CEBP / beta-targeting extracellular vesicle-ASO constructs

Abstract

The present disclosure relates to extracellular vesicles, 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 CEBP / beta transcript. Also provided herein are methods for producing the exosomes and methods for treating and / or preventing a disease or condition using the exosomes.

Description

Technical Field

[0001] This disclosure relates to extracellular vesicles (EVs), such as exosomes, that contain antisense oligonucleotides (ASOs) comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within the CEBP / β transcript. In some aspects of this disclosure, the extracellular vesicles further comprise scaffold proteins. Background Technology

[0002] Exosomes are small extracellular vesicles naturally produced by every eukaryotic cell. Exosomes contain a membrane that encloses their internal space (i.e., lumen). As drug delivery mediators, EVs (e.g., exosomes) offer numerous advantages over traditional drug delivery methods as novel therapeutic modalities in many therapeutic areas. In particular, exosomes possess inherently low immunogenicity, even when administered to different species.

[0003] Antisense oligonucleotides (ASOs) have become a powerful tool for regulating the expression of target genes in vitro and in vivo. However, there is still a need to improve the stability and targeting of ASOs in vivo. Therefore, there is a need for new and more effective engineered EVs (e.g., exosomes), especially those that can be used to deliver therapeutic agents that reduce the expression of disease-related genes (e.g., N for cancer), to better realize the therapeutic uses and other applications of EV-based technologies. Summary of the Invention

[0004] In some respects, this article provides an extracellular vesicle comprising an antisense oligonucleotide (ASO) comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, the continuous nucleotide sequence being complementary to a nucleic acid sequence within nucleotides 1800-1900 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO: 11; wherein the extracellular vesicle selectively delivers the ASO to myeloid-derived suppressor cells (MDSCs) and other bone marrow cells;

[0005] ASO can downregulate the expression of CEBP / β mRNA or CEBP / β protein; downregulation of CEBP / β expression can promote the immune regulation of MDSCs and other bone marrow cells, resulting in a pro-inflammatory phenotype.

[0006] Some aspects of this disclosure relate to an extracellular vesicle comprising an antisense oligonucleotide (ASO) comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, the continuous nucleotide sequence being complementary to a nucleic acid sequence within nucleotides 1438-2106 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO: 11.

[0007] In some aspects, the continuous nucleotide sequence is complementary to nucleic acid sequences within nucleotides 1500-2106, 1600-2106, 1700-2106, 1800-2106, 1500-2000, 1500-1900, 1600-2100, 1700-2000, or 1800-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to nucleic acid sequences within nucleotides 1800-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to nucleic acid sequences within nucleotides 1838-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1838-1857 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1853-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0008] In some respects, the continuous 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 CEBP / β transcript. In some respects, the continuous nucleotide sequence is completely complementary to the nucleotide sequence within the CEBP / β transcript.

[0009] In some aspects, the ASO comprises (i) a nucleotide sequence selected from the nucleotide sequences shown in SEQ ID NO:101-233, or (ii) a nucleotide sequence having one or two mismatches selected from the nucleotide sequences shown in SEQ ID NO:101-233. In some aspects, the ASO comprises the nucleotide sequence shown in SEQ ID NO:218. In some aspects, the ASO comprises the nucleotide sequence shown in SEQ ID NO:223.

[0010] Some aspects of this disclosure relate to an extracellular vesicle comprising an antisense oligonucleotide (ASO) comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, the continuous nucleotide sequence being complementary to a nucleic acid sequence within nucleotides 995-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO: 11.

[0011] In some respects, the continuous 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 nucleotides 995-1014 of the CEBP / β transcript. In some respects, the continuous nucleotide sequence is completely complementary to the nucleotide sequence within nucleotides 995-1014 of the CEBP / β transcript.

[0012] In some aspects, the ASO comprises the nucleotide sequence shown in SEQ ID NO:234, having one or two mismatched nucleotide sequences.

