence within an intron
  • the second ASO targets a sequence within an intron.
  • the EV comprises a first ASO and a second ASO, wherein the first ASO comprises a first nucleotide sequence that is complimentary to a first target sequence in a first transcript, and wherein the second ASO comprises a second nucleotide sequence that is complimentary to a second target sequence in a second transcript, wherein the first transcript is not the product of the same gene as the second transcript.
  • the ASO comprises a contiguous nucleotide sequence of from about 10 to about 30, such as 10–20, 14–20, 16–20, or 15–25, nucleotides in length. In certain aspects, the ASO is 20 nucleotides in length. In certain aspects, the ASO is 18 nucleotides in length.
  • the ASO is 19 nucleotides in length. In certain aspects, the ASO is 17 nucleotides in length. In certain aspects, the ASO is 16 nucleotides in length. In certain aspects, the ASO is 15 nucleotides in length.
  • the terms "antisense ASO,” “antisense oligonucleotide,” and “oligomer” as used herein are interchangeable with the term “ASO.” [0159] In various aspects, the ASO of the disclosure does not comprise RNA (units). In some aspects, the ASO comprises one or more DNA units. In one aspect, the ASO according to the disclosure is a linear molecule or is synthesized as a linear molecule.
  • the ASO is a single stranded molecule, and does not comprise short regions of, for example, at least 3, 4 or 5 contiguous nucleotides, which are complementary to equivalent regions within the same ASO (i.e. duplexes) - in this regard, the ASO is not (essentially) double stranded. In some aspects, the ASO is essentially not double stranded. In some aspects, the ASO is not a siRNA. In various aspects, the ASO of the disclosure can consist entirely of the contiguous nucleotide region. Thus, in some aspects the ASO is not substantially self-complementary. [0160] In other aspects, the present disclosure includes fragments of ASOs.
  • the disclosure includes at least one nucleotide, at least two contiguous nucleotides, at least three contiguous nucleotides, at least four contiguous nucleotides, at least five contiguous nucleotides, at least six contiguous nucleotides, at least seven contiguous nucleotides, at least eight contiguous nucleotides, or at least nine contiguous nucleotides of the ASOs disclosed herein. Fragments of any of the sequences disclosed herein are contemplated as part of the disclosure.
  • the ASOs for the present disclosure include a phosphorodiamidate Morpholino oligomer (PMO) or a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO).
  • PMO phosphorodiamidate Morpholino oligomer
  • PPMO peptide-conjugated phosphorodiamidate morpholino oligomer
  • the ASO of the disclosure is capable of down-regulating (e.g., reducing or removing) expression of the PMP22 mRNA or PMP22 protein.
  • the ASO of the disclosure can decrease the levels of PMP22 mRNA and/or PMP22 protein in a Schwann cell.
  • the present disclosure is directed to ASOs that target one or more regions of the PMP22 pre-mRNA (e.g., intron regions, exon regions, and/or exon-intron junction regions).
  • PMP22 can refer to PMP22 from one or more species (e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).
  • Peripheral myelin protein 22 (PMP22) is also known as growth arrest-specific protein 3 (GAS-3), is encoded by the PMP22 gene.
  • PMP22 is a 22 kDa transmembrane glycoprotein made up of 160 amino acids, and is mainly expressed in the Schwann cells of the peripheral nervous system. Schwann cells show high expression of PMP22, where it can constitute 2-5% of total protein content in compact myelin. Compact myelin is the bulk of the peripheral neuron's myelin sheath, a protective fatty layer that provides electrical insulation for the neuronal axon. The level of PMP22 expression is relatively low in the central nervous system of adults. [0165] In humans, the PMP22 gene is located on chromosome 17p11.2 and spans approximately 40kb.
  • the gene contains six exons conserved in both humans and rodents, two of which are 5’ untranslated exons (1a and 1b) and result in two different RNA transcripts with identical coding sequences.
  • the two transcripts differ in their 5' untranslated regions and have their own promoter regulating expression.
  • the remaining exons (2 to 5) include the coding region of the PMP22 gene, and are joined together after post-transcriptional modification (i.e. alternative splicing).
  • the PMP22 protein is characterized by four transmembrane domains, two extracellular loops (ECL1 and ECL2), and one intracellular loop.
  • ECL1 has been suggested to mediate a homophilic interaction between two PMP22 proteins
  • ECL2 has been shown to mediate a heterophilic interaction between PMP22 protein and Myelin protein zero (MPZ or MP0).
  • PMP22 plays an essential role in the formation and maintenance of compact myelin. When Schwann cells come into contact with a neuronal axon, expression of PMP22 is significantly up-regulated, whereas PMP22 is down-regulated during axonal degeneration or transection. PMP22 has shown association with zonula-occludens 1 and occludin, proteins that are involved in adhesion with other cells and the extracellular matrix, and also support functioning of myelin.
  • PMP22 is also up-regulated during Schwann cell proliferation, suggesting a role in cell-cycle regulation. PMP22 is detectable in non-neural tissues, where its expression has been shown to serve as growth-arrest-specific (gas- 3) function.
  • Improper gene dosage of the PMP22 gene can cause aberrant protein synthesis and function of myelin sheath. Since the components of myelin are stoichiometrically set, any irregular expression of a component can cause destabilization of myelin and neuropathic disorders.
  • Alterations of PMP22 gene expression are associated with a variety of neuropathies, such as Charcot–Marie–Tooth type 1A (CMT1A), Dejerine–Sottas disease, and Hereditary Neuropathy with Liability to Pressure Palsy (HNPP). Too much PMP22 (e.g. caused by gene duplication) results in CMT1A. Gene duplication of PMP22 is the most common genetic cause of CMT where the overproduction of PMP22 results in defects in multiple signaling pathways and dysfunction of transcriptional factors like KNOX20, SOX10 and EGR2. [0168] The sequence for the human PMP22 gene can be found under publicly available as NCBI RefSeq Acc. No. NM_000304.
  • Alternative RefSeq mRNA transcripts have accession numbers NM_001281455, NM-001281456, NM-153321, and NM_153322, respectively.
  • the human PMP22 gene is found at chromosome location 17p12 at 15,229,777-15,265,326.
  • the sequence for the human PMP22 pre-mRNA transcript corresponds to the reverse complement of residues 15,229,777-15,265,326, of chromosome location 17p12.
  • the PMP22 mRNA sequence (GenBank Accession No. NM_000304.4) is provided in SEQ ID NO: 164 (TABLE 1).
  • the sequence for human PMP22 protein can be found under publicly available Uniprot Accession Number Q01453 (canonical sequence, SEQ ID NO: 166; TABLE 1).
  • Potential PMP22 isoforms have Uniprot Accession Numbers A8MU75, J3KQW0, A0A2R8Y5L5, J3KT36, and J3QS08, respectively.
  • the publicly available contents of the database entries corresponding to accession numbers disclosed herein are incorporated by reference in their entireties. TABLE 1.
  • PMP22 mRNA and Protein Sequences [0170] Natural variants of the human PMP22 gene product are known.
  • natural variants of human PMP22 protein can contain one or more amino acid substitutions selected from L16P, S22F, ⁇ 25-26, D37V, V65F, S72L, S79C, G93R, L105R, G107V, T118N, L147R, H12Q, L16P, L19P, M69K, L71P, S72L, S72P, S72W, S76I, S79P, L80P, L80R, ⁇ 84, G100E, G100R, L105R, C109R, S149R, G150C, G150D, R157W, S22F, V30M, A67T, S23T, W28R, A67P, ⁇ 115-118, and any combination thereof.
  • the ASOs of the present disclosure can be designed to reduce or inhibit expression of the natural variants of the PMP22 protein.
  • An example of a target nucleic acid sequence of the ASOs is PMP22 pre- mRNA.
  • SEQ ID NO: 164 represents a human PMP22 genomic sequence (i.e., reverse complement of nucleotides 15,229,777-15,265,326, complement, of chromosome 17p12).
  • SEQ ID NO: 164 is identical to a PMP22 pre-mRNA sequence except that nucleotide "t" in SEQ ID NO: 164 is shown as "u" in pre-mRNA.
  • the ASO comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within nucleotides 1 to 1828 of a PMP22 transcript corresponding to a nucleotide sequence as set forth in SEQ ID NO: 164 (PMP22 full mRNA transcript) or nucleotides 208 to 690 of a PMP22 transcript corresponding to a nucleotide sequence as set forth in SEQ ID NO: 165 (PMP22 coding sequence).
  • the contiguous nucleotide sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% complementary to the nucleic acid sequence within the PMP22 transcript.
  • the ASO is capable of reducing PMP22 protein expression in a human cell (e.g., a Schwann cell), wherein the human cell expresses the PMP22 protein.
  • the PMP22 protein expression is reduced by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to PMP22 protein expression in a human cell that is not exposed to the ASO.
  • the ASO is capable of reducing a level of PMP22 mRNA in a human cell (e.g., an immune cell), wherein the human cell expresses the PMP22 mRNA.
  • the level of PMP22 mRNA is reduced by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to the level of the PMP22 mRNA in a human cell that is not exposed to the ASO.
  • the "target nucleic acid” comprises an intron of a PMP22 protein-encoding nucleic acids or naturally occurring variants thereof, and RNA nucleic acids derived therefrom, e.g., pre-mRNA.
  • the target nucleic acid comprises an exon region of a PMP22 protein-encoding nucleic acids or naturally occurring variants thereof, and RNA nucleic acids derived therefrom, e.g., pre-mRNA.
  • the target nucleic acid comprises an exon-intron junction of a PMP22 protein-encoding nucleic acids or naturally occurring variants thereof, and RNA nucleic acids derived therefrom, e.g., pre-mRNA.
  • the "target nucleic acid” can be a cDNA or a synthetic oligonucleotide derived from the above DNA or RNA nucleic acid targets.
  • the human PMP22 coding sequence is shows as SEQ ID NO:165, and protein sequence encoded by the coding sequence in the PMP22 pre-mRNA is shown as SEQ ID NO: 166.
  • the target nucleic acid comprises an untranslated region of a PMP22 protein- encoding nucleic acids or naturally occurring variants thereof, e.g., 5' UTR, 3' UTR, or both.
  • an ASO of the disclosure hybridizes to a region within the introns of a PMP22 transcript, e.g., SEQ ID NO: 164.
  • an ASO of the disclosure hybridizes to a region within the exons of a PMP22 transcript, e.g., SEQ ID NO: 164. In other aspects, an ASO of the disclosure hybridizes to a region within the exon-intron junction of a PMP22 transcript, e.g., SEQ ID NO: 164. [0179] In some aspects, the ASO comprises a contiguous nucleotide sequence (e.g., 10 to 30 nucleotides in length, e.g., 20 nucleotides in length) that are complementary to a nucleic acid sequence within a PMP22 transcript, e.g., a region corresponding to SEQ ID NO: 164.
  • the ASO comprises a contiguous nucleotide sequence that hybridizes to a nucleic acid sequence, or a region within the sequence, of a PMP22 transcript ("target region"), wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within (i) a 5' untranslated region (UTR); (ii) a coding region; or (iii) a 3' UTR of the PMP22 transcript.
  • target region a contiguous nucleotide sequence that hybridizes to a nucleic acid sequence, or a region within the sequence, of a PMP22 transcript ("target region"), wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within (i) a 5' untranslated region (UTR); (ii) a coding region; or (iii) a 3' UTR of the PMP22 transcript.
  • the contiguous nucleotide sequence is complementary to a nucleic acid sequence comprising (i) nucleotides 1 – 173 of SEQ ID NO: 164 (exon 1); (ii) nucleotides 174-285 of SEQ ID NO: 164 (exon 2); (iii) nucleotides 286 - 385 of SEQ ID NO: 164 (exon 3); (iv) nucleotides 386 – 526 of SEQ ID NO: 164 (exon 4); (v) 527 – 1828 of SEQ ID NO: 164 (exon 5); (vi) 200 – 300 of SEQ ID NO: 164, (vii) nucleotides 200 – 400 of SEQ ID NO: 164; (viii) nucleotides 500 -600 of SEQ ID NO: 164; (ix) nucleotides 600 - 700 of SEQ ID NO: 164; (x) nucleotides 600 – 800 of SEQ ID NO:
  • the contiguous nucleotide sequence is complementary to a nucleic acid sequence comprising nucleotides 152-168 (SEQ ID NO: 130), 225-244 (SEQ ID NO:131), 227-246 (SEQ ID NO:132), 235-254 (SEQ ID NO:133), 265-284 (SEQ ID NO:134), 271-290 (SEQ ID NO:135), 380-399 (SEQ ID NO:136), 383-402 (SEQ ID NO:137), 385-404 (SEQ ID NO:138), 418-437 (SEQ ID NO:139), 479-498 (SEQ ID NO:140), 583-602 (SEQ ID NO:141), 671-690 (SEQ ID NO:142), 672-691 (SEQ ID NO:143), 673-692 (SEQ ID NO:144), 674-693 (SEQ ID NO:145), 675-691 (SEQ ID NO:146), 676-691 (SEQ ID NO:147), 678-693 (SEQ ID NO:
  • the contiguous nucleotide sequence comprises a nucleotide sequence complementary to a nucleic acid sequence comprising nucleotides 152-168 (SEQ ID NO:130), 235-254 (SEQ ID NO:133), 385-404 (SEQ ID NO:138), 479-498 (SEQ ID NO:140), 672-691 (SEQ ID NO:143), 675-691 (SEQ ID NO:146), 939-958 (SEQ ID NO:149), 1130-1149 (SEQ ID NO:152), 1293-1312 (SEQ ID NO:153), 1365-1384 (SEQ ID NO:157), 1404-1423 (SEQ ID NO:158), or 1605-1624 (SEQ ID NO:160) of SEQ ID NO: 164.
  • the target region corresponds to a 16-mer nucleotide sequence corresponding to positions 208-223, 209-224, 210-225, 211-226, 212-227, 213-228, 214-229, 215-230, 216-231, 217-232, 218-233, 219-234, 220-235, 221-236, 222-237, 223-238, 224-239, 225-240, 226-241, 227-242, 228-243, 229-244, 230-245, 231-246, 232-247, 233-248, 234-249, 235-250, 236-251, 237-252, 238-253, 239-254, 240-255, 241-256, 242-257, 243-258, 244-259, 245-260, 246-261, 247-262, 248-263, 249-264, 250-265, 251-266, 252-267, 253-268, 254-269, 255-270, 256-271, 257-272, 258-273, 259
  • the target region corresponds to a 16-mer nucleotide between positions 208 and 690 of SEQ ID NO: 164. In some aspects, the target region corresponds to a 17-mer nucleotide between positions 208 and 690 of SEQ ID NO: 164. In some aspects, the target region corresponds to an 18-mer nucleotide sequence between positions 208 and 690 of SEQ ID NO: 164. In some aspects, the target region corresponds to a 19-mer nucleotide sequence between positions 208 and 690 of SEQ ID NO: 164. In some aspects, the target region corresponds to a 20-mer nucleotide sequence between positions 208 and 690 of SEQ ID NO: 164.
  • the target region corresponds to a 16-mer, 17-mer, 18-mer, 19-mer or 20- mer target region disclosed above ⁇ 10, ⁇ 20, ⁇ 30, ⁇ 40, ⁇ 50, ⁇ 60, ⁇ 70, ⁇ 80, or ⁇ 90 nucleotides at the 3' end and/or the 5' end.
  • the ASO is not (SEQ ID NO: 237).
  • the ASO is (SEQ ID NO: 237).
  • the ASO of the present disclosure hybridizes to multiple target regions within the PMP22 transcript (e.g., genomic sequence, SEQ ID NO: 164).
  • the ASO hybridizes to two different target regions within the PMP22 transcript. In some aspects, the ASO hybridizes to three different target regions within the PMP22 transcript. In some aspects, the ASOs that hybridizes to multiple regions within the PMP22 transcript (e.g., genomic sequence, SEQ ID NO: 164) are more potent (e.g., having lower EC50) at reducing PMP22 expression compared to ASOs that hybridizes to a single region within the PMP22 transcript (e.g., genomic sequence, SEQ ID NO: 164).
  • the ASO of the disclosure is capable of hybridizing to the target nucleic acid (e.g., PMP22 transcript) under physiological condition, i.e., in vivo condition. In some aspects, the ASO of the disclosure is capable of hybridizing to the target nucleic acid (e.g., PMP22 transcript) in vitro. In some aspects, the ASO of the disclosure is capable of hybridizing to the target nucleic acid (e.g., PMP22 transcript) in vitro under stringent conditions.
  • Stringency conditions for hybridization in vitro are dependent on, inter alia, productive cell uptake, RNA accessibility, temperature, free energy of association, salt concentration, and time (see, e.g., Stanley T Crooke, Antisense Drug Technology: Principles, Strategies and Applications, 2 nd Edition, CRC Press (2007)). Generally, conditions of high to moderate stringency are used for in vitro hybridization to enable hybridization between substantially similar nucleic acids, but not between dissimilar nucleic acids.
  • An example of stringent hybridization conditions includes hybridization in 5X saline-sodium citrate (SSC) buffer (0.75 M sodium chloride/0.075 M sodium citrate) for 1 hour at 40°C, followed by washing the sample 10 times in 1X SSC at 40°C and 5 times in 1X SSC buffer at room temperature.
  • SSC 5X saline-sodium citrate
  • In vivo hybridization conditions consist of intracellular conditions (e.g., physiological pH and intracellular ionic conditions) that govern the hybridization of antisense oligonucleotides with target sequences.
  • In vivo conditions can be mimicked in vitro by relatively low stringency conditions.
  • hybridization can be carried out in vitro in 2X SSC (0.3 M sodium chloride/0.03 M sodium citrate), 0.1% SDS at 37°C.
  • the ASO of the present disclosure is capable of targeting a PMP22 transcript from one or more species (e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).
  • the ASO disclosed herein is capable of targeting both human and rodent (e.g., mice or rats) PMP22 transcript.
  • the ASO is capable of down-regulating (e.g., reducing or removing) expression of the PMP22 mRNA or protein both in humans and in rodents (e.g., mice or rats).
  • any ASO described herein is part of a conjugate, comprising the ASO covalently linked to at least one non-nucleotide or non-polynucleotide.
  • Certain aspects of the present disclosure are directed to a conjugate comprising an ASO described herein.
  • the conjugate comprises an ASO covalently attached to at least one non-nucleotide.
  • the conjugate comprises an ASO covalently attached to at least non-polynucleotide moiety.
  • the non-nucleotide or non-polynucleotide moiety comprises a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or any combinations thereof.
  • the ASOs of the disclosure comprise a contiguous nucleotide sequence which corresponds to the complement of a region of PMP22 transcript, e.g., a nucleotide sequence corresponding to SEQ ID NO: 164.
  • the disclosure provides an ASO from 10 – 30, such as 10 – 15 nucleotides, 10 – 20 nucleotides, 10 – 25 nucleotides in length, or about 20 nucleotides in length, wherein the contiguous nucleotide sequence has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to a region within the complement of a PMP22 transcript, such as SEQ ID NO: 164 or naturally occurring variant thereof.
  • the ASO hybridizes to a single stranded nucleic acid molecule having the sequence of SEQ ID NO: 164 or a portion thereof.
  • the ASO can comprise a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to the equivalent region of a nucleic acid which encodes a mammalian PMP22 protein (e.g., SEQ ID NO: 164).
  • the ASO can comprise a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to a nucleic acid sequence, or a region within the sequence, corresponding to nucleotides X-Y of SEQ ID NO: 164, wherein X a start site corresponding to the “starting nucleotide” position in FIG.9 and Y is end site corresponding to (“starting position”+”length”+1), i.e., for example, a starting nucleotide of 152 and a length of 17 would correspond to a X start position of 152 and an Y end position of 168.
