EXTRACELLULAR VESICLES WITH STAT3-ANTISENSE OLIGONUCLEOTIDES
The present disclosure relates to modified extracellular vesicles, e.g., exosomes, comprising an antisense oligonucleotide (ASO), which is capable of reducing and/or inhibiting expression of STAT3 mRNA and/or STAT3 protein. ASOs that can be used with the modified extracellular vesicles are also disclosed. Also provided herein are methods for using the exosomes and ASOs to treat and/or prevent diseases, such as cancer.
1 . An extracellular vesicle comprising an antisense oligonucleotide (ASO) which is an antagonist and comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within a STAT3 transcript (SEQ ID NO: 1 or SEQ ID NO: 3).
2 . The extracellular vesicle of claim 1 , wherein the ASO is not
(SEQ ID NO: 5)
TAAGCTGATAATTCAACTCA.
3 - 9 . (canceled)
10 . The extracellular vesicle of claim 1 , wherein the ASO is a gapmer, a mixmer, or a totalmer.
11 . The extracellular vesicle of claim 1 , wherein the ASO comprises one or more nucleoside analogs.
12 . (canceled)
13 . The extracellular vesicle of claim 11 , wherein one or more of the nucleoside analogs is a sugar modified nucleoside.
14 - 21 . (canceled)
22 . The extracellular vesicle of claim 1 , wherein the contiguous nucleotide sequence comprises a nucleotide sequence complementary to a sequence selected from the sequences in FIG. 1 : or wherein the ASO comprises a nucleotide sequence from SEQ ID Nos: 100-199, with one or two mismatches.
23 - 24 . (canceled)
25 . The extracellular vesicle of claim 1 , wherein the ASO has a design selected from the group consisting of the designs in FIG. 1 , wherein the upper case letter is a sugar modified nucleoside and the lower case letter is DNA.
26 - 30 . (canceled)
31 . The extracellular vesicle of claim 1 , which further comprises
(i) an anchoring moiety, wherein the STAT3 antagonist is linked to the anchoring moiety;
(ii) an exogenous targeting moiety; or
(iii) both (i) and (ii).
32 - 40 . (canceled)
41 . The extracellular vesicle of claim 31 , wherein the exogenous targeting moiety binds to CD33 or a fragment thereof.
42 - 75 . (canceled)
76 . The extracellular vesicle of claim 31 , wherein the anchoring moiety comprises sterol, GM1, a lipid, a vitamin, a small molecule, a peptide, or a combination thereof.
77 - 78 . (canceled)
79 . The extracellular vesicle of claim 31 , wherein the STAT3 ASO is linked to the anchoring moiety by a linker.
80 . (canceled)
81 . The extracellular vesicle of claim 79 , wherein the linker:
(i) is a polypeptide;
(ii) is a non-polypeptide moiety;
(iii) comprises ethylene glycol;
(iv) comprises acrylic phosphoramidite (e.g., Acrydite™), adenylation, azide (NHS Ester), digoxigenin (NHS Ester), cholesterol-TEG, I-LINKER™, an amino modifier (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, or Uni-Link™ 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;
(v) comprises valine-alanine-p-aminobenzylcarbamate or valine-citrulline-p-aminobenzylcarbamate; or
(vi) any combination thereof.
82 - 88 . (canceled)
89 . The extracellular vesicle of claim 1 , wherein the EV is an exosome.
90 . An antisense oligonucleotide (ASO) comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within nucleotides 1 to 56982 of a STAT3 transcript corresponding to SEQ ID NO: 1, nucleotides 57003 to 75171 of a STAT3 transcript corresponding to SEQ ID NO: 1, nucleotides 1 to 1287 of a STAT3 transcript corresponding to SEQ ID NO: 3, or nucleotides 1306 to 2787 of a STAT3 transcript corresponding to SEQ ID NO: 3.
91 - 111 . (canceled)
112 . A conjugate comprising the ASO of claim 90 , wherein the ASO is covalently attached to at least one non-nucleotide or non-polynucleotide moiety.
113 - 114 . (canceled)
115 . A pharmaceutical composition comprising the extracellular vesicle of claim 1 , and a pharmaceutically acceptable diluent, carrier, salt, or adjuvant.
116 - 120 . (canceled)
121 . A kit comprising the extracellular vesicle of claim 1 , and instructions for use.
122 . A diagnostic kit comprising the extracellular vesicle of claim 1 , and instructions for use.
123 . A method of inhibiting or reducing STAT3 protein expression in a cell, comprising administering the extracellular vesicle of claim 1 to the cell expressing STAT3 protein, wherein the STAT3 protein expression in the cell is inhibited or reduced after the administration.
124 - 126 . (canceled)
127 . A method of treating a cancer in a subject in need thereof, comprising administering an effective amount of the extracellular vesicle of claim 1 to the subject.
128 - 131 . (canceled)