[0013] In some respects, extracellular vesicles target cells selected from the group consisting of macrophages, myeloid-derived suppressor cells (MDSCs), monocytes, basophils, neutrophils, eosinophils, and any combination thereof.

[0014] In some respects, ASO can reduce CEBP / β protein expression in human cells (e.g., immune cells) that express CEBP / β protein. In some respects, CEBP / β 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 CEBP / β protein expression in human cells not exposed to ASO.

[0015] In some respects, ASO can reduce the level of CEBP / β mRNA in human cells (e.g., immune cells) that express CEBP / β mRNA. In some respects, the level of CEBP / β 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 CEBP / β mRNA in human cells not exposed to ASO.

[0016] In some respects, ASO is a gapmer, a mixmer, or a totalmer.

[0017] In some aspects, ASO comprises one or more nucleoside analogs. In some aspects, one or more of the nucleoside analogs include 2'-O-alkyl-RNA; 2'-O-methylRNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; Arabica nucleic acid (ANA); 2'-fluoro-ANA; or bicyclic nucleoside analogs. In some aspects, one or more of the nucleoside analogs are sugar-modified nucleosides. In some aspects, the sugar-modified nucleosides are 2'-sugar-modified nucleosides that enhance affinity. In some aspects, one or more of the nucleoside analogs include nucleosides containing bicyclic sugars. In some aspects, one or more of the nucleoside analogs include LNA. In some aspects, one or more of the nucleotide analogs are selected from the group consisting of: restricted ethyl nucleosides (cEt), 2',4'-restricted 2'-O-methoxyethyl (cMOE), α-L-LNA, β-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 contains one or more 5'-methyl-cytosine nucleobases. In some aspects, the ASO has a design selected from the group consisting of designs in Figure 1, where uppercase letters are sugar-modified nucleosides and lowercase letters are DNA.

[0018] In some respects, ASO is 14 to 20 nucleotides in length.

[0019] In some respects, the continuous nucleotide sequence contains one or more modified internucleotide links. In some respects, one or more modified internucleotide links are phosphate thioester links. In some respects, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the internucleotide links are modified. In some respects, each of the internucleotide links in the ASO is a phosphate thioester link.

[0020] In some respects, extracellular vesicles further include an anchoring portion. In some respects, the ASO is attached to the anchoring portion.

[0021] In some respects, extracellular vesicles further comprise an exogenous targeting portion. In some respects, the exogenous targeting portion comprises a peptide, an antibody or an antigen-binding fragment thereof, a chemical compound, an RNA aptamer, or any combination thereof. In some respects, the exogenous targeting portion comprises a peptide. In some respects, the exogenous targeting portion comprises a microprotein, a designed ankyrin repeat protein (darpin), a lipid carrier protein, an antibody mimicry protein, an aptamer, a peptide mimic molecule, a natural ligand of a receptor, a camelid nanobody, or any combination thereof. In some respects, the exogenous targeting portion 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), scFv, Fv, Fab, Fab', F(ab')2, or any combination thereof. In some respects, the antibody is a single-chain antibody.

[0022] In some aspects, the exogenous targeting portion enables the exosome to target the liver, heart, lungs, brain, kidneys, central nervous system, peripheral nervous system, muscles, bones, joints, skin, intestines, bladder, pancreas, lymph nodes, spleen, blood, bone marrow, or any combination thereof. In some aspects, the exogenous targeting portion enables the exosome to target tumor cells, dendritic cells, T cells, B cells, macrophages, neurons, hepatocytes, Kupffer cells, myeloid cells (e.g., neutrophils, monocytes, macrophages, hematopoietic stem cells, MDSCs (e.g., monocytic MDSCs or granulocytic MDSCs)) or any combination thereof.

[0023] In some aspects, the EV includes a stent portion that connects the exogenous targeting portion to the EV. In some aspects, the anchoring portion and / or stent portion is a stent X. In some aspects, the anchoring portion and / or stent portion is a stent Y.