  • the ASO can comprise a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to the equivalent region of a mRNA which encodes a mammalian PMP22 protein (e.g., SEQ ID NO: 164).
  • the ASO can comprise a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to a mRNA sequence, or a region within the sequence, corresponding to nucleotides X-Y of SEQ ID NO: 164, wherein X a start site corresponding to the “starting nucleotide” position in FIG.9 and Y is end site corresponding to (“starting position”+”length”+1), i.e., for example, a starting nucleotide of 152 and a length of 17 would correspond to a X start position of 152 and an Y end position of 168.
  • the nucleotide sequence of the ASOs of the disclosure or the contiguous nucleotide sequence has at least about 80% sequence identity to a sequence selected from SEQ ID NOs: 130 to 163 (i.e., the sequences in FIG.10) or 302 to 336 (i.e., the sequences in FIG. 5), such as at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, such as about 100% sequence identity (homologous).
  • the ASO (or contiguous nucleotide portion thereof) is selected from, or comprises, one of the sequences selected from the group consisting of SEQ ID NOs: 130 to 163 or 202 to 236 or a region of at least 10 contiguous nucleotides thereof, wherein the ASO (or contiguous nucleotide portion thereof) can optionally comprise one, two, three, or four mismatches when compared to the corresponding sequence in the PMP22 transcript.
  • the ASO comprises the sequence as set forth in SEQ ID NO: 130.
  • the ASO comprises the sequence as set forth in SEQ ID NO: 131.
  • the ASO comprises the sequence as set forth in SEQ ID NO: 132. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 133. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 134. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 135. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 136. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 137. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 138. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 139.
  • the ASO comprises the sequence as set forth in SEQ ID NO: 140. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 141. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 142. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 143. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 144. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 145. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 146. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 147.
  • the ASO comprises the sequence as set forth in SEQ ID NO: 148. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 149. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 150. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 151. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 152. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 153. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 154. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 155.
  • the ASO comprises the sequence as set forth in SEQ ID NO: 156. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 157. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 158. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 159. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 160. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 161. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 162. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 163.
  • the ASO comprises the sequence as set forth in SEQ ID NO: 202. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 203. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 204. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 205. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 206. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 207. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 208. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 209.
  • the ASO comprises the sequence as set forth in SEQ ID NO: 210. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 211. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 212. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 213. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO: 214. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:215. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:216. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:217.
  • the ASO comprises the sequence as set forth in SEQ ID NO:218. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:219. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:220. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:221. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:222. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:223. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:224. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:225. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:226.
  • the ASO comprises the sequence as set forth in SEQ ID NO:227. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:228. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:229. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:230. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:231. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:232. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:233. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:234. In some aspects, the ASO comprises the sequence as set forth in SEQ ID NO:235.
  • the ASO comprises the sequence as set forth in SEQ ID NO:236. [0196] In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 130. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 131. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 132. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 133. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 134.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 135. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 136. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 137. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 138. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 139. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 140.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 141. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 142. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 143. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 144. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 145. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 146.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 147. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 148. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 149. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 150. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 151. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 152.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 153. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 154. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 155. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 156. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 157. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 158.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 159. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 160. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 161. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 162. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 163. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 202.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 203. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 204. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 205. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 206. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 207. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 208.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 209. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 210. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 211. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 212. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 213. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO: 214.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:215. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:216. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:217. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:218. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:219. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:220.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:221. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:222. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:223. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:224. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:225. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:226.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:227. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:228. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:229. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:230. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:231. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:232.
  • the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:233. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:234. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:235. In some aspects, the ASO consists or consists essentially of the sequence as set forth in SEQ ID NO:236. [0197] In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 130 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 131 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 132 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 133 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 34 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 135 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 136 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 137 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 138 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 139 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 140 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 141 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 142 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 143 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 144 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 145 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 146 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 147 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 148 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 149 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 150 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 151 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 152 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 153 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 154 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 155 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 156 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 157 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 158 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 159 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 160 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 161 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 162 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 163 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 202 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 203 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 204 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 205 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 206 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 207 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 208 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 209 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 210 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 211 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 212 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 213 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 214 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:215 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:216 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:217 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:218 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:219 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:220 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:221 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:222 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:223 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:224 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:225 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:226 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:227 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:228 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:229 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:230 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:231 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:232 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:233 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:234 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:235 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:236 plus 1, 2, 3, 4, or 5 additional 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 130 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 131 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 132 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 133 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 134 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 135 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 136 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 137 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 138 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 139 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 140 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 141 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 142 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 143 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 144 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 145 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 146 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 147 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 148 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 149 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 150 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 151 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 152 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 153 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 154 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 155 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 156 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 157 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 158 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 159 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 160 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 161 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 162 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 163 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 202 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 203 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 204 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 205 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 206 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 207 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 208 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 209 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 210 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 211 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 212 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 213 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 214 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:215 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:216 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:217 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:218 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:219 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:220 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:221 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:222 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:223 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:224 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:225 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:226 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:227 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:228 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:229 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:230 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:231 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:232 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:233 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:234 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:235 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:236 plus 1, 2, 3, 4, or 5 additional 5’ nucleotides complementary to SEQ ID NO:164. [0199] In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 130 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 131 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 132 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 133 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 134 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 135 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 136 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 137 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 138 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 139 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 140 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 141 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 142 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 143 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 144 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 145 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 146 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 147 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 148 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 149 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 150 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 151 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 152 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 153 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 154 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 155 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 156 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 157 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 158 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 159 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 160 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 161 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 162 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 163 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 202 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 203 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 204 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 205 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 206 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 207 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 208 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 209 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 210 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 211 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO: 212 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 213 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO: 214 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:215 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:216 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:217 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:218 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:219 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:220 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:221 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:222 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:223 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:224 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:225 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:226 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:227 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:228 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:229 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:230 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:231 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:232 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:233 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:234 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164. In some aspects, the ASO consists of the sequence as set forth in SEQ ID NO:235 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASO consists of the sequence as set forth in SEQ ID NO:236 plus 1, 2, 3, 4, or 5 additional 5’ and/or 3’ nucleotides complementary to SEQ ID NO:164.
  • the ASOs of the disclosure bind to the target nucleic acid sequence (e.g., PMP22 transcript) and are capable of inhibiting or reducing expression of the PMP22 transcript by at least 10% or 20% compared to the normal (i.e., control) expression level in the cell, e.g., at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% compared to the normal expression level (e.g., expression level in cells that have not been exposed to the ASO).
  • the normal expression level e.g., expression level in cells that have not been exposed to the ASO.
  • the ASOs of the disclosure are capable of reducing expression of PMP22 mRNA in vitro by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% in target cells when the cells are in contact with the ASO compared to cells that are not in contact with the ASO (e.g., contact with saline).
  • the ASO can tolerate 1, 2, 3, or 4 (or more) mismatches, when hybridizing to the target sequence and still sufficiently bind to the target to show the desired effect, i.e., down-regulation of the target mRNA and/or protein. Mismatches can, for example, be compensated by increased length of the ASO nucleotide sequence and/or an increased number of nucleotide analogs, which are disclosed elsewhere herein.
  • the ASO of the disclosure comprises no more than three mismatches when hybridizing to the target sequence. In other aspects, the contiguous nucleotide sequence comprises no more than two mismatches when hybridizing to the target sequence.
  • the contiguous nucleotide sequence comprises no more than one mismatch when hybridizing to the target sequence.
  • the ASOs can comprise a contiguous nucleotide sequence of a total of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides in length. It should be understood that when a range is given for an ASO, or contiguous nucleotide sequence length, the range includes the lower and upper lengths provided in the range, for example from (or between) 10–30, includes both 10 and 30.
  • the ASOs comprise a contiguous nucleotide sequence of a total of about 14-20, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides in length. In certain aspects, the ASOs comprise a contiguous nucleotide sequence of a total of about 20 contiguous nucleotides in length. In certain aspects, ASOs of the present disclosure are 14 nucleotides in length. In certain aspects, ASOs of the present disclosure are 15 nucleotides in length. In certain aspects, ASOs of the present disclosure are 16 nucleotides in length. In certain aspects, ASOs of the present disclosure are 17 nucleotides in length.
  • ASOs of the present disclosure are 18 nucleotides in length. In certain aspects, ASOs of the present disclosure are 19 nucleotides in length. In certain aspects, ASOs of the present disclosure are 20 nucleotides in length. II.D. Nucleosides and Nucleoside analogs [0206] In one aspect of the disclosure, the ASOs comprise one or more non-naturally occurring nucleoside analogs. "Nucleoside analogs" as used herein are variants of natural nucleosides, such as DNA or RNA nucleosides, by virtue of modifications in the sugar and/or base moieties.
  • Analogs could in principle be merely "silent” or “equivalent” to the natural nucleosides in the context of the oligonucleotide, i.e. have no functional effect on the way the oligonucleotide works to inhibit target gene expression.
  • Such "equivalent” analogs can nevertheless be useful if, for example, they are easier or cheaper to manufacture, or are more stable to storage or manufacturing conditions, or represent a tag or label.
  • the analogs will have a functional effect on the way in which the ASO works to inhibit expression; for example, by producing increased binding affinity to the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into the cell.
  • nucleoside analogs are described by e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and in Scheme 1.
  • the ASOs of the present disclosure can contain more than one, more than two, more than three, more than four, more than five, more than six, more than seven, more than eight, more than nine, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 18, more than 19, or more than 20 nucleoside analogs.
  • the nucleoside analogs in the ASOs are the same. In other aspects, the nucleoside analogs in the ASOs are different.
  • nucleoside analogs in the ASOs can be any one of or combination of the following nucleoside analogs.
  • the nucleoside analog comprises a 2'-O-alkyl-RNA; 2'-O- methyl RNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluro-RNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro-ANA; bicyclic nucleoside analog; or any combination thereof.
  • the nucleoside analog comprises a sugar modified nucleoside.
  • the nucleoside analog comprises a nucleoside comprising a bicyclic sugar. In some aspects, the nucleoside analog comprises an LNA. [0208] In some aspects, the nucleoside analog is selected from the group consisting of constrained ethyl nucleoside (cEt), 2',4'-constrained 2′-O-methoxyethyl (cMOE), ⁇ -L-LNA, ⁇ - D-LNA, 2'-O,4'-C-ethylene-bridged nucleic acids (ENA), amino-LNA, oxy-LNA, thio-LNA, and any combination thereof. In some aspects, the ASO comprises one or more 5'-methyl- cytosine nucleobases.
  • nucleobase includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization.
  • pyrimidine e.g., uracil, thymine and cytosine
  • nucleobase also encompasses modified nucleobases which can differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization.
  • the nucleobase moiety is modified by modifying or replacing the nucleobase.
  • nucleobase refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al., (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.371.4.1.
  • the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl- cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5- thiazolo-uracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2- aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.
  • a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl- cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil,
  • the nucleobase moieties can be indicated by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, wherein each letter can optionally include modified nucleobases of equivalent function.
  • the nucleobase moieties are selected from A, T, G, C, and 5-methyl-cytosine.
  • 5-methyl-cytosine LNA nucleosides can be used.
  • the ASO of the disclosure can comprise one or more nucleosides which have a modified sugar moiety, i.e.
  • modifications include those where the ribose ring structure is modified, e.g.
  • HNA hexose ring
  • LNA ribose ring
  • UPA unlinked ribose ring which typically lacks a bond between the C2' and C3' carbons
  • Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798).
  • Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
  • Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2'-OH group naturally found in RNA nucleosides. Substituents can, for example be introduced at the 2', 3', 4', or 5' positions.
  • Nucleosides with modified sugar moieties also include 2' modified nucleosides, such as 2' substituted nucleosides.
  • a 2' sugar modified nucleoside is a nucleoside which has a substituent other than H or –OH at the 2' position (2' substituted nucleoside) or comprises a 2' linked biradical, and includes 2' substituted nucleosides and LNA (2' – 4' biradical bridged) nucleosides.
  • the 2' modified sugar can provide enhanced binding affinity (e.g., affinity enhancing 2' sugar modified nucleoside) and/or increased nuclease resistance to the oligonucleotide.
  • 2' substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'- alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA, 2'-Fluro- DNA, arabino nucleic acids (ANA), and 2'-Fluoro-ANA nucleoside.
  • MOE 2'-amino-DNA
  • 2'-Fluoro-RNA 2'-Fluro- DNA
  • ANA arabino nucleic acids
  • 2'-Fluoro-ANA nucleoside please see, e.g., Freier & Altmann; Nucl.
  • LNA nucleosides are modified nucleosides which comprise a linker group (referred to as a biradical or a bridge) between C2' and C4' of the ribose sugar ring of a nucleoside (i.e., 2'-4' bridge), which restricts or locks the conformation of the ribose ring.
  • a linker group referred to as a biradical or a bridge
  • nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.
  • BNA bicyclic nucleic acid
  • the locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.
  • Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et al., Bioorganic & Med.Chem. Lett. 12, 73-76, Seth et al., J. Org. Chem. 2010, Vol 75(5) pp.
  • Nuclease mediated degradation refers to an oligonucleotide capable of mediating degradation of a complementary nucleotide sequence when forming a duplex with such a sequence.
  • the oligonucleotide can function via nuclease mediated degradation of the target nucleic acid, where the oligonucleotides of the disclosure are capable of recruiting a nuclease, particularly and endonuclease, preferably endoribonuclease (RNase), such as RNase H.
  • RNase endoribonuclease
  • oligonucleotide designs which operate via nuclease mediated mechanisms are oligonucleotides which typically comprise a region of at least 5 or 6 DNA nucleosides and are flanked on one side or both sides by affinity enhancing nucleosides, for example gapmers.
  • II.F. RNase H Activity and Recruitment [0220] The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule and induce degradation of the complementary RNA molecule.
  • WO01/23613 provides in vitro methods for determining RNaseH activity, which can be used to determine the ability to recruit RNaseH.
  • an oligonucleotide is deemed capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, it has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10% or more than 20% of the of the initial rate determined when using a oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers, with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Example 91 - 95 of WO01/23613.
  • an oligonucleotide is deemed essentially incapable of recruiting RNaseH if, when provided with the complementary target nucleic acid, the RNaseH initial rate, as measured in pmol/l/min, is less than 20%, such as less than 10%,such as less than 5% of the initial rate determined when using a oligonucleotide having the same base sequence as the oligonucleotide being tested, but containing only DNA monomers, with no 2' substitutions, with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Example 91 - 95 of WO01/23613. II.G.
  • the ASO of the disclosure can comprise a nucleotide sequence which comprises both nucleosides and nucleoside analogs, and can be in the form of a gapmer. Examples of configurations of a gapmer that can be used with the ASO of the disclosure are described in U.S. Patent Appl. Publ. No.2012/0322851.
  • the term "gapmer” as used herein refers to an antisense oligonucleotide which comprises a region of RNase H recruiting oligonucleotides (gap) which is flanked 5' and 3' by one or more affinity enhancing modified nucleosides (flanks).
  • LNA gapmer is a gapmer oligonucleotide wherein at least one of the affinity enhancing modified nucleosides is an LNA nucleoside.
  • mixed wing gapmer refers to an LNA gapmer wherein the flank regions comprise at least one LNA nucleoside and at least one DNA nucleoside or non- LNA modified nucleoside, such as at least one 2' substituted modified nucleoside, such as, for example, 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA, 2'-Fluro-DNA, arabino nucleic acid (ANA), and 2'- Fluoro-ANA nucleoside(s).
  • the ASO of the disclosure can be in the form of a mixmer. In some aspects, the ASO of the disclosure can be in the form of a totalmer. In some aspects, in addition to enhancing affinity of the ASO for the target region, some nucleoside analogs also mediate RNase (e.g., RNaseH) binding and cleavage. Since ⁇ -L-LNA monomers recruit RNaseH activity to a certain extent, in some aspects, gap regions (e.g., region B as referred to herein) of ASOs containing ⁇ -L-LNA monomers consist of fewer monomers recognizable and cleavable by the RNaseH, and more flexibility in the mixmer construction is introduced. II.G.1.
  • RNaseH RNaseH
  • the ASO of the disclosure is a gapmer and comprises a contiguous stretch of nucleotides (e.g., one or more DNA) which is capable of recruiting an RNase, such as RNaseH, referred to herein in as region B (B), wherein region B is flanked at both 5' and 3' by regions of nucleoside analogs 5' and 3' to the contiguous stretch of nucleotides of region B– these regions are referred to as regions A (A) and C (C), respectively.
  • the nucleoside analogs are sugar modified nucleosides (e.g., high affinity sugar modified nucleosides).
  • the sugar modified nucleosides of regions A and C enhance the affinity of the ASO for the target nucleic acid (i.e., affinity enhancing 2' sugar modified nucleosides).
  • the sugar modified nucleosides are 2' sugar modified nucleosides, such as high affinity 2' sugar modifications, such as LNA and/or 2'-MOE.
  • the 5' and 3' most nucleosides of region B are DNA nucleosides, and are positioned adjacent to nucleoside analogs (e.g., high affinity sugar modified nucleosides) of regions A and C, respectively.
  • regions A and C can be further defined by having nucleoside analogs at the end most distant from region B (i.e., at the 5' end of region A and at the 3' end of region C).
  • the ASOs of the present disclosure comprise a nucleotide sequence of formula (5' to 3') A-B-C, wherein: (A) (5' region or a first wing sequence) comprises at least one nucleoside analog (e.g., 3-5 LNA units); (B) comprises at least four consecutive nucleosides (e.g., 4-24 DNA units), which are capable of recruiting RNase (when formed in a duplex with a complementary RNA molecule, such as the pre-mRNA or mRNA target); and (C) (3' region or a second wing sequence) comprises at least one nucleoside analog (e.g., 3-5 LNA units).
  • region A comprises 3-5 nucleoside analogs, such as LNA
  • region B consists of 6-24 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) DNA units
  • region C consists of 3 or 4 nucleoside analogs, such as LNA.
  • Such designs include (A-B-C) 3-14-3, 3-11-3, 3- 12-3, 3-13-3, 4-9-4, 4-10-4, 4-11-4, 4-12-4, and 5-10-5.
  • the ASO has a design of LLLDnLLL, LLLLDnLLLL, or LLLLLDnLLLLL, wherein the L is a nucleoside analog, the D is DNA, and n can be any integer between 4 and 24.
  • n can be any integer between 6 and 14. In some aspects, n can be any integer between 8 and 12.
  • the ASO has a design of LLLMMDnMMLLL, LLLMDnMLLL, LLLLMMDnMMLLLL, LLLLMDnMLLLL, LLLLLLMMDnMMLLLLL, or LLLLLLMDnMLLLLL, wherein the D is DNA, n can be any integer between 3 and 15, the L is LNA, and the M is 2'MOE.
  • Further gapmer designs are disclosed in WO2004/046160, WO2007/146511, and WO2008/113832, each of which is hereby incorporated by reference in its entirety. II.H.
  • Internucleotide Linkages [0230] The monomers of the ASOs described herein are coupled together via linkage groups. Suitably, each monomer is linked to the 3' adjacent monomer via a linkage group. [0231] The person having ordinary skill in the art would understand that, in the context of the present disclosure, the 5' monomer at the end of an ASO does not comprise a 5' linkage group, although it can or can not comprise a 5' terminal group. [0232] In some aspects, the contiguous nucleotide sequence comprises one or more modified internucleoside linkages.
  • linkage group or "internucleoside linkage” are intended to mean a group capable of covalently coupling together two nucleosides.
  • Non- limiting examples include phosphate groups and phosphorothioate groups.
  • the nucleosides of the ASO of the disclosure or contiguous nucleosides sequence thereof are coupled together via linkage groups.
  • each nucleoside is linked to the 3' adjacent nucleoside via a linkage group.
  • the internucleoside linkage is modified from its normal phosphodiester to one that is more resistant to nuclease attack, such as phosphorothioate, which is cleavable by RNaseH, also allows that route of antisense inhibition in reducing the expression of the target gene.
  • At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of internucleoside linkages are modified.
  • Extracellular Vesicles, e.g., Exosomes [0235] Disclosed herein are EVs comprising an ASO.
  • the ASO can be any ASO described herein or a functional fragment thereof.
  • the ASO reduces the level of an PMP22 mRNA or an PMP22 protein in a target cell, e.g., a Schwann cell.
  • the EV e.g., the exosome
  • the EV targets the peripheral nervous system.
  • the EV reduces the expression of one or more gene that is regulated by PMP22, e.g., transcriptional factors like KNOX20, SOX10 and EGR2.
  • the EV treats a neuropathy in a subject in need thereof.
  • the neuropathy is a peripheral neuropathy.
  • the neuropathy is Charcot- Marie-Tooth disease 1A (CMT1A).
  • CMT1A Charcot- Marie-Tooth disease 1A
  • the EV treats a neuropathy in a subject in need thereof.
  • PMP22 overexpression causes neuropathy with an early onset (about 75% in the first decade, about 10% in the second decade).
  • Initial symptoms are gait disorder and foot deformity.
  • Initial signs in the first decade are leg areflexia (100%), disordered heel walking (66%), foot muscle atrophy (50%), nerve enlargement (50%), pes cavus (33%) or short Achilles tendon (25%).
  • the weakness is related to axonal loss, particularly in distal muscles. Muscle wasting is common. Tendon reflexes tend to be reduced or absent.
  • the neuropathy is Charcot-Marie-Tooth disease 1A (CMT1A).
  • the EV treats Charcot-Marie-Tooth disease 1A (CMT1A).
  • CMT1A Charcot-Marie-Tooth disease 1A
  • the present disclosure provides a method to treat Charcot-Marie-Tooth disease 1A (CMT1A), or to ameliorate and/or delay the onset of at least one symptom, or prevent and/or mitigates at least one sequela of Charcot-Marie-Tooth disease 1A (CMT1A), or any combination thereof.
  • EVs described herein are extracellular vesicles with a diameter between about 20-300 nm.
  • an EV of the present disclosure has a diameter between about 20-290 nm, 20-280 nm, 20-270 nm, 20-260 nm, 20-250 nm, 20-240 nm, 20-230 nm, 20-220 nm, 20-210 nm, 20-200 nm, 20-190 nm, 20-180 nm, 20-170 nm, 20- 160 nm, 20-150 nm, 20-140 nm, 20-130 nm, 20-120 nm, 20-110 nm, 20-100 nm, 20-90 nm, 20-80 nm, 20-70 nm, 20-60 nm, 20-50 nm, 20-40 nm, 20-30 nm, 30-300 nm, 30-290 nm, 30- 280 nm, 30-270 nm, 30-260 nm, 30-250 nm, 30-240 nm, 30-230 nm, 30-220 nm,
  • an EV of the present disclosure comprises a bi-lipid membrane ("EV membrane"), comprising an interior (luminal) surface and an exterior surface.
  • the interior (luminal) surface faces the inner core (i.e., lumen) of the EV.
  • the exterior surface can be in contact with the endosome, the multivesicular bodies, or the membrane/cytoplasm of a producer cell or a target cell
  • the EV membrane comprises lipids and fatty acids.
  • the EV membrane comprises phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterols, and phosphatidylserines.
  • the EV membrane comprises an inner leaflet and an outer leaflet.
  • the composition of the inner and outer leaflet can be determined by transbilayer distribution assays known in the art, see, e.g., Kuypers et al., Biohim Biophys Acta 1985 819:170.
  • the composition of the outer leaflet is between approximately 70- 90% choline phospholipids, between approximately 0-15% acidic phospholipids, and between approximately 5-30% phosphatidylethanolamine.
  • the composition of the inner leaflet is between approximately 15-40% choline phospholipids, between approximately 10- 50% acidic phospholipids, and between approximately 30-60% phosphatidylethanolamine.
  • the EV membrane comprises one or more polysaccharide, such as glycan.
  • the EV of the present disclosure comprises an ASO, wherein the ASO is linked to the EV via a scaffold moiety, either on the exterior surface of the EV or on the luminal surface of the EV.
  • the EV comprising an ASO comprises an anchoring moiety, which optionally comprising a linker, between the ASO and the exosome membrane.
  • Anchoring moieties can be used to anchor an ASO to the EV of the present disclosure.
  • the ASO is linked directly to the anchoring moiety or via a linker.
  • the ASO can be attached to an anchoring moiety or linker combination via reaction between a "reactive group” (RG; e.g., amine, thiol, hydroxy, carboxylic acid, or azide) with a "reactive moiety” (RM; e.g., maleimide, succinate, NHS).
  • RG reactive group
  • RM reactive moiety
  • the modifications increase the hydrophobicity of the an ASO by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10-fold relative to native (non-modified) ASO. In some aspects, the modifications increase the hydrophobicity of the ASO by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 orders of magnitude relative to native (non-modified) ASO.
  • the modifications increase the hydrophobicity of the ASO by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000% relative to native (non-modified) ASO, e.g., the corresponding unmodified ASO.
  • hydrophobicity can be determined by measuring the percentage solubility in an organic solvent, such as octanol, as compared to solubility in an aqueous solvent, such as water.
  • an anchoring moiety can be chemically conjugated to an ASO to enhance its hydrophobic character.
  • the anchoring moiety is a sterol (e.g., cholesterol), GM1, a lipid, a vitamin, a small molecule, a peptide, or a combination thereof.
  • the moiety is a lipid.
  • the anchoring moiety is a sterol, e.g., cholesterol.
  • Additional hydrophobic moieties include, for example, phospholipids, lysophospholipids, fatty acids, or vitamins (e.g., vitamin D or vitamin E).
  • the anchoring moiety is conjugated at the termini of the ASO either directly or via one or more linkers (i.e., "terminal modification"). In other aspects, the anchoring moiety is conjugated to other portions of the ASO.
  • the ASO can include a detectable label. Exemplary labels include fluorescent labels and/or radioactive labels. In some aspects, where ASOs are fluorescently labeled, the detectable label can be, for example, Cy3.
  • Adding a detectable label to ASOs can be used as a way of labeling exosomes, and following their biodistribution.
  • a detectable label can be attached to exosomes directly, for example, by way of labeling an exosomal lipid and/or an exosomal peptide.
  • the different components of an ASO i.e., anchoring moieties, linkers and linker combinations, and ASOs
  • the different components of an ASO can be linker using bifunctional linkers (i.e., linkers containing two functional groups), such as N-succinimidyl-3- (2-pyridyldithio)propionate, N-4-maleimide butyric acid, S-(2-pyridyldithio)cysteamine, iodoacetoxysuccinimide, N-(4-maleimidebutyloxy) succinimide, N-[5-(3 ′ -maleimide propylamide)-1-carboxypentyl]iminodiacetic acid, N-(5-aminopentyl)-iminodiacetic acid, and the like.
  • bifunctional linkers i.e., linkers containing two functional groups
  • linkers containing two functional groups such as N-succinimidyl-3- (2-pyridyldithio)propionate, N-4-maleimide butyric acid, S-(2-pyr
  • Anchoring moieties capable of anchoring an ASO to the surface of an EV comprise for example sterols (e.g., cholesterol), lipids, lysophospholipids, fatty acids, or fat-soluble vitamins, as described in detail below.
  • the anchoring moiety can be a lipid.
  • a lipid anchoring moiety can be any lipid known in the art, e.g., palmitic acid or glycosylphosphatidylinositols.
  • the lipid is a fatty acid, phosphatide, phospholipid (e.g., phosphatidyl choline, phosphatidyl serine, or phosphatidyl ethanolamine), or analogue thereof (e.g. phosphatidylcholine, lecithin, phosphatidylethanolamine, cephalin, or phosphatidylserine or analogue or portion thereof, such as a partially hydrolyzed portion thereof).
  • anchoring moieties are chemically attached.
  • an anchoring moiety can be attached to an ASO enzymatically. In some aspects, in the possible to attach an anchoring moiety to an ASO via modification of cell culture conditions.
  • the anchoring moiety can be conjugated to an ASO directly or indirectly via a linker combination, at any chemically feasible location, e.g., at the 5′ and/or 3′ end of the ASO. In one aspect, the anchoring moiety is conjugated only to the 3′ end of the ASO.
  • an anchoring moiety of the present disclosure can comprise two or more types of anchoring moieties disclosed herein.
  • an anchoring moiety can comprise two lipids, e.g., a phospholipids and a fatty acid, or two phospholipids, or two fatty acids, or a lipid and a vitamin, or cholesterol and a vitamin, etc.
  • the combination of anchoring moieties e.g., a combination of the lipids (e.g., fatty acids) has an ECN of 6-80, 8-80, 10-80, 12-80, 14-80, 16-80, 18-80, 20- III.A.1.a. Cholesterol and other sterols
  • the anchoring moiety comprises a sterol, steroid, hopanoid, hydroxysteroid, secosteroid, or analog thereof with lipophilic properties.
  • the anchoring moiety comprises a sterol, such as a phytosterol, mycosterol, or zoosterol.
  • exemplary zoosterols include cholesterol and 24S-hydroxycholesterol;
  • exemplary phytosterols include ergosterol (mycosterol), campesterol, sitosterol, and stigmasterol.
  • the sterol is selected from ergosterol, 7-dehydrocholesterol, cholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fucosterol, saringosterol, campesterol, ⁇ -sitosterol, sitostanol, coprostanol, avenasterol, or stigmasterol.
  • Sterols can be found either as free sterols, acylated (sterol esters), alkylated (steryl alkyl ethers), sulfated (sterol sulfate), or linked to a glycoside moiety (steryl glycosides), which can be itself acylated (acylated sterol glycosides).
  • the anchoring moiety comprises a steroid.
  • the steroid is selected from dihydrotestosterone, uvaol, hecigenin, diosgenin, progesterone, or cortisol.
  • sterols can be conjugated to the ASO directly or via a linker combination at the available —OH group of the sterol.
  • Exemplary sterols have the general skeleton shown below: [0267]
  • ergosterol has the structure below: [0268]
  • Cholesterol has the structure below: [0269] Accordingly, in some aspects, the free —OH group of a sterol or steroid is used to conjugate the ASO directly or via a linker combination, to the sterol (e.g., cholesterol) or steroid. III.A.1.b.
  • Fatty acids [0270]
  • the anchoring moiety is a fatty acid.
  • the fatty acid is a short-chain, medium-chain, or long-chain fatty acid. In some aspects, the fatty acid is a saturated fatty acid. In some aspects, the fatty acid is an unsaturated fatty acid. In some aspects, the fatty acid is a monounsaturated fatty acid. In some aspects, the fatty acid is a polyunsaturated fatty acid, such as an ⁇ -3 (omega-3) or ⁇ -6 (omega-6) fatty acid. [0271] In some aspects, the lipid, e.g., fatty acid, has a C 2 -C 60 chain. In some aspects, the lipid, e.g., fatty acid, has a C 2 -C 28 chain.
  • the fatty acid has a C 2 -C 40 chain. In some aspects, the fatty acid, has a C2-C12 or C4-C12 chain.
  • the anchoring moiety comprises two fatty acids, each of which is independently selected from a fatty acid having a chain with any one of the foregoing ranges or numbers of carbon atoms. In some aspects, one of the fatty acids is independently a fatty acid with a C6-C21 chain and one is independently a fatty acid with a C12-C36 chain. In some aspects, each fatty acid independently has a chain of 11, 12, 13, 14, 15, 16, or 17 carbon atoms. III.A.1.c.
  • the anchoring moiety comprises a phospholipid.
  • Phospholipids are a class of lipids that are a major component of all cell membranes. They can form lipid bilayers because of their amphiphilic characteristic.
  • the structure of the phospholipid molecule generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head” consisting of a phosphate group.
  • a phospholipid can be a lipid according to the following formula: in which Rp represents a phospholipid moiety and R1 and R2 represent fatty acid moieties with or without unsaturation that can be the same or different.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2 lysophosphatidyl choline, and a sphingomyelin. III.A.1.d. Lysolipids (e.g., lysophospholipids) [0275] In some aspects, the anchoring moiety comprises a lysolipid, e.g., a lysophospholipid.
  • Lysolipids are derivatives of a lipid in which one or both fatty acyl chains have been removed, generally by hydrolysis.
  • Lysophospholipids are derivatives of a phospholipid in which one or both fatty acyl chains have been removed by hydrolysis.
  • the anchoring moiety comprises any of the phospholipids disclosed above, in which one or both acyl chains have been removed via hydrolysis, and therefore the resulting lysophospholipid comprises one or no fatty acid acyl chain.
  • the anchoring moiety comprises a lysoglycerophospholipid, a lysoglycosphingoliopid, a lysophosphatidylcholine, a lysophosphatidylethanolamine, a lysophosphatidylinositol, or a lysophosphatidylserine.
  • the anchoring moiety comprises a lipophilic vitamin, e.g., folic acid, vitamin A, vitamin E, or vitamin K
  • the anchoring moiety comprises vitamin A.
  • Vitamin A is a group of unsaturated nutritional organic compounds that includes retinol, retinal, retinoic acid, and several provitamin A carotenoids (most notably beta-carotene).
  • the anchoring moiety comprises retinol.
  • the anchoring moiety comprises a retinoid.
  • Retinoids are a class of chemical compounds that are vitamers of vitamin A or are chemically related to it.
  • the anchoring moiety comprises a first generation retinoid (e.g., retinol, tretinoin, isotreatinoin, or alitretinoin), a second-generation retinoid (e.g., etretinate or acitretin), a third-generation retinoid (e.g., adapalene, bexarotene, or tazarotene), or any combination thereof.
  • a first generation retinoid e.g., retinol, tretinoin, isotreatinoin, or alitretinoin
  • a second-generation retinoid e.g., etretinate or acitretin
  • a third-generation retinoid e.g., adapalene, bexarotene, or tazarotene
  • an ASO is linked to a hydrophobic membrane anchoring moiety disclosed herein via a linker combination,
  • linker combination The main function of a linker combination is to provide the optimal spacing between the anchoring moiety or moieties and the BAM target.
  • the linker combination should reduce steric hindrances and position the ASO so it can interact with a target nucleic acid, e.g., a mRNA or a miRNA.
  • Linkers can be susceptible to cleavage ("cleavable linker") thereby facilitating release of the biologically active molecule.
  • a linker combination disclosed herein can comprise a cleavable linker.
  • cleavable linkers can be susceptible, for example, to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the biologically active molecule remains active.
  • linkers can be substantially resistant to cleavage ("non-cleavable linker").
  • the cleavable linker comprises a spacer.
  • the spacer is PEG.
  • a linker combination comprises at least 2, at least 3, at least 4, at least 5, or at least 6 or more different linkers disclosed herein.
  • linkers in a linker combination can be linked by an ester linkage (e.g., phosphodiester or phosphorothioate ester).
  • the linker is direct bond between an anchoring moiety and a BAM, e.g., an ASO. III.A.2.a.
  • Non-cleavable linkers [0284]
  • the linker combination comprises a "non-cleavable liker.”
  • Non-cleavable linkers are any chemical moiety capable of linking two or more components of a modified biologically active molecule of the present disclosure (e.g., a biologically active molecule and an anchoring moiety; a biologically active molecule and a cleavable linker; an anchoring moiety and a cleavable linker) in a stable, covalent manner and does not fall off under the categories listed above for cleavable linkers.
  • non-cleavable linkers are substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage.
  • non-cleavable refers to the ability of the chemical bond in the linker or adjoining to the linker to withstand cleavage induced by an acid, photolabile-cleaving agent, a peptidase, an esterase, or a chemical or physiological compound that cleaves a disulfide bond, at conditions under which a cyclic dinucleotide and/or the antibody does not lose its activity.
  • the biologically active molecule is attached to the linker via another linker, e.g., a self-immolative linker.
  • the linker combination comprises a non-cleavable linker comprising, e.g., tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), succinimide, or any combination thereof.
  • the non-cleavable linker comprises a spacer unit to link the biologically active molecule to the non-cleavable linker.
  • one or more non-cleavable linkers comprise smaller units (e.g., HEG, TEG, glycerol, C2 to C12 alkyl, and the like) linked together.
  • the linkage is an ester linkage (e.g., phosphodiester or phosphorothioate ester) or other linkage.
  • the linker combination comprises a non-cleavable linker, wherein the non-cleavable linker comprises a polyethylene glycol (PEG) characterized by a formula R 3 -(O-CH2-CH2)n- or R 3 -(0-CH2-CH2)n-O- with R 3 being hydrogen, methyl or ethyl and n having a value from 2 to 200.
  • the linker comprises a spacer, wherein the spacer is PEG.
  • the PEG linker is an oligo-ethylene glycol, e.g., diethylene glycol, triethylene glycol, tetra ethylene glycol (TEG), pentaethylene glycol, or a hexaethylene glycol (HEG) linker.
  • TEG tetra ethylene glycol
  • HOG hexaethylene glycol
  • n has a value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114
  • n is between 2 and 10, between 10 and 20, between 20 and 30, between 30 and 40, between 40 and 50, between 50 and 60, between 60 and 70, between 70 and 80, between 80 and 90, between 90 and 100, between 100 and 110, between 110 and 120, between 120 and 130, between 130 and 140, between 140 and 150, between 150 and 160, between 160 and 170, between 170 and 180, between 180 and 190, or between 190 and 200.
  • n has a value from 3 to 200, from 3 to 20, from 10 to 30, or from 9 to 45.
  • a linker combination of the present disclosure can comprise several PEG linkers, e.g., a cleavable linker flanked by PEG, HEG, or TEG linkers.
  • the linker combination comprises (HEG)n and/or (TEG)n, wherein n is an integer between 1 and 50, and each unit is connected, e.g., via a phosphate ester linker, a phosphorothioate ester linkage, or a combination thereof.
  • the linker combination comprises a non-cleavable linker comprising a glycerol unit or a polyglycerol (PG) described by the formula ((R 3 —O—(CH 2 — CHOH—CH 2 O) n —) with R3 being hydrogen, methyl or ethyl, and n having a value from 3 to 200.
  • n has a value from 3 to 20.
  • n has a value from 10 to 30.
  • the PG linker is a diglycerol, triglycerol, tetraglycerol (TG), pentaglycerol, or a hexaglycerol (HG) linker.
  • Aliphatic (Alkyl) linkers [0297]
  • the linker combination comprises at least one aliphatic (alkyl) linker, e.g., propyl, butyl, hexyl, or C2-C12 alkyl, such as C2-C10 alkyl or C2-C6 alkyl.
  • the linker combination comprises an alkyl chain, e.g., an unsubstituted alkyl. III.A.3.
  • Cleavable linkers [0298]
  • different components of an ASO disclosed herein can be linker by a cleavable linker.
  • cleavable linker refers to a linker comprising at least one linkage or chemical bond that can be broken or cleaved.
  • cleave refers to the breaking of one or more chemical bonds in a relatively large molecule in a manner that produces two or more relatively smaller molecules.
  • Cleavage can be mediated, e.g., by a nuclease, peptidase, protease, phosphatase, oxidase, or reductase, for example, or by specific physicochemical conditions, e.g., redox environment, pH, presence of reactive oxygen species, or specific wavelengths of light.