[0024] In some respects, scaffold X is a scaffold protein capable of anchoring ASO to the luminal surface and / or the outer surface of the EV. In some respects, scaffold X is selected from the group consisting of: prostaglandin F2 receptor negative regulator (PTGFRN protein); basic glycoprotein (BSG protein); immunoglobulin superfamily member 2 (IGSF2 protein); immunoglobulin superfamily member 3 (IGSF3 protein); immunoglobulin superfamily member 8 (IGSF8 protein); integrin β-1 (ITGB1 protein); integrin α-4 (ITGA4 protein); 4F2 cell surface antigen heavy chain (SLC3A2 protein); a class of ATP transporters (ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4 proteins); their functional fragments; and any combination thereof. In some respects, the anchoring portion and / or the scaffold portion is PTGFRN protein or a functional fragment thereof. In some aspects, the anchoring portion and / or the scaffold portion comprises an amino acid sequence as shown in SEQ ID NO:302. In some aspects, the anchoring portion and / or the scaffold portion comprises an amino acid sequence that is 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 that in SEQ ID NO:301.

[0025] In some respects, scaffold Y is a scaffold protein capable of anchoring ASO to the luminal surface and / or outer surface of the EV. In some respects, scaffold Y is selected from the group consisting of: myristylated alanine-rich protein kinase C substrate (MARCKS protein), myristylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1 protein), brain acid-soluble protein 1 (BASP1 protein), functional fragments thereof, and any combination thereof. In some respects, scaffold Y is BASP1 protein or a functional fragment thereof.

[0026] In some aspects, scaffold Y comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND and / or ED associate with the luminal surface of the exosome. In some aspects, the ND associates with the luminal surface of the exosome via myristylation. In some aspects, the ED associates with the luminal surface of the exosome via ion interactions.

[0027] In some aspects, ND comprises an amino acid sequence selected from the group consisting of: (i) GGKLSKK (SEQ ID NO:411), (ii) GAKLSKK (SEQ ID NO:412), (iii) GGKQSKK (SEQ ID NO:413), (iv) GGKLAKK (SEQ ID NO:414), (v) GGKLSK (SEQ ID NO:415), or (vi) any combination thereof. In some aspects, ND comprises the amino acid sequence GGKLSKK (SEQ ID NO:411).

[0028] In some aspects, the ASO is attached to an anchoring portion and / or a support portion on the outer surface of the EV. In some aspects, the ASO is attached to an anchoring portion and / or a support portion on the luminal surface of the EV. In some aspects, the anchoring portion comprises sterols, GM1, lipids, vitamins, small molecules, peptides, or combinations thereof. In some aspects, the anchoring portion comprises cholesterol. In some aspects, the anchoring portion comprises phospholipids, lysophospholipids, fatty acids, vitamins (e.g., vitamin D and / or vitamin E), or any combination thereof.

[0029] In some aspects, the ASO is connected to the anchoring portion and / or the scaffold portion via a connector. In some aspects, the ASO is connected to the EV via a connector. In some aspects, the connector is a peptide. In some aspects, the connector is a non-peptide molecule. In some aspects, the connector contains ethylene glycol. In some aspects, the connector contains HEG, TEG, PEG, or any combination thereof. In some aspects, the connector contains phosphorous acrylate (e.g., ACRYDITE). TM Adenosine, azide (NHS ester), digoxin (NHS ester), cholesterol-TEG, I-linker TM Amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, or Uni-Link) TM The linker may contain amino-modified compounds, alkynes, 5'-hexynyl, 5-octadiynyl dU, biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, or desulfurized biotin), thiol modification (thiol modifier C3 SS, dithiol, or thiol modifier C6 SS), or any combination thereof. In some aspects, the linker is a pyrolytic linker. In some aspects, the linker comprises valine-alanine-p-aminobenzylcarbamate or valine-citrulline-p-aminobenzylcarbamate. In some aspects, the linker comprises (i) a maleimide moiety and (ii) valine-alanine-p-aminobenzylcarbamate or valine-citrulline-p-aminobenzylcarbamate.