  • a nuclease e.g., a nuclease, peptidase, protease, phosphatase, oxidase, or reductase
  • specific physicochemical conditions e.g., redox environment, pH, presence of reactive oxygen species, or specific wavelengths of light.
  • the term "cleavable,” as used herein, refers, e.g., to rapidly degradable linkers, such as, e.g., phosphodiester and disulfides, while the term “non-cleavable” refers, e.g., to more stable linkages, such as, e.g., nuclease-resistant phosphorothioates.
  • the cleavable linker is a dinucleotide or trinucleotide linker, a disulfide, an imine, a thioketal, a val-cit dipeptide, or any combination thereof.
  • the cleavable linker comprises valine-alanine-p- aminobenzylcarbamate or valine-citrulline-p-aminobenzylcarbamate.
  • the linker combination comprises a redox cleavable linker.
  • one type of cleavable linker is a redox cleavable linking group that is cleaved upon reduction or upon oxidation.
  • the linker combination can comprise a cleavable linker which can be cleaved by a reactive oxygen species (ROS), such as superoxide (Of) or hydrogen peroxide (H2O2), generated, e.g., by inflammation processes such as activated neutrophils.
  • ROS reactive oxygen species
  • the ROS cleavable linker is a thioketal cleavable linker. See, e.g., U.S. Pat. 8,354,455B2, which is herein incorporated by reference in its entirety.
  • the linker is an "acid labile linker" comprising an acid cleavable linking group, which is a linking group that is selectively cleaved under acidic conditions (pH ⁇ 7).
  • the linker combination comprises a self-immolative linker.
  • the self-immolative linker in the EV (e.g., exosome) of the present disclosure undergoes 1,4 ellimination after the enzymatic cleavage of the protease-cleavable linker.
  • the self-immolative linker in the EV (e.g., exosome) of the present disclosure undergoes 1,6 ellimination after the enzymatic cleavage of the protease-cleavable linker.
  • the self-immolative linker is, e.g., a p-aminobenzyl (pAB) derivative, such as a p-aminobenzyl carbamate (pABC), a p-amino benzyl ether (PABE), a p-amino benzyl carbonate, or a combination thereof.
  • pAB p-aminobenzyl
  • Reactive moieties are generated either via chemical synthesis or via chemical reaction between their components.
  • an anchoring moiety comprising a reactive group e.g., maleimide
  • an ASO comprising a maleimide-reacting group can react with an ASO comprising a maleimide-reacting group, to yield a hydrophobically modified ASO of the present disclosure, where the anchoring moiety can insert into the lipid bilayer of the membrane of an exosome, thereby attaching the ASO to the surface of the exosome.
  • any of the anchoring moieties, linker or linker combinations, or ASO disclosed herein can be conjugated to a reactive moiety, e.g., an amino reactive moiety (e.g., NHS-ester, p-nitrophenol, isothiocyanate, isocyanate, or aldehyde), a thiol reactive moiety (e.g., acrylate, maleimide, or pyridyl disulfide), a hydroxy reactive moiety (e.g., isothiocyanate or isocyanate), a carboxylic acid reactive moiety (e.g., epoxyde), or an azide reactive moiety (e.g., alkyne).
  • a reactive moiety e.g., an amino reactive moiety (e.g., NHS-ester, p-nitrophenol, isothiocyanate, isocyanate, or aldehyde), a thiol reactive moiety (e.g., acrylate, maleimide, or
  • Exemplary reactive moieties that can be used to covalent bind two components disclosed herein include, e.g., N-succinimidyl-3-(2-pyridyldithio)propionate, N-4-maleimide butyric acid, S-(2-pyridyldithio)cysteamine, iodoacetoxysuccinimide, N-(4-maleimidebutyryl oxy)succinimide, N-[5-(3′-maleimide propylamide)-1-carboxypentyl]iminodiacetic acid, N- (5-aminopentyl)iminodiacetic acid, and 1 ′ -[(2-cyanoethyl
  • an anchoring moiety, linker, or ASO can comprise a terminal oxyamino group, e.g., —ONH2, an hydrazino group, —NHNH2, a mercapto group (i.e., SH or thiol), or an olefin (e.g., CH ⁇ CH2).
  • an anchoring moiety, linker, or ASO can comprise an electrophilic moiety, e.g., at a terminal position, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, a phosphoramidite, or a pentafluorophenyl ester.
  • a covalent bond can be formed by coupling a nucleophilic group of a ligand, e.g., a hydroxyl, a thiol or amino group, with an electrophilic group.
  • the present invention is amenable to all manner of reactive groups and reactive moieties including but not limited to those known in the art.
  • the term "protecting group,” as used herein, refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures. Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions. Protecting groups as known in the art are described generally in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).
  • Solid phase synthesis known in the art can additionally or alternatively be employed. Suitable solid phase techniques, including automated synthesis techniques, are described in F. Eckstein (ed.), Oligonucleotides and Analogues, a Practical Approach, Oxford University Press, New York (1991) and Toy, P.H.; Lam, Y (ed.), Solid-Phase Organic synthesis, concepts, Strategies, and Applications, John Wiley & Sons, Inc. New Jersey (2012).
  • the reactive group can alternatively react with more than one of the reactive moieties described below. III.A.5.
  • the linker combination consists of a linker of formula [Alkyl linker]m-[PEG1]n-[PEG2]o wherein m, n, and o are 0 or 1, and at least one of m, n, or o is not zero.
  • Exemplary linker combinations according to such formula are C6-TEG-HEG, C6-HEG, C6-TEG, C6, TEG- HEG, TEG, C8-TEG-HEG, C8-HEG, C8-TEG, and C8.
  • the linker combination comprises a non-cleavable linker (e.g., TEG or HEG) in combination with one or more cleavable linkers, e.g., an enzymatic cleavable linker and a self immolative linker.
  • the linker combination comprises the linker combination TEG (non-cleavable linker)-Val-Cit(cleavable linker)-pAB(self- immolative linker), as shown below
  • Specific combinations of anchoring moieties and linker combinations are illustrated in the tables below. TABLE 2. TABLE 3.
  • [Cholesterol] is a cholesterol anchoring moiety
  • [TEG] is a TEG non-cleavable linker
  • [HEG] is a HEG non-cleavable linker
  • [SS] is a disulfide redox cleavable linker
  • [C6] is an alkyl non-cleavable linker
  • [SMal] is S-maleimide
  • [Val-Cit] is a valine-citrulline cleavable linker
  • [pAB] is a pAB self-immolative linker.
  • an ASO of the present disclosure has a structure according to the exemplary structures provided above, in which one or more components has been replaced by a component in the same class as those depicted in the example.
  • the [cholesterol] anchoring moiety can be substituted by another anchoring moiety disclosed herein
  • a [TEG] can be substituted by another polymeric non- cleavable linker disclosed herein (e.g., HEG, PEG, PG)
  • [Val-Cit] can be replaced by another peptidase cleavable linker
  • [pAB] can be substituted by another self-immolative linker.
  • one or more scaffold moieties are used to anchor an ASO to the EV of the present disclosure.
  • one or more scaffold moieties are used to anchor a protein or a molecule to the EVs in addition to the ASOs. Therefore, an EV of the present disclosure comprises an anchoring moiety linking an ASO and a scaffold moiety linking a protein or a molecule, e.g., a targeting moiety, a tropism moiety, and antiphagocytic moiety (e.g., CD47), or a combination thereof.
  • the ASO is linked to the scaffold moiety.
  • the EV comprises more than one scaffold moiety.
  • a first ASO is linked to a first scaffold moiety and a second ASO is linked to a second scaffold moiety.
  • the first scaffold moiety and the second scaffold moiety are the same type of scaffold moiety, e.g., the first and second scaffold moieties are both a Scaffold X protein.
  • the first scaffold moiety and the second scaffold moiety are different types of scaffold moiety, e.g., the first scaffold moiety is a Scaffold Y protein and the second scaffold moiety is a Scaffold X protein.
  • the first scaffold moiety is a Scaffold Y, disclosed herein.
  • the first scaffold moiety is a Scaffold X, disclosed herein.
  • the second scaffold moiety is a Scaffold Y, disclosed herein. In some aspects, the second scaffold moiety is a Scaffold X, disclosed herein.
  • the EV comprises one or more scaffold moieties, which are capable of anchoring an ASO to the EV (e.g., either on the luminal surface or on the exterior surface).
  • the scaffold moiety is a polypeptide ("scaffold protein").
  • the scaffold protein comprises an exosome protein or a fragment thereof.
  • scaffold moieties are non-polypeptide moieties.
  • scaffold proteins include various membrane proteins, such as transmembrane proteins, integral proteins and peripheral proteins, enriched on the exosome membranes.
  • a scaffold moiety (e.g., scaffold protein) comprises Scaffold X.
  • a scaffold moiety (e.g., exosome protein) comprises Scaffold Y.
  • a scaffold moiety (e.g., exosome protein) comprises both a Scaffold X and a Scaffold Y. III.B.1.
  • Scaffold X-Engineered EVs [0322]
  • EVs of the present disclosure comprise a membrane modified in its composition. For example, their membrane compositions can be modified by changing the protein, lipid, or glycan content of the membrane.
  • the surface-engineered EVs are generated by chemical and/or physical methods, such as PEG-induced fusion and/or ultrasonic fusion.
  • the surface-engineered EVs are generated by genetic engineering. EVs produced from a genetically-modified producer cell or a progeny of the genetically-modified cell can contain modified membrane compositions.
  • surface-engineered EVs have scaffold moiety (e.g., exosome protein, e.g., Scaffold X) at a higher or lower density (e.g., higher number) or include a variant or a fragment of the scaffold moiety.
  • surface (e.g., Scaffold X)-engineered EVs can be produced from a cell (e.g., HEK293 cells) transformed with an exogenous sequence encoding a scaffold moiety (e.g., exosome proteins, e.g., Scaffold X) or a variant or a fragment thereof.
  • EVs including scaffold moiety expressed from the exogenous sequence can include modified membrane compositions.
  • Various modifications or fragments of the scaffold moiety can be used for the aspects of the present disclosure.
  • scaffold moiety modified to have enhanced affinity to a binding agent can be used for generating surface-engineered EV that can be purified using the binding agent.
  • Scaffold moieties modified to be more effectively targeted to EVs and/or membranes can be used. Scaffold moieties modified to comprise a minimal fragment required for specific and effective targeting to exosome membranes can be also used.
  • Scaffold moieties can be engineered to be expressed as a fusion molecule, e.g., fusion molecule of Scaffold X to an ASO.
  • the fusion molecule can comprise a scaffold moiety disclosed herein (e.g., Scaffold X, e.g., PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter, or a fragment or a variant thereof) linked to an ASO.
  • the surface (e.g., Scaffold X)-engineered EVs described herein demonstrate superior characteristics compared to EVs known in the art.
  • surface (e.g., Scaffold X)-engineered contain modified proteins more highly enriched on their surface than naturally occurring EVs or the EVs produced using conventional exosome proteins.
  • the surface (e.g., Scaffold X)-engineered EVs of the present disclosure can have greater, more specific, or more controlled biological activity compared to naturally occurring EVs or the EVs produced using conventional exosome proteins.
  • the scaffold moiety e.g., Scaffold X
  • the PTGFRN polypeptide can be also referred to as CD9 partner 1 (CD9P-1), Glu-Trp-Ile EWI motif-containing protein F (EWI- F), Prostaglandin F2-alpha receptor regulatory protein, Prostaglandin F2-alpha receptor- associated protein, or CD315.
  • CD9 partner 1 CD9P-1
  • EWI- F Glu-Trp-Ile EWI motif-containing protein F
  • Prostaglandin F2-alpha receptor regulatory protein Prostaglandin F2-alpha receptor- associated protein
  • the full-length amino acid sequence of the human PTGFRN polypeptide (Uniprot Accession No. Q9P2B2) is shown at TABLE 4 as SEQ ID NO: 1.
  • the PTGFRN polypeptide contains a signal peptide (amino acids 1 to 25 of SEQ ID NO: 1), the extracellular domain (amino acids 26 to 832 of SEQ ID NO: 1), a transmembrane domain (amino acids 833 to 853 of SEQ ID NO: 1), and a cytoplasmic domain (amino acids 854 to 879 of SEQ ID NO: 1).
  • the mature PTGFRN polypeptide consists of SEQ ID NO: 1 without the signal peptide, i.e., amino acids 26 to 879 of SEQ ID NO: 1.
  • a PTGFRN polypeptide fragment useful for the present disclosure comprises a transmembrane domain of the PTGFRN polypeptide.
  • a PTGFRN polypeptide fragment useful for the present disclosure comprises the transmembrane domain of the PTGFRN polypeptide and (i) at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150 amino acids at the N terminus of the transmembrane domain, (ii) at least about five, at least about 10, at least about 15, at least about 20, or at least about 25 amino acids at the C terminus of the transmembrane domain, or both (i) and (ii).
  • the fragments of PTGFRN polypeptide lack one or more functional or structural domains, such as IgV.
  • the scaffold moiety e.g., Scaffold X, comprises an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 26 to 879 of SEQ ID NO: 1.
  • the scaffold moiety e.g., Scaffold X
  • the scaffold moiety e.g., Scaffold X
  • the mutations can be a substitution, an insertion, a deletion, or any combination thereof.
  • the scaffold moiety e.g., Scaffold X
  • the scaffold moiety e.g., Scaffold X
  • the Scaffold X comprises the amino acid sequence of amino acids 26 to 879 of SEQ ID NO: 1, amino acids 833 to 853 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 1, except one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations.
  • the mutations can be a substitution, an insertion, a deletion, or any combination thereof.
  • the scaffold moiety e.g., Scaffold X
  • the scaffold moiety e.g., Scaffold X
  • the scaffold moiety e.g., Scaffold X
  • the scaffold moiety comprises the amino acid sequence of SEQ ID NO: 103, 104, 105, 106, 107, or 108, except one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations.
  • the mutations can be a substitution, an insertion, a deletion, or any combination thereof.
  • the scaffold moiety e.g., Scaffold X
  • the scaffold moiety comprises the amino acid sequence of SEQ ID NO: 103, 104, 105, 106, 107, or 108, and 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or longer at the N terminus and/or C terminus of SEQ ID NO: 103, 104, 105, 106, 107, or 108. TABLE 4. Exemplary Scaffold Protein Sequences
  • Non-limiting examples of other scaffold moieties e.g., Scaffold X proteins
  • the sequence encodes a fragment of the scaffold moiety lacking at least about 5, at least about 10, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, or at least about 800 amino acids from the N-terminus of the native protein.
  • the sequence encodes a fragment of the scaffold moiety lacking at least about 5, at least about 10, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, or at least about 800 amino acids from the C-terminus of the native protein. In some aspects, the sequence encodes a fragment of the scaffold moiety lacking at least about 5, at least about 10, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, or at least about 800 amino acids from both the N-terminus and C-terminus of the native protein.
  • the sequence encodes a fragment of the scaffold moiety lacking one or more functional or structural domains of the native protein.
  • the scaffold moiety e.g., Scaffold X, e.g., a PTGFRN protein
  • the scaffold moiety is linked to one or more heterologous proteins.
  • the one or more heterologous proteins can be linked to the N-terminus of the scaffold moiety.
  • the one or more heterologous proteins can be linked to the C-terminus of the scaffold moiety.
  • the one or more heterologous proteins are linked to both the N-terminus and the C-terminus of the scaffold moiety.
  • the heterologous protein is a mammalian protein.
  • the heterologous protein is a human protein.
  • the scaffold moiety e.g., Scaffold X
  • the PTGFRN polypeptide can be used to link one or more biologically active molecules indirectly through a maleimide moiety or directly to a maleimide moiety or a linker to the luminal surface in addition to the external surface of the EV. Therefore, in certain aspects, Scaffold X can be used for dual purposes.
  • the EVs of the present disclosure comprise a higher number of Scaffold X proteins compared to the naturally-occurring EVs.
  • the EVs of the disclosure comprise at least about 5 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 110 fold, at least about 120 fold, at least about 130 fold, at least about 140 fold, at least about 150 fold, at least about 160 fold, at least about 170 fold, at least about 180 fold, at least about 190 fold, at least about 200 fold, at least about 210 fold, at least about 220 fold, at least about 230 fold, at least about 240 fold, at least about 250 fold, at least about 260 fold, at least about 270 fold higher number of Scaffold X (e.g., a PTGFRN polypeptide) compared to the naturally- occurring EV (e.g., exosome).
  • Scaffold X e.g., a PTGFRN polypeptide
  • the number of scaffold moieties, e.g., Scaffold X, such as, a PTGFRN polypeptide, on the EV (e.g., exosome) of the present disclosure is at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, at least about 2500, at least about 2600, at least about 2700, at least about 2800, at least about 2900, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about
  • the number of scaffold moieties, e.g., Scaffold X, such as, a PTGFRN polypeptide, on the EV of the present disclosure is from about 100 to about 100,000, from about 200 to about 9000, from about 300 to about 9000, from about 400 to about 9000, from about 500 to about 9000, from about 600 to about 8000, from about 800 to about 8000, from about 900 to about 8000, from about 1000 to about 8000, from about 1100 to about 8000, from about 1200 to about 8000, from about 1300 to about 8000, from about 1400 to about 8000, from about 1500 to about 8000, from about 1600 to about 8000, from about 1700 to about 8000, from about 1800 to about 8000, from about 1900 to about 8000, from about 2000 to about 8000, from about 2100 to about 8000, from about 2200 to about 8000, from about 2300 to about 8000, from about 2400 to about 8000, from about 2500 to about 8000, from about 2
  • the number of scaffold moieties, e.g., Scaffold X, such as, a PTGFRN polypeptide, on the EV (e.g., exosome) of the present disclosure is from about 5000 to about 8000, e.g., about 5000, about 6000, about 7000, or about 8000. In some aspects, the number of scaffold moieties, e.g., Scaffold X, such as, a PTGFRN polypeptide, on the EV (e.g., exosome) of the present disclosure is from about 6000 to about 8000, e.g., about 6000, about 7000, or about 8000.
  • the number scaffold moieties, e.g., Scaffold X, such as, a PTGFRN polypeptide, on the EV (e.g., exosome) of the present disclosure is from about 4000 to about 9000, e.g., about 4000, about 5000, about 6000, about 7000, about 8000, about 9000. III.B.2. Scaffold Y-Engineered EVs [0343]
  • EVs of the present disclosure comprise an internal space (i.e., lumen) that is different from that of the naturally occurring EVs.
  • engineered EVs can be produced from a cell transformed with an exogenous sequence encoding a scaffold moiety (e.g., exosome proteins, e.g., Scaffold Y) or a modification or a fragment of the scaffold moiety that changes the composition or content of the luminal surface of the exosome.
  • a scaffold moiety e.g., exosome proteins, e.g., Scaffold Y
  • modification or a fragment of the scaffold moiety that changes the composition or content of the luminal surface of the exosome.
  • modifications or fragments of the EV protein that can be expressed on the luminal surface of the EV can be used for the aspects of the present disclosure.
  • the EV proteins that can change the luminal surface of the EV include, but are not limited to the MARCKS protein, MARCKSL1 protein, BASP1 protein, or any combination thereof.
  • the scaffold moiety e.g., Scaffold Y, comprises Brain Acid Soluble Protein 1 (the BASP1 protein).
  • the BASP1 protein is also known as 22 kDa neuronal tissue-enriched acidic protein or neuronal axonal membrane protein NAP-22.
  • the full-length human BASP1 protein sequence (isomer 1) is shown in TABLE 5.
  • An isomer produced by an alternative splicing is missing amino acids 88 to 141 from the BASP1 protein in TABLE 5 (isomer 1). TABLE 5.
  • the scaffold moiety comprises a protein is selected from the group consisting of MARCKS, MARKSL1, BASP1, any functional fragment, variant, or derivative thereof, or any combination thereof.
  • the scaffold moiety, e.g., Scaffold Y comprises an Src protein or a fragment thereof.
  • the scaffold moiety, e.g., Scaffold Y comprises a sequence disclosed, e.g., in U.S. Patent No.9,611,481.
  • the scaffold moiety, e.g., Scaffold Y, of the present disclosure comprises the MARCKS protein, or a fragment, variant, or derivative thereof.
  • the MARCKS protein (Uniprot accession no. P29966) is also known as protein kinase C substrate, 80 kDa protein, light chain.
  • the full-length human MARCKS protein is 332 amino acids in length and comprises a calmodulin-binding domain at amino acid residues 152-176.
  • the scaffold moiety, e.g., Scaffold Y, of the present disclosure comprises a mature MARCKS protein (i.e., without N-terminal methionine).
  • the scaffold moiety, e.g., Scaffold Y, of the present disclosure is derived from a mature MARCKS protein, i.e., it is a fragment, variant, or derivate of a mature MARCKS protein and therefore it lacks the N- terminal protein present in the nonmature protein.
  • the scaffold moiety, e.g., Scaffold Y, of the present disclosure comprises the MARCKSL1 protein (Uniprot accession no. P49006), also known as MARCKS- like protein 1, and macrophage myristoylated alanine-rich C kinase substrate.
  • the full-length human MARCKSL1 protein is 195 amino acids in length.
  • the MARCKSL1 protein has an effector domain involved in lipid-binding and calmodulin-binding at amino acid residues 87- 110.
  • the scaffold moiety, e.g., Scaffold Y, of the present disclosure comprises a mature MARCKSL1 protein (i.e., without N-terminal methionine).
  • the scaffold moiety, e.g., Scaffold Y, of the present disclosure is derived from a mature MARCKSL1 protein, i.e., it is a fragment, variant, or derivate of a mature MARCKSL1 protein and therefore it lacks the N-terminal protein present in the non-mature protein.
  • the scaffold moiety e.g., Scaffold Y
  • the scaffold moiety comprises the BASP1 protein (Uniprot accession number P80723), also known as 22 kDa neuronal tissue-enriched acidic protein or neuronal axonal membrane protein NAP-22.
  • the full-length human BASP1 protein sequence is 227 amino acids in length.
  • An isomer produced by an alternative splicing is missing amino acids 88 to 141 from isomer 1.
  • the scaffold moiety, e.g., Scaffold Y, of the present disclosure comprises a mature BASP1 protein (i.e., without N-terminal methionine).
  • the scaffold moiety e.g., Scaffold Y
  • the scaffold moiety is derived from a mature BASP1 protein, i.e., it is a fragment, variant, or derivate of a mature BASP1 protein and therefore it lacks the N-terminal protein present in the non-mature protein.
  • the mature BASP1 protein sequence is missing the first Met from SEQ ID NO: 10 and thus contains amino acids 2 to 227 of SEQ ID NO: 10.
  • a scaffold moiety e.g., Scaffold Y
  • the scaffold moiety e.g., a Scaffold X protein
  • BASP1 functional fragment of the mature form of SEQ ID NO: 10
  • a scaffold moiety, e,g, Scaffold, Y useful for the present disclosure comprises the amino acid sequence of SEQ ID NO: 10 except one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations.
  • the mutations can be a substitution, an insertion, a deletion, or any combination thereof.
  • a scaffold moiety e.g., Scaffold Y
  • the protein sequence of any of SEQ ID NOs: 1-109 disclosed in WO2019099942A1 is sufficient to be a Scaffold Y for the present disclosure (e.g., scaffold moiety linked to a linker).
  • Non-limiting examples of scaffold proteins can be found at WO/2019/099942, published May 23, 2019 and WO/2020/101740, published May 22, 2020, which are incorporated by reference in their entireties.
  • Scaffold Y-engineered exosomes described herein can be produced from a cell transformed with any sequence set forth in WO2019099942A1 (SEQ ID NO: 4-109 from WO2019099942A1).
  • the EVs of the present disclosure comprise a higher number of Scaffold Y proteins compared to the naturally-occurring EVs.
  • the EVs of the disclosure comprise at least about 5 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 110 fold, at least about 120 fold, at least about 130 fold, at least about 140 fold, at least about 150 fold, at least about 160 fold, at least about 170 fold, at least about 180 fold, at least about 190 fold, at least about 200 fold, at least about 210 fold, at least about 220 fold, at least about 230 fold, at least about 240 fold, at least about 250 fold, at least about 260 fold, at least about 270 fold higher number of Scaffold Y (e.g., a BASP-1 polypeptide) compared to the
  • the number of Scaffold Y, e.g., BASP-1 polypeptide, on the EV of the present disclosure is at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, at least about 2500, at least about 2600, at least about 2700, at least about 2800, at least about 2900, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, or at least about 10000.
  • the number of Scaffold Y, e.g., a BASP-1 polypeptide, on the EV of the present disclosure is from about 100 to about 100,000, from about 200 to about 9000, from about 300 to about 9000, from about 400 to about 9000, from about 500 to about 9000, from about 600 to about 8000, from about 800 to about 8000, from about 900 to about 8000, from about 1000 to about 8000, from about 1100 to about 8000, from about 1200 to about 8000, from about 1300 to about 8000, from about 1400 to about 8000, from about 1500 to about 8000, from about 1600 to about 8000, from about 1700 to about 8000, from about 1800 to about 8000, from about 1900 to about 8000, from about 2000 to about 8000, from about 2100 to about 8000, from about 2200 to about 8000, from about 2300 to about 8000, from about 2400 to about 8000, from about 2500 to about 8000, from about 2600, from about 2700 to about 8000, from about
  • the number of Scaffold Y, e.g., a BASP-1 polypeptide, on the EV of the present disclosure is from about 5000 to about 8000, e.g., about 5000, about 6000, about 7000, or about 8000. In some aspects, the number of Scaffold Y, e.g., a BASP-1 polypeptide, on the EV of the present disclosure is from about 6000 to about 8000, e.g., about 6000, about 7000, or about 8000.
  • the number of Scaffold Y, e.g., a BASP-1 polypeptide, on the EV of the present disclosure is from about 4000 to about 9000, e.g., about 4000, about 5000, about 6000, about 7000, about 8000, about 9000.
  • the peptide linker is synthetic, i.e., non-naturally occurring.
  • a peptide linker includes peptides (or polypeptides) (e.g., natural or non-naturally occurring peptides) which comprise an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids to which it is not naturally linked or genetically fused in nature.
  • the peptide linker can comprise non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion).
  • the peptide linker can comprise non-naturally occurring amino acids.
  • the peptide linker can comprise naturally occurring amino acids occurring in a linear sequence that does not occur in nature.
  • the peptide linker can comprise a naturally occurring polypeptide sequence.
  • the scaffold moiety e.g., Scaffold Y, useful for the present disclosure does not contain an N-terminal Met.
  • the scaffold moiety e.g., Scaffold Y
  • the amino acid residue at the N-terminus of the scaffold protein is Gly. The presence of an N-terminal Gly is an absolute requirement for N-myristoylation.
  • the amino acid residue at the N-terminus of the scaffold protein is synthetic.
  • the amino acid residue at the N-terminus of the scaffold protein is a glycine analog, e.g., allylglycine, butylglycine, or propargylglycine. III.C.
  • the EV comprises at least one targeting moiety, e.g., an exogenous targeting moiety.
  • the exogenous targeting moiety comprises a peptide, an antibody or an antigen-binding fragment thereof, a chemical compound, an RNA aptamer, or any combination thereof.
  • the targeting moiety comprises a microprotein, a designed ankyrin repeat protein (darpin), an anticalin, an adnectin, an aptamer, a peptide mimetic molecule, a natural ligand for a receptor, a camelid nanobody, or any combination thereof.
  • the exogenous targeting moiety comprises a full-length antibody, a single domain antibody, a heavy chain only antibody (VHH), a single chain antibody, a shark heavy chain only antibody (VNAR), an scFv, a Fv, a Fab, a Fab', a F(ab')2, or any combination thereof.
  • the antibody is a single chain antibody.
  • the targeting moiety targets the exosome to the peripheral nervous system.
  • the targeting moiety targets the exosome to a Schwann cell.
  • the targeting moiety comprises a tissue or cell-specific target ligand which increases EV tropism to a specific peripheral nerves.
  • the EV comprises a tissue or cell-specific target ligand which increases EV tropism to Schwann cells.
  • the cell-specific target ligand which increases EV tropism to a Schwann cells binds to a Schwann cell surface marker.
  • the Schwann cell surface marker is selected from Myelin Basic Protein (MBP) and isoforms thereof, Myelin Protein Zero (P0), P75NTR, NCAM, PMP22, and any combination thereof.
  • the cell-specific target ligand comprises an antibody or an antigen-binding portion thereof, an aptamer, or an agonist or antagonist of a receptor expressed on the surface of the Schwann cell.
  • the targeting moiety is linked to the EV by a scaffold protein.
  • the scaffold protein is any scaffold protein disclosed herein.
  • the scaffold protein is a Scaffold X.
  • the scaffold protein is a Scaffold Y.
  • an EV of the present disclosure comprises a targeting moiety, and optionally further comprises a tropism moiety, and antiphagocytic moiety (e.g., CD47), or a combination thereof. III.D.
  • extracellular vesicles (EVs) of the present disclosure can comprise one or more linkers that link a molecule of interest (e.g., an ASO) to the EVs (e.g., to the exterior surface or on the luminal surface).
  • an ASO is linked to the EVs directly or via a scaffold moiety (e.g., Scaffold X or Scaffold Y).
  • the ASO is linked to the scaffold moiety by a linker.
  • the ASO is linked to the second scaffold moiety by a linker.
  • an ASO is linked to the exterior surface of an exosome via Scaffold X. In further aspects, an ASO is linked to the luminal surface of an exosome via Scaffold X or Scaffold Y.
  • the linker can be any chemical moiety known in the art.
  • the term "linker" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or to a non-polypeptide, e.g., an alkyl chain. In some aspects, two or more linkers can be linked in tandem. When multiple linkers are present, each of the linkers can be the same or different.
  • linkers provide flexibility or prevent/ameliorate steric hindrances. Linkers are not typically cleaved; however, in certain aspects, such cleavage can be desirable. Accordingly, in some aspects, a linker can comprise one or more protease-cleavable sites, which can be located within the sequence of the linker or flanking the linker at either end of the linker sequence. [0365] In some aspects, the linker is a peptide linker.
  • the peptide linker can comprise at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids.
  • the peptide linker is synthetic, i.e., non-naturally occurring.
  • a peptide linker includes peptides (or polypeptides) (e.g., natural or non-naturally occurring peptides) which comprise an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids to which it is not naturally linked or genetically fused in nature.
  • the peptide linker can comprise non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion).
  • Linkers can be susceptible to cleavage ("cleavable linker") thereby facilitating release of the biologically active molecule (e.g., an ASO).
  • the linker is a "reduction-sensitive linker.” In some aspects, the reduction-sensitive linker contains a disulfide bond. In some aspects, the linker is an "acid labile linker.” In some aspects, the acid labile linker contains hydrazone. Suitable acid labile linkers also include, for example, a cis-aconitic linker, a hydrazide linker, a thiocarbamoyl linker, or any combination thereof. [0369] In some aspects, the linker comprises a non-cleavable linker.
  • the linker comprises acrylic phosphoramidite (e.g., ACRYDITETM), adenylation, azide (NHS Ester), digoxigenin (NHS Ester), cholesterol-TEG, I-LINKERTM, an amino modifier (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, or Uni-LinkTM amino modifier), alkyne, 5' Hexynyl, 5-Octadiynyl dU, biotinylation (e.g., biotin, biotin (Azide), biotin dT, biotin-TEG, dual biotin, PC biotin, or desthiobiotin), thiol modification (thiol modifier C3 S-S, dithiol or thiol modifier C6 S-S), or any combination thereof.
  • acrylic phosphoramidite e.g., ACRYDITETM
  • adenylation azide
  • NHS Ester digoxigenin
  • the linker comprises a terpene such as nerolidol, farnesol, limonene, linalool, geraniol, carvone, fenchone, or menthol; a lipid such as palmitic acid or myristic acid; cholesterol; oleyl; retinyl; cholesteryl residues; cholic acid; adamantane acetic acid; 1-pyrene butyric acid; dihydrotestosterone; 1,3-Bis-O(hexadecyl)glycerol; geranyloxyhexyl group; hexadecylglycerol; borneol; 1,3-propanediol; heptadecyl group; O3- (oleoyl)lithocholic acid; O3-(oleoyl)cholenic acid; dimethoxytrityl; phenoxazine, a male
  • the surface of the EV is modified to limit or block uptake of the EV by cells of the immune system, e.g., macrophages.
  • the surface of the EV is modified to express one or more surface antigen that inhibits uptake of the EV by a macrophage, i.e., an "antiphagocytic signal.”
  • the surface antigen is associated with the exterior surface of the EV.
  • Surface antigens useful in the present disclosure that can function as antiphagocytic signals include, but are not limited to, antigens that label a cell as a "self" cell.
  • the surface antigen (antiphagocytic signal) is selected from CD47, CD24, a fragment thereof, and any combination thereof.
  • the surface antigen comprises CD24, e.g., human CD24.
  • the surface antigen comprises a fragment of CD24, e.g., human CD24.
  • the EV is modified to express CD47 or a fragment thereof on the exterior surface of the EV.
  • CD47 also referred to as leukocyte surface antigen CD47 and integrin associated protein (IAP), as used herein, is a transmembrane protein that is found on many cells in the body.
  • CD47 is often referred to as the "don't eat me” signal, as it signals to immune cells, in particular myeloid cells, that a particular cell expressing CD47 is not a foreign cell.
  • CD47 is the receptor for SIRPA, binding to which prevents maturation of immature dendritic cells and inhibits cytokine production by mature dendritic cells. Interaction of CD47 with SIRPG mediates cell-cell adhesion, enhances superantigen-dependent T-cell-mediated proliferation and costimulates T-cell activation.
  • CD47 is also known to have a role in both cell adhesion, by acting as an adhesion receptor for THBS1 on platelets, and in the modulation of integrins. CD47 also plays an important role in memory formation and synaptic plasticity in the hippocampus (by similarity). In addition, CD47 can play a role in membrane transport and/or integrin dependent signal transduction, prevent premature elimination of red blood cells, and be involved in membrane permeability changes induced following virus infection. [0375] In some aspects, an EV disclosed herein is modified to express a human CD47 on the surface of the EV.
  • the EV is modified to express a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 70 or a fragment thereof. In some aspects, the EV is modified to express a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 71 or a fragment thereof. In some aspects, the EV is modified to express a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 72 or a fragment thereof. In some aspects, the EV is modified to express a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 73 or a fragment thereof. TABLE 6: Human CD47 Amino Acid Sequences
  • the EV is modified to express full length CD47 on the surface of the EV.
  • the EV is modified to express a fragment of CD47 on the surface of the EV wherein the fragment comprises the extracellular domain of CD47, e.g., human CD47. Any fragment of CD47 that retains an ability to block and/or inhibit phagocytosis by a macrophage can be used in the EVs disclosed herein.
  • the fragment comprises amino acids 19 to about 141 of the canonical human CD47 sequence (e.g., amino acids 19-141 of SEQ ID NO: 70).
  • the fragment comprises amino acids 19 to about 135 of the canonical human CD47 sequence (e.g., amino acids 19-135 of SEQ ID NO: 70). In some aspects, the fragment comprises amino acids 19 to about 130 of the canonical human CD47 sequence (e.g., amino acids 19-130 of SEQ ID NO: 70). In some aspects, the fragment comprises amino acids 19 to about 125 of the canonical human CD47 sequence (e.g., amino acids 19-125 of SEQ ID NO: 70). [0377] In some aspects, an EV expressing CD47 or a fragment thereof has an altered biodistribution when compared with an exosome that does not express CD47 or a fragment.
  • an EV disclosed herein can be surface engineered to adjust its properties, e.g., biodistribution, e.g., via incorporation of immuno-affinity ligands or cognate receptor ligands.
  • EV disclosed herein can be surface engineered to direct them to a specific cellular type, e.g., Schwann cells, sensory neurons, motor neurons, or meningeal macrophages, or can be surface engineered to enhance their migration to a specific compartment, e.g., to the CNS in order to improve intrathecal compartment retention.
  • a specific cellular type e.g., Schwann cells, sensory neurons, motor neurons, or meningeal macrophages
  • an EV for delivery to the CNS disclosed herein comprises a bio-distribution modifying agent or targeting moiety.
  • the terms “bio-distribution modifying agent” and “targeting moiety” are used interchangeably and refer to an agent that can modify the distribution of extracellular vesicles (e.g., exosomes, nanovesicles) in vivo or in vitro (e.g., in a mixed culture of cells of different varieties).
  • the targeting moiety alters the tropism of the EV, i.e., the target moiety is a "tropism moiety”.
  • the term “tropism moiety” refers to a targeting moiety that when expressed on an EV alters and/or enhances the natural movement of the EV.
  • a tropism moiety can promote the EV to be taken up by a particular cell, tissue, or organ.
  • EVs exhibit preferential uptake in discrete cell types and tissues, and their tropism can be directed by adding proteins to their surface that interact with receptors on the surface of target cells.
  • the tropism moiety can comprise a biological molecule, such as a protein, a peptide, a lipid, or a carbohydrate, or a synthetic molecule.
  • the tropism moiety can comprise an affinity ligand, e.g., an antibody (such as an anti- CD19 nanobody, an anti-CD22 nanobody, an anti-CLEC9A nanobody, or an anti-CD3 nanobody), a VHH domain, a phage display peptide, a fibronectin domain, a camelid nanobody, and/or a vNAR.
  • an affinity ligand e.g., an antibody (such as an anti- CD19 nanobody, an anti-CD22 nanobody, an anti-CLEC9A nanobody, or an anti-CD3 nanobody), a VHH domain, a phage display peptide, a fibronectin domain, a camelid nanobody, and/or a vNAR.
  • the tropism moiety can comprise, e.g., a synthetic polymer (e.g., PEG), a natural ligand/molecule (e.g., CD40L, albumin, CD47, CD24, CD55, CD59), and/or a recombinant protein (e.g., XTEN).
  • a tropism moiety can increase uptake of the EV by a cell.
  • the tropism moiety that can increase uptake of the EV by a cell comprises a lymphocyte antigen 75 (also known as DEC205 or CD205), C-type lectin domain family 9 member A (CLEC9A), C-type lectin domain family 6 (CLEC6), C-type lectin domain family 4 member A (also known as DCIR or CLEC4A), Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (also known as DC-SIGN or CD209), lectin-type oxidized LDL receptor 1(LOX-1), macrophage receptor with collagenous structure (MARCO), C-type lectin domain family 12 member A (CLEC12A), C-type lectin domain family 10 member A (CLEC10A), DC-asialoglycoprotein receptor (DC-ASGPR), DC immunoreceptor 2 (DCIR2), Dectin-1, macrophage mannose receptor (MMR), BDCA-2 (CD303, CLEC4C), Dectin-2,
  • an EV of the present disclosure can comprise a tissue or cell-specific target ligand, which increases EV tropism to a specific central nervous system tissue or cell.