[0030] In some respects, EVs are exosomes.

[0031] Some aspects of this disclosure relate to an antisense oligonucleotide (ASO) comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to a nucleic acid sequence within nucleotides 1438-2106 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11.

[0032] In some aspects, the continuous nucleotide sequence is complementary to nucleic acid sequences within nucleotides 1500-2106, 1600-2106, 1700-2106, 1800-2106, 1500-2000, 1500-1900, 1600-2100, 1700-2000, or 1800-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to nucleic acid sequences within nucleotides 1800-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to nucleic acid sequences within nucleotides 1838-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1838-1857 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1853-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0033] In some respects, the continuous 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 CEBP / β transcript. In some respects, the continuous nucleotide sequence is completely complementary to the nucleotide sequence within the CEBP / β transcript.

[0034] In some aspects, the ASO comprises (i) a nucleotide sequence selected from the nucleotide sequences shown in SEQ ID NO:101-223, or (ii) a nucleotide sequence having one or two mismatches selected from the nucleotide sequences shown in SEQ ID NO:101-223. In some aspects, the ASO comprises the nucleotide sequence shown in SEQ ID NO:218. In some aspects, the ASO comprises the nucleotide sequence shown in SEQ ID NO:233.

[0035] Some aspects of this disclosure relate to an ASO comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to a nucleic acid sequence within nucleotides 995-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the nucleic acid sequence within nucleotides 995-1014 of the CEBP / β transcript. In some aspects, the continuous nucleotide sequence is completely complementary to the nucleotide sequence within nucleotides 995-1014 of the CEBP / β transcript.

[0036] In some aspects, the ASO comprises the nucleotide sequence shown in SEQ ID NO:234, having one or two mismatched nucleotide sequences.

[0037] In some respects, ASO can reduce CEBP / β protein expression in human cells (e.g., immune cells) that express CEBP / β protein. In some respects, CEBP / β 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 CEBP / β protein expression in human cells not exposed to ASO.

[0038] In some respects, ASO can reduce the level of CEBP / β mRNA in human cells (e.g., immune cells) that express CEBP / β mRNA. In some respects, the level of CEBP / β 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 CEBP / β mRNA in human cells not exposed to ASO.

[0039] In some respects, ASO is a gapmer, a mixmer, or a totalmer.

[0040] In some aspects, ASO comprises one or more nucleoside analogs. In some aspects, one or more of the nucleoside analogs include 2'-O-alkyl-RNA; 2'-O-methylRNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; Arabica nucleic acid (ANA); 2'-fluoro-ANA; or bicyclic nucleoside analogs. In some aspects, one or more of the nucleoside analogs are sugar-modified nucleosides. In some aspects, the sugar-modified nucleosides are 2'-sugar-modified nucleosides that enhance affinity. In some aspects, one or more of the nucleoside analogs include nucleosides containing bicyclic sugars. In some aspects, one or more of the nucleoside analogs include LNA. In some aspects, one or more of the nucleotide analogs are selected from the group consisting of: restricted ethyl nucleosides (cEt), 2',4'-restricted 2'-O-methoxyethyl (cMOE), α-L-LNA, β-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 contains one or more 5'-methyl-cytosine nucleobases. In some aspects, the ASO has a design selected from the group consisting of designs in Figure 1, where uppercase letters are sugar-modified nucleosides and lowercase letters are DNA.

[0041] In some respects, ASO is 14 to 20 nucleotides in length.

[0042] In some respects, the continuous nucleotide sequence contains one or more modified internucleotide links. In some respects, one or more modified internucleotide links are phosphate thioester links. In some respects, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the internucleotide links are modified. In some respects, each of the internucleotide links in the ASO is a phosphate thioester link.