  • the cell is a glial cell.
  • the glial cell is an oligodendrocyte, an astrocyte, an ependymal cell, a microglia cell, a Schwann cell, a satellite glial cell, an olfactory ensheathing cell, or a combination thereof.
  • the cell is a neural stem cell.
  • the cell-specific target ligand which increases EV tropism to a Schwann cells binds to a Schwann cell surface marker such as Myelin Basic Protein (MBP), Myelin Protein Zero (P0), P75NTR, NCAM, PMP22, or any combination thereof.
  • MBP Myelin Basic Protein
  • P0 Myelin Protein Zero
  • P75NTR NCAM
  • PMP22 P75NTR
  • NCAM NCAM
  • PMP22 binds to a Schwann cell surface marker
  • the cell-specific tropism moiety comprises an antibody or an antigen-binding portion thereof, an aptamer, or an agonist or antagonist of a receptor expressed on the surface of the Schwann cell.
  • the EVs of the present disclosure comprising at least one tropism moiety that can direct the EV to a specific target cell or tissue (e.g., a cell in the CNS or a Schwann cell in peripheral nerves) can be administered using any suitable administration method known in the art (e.g., intravenous injection or infusion) since the presence of the tropism moiety (alone or in combination with the presence of an antiphagocytic signal such as CD47 and the use of a specific administration route) will induce a tropism of the EVs towards the desired target cell or tissue.
  • a specific target cell or tissue e.g., a cell in the CNS or a Schwann cell in peripheral nerves
  • any suitable administration method known in the art e.g., intravenous injection or infusion
  • an antiphagocytic signal such as CD47 and the use of a specific administration route
  • the tropism moiety is linked, e.g., chemically linked via a maleimide moiety, to a scaffold moiety, e.g., a Scaffold X protein or a fragment thereof, on the exterior surface of the EV.
  • a scaffold moiety e.g., a Scaffold X protein or a fragment thereof
  • Tropism can be further improved by the attachment of an anti- phagocytic signal (e.g., CD47 and/or CD24), a half-life extension moiety (e.g., albumin or PEG), or any combination thereof to the external surface of an EV of the present disclosure.
  • an anti- phagocytic signal e.g., CD47 and/or CD24
  • a half-life extension moiety e.g., albumin or PEG
  • the anti-phagocytic signal is linked, e.g., chemically linked via a maleimide moiety, to a scaffold moiety, e.g., a Scaffold X protein or a fragment thereof, on the exterior surface of the EV.
  • a scaffold moiety e.g., a Scaffold X protein or a fragment thereof.
  • Pharmacokinetics, biodistribution, and in particular tropism and retention in the desired tissue or anatomical location can also be accomplish by selecting the appropriate administration route (e.g., intrathecal administration or intraocular administration to improve tropism to the central nervous system).
  • the EV comprises at least two different tropism moieties. In some aspects, the EV comprises three different tropism moieties.
  • the EV comprises four different tropism moieties. In some aspects, the EV comprises five or more different tropism moieties. In some aspects, one or more of the tropism moieties increases uptake of the EV by a cell.
  • each tropism moiety is attached to a scaffold moiety, e.g., a Scaffold X protein or a fragment thereof.
  • multiple tropism moieties can be attached to the same scaffold moiety, e.g., a Scaffold X protein or a fragment thereof.
  • several tropism moieties can be attached in tandem to a scaffold moiety, e.g., a Scaffold X protein or a fragment thereof.
  • a tropism moiety disclosed herein or a combination thereof is attached to a scaffold moiety, e.g., a Scaffold X protein or a fragment thereof, via a linker or spacer.
  • a linker or spacer or a combination thereof is interposed between two tropism moieties disclosed herein.
  • Non-limiting examples of tropism moieties capable of directing EVs of the present disclosure to different nervous system cell types are disclosed below.
  • Tropism moieties targeting Schwann cells In some aspects, a tropism moiety can target a Schwann cell.
  • the tropism moiety that directs an EV disclosed herein to a Schwann cell targets, e.g., a transferrin receptor (TfR), apolipoprotein D (ApoD), Galectin 1 (LGALS1), Myelin proteolipid protein (PLP), Glypican 1, or Syndecan 3.
  • the tropism moiety directing an EV of the present disclosure to a Schwann cell is a transferrin, or a fragment, variant or derivative thereof.
  • a tropism moiety of the present disclosure targets a transferring receptor (TfR).
  • Transferrin receptors e.g., TfR1 or TfR2 are carrier proteins for transferrin.
  • TfR1 (see, e.g., UniProt P02786 TFR1_Human) or transferrin receptor 1 (also known as cluster of differentiation 71 or CD71) is expressed on the endothelial cells of the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • TfR1 is known to be expressed in a variety of cells such as red blood cells, monocytes, hepatocytes, intestinal cells, and erythroid cells, and is upregulated in rapidly dividing cells such as tumor cells (non small cell lung cancer, colon cancer, and leukemia) as well as in tissue affected by disorders such as acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • TfR2 is primarily expressed in liver and erythroid cells, is found to a lesser extent in lung, spleen and muscle, and has a 45% identity and 66% similarity with TfR1.
  • TfR1 is a transmembrane receptor that forms a homodimer of 760 residues with disulfide bonds and a molecular weight of 90 kDa. Affinity for transferrin varies between the two receptor types, with the affinity for TfR1 being at least 25-30 fold higher than that of TfR2. [0391] Binding to TfR1 allows the transit of large molecules, e.g., antibodies, into the brain.
  • TfR1-targeting antibodies have been shown to cross the blood-brain barrier, without interfering with the uptake of iron.
  • those are the mouse anti rat-TfR antibody OX26 and the rat anti mouse-TfR antibody 8D3.
  • the affinity of the antibody-TfR interaction is important to determine the success of transcytotic transport over endothelial cells of the BBB.
  • Monovalent TfR interaction favors BBB transport due to altered intracellular sorting pathways. Avidity effects of bivalent interactions redirecting transport to the lysosome.
  • reducing TfR binding affinity directly promote dissociation from the TfR which increase brain parenchymal exposure of the TfR binding antibody. See, e.g., U.S.
  • a tropism moiety of the present disclosure can comprise a ligand that can target TfR, e.g., target TfR1, such as transferrin, or an antibody or other binding molecule capable of specifically binding to TfR.
  • the antibody targeting a transferrin receptor is a low affinity anti- transferring receptor antibody (see, e.g., US20190202936A1 which is herein incorporated by reference in its entirety).
  • the tropism moiety comprises all or a portion (e.g., a binding portion) of a ligand for a transferrin receptor, for example a human transferrin available in GenBank as Accession numbers NM001063, XM002793, XM039847, NM002343 or NM013900, among others, or a variant, fragment, or derivative thereof.
  • the tropism moiety comprises a transferrin-receptor-targeting moiety, i.e., a targeting moiety directed to a transferrin receptor.
  • Suitable transferrin-receptor- targeting moieties include a transferrin or transferrin variant, such as, but not limited to, a serum transferrin, lacto transferrin (lactoferrin) ovotransferrin, or melanotransferrin.
  • Transferrins are a family of nonheme iron-binding proteins found in vertebrates, including serum transferrins, lacto transferrins (lactoferrins), ovotransferrins, and melanotransferrins.
  • Serum transferrin is a glycoprotein with a molecular weight of about 80 kDa, comprising a single polypeptide chain with two N-linked polysaccharide chains that are branched and terminate in multiple antennae, each with terminal sialic acid residues.
  • the tropism moiety is a serum transferrin or transferrin variant such as, but not limited to a hexasialo transferrin, a pentasialo transferrin, a tetrasialo transferrin, a trisialo transferrin, a disialo transferrin, a monosialo transferrin, or an asialo transferrin, or a carbohydrate-deficient transferrin (CDT) such as an asialo, monosialo or disialo transferrin, or a carbohydrate-free transferrin (CFT) such as an asialo transferrin.
  • CDT carbohydrate-deficient transferrin
  • CFT carbohydrate-free transferrin
  • the tropism moiety is a transferrin variant having the N-terminal domain of transferrin, the C-terminal domain of transferrin, the glycosylation of native transferrin, reduced glycosylation as compared to native (wild-type) transferrin, no glycosylation, at least two N terminal lobes of transferrin, at least two C terminal lobes of transferrin, at least one mutation in the N domain, at least one mutation in the C domain, a mutation wherein the mutant has a weaker binding avidity for transferrin receptor than native transferrin, and/or a mutation wherein the mutant has a stronger binding avidity for transferrin receptor than native transferrin, or any combination of the foregoing.
  • the tropism moiety targeting a transferrin receptor comprises an anti-trasferrin receptor variable new antigen receptor (vNAR), e.g., a binding domain with a general motif structure (FW1-CDR1-FW2-3-CDR3-FW4).
  • vNARs are key component of the adaptive immune system of sharks. At only 11 kDa, these single-domain structures are the smallest IgG-like proteins in the animal kingdom and provide an excellent platform for molecular engineering and biologics drug discovery.
  • the tropism moiety comprises a vNAR domain capable of specifically binding to TfR1, wherein the vNAR domain comprises or consists essentially of a vNAR scaffold with any one CDR1 peptide in Table 1 of U.S.2017-0348416 in combination with any one CDR3 peptide in Table 1 of U.S.2017-0348416.
  • a tropism moiety of the present disclosure targets ApoD.
  • apolipoprotein D is mainly produced in the brain, cerebellum, and peripheral nerves.
  • ApoD is 169 amino acids long, including a secretion peptide signal of 20 amino acids. It contains two glycosylation sites (aspargines 45 and 78) and the molecular weight of the mature protein varies from 20 to 32 kDa.
  • ApoD binds steroid hormones such as progesterone and pregnenolone with a relatively strong affinity, and to estrogen with a weaker affinity.
  • Arachidonic acid (AA) is an ApoD ligand with a much better affinity than that of progesterone or pregnenolone.
  • a tropism moiety of the present disclosure comprises a ligand that can target ApoD, e.g., an antibody or other binding molecule capable of specifically binding to ApoD.
  • a tropism moiety of the present disclosure targets Galectin 1.
  • the galectin-1 protein is 135 amino acids in length.
  • a tropism moiety of the present disclosure comprises a ligand that can target Galectin 1, e.g., an antibody or other binding molecule capable of specifically binding to Galectin 1.
  • a tropism moiety of the present disclosure targets PLP.
  • PLP is the major myelin protein from the CNS. It plays an important role in the formation or maintenance of the multilamellar structure of myelin.
  • the myelin sheath is a multi-layered membrane, unique to the nervous system that functions as an insulator to greatly increase the efficiency of axonal impulse conduction.
  • PLP is a highly conserved hydrophobic protein of 276 to 280 amino acids which contains four transmembrane segments, two disulfide bonds and which covalently binds lipids (at least six palmitate groups in mammals).
  • a tropism moiety of the present disclosure comprises a ligand that can target PLP, e.g., an antibody or other binding molecule capable of specifically binding to PLP.
  • a tropism moiety of the present disclosure targets Glypican 1.
  • a tropism moiety of the present disclosure comprises a ligand that can target Glypican 1, e.g, an antibody or other binding molecule capable of specifically binding to Glypican 1.
  • a tropism moiety of the present disclosure targets Syndecan 3.
  • a tropism moiety of the present disclosure comprises a ligand that can target Syndecan 3, e.g., an antibody or other binding molecule capable of specifically binding to Syndecan 3.
  • Tropism moieties targeting sensory neurons In some aspects, a tropism moiety disclosed herein can direct an EV disclosed herein to a sensory neuron. In some aspects, the tropism moiety that directs an EV disclosed herein to a sensory neuron targets a Trk receptor, e.g., TrkA, TrkB, TrkC, or a combination thereof.
  • Trk (tropomyosin receptor kinase) receptors are a family of tyrosine kinases that regulates synaptic strength and plasticity in the mammalian nervous system.
  • the common ligands of Trk receptors are neurotrophins, a family of growth factors critical to the functioning of the nervous system. The binding of these molecules is highly specific. Each type of neurotrophin has different binding affinity toward its corresponding Trk receptor. Accordingly, in some aspects, the tropism moiety directing an EV disclosed herein to a sensory neuron, comprises a neurotrophin.
  • Neurotrophins bind to Trk receptors as homodimers.
  • the tropism moiety comprises at least two neurotrophins disclosed herein, e.g., in tandem.
  • the tropism moiety comprises at least two neurotrophins disclosed herein, e.g., in tandem, that are attached to a scaffold protein, for example, Protein X, via a linker.
  • the linker connecting the scaffold protein, e.g., Protein X, to the neurotrophin has a length of at least 10 amino acids.
  • the linker connecting the scaffold protein, e.g., Protein X, to the neurotrophin has a length of at least about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, or about 50 amino acids.
  • the neurotrophin is a neurotrophin precursor, i.e., a proneurotrophin, which is later cleaved to produce a mature protein.
  • Nerve growth factor is the first identified and probably the best characterized member of the neurotrophin family. It has prominent effects on developing sensory and sympathetic neurons of the peripheral nervous system.
  • Neurotrophin-3 is the third member of the NGF family and is expressed predominantly in a subset of pyramidal and granular neurons of the hippocampus, and has been detected in the cerebellum, cerebral cortex and peripheral tissues such as liver and skeletal muscles (Ernfors, P. et al., Neuron 1: 983-996 (1990)).
  • Neurotrophin-4 also called NT-415 is the most variable member of the neurotrophin family.
  • Neurotrophin-6 (NT-5) was found in teleost fish and binds to p75 receptor.
  • the neurotrophin targeting TrkB comprises, e.g., NT-4 or BDNF, or a fragment, variant, or derivative thereof.
  • the neurotrophin targeting TrkA comprises, e.g., NGF or a fragment, variant, or derivative thereof.
  • the neurotrophin targeting TrkC comprises, e.g., NT-3 or a fragment, variant, or derivative thereof.
  • the tropism moiety comprises brain derived neurotrophic factor (BDNF).
  • the BDNF is a variant of native BDNF, such as a two amino acid carboxyl-truncated variant.
  • the tropism moiety comprises the full length 119 amino acid sequence of BDNF TLTIKRGR; SEQ ID NO: 74).
  • a one amino-acid carboxy-truncated variant of BDNF is utilized (amino acids 1-118 of SEQ ID NO: 74).
  • the tropism moiety comprises a carboxy-truncated variant of the native BDNF, e.g., a variant in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acids are absent from the carboxy-terminus of the BDNF.
  • BDNF variants include the complete 119 amino acid BDNF, the 117 or 118 amino acid variant with a truncated carboxyl terminus, variants with a truncated amino terminus, or variants with up to about 20%, about 30, or about 40% change in amino acid composition, as long as the protein variant still binds to the TrkB receptor with high affinity.
  • the tropism moiety comprises a two amino-acid carboxy- truncated variant of BDNF (amino acids 1-117 of SEQ ID NO: 74).
  • the tropism moiety comprises a three amino-acid carboxy-truncated variant of BDNF (amino acids 1-116 of SEQ ID NO: 74).
  • the tropism moiety comprises a four amino-acid carboxy-truncated variant of BDNF (amino acids 1-115 of SEQ ID NO: 74). In some aspects, the tropism moiety comprises a five amino-acid carboxy-truncated variant of BDNF (amino acids 1-114 of SEQ ID NO: 74).
  • the tropism moiety comprises a BDNF that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical with the sequence of SEQ ID NO: 74, or a truncated version thereof, e.g., the 117 or 118 amino acid variant with a one- or two-amino acid truncated carboxyl terminus, or variants with a truncated amino terminus. See, e.g., U.S. Pat. No.8,053,569B2, which is herein incorporated by reference in its entirety.
  • the tropism moiety comprises nerve growth factor (NGF).
  • NGF nerve growth factor
  • the NGF is a variant of native NGF, such as a truncated variant.
  • the tropism moiety comprises the 26-kDa beta subunit of protein, the only component of the 7S NGF complex that is biologically active.
  • the tropism moiety comprises the full length 120 amino acid sequence of beta NGF (SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETK RKAVRRA; SEQ ID NO: 75).
  • the tropism moiety comprises a carboxy- truncated variant of the native NGF, e.g., a variant in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acids are absent from the carboxy-terminus of NGF.
  • NGF variants include the complete 120 amino acid NGF, the shorter amino acid variants with a truncated carboxyl terminus, variants with a truncated amino terminus, or variants with up to about 20%, about 30%, or about 40% change in amino acid composition, as long as the tropism moiety still binds to the TrkB receptor with high affinity.
  • the tropism moiety comprises an NGF that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical with the sequence of SEQ ID NO: 75, or a truncated version thereof.
  • the tropism moiety comprises neurotrophin-3 (NT-3).
  • the NT-3 is a variant of native NT-3, such as a truncated variant.
  • the tropism moiety comprises the full length 119 amino acid sequence of NT-3 (YAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRC RKIGRT; SEQ ID NO: 76).
  • the tropism moiety comprises a carboxy-truncated variant of the native NT-3, e.g., a variant in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acids are absent from the carboxy-terminus of NT-3.
  • NT-3 variants include the complete 119 amino acid NT-3, the shorter amino acid variants with a truncated carboxyl terminus, variants with a truncated amino terminus, or variants with up to about 20%, about 30%, or about 40% change in amino acid composition, as long as the tropism moiety still binds to the TrkC receptor with high affinity.
  • the tropism moiety comprises an NT-3 that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical with the sequence of SEQ ID NO: 76, or a truncated version thereof.
  • the tropism moiety comprises neurotrophin-4 (NT-4).
  • the NT-4 is a variant of native NT-4, such as a truncated variant.
  • the tropism moiety comprises the full length 130 amino acid sequence of NT-4 WRWIRIDTACVCTLLSRTGRA; SEQ ID NO: 77).
  • the tropism moiety comprises a carboxy-truncated variant of the native NT-4, e.g., a variant in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acids are absent from the carboxy-terminus of NT-4.
  • NT- 4 variants include the complete 130 amino acid NT-4, the shorter amino acid variants with a truncated carboxyl terminus, variants with a truncated amino terminus, or variants with up to about 20%, about 30%, or about 40% change in amino acid composition, as long as the tropism moiety still binds to the TrkB receptor with high affinity.
  • the tropism moiety comprises an NT-4 that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical with the sequence of SEQ ID NO: 77, or a truncated version thereof.
  • NGF region 25-36 structure/function relationship studies of NGF and NGF-related recombinant molecules demonstrated that mutations in NGF region 25-36, along with other ⁇ -hairpin loop and non-loop regions, significantly influenced NGF/NGF-receptor interactions (Ibanez et al., EMBO J., 10, 2105-2110, (1991)). Small peptides derived from this region have been demonstrated to mimic NGF in binding to Mock receptor and affecting biological responses (LeSêt et al. J. Biol. Chem. 270, 6564-6569, 1995).
  • a tropism moiety of the present disclosure comprises such peptides.
  • Cyclic peptides have also been designed and synthesized to mimic the ⁇ -loop regions of NGF, BDNF, NT3 and NT-4/5.
  • cyclic peptides can have a three-dimensional structure, which binds to neurotrophin receptors under physiological conditions. All of these structural analogs of neurotrophins that bind to nerve cell surface receptors and are internalized can serve as the binding agent B of the compound according to the present disclosure to deliver the conjugated therapeutic moiety TM to the nervous system. Accordingly, in some aspects, a tropism moiety of the present disclosure comprises such cyclic peptides or combinations thereof. [0415] In some aspects, antibodies against nerve cell surface receptors that are capable of binding to the receptors and being internalized can also serve as tropism moieties binding to a Trk receptor.
  • monoclonal antibody (MAb) 5C3 is specific for the NGF docking site of the human p140 TrkA receptor, with no cross-reactivity with human TrkB receptor.
  • MAb 5C3 and its Fab mimic the effects of NGF in vitro, and image human Trk-A positive tumors in vivo (Kramer et al., Eur. J. Cancer, 33, 2090-2091, (1997)).
  • Molecular cloning, recombination, mutagenesis and modeling studies of Mab 5C3 variable region indicated that three or less of its complementarity determining regions (CDRs) are relevant for binding to TrkA.
  • the target moiety comprises a neurotrophin selected from the group consisting of fibroblast growth factor (FGF)-2 and other FGFs, erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-a, TGF-(3, vascular endothelial growth factor (VEGF), interleukin-1 receptor antagonist (IL- lra), ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), neurturin, platelet- derived growth factor (PDGF), heregulin, neuregulin, artemin, persephin, interleukins, granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF), midlcine, pleiotrophin, bone morphogenetic proteins (BMPs), net
  • the tropism moiety directing an EV disclosed herein to a sensory neuron comprises a varicella zoster virus (VZV) peptide.
  • VZV varicella zoster virus
  • Tropism moieties targeting motor neurons In some aspects, a tropism moiety disclosed herein can direct an EV disclosed herein to a motor neuron. In some aspects, the tropism moiety that directs an EV disclosed herein to a motor comprises a Rabies Virus Glycoprotein (RVG) peptide, a Targeted Axonal Import (TAxI) peptide, a P75R peptide, or a Tet-C peptide.
  • RVG Rabies Virus Glycoprotein
  • TxI Targeted Axonal Import
  • the tropism moiety comprises a Rabies Virus Glycoprotein (RVG) peptide.
  • RVG peptide comprises amino acid residues 173-202 of the RVG (Y ; SEQ ID NO:78) or a variant, fragment, or derivative thereof.
  • the tropism moiety is a fragment of SEQ ID NO:78. Such a fragment of SEQ ID NO:78 can have, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted from the N-terminal and/or the C-terminal of SEQ ID NO:78.
  • a functional fragment derived from SEQ ID NO:78 can be identified by sequentially deleting N- and/or C-terminal amino acids from SEQ ID NO:78 and assessing the function of the resulting peptide fragment, such as function of the peptide fragment to bind acetylcholine receptor and/or ability to transmit through the blood brain barrier.
  • the tropism moiety comprises a fragment of SEQ ID NO:34628, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16 or 15 amino acids in length.
  • the tropism moiety comprises a fragment of SEQ ID NO:78 less than 15 peptides in length.
  • a “variant” of a RGV peptide for example SEQ ID NO:78, is meant to refer to a molecule substantially similar in structure and function, i.e., where the function is the ability to pass or transit through the BBB, to either the entire molecule, or to a fragment thereof.
  • a variant of an RVG peptide can contain a mutation or modification that differs from a reference amino acid in SEQ ID NO:78.
  • a variant of SEQ ID NO:78 is a fragment of SEQ ID NO:78 as disclosed herein.
  • an RVG variant can be a different isoform of SEQ ID NO:78 or can comprise different isomer amino acids.
  • Variants can be naturally- occurring, synthetic, recombinant, or chemically modified polynucleotides or polypeptides isolated or generated using methods well known in the art.
  • RVG variants can include conservative or non-conservative amino acid changes. See, e.g., U.S. Pat. No.9,757,470, which is herein incorporated by reference in its entirety.
  • the tropism moiety comprises a Targeted Axonal Import (TAxI) peptide.
  • the TAxI peptide is cyclized TAxI peptide of sequence SACQSQSQMRCGGG (SEQ ID NO:79). See, e.g., Sellers et al. (2016) Proc.
  • TAxI transport peptides as described herein can be of any length. Typically, the transport peptide will be between 6 and 50 amino acids in length, more typically between 10 and 20 amino acids in length. In some aspects, the TAxI transport peptide comprises the amino acid sequence QSQSQMR (SEQ ID NO: 80), ASGAQAR (SEQ ID NO: 81), PF, or TSTAPHLRLRLTSR (SEQ ID NO: 82).
  • the TAxI transport peptide further includes a flanking sequence to facilitate incorporation into a delivery construct or carrier, e.g., a linker.
  • the peptide is flanked with cysteines.
  • the TAxI transport peptide further comprises additional sequence selected to facilitate delivery into nuclei.
  • a peptide that facilitates nuclear delivery is a nuclear localizing signal (NLS).
  • NLS nuclear localizing signal
  • this signal consists of a few short sequences of positively charged lysines or arginines, such as PPKKRKV (SEQ ID NO: 83).
  • the NLS has the amino acid sequence PKKRKV (SEQ ID NO: 84).
  • a tropism moiety of the present disclosure comprises a peptide BBB shuttle disclosed in the table below. See, e.g., Oller-Salvia et al. (2016) Chem. Soc. Rev. 45, 4690-4707, and Jafari et al. (2019) Expert Opinion on Drug Delivery 16:583-605 which are herein incorporated by reference in their entireties. TABLE 7. Nomenclature for cyclic peptides (&) is adapted to the 3-letter amino acid code from the one described by Spengler et al . Pept. Res., 2005, 65, 550–555 [Dap] stands for diaminopropionic acid. III.G.
  • the EVs are administered by intrathecal administration, followed by application of a mechanical convective force to the torso.
  • a mechanical convective force to the torso.
  • certain aspects of the present disclosure are directed to methods of administering an EV to a subject in need thereof, comprising administering the EV to the subject by intrathecal injection, followed by applying a mechanical convective force to the torso of the subject.
  • the mechanical convective force is achieved using a high frequency chest wall or lumbothoracic oscillating respiratory clearance device (e.g., a Smart Vest or Smart Wrap, ELECTROMED INC, New Prague, MN, USA).
  • the mechanical convective force e.g., the oscillating vest, facilitates spread of the intrathecally dosed EVs further down the nerve thus allowing for better EV delivery to nerves.
  • the intra- and trans-compartmental biodistribution of exosomes can be manipulated by exogenous extracorporeal forces acting upon a subject after compartmental delivery of exosomes.
  • the application of chest wall vibrations by several means including an oscillating mechanical jacket can spread the biodistribution of exosomes along the neuraxis or along cranial and spinal nerves, which can be helpful in the treatment of nerve disorders by drug carrying exosomes.
  • the application of external mechanical convective forces via an oscillating jacket or other similar means can be used to remove exosomes and other material from the cerebrospinal fluid of the intrathecal space and out to the peripheral circulation.
  • exosomes delivered via the intracebroventricular route can be made to translocate throughout the neuraxis by simultaneously incorporating a lumbar puncture and allowing for ventriculo-lumbar perfusion wherein additional fluid is infused into the ventricles after exosome dosing, while allowing the existing neuraxial column of CSF to exit is the lumbar puncture.
  • Ventriculo-lumbar perfusion can allow ICV dosed exosome to spread along the entire neuraxis and completely cover the subarachnoid space in order to treat leptomeningeal cancer and other diseases.
  • the application of external extracorporeal focused ultrasound, thermal energy (heat) or cold can be used to manipulate the compartmental pharmacokinetics and drug release properties of exosomes engineered to be sensitive to these phenomena.
  • the intracompartmental behavior and biodistribution of exosomes engineered to contain paramagnetic material can be manipulated by the external application of magnets or a magnetic field. IV.
  • EVs of the present disclosure can be produced from a cell grown in vitro or a body fluid of a subject.
  • various producer cells e.g., HEK293 cells, CHO cells, and MSCs, can be used.
  • a producer cell is not a dendritic cell, macrophage, B cell, mast cell, neutrophil, Kupffer-Browicz cell, cell derived from any of these cells, or any combination thereof.
  • Human embryonic kidney 293 cells also often referred to as HEK 293, HEK- 293, 293 cells, or less precisely as HEK cells, are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture.
  • HEK 293 cells were generated in 1973 by transfection of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA in Alex van der Eb's laboratory in Leiden, the Netherlands. The cells were cultured and transfected by adenovirus. Subsequent analysis has shown that the transformation was brought about by inserting ⁇ 4.5 kilobases from the left arm of the viral genome, which became incorporated into human chromosome 19.
  • HEK 293 cells have a complex karyotype, exhibiting two or more copies of each chromosome and with a modal chromosome number of 64. They are described as hypotriploid, containing less than three times the number of chromosomes of a haploid human gamete.
  • Chromosomal abnormalities include a total of three copies of the X chromosome and four copies of chromosome 17 and chromosome 22.
  • Variants of HEK293 cells useful to produce EVs include, but are not limited to, HEK 293F, HEK 293FT, and HEK 293T.
  • the producer cell can be genetically modified to comprise exogenous sequences encoding an ASO to produce EVs described herein.
  • the genetically-modified producer cell can contain the exogenous sequence by transient or stable transformation.
  • the exogenous sequence can be transformed as a plasmid.
  • the exogenous sequence is a vector.
  • the exogenous sequences can be stably integrated into a genomic sequence of the producer cell, at a targeted site or in a random site. In some aspects, a stable cell line is generated for production of lumen-engineered exosomes. [0437]
  • the exogenous sequences can be inserted into a genomic sequence of the producer cell, located within, upstream (5’-end) or downstream (3’-end) of an endogenous sequence encoding an exosome protein.
  • Various methods known in the art can be used for the introduction of the exogenous sequences into the producer cell.
  • exogenous sequences can comprise a sequence encoding a scaffold moiety disclosed herein or a fragment or variant thereof.
  • An extra copy of the sequence encoding a scaffold moiety can be introduced to produce an exosome described herein (e.g., having a higher density of a scaffold moiety on the surface or on the luminal surface of the EV).
  • An exogenous sequence encoding a modification or a fragment of a scaffold moiety can be introduced to produce a lumen-engineered and/or surface-engineered exosome containing the modification or the fragment of the scaffold moiety.
  • a producer cell can be modified, e.g., transfected, with one or more vectors encoding a scaffold moiety linked to an ASO.
  • EVs of the present disclosure e.g., surface-engineered and/or lumen-engineered exosomes
  • compositions comprising an EV of the present disclosure having the desired degree of purity, and a pharmaceutically acceptable carrier or excipient, in a form suitable for administration to a subject.
  • Pharmaceutically acceptable excipients or carriers can be determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions comprising a plurality of extracellular vesicles.
  • a pharmaceutical composition comprises one or more therapeutic agents and an exosome described herein.
  • the EVs are co- administered with one or more additional therapeutic agents in a pharmaceutically acceptable carrier.
  • the ASO and the one or more additional therapeutic agents for the present disclosure can be administered in the same EV.
  • the ASO and the one or more additional therapeutic agents for the present disclosure are administered in different EVs.
  • the present disclosure includes a pharmaceutical composition comprising an EV comprising an ASO and an EV comprising an additional therapeutic agent.
  • the pharmaceutical composition comprising the EV is administered prior to administration of the additional therapeutic agent(s).
  • the pharmaceutical composition comprising the EV is administered after the administration of the additional therapeutic agent(s).
  • the pharmaceutical composition comprising the EV is administered concurrently with the additional therapeutic agent(s).
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine,
  • Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin.
  • the use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the extracellular vesicles described herein, use thereof in the compositions is contemplated. Supplementary therapeutic agents can also be incorporated into the compositions.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • the EVs can be administered by intrathecal, parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intratumoral, intramuscular route or as inhalants.
  • the pharmaceutical composition comprising exosomes is administered intravenously, e.g. by injection.
  • the EVs can optionally be administered in combination with other therapeutic agents that are at least partly effective in treating the disease, disorder or condition for which the EVs are intended.
  • Solutions or suspensions can include the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition is generally sterile and fluid to the extent that easy syringeability exists.
  • the carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic compounds e.g., sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride can be added to the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g., aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the EVs in an effective amount and in an appropriate solvent with one or more ingredients enumerated herein or known in the art, as desired.
  • dispersions are prepared by incorporating the EVs into a sterile vehicle that contains a basic dispersion medium and any desired other ingredients.
  • compositions comprising exosomes can also be by transmucosal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • the pharmaceutical composition comprising EVs is administered intravenously into a subject that would benefit from the pharmaceutical composition.
  • the pharmaceutical composition comprising EVs is administered intrathecally into a subject that would benefit from the pharmaceutical composition.
  • the pharmaceutical composition comprising EVs is administered perineurally into a subject that would benefit from the pharmaceutical composition.
  • the pharmaceutical composition comprising EVs is administered intraneurally into a subject that would benefit from the pharmaceutical composition.
  • the pharmaceutical composition comprising exosomes is administered as a liquid suspension.
  • the pharmaceutical composition is administered as a formulation that is capable of forming a depot following administration.
  • the depot slowly releases the EVs into circulation, or remains in depot form.
  • pharmaceutically-acceptable compositions are highly purified to be free of contaminants, are biocompatible and not toxic, and are suited to administration to a subject. If water is a constituent of the carrier, the water is highly purified and processed to be free of contaminants, e.g., endotoxins.
  • the pharmaceutically-acceptable carrier can be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and/or mineral oil, but is not limited thereto.
  • the pharmaceutical composition can further include a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and/or a preservative.
  • the pharmaceutical compositions described herein comprise a pharmaceutically acceptable salt.
  • the pharmaceutically acceptable salt comprises a sodium salt, a potassium salt, an ammonium salt, or any combination thereof.
  • the pharmaceutical compositions described herein comprise the EVs described herein and optionally an additional pharmaceutically active or therapeutic agent.
  • the additional therapeutic agent can be a biological agent, a small molecule agent, or a nucleic acid agent.
  • the additional therapeutic agent is an additional PMP22 antagonist.
  • the PMP22 antagonist is any PMP22 antagonist disclosed herein.
  • the additional PMP22 antagonist is an anti-PMP22 antibody.
  • the additional PMP22 antagonist is a small molecule.
  • the additional PMP22 antagonist is a chemical compound, an siRNA, an shRNA, an antisense oligonucleotide, a protein, or any combination thereof.
  • the PMP22 antagonist is a progesterone antagonist.
  • the progesterone antagonist reduces the expression of PMP22.
  • the progesterone antagonist is a progesterone receptor antagonist.
  • the progesterones receptor antagonist is a steroidal antiprogestogen.
  • the progesterone receptor antagonist selected from the group consisting of onapristone, aglepristone, lilopristone, telapristone, toripristone, mifepristone, ulipristal cetate, or any combination thereof.
  • the progesterone antagonist is onapristone (11 ⁇ -(4- (dimethylamino)phenyl)-17 ⁇ -hydroxy-17 ⁇ -(3-hydroxypropyl)-13 ⁇ -estra-4,9-dien-3-one)
  • the additional PMP22 antagonist comprises an ASO.
  • the additional PMP22 antagonist comprises any ASO described herein.
  • Dosage forms are provided that comprise a pharmaceutical composition comprising the EVs described herein.
  • the dosage form is formulated as a liquid suspension for intravenous injection.
  • the dosage form is formulated as a liquid suspension for intrathecal administration, e.g., intrathecal injection.
  • the preparation of exosomes is subjected to radiation, e.g., X rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, UV rays in order to damage residual replication-competent nucleic acids.
  • the preparation of exosomes is subjected to gamma irradiation using an irradiation dose of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more than 100 kGy.
  • the preparation of exosomes is subjected to X-ray irradiation using an irradiation dose of more than 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or greater than 10000 mSv.
  • kits comprising one or more exosomes described herein.
  • kits comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more exosomes provided herein, optional an instruction for use.
  • the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein.
  • the kit further comprises instructions to administer the EV according to any method disclosed herein.
  • the kit is for use in the treatment of a disease or condition associated with hematopoiesis.
  • the kit is a diagnostic kit. VII. Methods of Producing EVs [0462]
  • the present disclosure is also directed to methods of producing EVs described herein.
  • the method comprises: obtaining the EV from a producer cell, wherein the producer cell contains one or more components of the EV (e.g., an ASO); and optionally isolating the obtained EV.
  • the method comprises: modifying a producer cell by introducing one or more components of an EV disclosed herein (e.g., an ASO); obtaining the EV from the modified producer cell; and optionally isolating the obtained EV.
  • the method comprises: obtaining an EV from a producer cell; isolating the obtained EV; and modifying the isolated EV.
  • the method further comprises formulating the isolated EV into a pharmaceutical composition. VII.A.
  • a method of producing an EV comprises modifying a producer cell with one or more moieties (e.g., an ASO).
  • the one or more moieties comprise an ASO.
  • the one or more moieties further comprise a scaffold moiety disclosed herein (e.g., Scaffold X or Scaffold Y).
  • the producer cell can be a mammalian cell line, a plant cell line, an insect cell line, a fungi cell line, or a prokaryotic cell line.
  • the producer cell is a mammalian cell line.
  • Non-limiting examples of mammalian cell lines include: a human embryonic kidney (HEK) cell line, a Chinese hamster ovary (CHO) cell line, an HT- 1080 cell line, a HeLa cell line, a PERC-6 cell line, a CEVEC cell line, a fibroblast cell line, an amniocyte cell line, an epithelial cell line, a mesenchymal stem cell (MSC) cell line, and combinations thereof.
  • HEK human embryonic kidney
  • CHO Chinese hamster ovary
  • the mammalian cell line comprises HEK-293 cells, BJ human foreskin fibroblast cells, fHDF fibroblast cells, AGE.HN ® neuronal precursor cells, CAP ® amniocyte cells, adipose mesenchymal stem cells, RPTEC/TERT1 cells, or combinations thereof.
  • the producer cell is a primary cell.
  • the primary cell can be a primary mammalian cell, a primary plant cell, a primary insect cell, a primary fungi cell, or a primary prokaryotic cell.
  • the producer cell is not an immune cell, such as an antigen presenting cell, a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a T helper cell, or a regulatory T cell (Treg cell).
  • the producer cell is not an antigen presenting cell (e.g., dendritic cells, macrophages, B cells, mast cells, neutrophils, Kupffer-Browicz cell, or a cell derived from any such cells).
  • the one or more moieties can be a transgene or mRNA, and introduced into the producer cell by transfection, viral transduction, electroporation, extrusion, sonication, cell fusion, or other methods that are known to the skilled in the art.
  • the one or more moieties is introduced to the producer cell by transfection.
  • the one or more moieties can be introduced into suitable producer cells using synthetic macromolecules, such as cationic lipids and polymers (Papapetrou et al., Gene Therapy 12: S118-S130 (2005)).
  • the cationic lipids form complexes with the one or more moieties through charge interactions.
  • the positively charged complexes bind to the negatively charged cell surface and are taken up by the cell by endocytosis.
  • a cationic polymer can be used to transfect producer cells.
  • the cationic polymer is polyethylenimine (PEI).
  • chemicals such as calcium phosphate, cyclodextrin, or polybrene, can be used to introduce the one or more moieties to the producer cells.
  • the one or more moieties can also be introduced into a producer cell using a physical method such as particle-mediated transfection, "gene gun", biolistics, or particle bombardment technology (Papapetrou et al., Gene Therapy 12: S118-S130 (2005)).
  • a reporter gene such as, for example, beta- galactosidase, chloramphenicol acetyltransferase, luciferase, or green fluorescent protein can be used to assess the transfection efficiency of the producer cell.
  • the one or more moieties are introduced to the producer cell by viral transduction.
  • viruses can be used as gene transfer vehicles, including moloney murine leukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), lentiviruses, and spumaviruses.
  • the viral mediated gene transfer vehicles comprise vectors based on DNA viruses, such as adenovirus, adeno-associated virus and herpes virus, as well as retroviral based vectors.
  • the one or more moieties are introduced to the producer cell by electroporation. Electroporation creates transient pores in the cell membrane, allowing for the introduction of various molecules into the cell.
  • DNA and RNA as well as polypeptides and non-polypeptide therapeutic agents can be introduced into the producer cell by electroporation.
  • a glass micropipette can be used to inject the one or more moieties into the producer cell at the microscopic level.
  • the one or more moieties are introduced to the producer cell by extrusion.
  • the one or more moieties are introduced to the producer cell by sonication.
  • the producer cell is exposed to high intensity sound waves, causing transient disruption of the cell membrane allowing loading of the one or more moieties.
  • the one or more moieties are introduced to the producer cell by cell fusion.
  • the one or more moieties are introduced by electrical cell fusion.
  • PEG polyethylene glycol
  • sendai virus is used to fuse the producer cells.
  • the one or more moieties are introduced to the producer cell by hypotonic lysis.
  • the producer cell can be exposed to low ionic strength buffer causing them to burst allowing loading of the one or more moieties.
  • controlled dialysis against a hypotonic solution can be used to swell the producer cell and to create pores in the producer cell membrane. The producer cell is subsequently exposed to conditions that allow resealing of the membrane.
  • the one or more moieties are introduced to the producer cell by detergent treatment.
  • producer cell is treated with a mild detergent which transiently compromises the producer cell membrane by creating pores allowing loading of the one or more moieties. After producer cells are loaded, the detergent is washed away thereby resealing the membrane.
  • the one or more moieties introduced to the producer cell by receptor mediated endocytosis.
  • producer cells have a surface receptor which upon binding of the one or more moieties induces internalization of the receptor and the associated moieties.
  • the one or more moieties are introduced to the producer cell by filtration.
  • the producer cells and the one or more moieties can be forced through a filter of pore size smaller than the producer cell causing transient disruption of the producer cell membrane and allowing the one or more moieties to enter the producer cell.
  • the producer cell is subjected to several freeze thaw cycles, resulting in cell membrane disruption allowing loading of the one or more moieties.
  • VII.B. Methods of Modifying EV [0479] In some aspects, a method of producing an EV comprises modifying the isolated EV by directly introducing one or more moieties into the EVs. In certain aspects, the one or more moieties comprise an ASO.
  • the one or more moieties comprise a scaffold moiety disclosed herein (e.g., Scaffold X or Scaffold Y).
  • the one or more moieties are introduced to the EV by transfection.
  • the one or more moieties can be introduced into the EV using synthetic macromolecules such as cationic lipids and polymers (Papapetrou et al., Gene Therapy 12: S118-S130 (2005)).
  • chemicals such as calcium phosphate, cyclodextrin, or polybrene, can be used to introduce the one or more moieties to the EV.
  • the one or more moieties are introduced to the EV by electroporation.
  • EVs are exposed to an electrical field which causes transient holes in the EV membrane, allowing loading of the one or more moieties.
  • the one or more moieties are introduced to the EV by microinjection.
  • a glass micropipette can be used to inject the one or more moieties directly into the EV at the microscopic level.
  • the one or more moieties are introduced to the EV by extrusion.
  • the one or more moieties are introduced to the EV by sonication.
  • EVs are exposed to high intensity sound waves, causing transient disruption of the EV membrane allowing loading of the one or more moieties.
  • one or more moieties can be conjugated to the surface of the EV. Conjugation can be achieved chemically or enzymatically, by methods known in the art.
  • the EV comprises one or more moieties that are chemically conjugated. Chemical conjugation can be accomplished by covalent bonding of the one or more moieties to another molecule, with or without use of a linker. The formation of such conjugates is within the skill of artisans and various techniques are known for accomplishing the conjugation, with the choice of the particular technique being guided by the materials to be conjugated.
  • polypeptides are conjugated to the EV.
  • non- polypeptides such as lipids, carbohydrates, nucleic acids, and small molecules, are conjugated to the EV.
  • the one or more moieties are introduced to the EV by hypotonic lysis.
  • the EVs can be exposed to low ionic strength buffer causing them to burst allowing loading of the one or more moieties.
  • controlled dialysis against a hypotonic solution can be used to swell the EV and to create pores in the EV membrane. The EV is subsequently exposed to conditions that allow resealing of the membrane.
  • the one or more moieties are introduced to the EV by detergent treatment.
  • extracellular vesicles are treated with a mild detergent which transiently compromises the EV membrane by creating pores allowing loading of the one or more moieties. After EVs are loaded, the detergent is washed away thereby resealing the membrane.
  • the one or more moieties are introduced to the EV by receptor mediated endocytosis.
  • EVs have a surface receptor which upon binding of the one or more moieties induces internalization of the receptor and the associated moieties.
  • the one or more moieties are introduced to the EV by mechanical firing.
  • extracellular vesicles can be bombarded with one or more moieties attached to a heavy or charged particle such as gold microcarriers. In some of these aspects, the particle can be mechanically or electrically accelerated such that it traverses the EV membrane. [0491] In some aspects, extracellular vesicles are subjected to several freeze thaw cycles, resulting in EV membrane disruption allowing loading of the one or more moieties. VII.C. Methods of Isolating EV [0492] In some aspects, methods of producing EVs disclosed herein comprises isolating the EV from the producer cells. In certain aspects, the EVs released by the producer cell into the cell culture medium.
  • EVs are deemed suitable for use herein.
  • physical properties of EVs can be employed to separate them from a medium or other source material, including separation on the basis of electrical charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieving, etc.), density (e.g., regular or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external force, etc.).
  • electrical charge e.g., electrophoretic separation
  • size e.g., filtration, molecular sieving, etc.
  • density e.g., regular or gradient centrifugation
  • Svedberg constant e.g., sedimentation with or without external force, etc.
  • isolation can be based on one or more biological properties, and include methods that can employ surface markers (e.g., for precipitation, reversible binding to solid phase, FACS separation, specific ligand binding, non- specific ligand binding, affinity purification etc.).
  • surface markers e.g., for precipitation, reversible binding to solid phase, FACS separation, specific ligand binding, non- specific ligand binding, affinity purification etc.
  • Isolation and enrichment can be done in a general and non-selective manner, typically including serial centrifugation.
  • isolation and enrichment can be done in a more specific and selective manner, such as using EV or producer cell-specific surface markers.
  • specific surface markers can be used in immunoprecipitation, FACS sorting, affinity purification, and magnetic separation with bead-bound ligands.
  • size exclusion chromatography can be utilized to isolate the EVs. Size exclusion chromatography techniques are known in the art. Exemplary, non-limiting techniques are provided herein.
  • a void volume fraction is isolated and comprises the EVs of interest.
  • the EVs can be further isolated after chromatographic separation by centrifugation techniques (of one or more chromatography fractions), as is generally known in the art.
  • density gradient centrifugation can be utilized to further isolate the extracellular vesicles.
  • the producer cell-derived EVs can be separated from non-producer cell-derived EVs by immunosorbent capture using an antigen antibody specific for the producer cell.
  • the isolation of EVs can involve combinations of methods that include, but are not limited to, differential centrifugation, size-based membrane filtration, immunoprecipitation, FACS sorting, and magnetic separation.
  • the practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed.
  • the present disclosure provides methods of preventing and/or treating a disease or disorder in a subject in need thereof, comprising administering an EV (e.g., exosome) disclosed herein (e.g., comprising an ASO of the present disclosure) to the subject.
  • an EV e.g., exosome
  • ASOs useful for the present disclosure can specifically hybridize to one or more regions of a PMP22 transcript (e.g., pre-mRNA or mRNA), resulting in reduction and/or inhibition of PMP22 protein expression in a cell.
  • EVs e.g., exosomes
  • ASO e.g., EVs disclosed herein
  • a disease or disorder that can be treated with the present methods comprises a neuropathy.
  • the cancer is associated with increased expression of a PMP22 protein.
  • Neuropathies that can be treated with the present disclosure include Charcot-Marie-Tooth disease 1A (CMT1A).
  • CMT1A Charcot-Marie-Tooth disease 1A
  • Alterations of PMP22 gene expression are associated with a variety of neuropathies.
  • CMT1A Charcot- Marie-Tooth disease 1A
  • Charcot-Marie-Tooth disease is dominant demyelinating form of Charcot-Marie-Tooth disease, a disorder of the peripheral nervous system, characterized by progressive weakness and atrophy, initially of the peroneal muscles and later of the distal muscles of the arms.
  • Charcot-Marie-Tooth disease is classified in two main groups on the basis of electrophysiologic properties and histopathology: primary peripheral demyelinating neuropathies (designated CMT1 when they are dominantly inherited).
  • Demyelinating neuropathies such as CMT1A are characterized by severely reduced nerve conduction velocities (less than 38 m/sec), segmental demyelination and remyelination with onion bulb formations on nerve biopsy, slowly progressive distal muscle atrophy and weakness, absent deep tendon reflexes, and hollow feet.
  • PMP22 mutations associated with CMT1A include L16P, S22F, ⁇ 25-26, D37V, V65F, S72L, S79C, G93R, L105R, G107V, T118N, L147R.
  • the EVs are administered to the subject by intrathecal administration.
  • the EVs are administered via an injection into the spinal canal, or into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • the EVs of the present disclosure comprising at least one targeting moiety that can direct the exosome and its payload (e.g., an ASO that specifically targets PMP22) to a specific target cell or tissue (e.g., Schwann cells in peripheral nerves) can be administered using any suitable administration method known in the art (e.g., intravenous injection or infusion) since the presence of the targeting moiety will induce a tropism of the exosomes towards the target cell or tissue.
  • Selection Method 1 (see FIGS.1 and 2): Selection criteria were (i) top specific ASOs in human, cynomolgus and rhesus monkeys (0 hits with 0, 1, or 2 mismatches for 20- mers, 0 hits with 0 or 1 mismatches for 16- and 17-mers, 0 hits with 0 mismatches and ⁇ 101 mismatches for 15-mers), (ii) cross reactive with relevant human, cynomolgus monkey and rhesus monkey target transcripts, and (iii) do not match target sites in human mRNA harboring SNPs with a MAF ⁇ 1%.
  • Selection Method 2 (see FIGS.3 and 4): Selection criteria were (i) top specific ASOSs in human (0 hits with 0, 1, or 2 mismatches for 20-mers, 0 hits with 0 or 2 mismatches and ⁇ 102 mismatches for 17-mer, 0 hits with 0, 1 mismatches for 15- and 16-mers), and (ii) do not match target sites in human mRNAs harboring SNPs with a MAF ⁇ 1%.
  • FIG. 4 shows that there are multiple naturally occurring PMP22 transcripts. There are multiple transcription start sites which are cell type specific.
  • the PMP22 protein coding sequence is identical among all transcripts.
  • FIG.4 also show the location of exons (1A, 1B, 2, 3, 4, 5).
  • Selection Method 3 (see FIGS. 4 and 5): An additional set of ASOs was selected, all of them located in exon 1A. Selection criteria were (i) top specific ASOs in human (0 hits with 0, 1, or 2 mismatches, maximum of 5 hits with 3 mismatches), (ii) number of toxic motifs (less than 6 toxic motifs AA, AG, TA, GG, or GA), and (iii) do not match target sites in human target sites in human mRNAs harboring SNPs with a MAF ⁇ 1%.
  • ASOs selected according to the method described above were tested in vitro in HEK293 cells at 15,000 cells/well. ASOs were introduced in the cells using Lipofectamine 2000 (0.5ul/well) for 24 hours.2nM and 20 nM ASO doses were used, with 4 replicated well per dose.
  • FIG.6 shows the results of this test, and the box in FIG.6 indicates which ASOs were selected for further testing.
  • FIG. 7 compares the results obtained when administered ASO at 2nM and at 20 nM.
  • the box in FIG.7 indicates also the ASOs that were selected for further testing.
  • ASOs those with >80% inhibition of PMP22 and ⁇ 20% inhibition of GAPDH at one of the doses tested were selected. See FIG. 8.
  • Species cross- reactivity (FIG.9) and KD activity (FIG.10) were evaluated and a subset of the best performing ASOs comprising 16 ASOs was selected for dose response analysis (see FIG.11).
  • the top two ASOs, corresponding to SEQ ID NO: 146 and SEQ ID NO: 157 had IC50 values of approximately 50 pM, and were cynomolgus monkey, rhesus monkey, mouse and rat cross reactive as determined via sequence alignment.
  • Example 2 In vitro analysis of mRNA and/or protein reduction
  • the disclosed ASOs will be tested for their ability to knockdown PMP22 mRNA and/or PMP22 protein expression in reporter cell lines, e.g., the Panc-1 cell line.
  • Experimental data presented in FIG.14 confirmed the presence of acceptable levels of PMP22 expression in Panc-1, which are therefore a suitable in vitro model for screening and characterization of ASOs targeting PMP22.
  • the reporter cell lines expressing PMP22 e.g., Panc-1 are in cell culture media and seeded onto a 96 well plate.
  • EVs e.g., exosomes
  • EV-ASO e.g., exosomes
  • Methods for producing such EVs are provided elsewhere in the present disclosure.
  • Approximately 3 days after EV-ASO treatment the cells are harvested and RNA and/or protein is purified from the cells.
  • the PMP22 mRNA and/or PMP22 protein expression levels in the cells are quantified using assays such as, qPCR and Western blot.
  • Example 3 Construction of an Exosome [0514] To generate exosomes described herein, human embryonic kidney (HEK) cell line (e.g., HEK293SF) are used.
  • HEK human embryonic kidney
  • the cells are stably transfected with Scaffold X, Scaffold Y, and/or anchoring moiety linked to an agent of interest.
  • HEK cells are grown to high density in chemically defined medium for 7 days. Conditioned cell culture media is then collected and centrifuged at 300 – 800 x g for 5 minutes at room temperature to remove cells and large debris. Media supernatant is supplemented with 1000 U/L BENZONASE ® and incubated at 37 °C for 1 hour in a water bath. Supernatant is collected and centrifuged at 16,000 x g for 30 minutes at 4 °C to remove residual cell debris and other large contaminants.
  • Supernatant is then ultracentrifuged at 133,900 x g for 3 hours at 4 °C to pellet the exosomes. Supernatant is discarded and any residual media is aspirated from the bottom of the tube.
  • the pellet is resuspended in 200 – 1000 ⁇ L PBS (-Ca -Mg).
  • the pellet is processed via density gradient purification (sucrose or OPTIPREP TM ).
  • the gradient is spun at 200,000 x g for 16 hours at 4 °C in a 12 mL Ultra-Clear (344059) tube placed in a SW 41 Ti rotor to separate the exosome fraction.
  • the exosome layer is then gently removed from the top layer and diluted in ⁇ 32.5 mL PBS in a 38.5 mL Ultra-Clear (344058) tube and ultracentrifuged again at 133,900 x g for 3 hours at 4 °C to pellet the purified exosomes. The resulting pellet is resuspended in a minimal volume of PBS ( ⁇ 200 ⁇ L) and stored at 4 °C.
  • OPTIPREP TM gradient a 3-tier sterile gradient is prepared with equal volumes of 10%, 30%, and 45% OPTIPREP TM in a 12 mL Ultra-Clear (344059) tube for a SW 41 Ti rotor.
  • the pellet is added to the OPTIPREP TM gradient and ultracentrifuged at 200,000 x g for 16 hours at 4 °C to separate the exosome fraction.
  • the exosome layer is then gently collected from the top ⁇ 3 mL of the tube.
  • the exosome fraction is diluted in ⁇ 32 mL PBS in a 38.5 mL Ultra-Clear (344058) tube and ultracentrifuged at 133,900 x g for 3 hours at 4 °C to pellet the purified exosomes.
  • the pelleted exosomes is then resuspended in a minimal volume of PBS ( ⁇ 200 ⁇ L) and stored at 4°C until ready to be used.
  • Example 4 Construction and Characterization of Exosomes Expressing CD47 [0521]
  • various constructs were created to express human CD47 or a fragment thereof on the surface of exosomes.
  • the extracellular domain of human wild type CD47, having a C15S substitution, or Velcro-CD47 was fused to Scaffold X or a fragment thereof and expressed in exosome-producing cells (FIGs.15A-15B).
  • exosomes were produced expressing a modified CD47 having a truncated Scaffold X protein inserted in the first domain of human wild type CD47, having a C15S substitution, or Velcro-CD47 (FIG.15C).
  • Exosomes were generated expressing a minimal "self" peptide (GNYTCEVTELTREGETIIELK; SEQ ID NO: 102) fused to Scaffold X or a fragment thereof (FIG. 15D; see, e.g., Rodriguez et al., Science 339:971-75 (Feb.2013)).
  • Exosomes expressing each construct were assayed for CD47 expression by ELISA using an anti-CD47 antibody targeted to a specific epitope of CD47 or by binding to SIRP ⁇ using a SIRP ⁇ (human) signaling reporter cell bioassay (DiscoverX) or using Octet analysis.
  • Exosomes comprising the ASOs of the present disclosure can be targeted to cells expressing transferrin receptor on their surface.
  • Western blots using a monoclonal antibody against human transferrin receptor showed that transferrin is expressed in cells lines such as Panc-1, HepG2, Hep3B2.1-7, Neuro- 2A, HeLa, HEK293, Sol8, and C2C12, as well as in human and murine primary Schwann cell cultures (FIG.18 and FIG.19).
  • Example 7 PMP22 Knockdown – ASO Exosome Delivery
  • X61832 i.e., a PMP22 ASO of sequence (SEQ ID NO: 146) was delivered to mouse Schwann cells using exosomes expressing mouse transferrin on their surfaces (exomTF exosomes).
  • FIG.28 shows that delivery of the PMP22 ASO using exomTF exosomes resulted in a significantly higher knockdown that the knockdown observed when the ASO was administeres without exosomes, or when the ASO was administering using exosomes that did not express transferrin.
  • Example 8 Panc-1 Cells as In Vitro Model for PMP22 ASO Screening [0528] In order to determine whether an established cell line could be used as a model system, as an alternative to the use of primary cultures of Schwann cells, levels of PMP22 were quantitated in a number of cell lines, as shown for example in FIG.18. The presence of high levels of PMP22 expression in Panc-1 was confirmed (see FIG. 29), suggesting that Panc-1 cells are a suitable model system to screen PMP22 ASOs, and other agents capable of modulating PMP22 expression levels.

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Abstract

La présente invention concerne des vésicules extracellulaires, par exemple des exosomes, comprenant un oligonucléotide antisens (ASO), l'ASO comprenant une séquence nucléotidique contiguë de 10 à 30 nucléotides de longueur qui est complémentaire à une séquence d'acide nucléique à l'intérieur d'un transcrit de protéine de myéline périphérique 22 (PMP22) . L'invention concerne également des procédés de production des exosomes et des procédés d'utilisation des exosomes pour traiter et/ou prévenir des maladies ou des troubles.
PCT/US2021/022433 2020-03-13 2021-03-15 Constructions de vésicules extracellulaires - aso ciblant pmp22 WO2021184021A1 (fr)

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WO2024013563A1 (fr) * 2022-07-12 2024-01-18 Potentia Therapeutics Ltd Administration ciblée dans les cellules de schwann et méthodes de traitement dans des maladies associées aux cellules de schwann
CN117024557A (zh) * 2023-10-10 2023-11-10 天津外泌体科技有限公司 14-3-3蛋白theta异构体作为细胞外囊泡支架蛋白的应用和细胞外囊泡
CN117024557B (zh) * 2023-10-10 2024-01-30 天津外泌体科技有限公司 14-3-3蛋白theta异构体作为细胞外囊泡支架蛋白的应用和细胞外囊泡

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