[0043] Some aspects of this disclosure relate to a conjugate comprising the ASO disclosed herein, wherein the ASO is covalently attached to at least one nonnucleotide or nonpolynucleotide moiety. In some aspects, the nonnucleotide or nonpolynucleotide moiety comprises a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or any combination thereof.

[0044] Some aspects of this disclosure relate to an extracellular vesicle containing the ASO disclosed herein or a conjugate disclosed herein.

[0045] Some aspects of this disclosure relate to a pharmaceutical composition comprising the extracellular vesicles disclosed herein, the ASO disclosed herein, or the conjugates disclosed herein, and a pharmaceutically acceptable diluent, carrier, salt, or adjuvant. In some aspects, a pharmaceutically acceptable salt includes sodium, potassium, ammonium, or any combination thereof.

[0046] In some aspects, the pharmaceutical composition further comprises at least one additional therapeutic agent. In some aspects, the additional therapeutic agent is a CEBP / β antagonist. In some aspects, the CEBP / β antagonist is a chemical compound, siRNA, shRNA, antisense oligonucleotide, protein, or any combination thereof. In some aspects, the CEBP / β antagonist is an anti-CEBP / β antibody or a fragment thereof. In some aspects, the CEBP / β antagonist comprises an antisense oligonucleotide (ASO).

[0047] Some aspects of this disclosure relate to a kit comprising the extracellular vesicles disclosed herein, the ASO disclosed herein, the conjugates disclosed herein, or the pharmaceutical compositions disclosed herein, and instructions for use.

[0048] Some aspects of this disclosure relate to a diagnostic kit comprising the extracellular vesicles disclosed herein, the ASO disclosed herein, the conjugates disclosed herein, or the pharmaceutical compositions disclosed herein, and instructions for use.

[0049] Some aspects of this disclosure relate to a method for inhibiting or reducing the expression of CEBP / β protein in cells, the method comprising administering to cells expressing CEBP / β protein the extracellular vesicles disclosed herein, the ASO disclosed herein, the conjugates disclosed herein, or the pharmaceutical compositions disclosed herein, wherein the expression of CEBP / β protein in the cells is inhibited or reduced upon administration.

[0050] Some aspects of this disclosure relate to a method of treating cancer in a subject who requires such treatment, the method comprising administering to the subject an effective amount of the extracellular vesicles disclosed herein, the ASO disclosed herein, the conjugates disclosed herein, or the pharmaceutical compositions disclosed herein.

[0051] Some aspects of this disclosure relate to the use of the extracellular vesicles, ASOs, conjugates, or pharmaceutical compositions disclosed herein in the manufacture of a medicament for treating cancer in a subject with such need.

[0052] Some aspects of this disclosure relate to a method of treating a disease or condition in a subject who requires such treatment, the method comprising administering to the subject an effective amount of the extracellular vesicle disclosed herein, the ASO disclosed herein, the conjugate disclosed herein, or the pharmaceutical composition disclosed herein, wherein the disease or condition is selected from fibrosis, inflammation, neurodegenerative diseases, metabolic disorders / CVD, and any combination thereof.

[0053] Some aspects of this disclosure relate to the use of the extracellular vesicles, ASOs, conjugates, or pharmaceutical compositions disclosed herein in the manufacture of a medicament for treating a disease or condition in a subject with such need, wherein the disease or condition is selected from fibrosis, inflammation, neurodegenerative diseases, metabolic disorders / CVD, and any combination thereof.

[0054] In some respects, ASO inhibits or reduces the expression of CEBP / β mRNA in cells after administration.

[0055] In some aspects, compared with the level of CEBP / β mRNA in cells not exposed to ASO, the level of CEBP / β mRNA decreased 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. In some aspects, compared with the expression of CEBP / β protein in cells not exposed to ASO, the expression of CEBP / β protein decreased 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.

[0056] In some respects, extracellular vesicles, ASOs, conjugates, or pharmaceutical compositions are administered intracardiacly, orally, parenterally, intrathecally, intrapulmonaryly, locally, or intracardiacly.

[0057] In some respects, cancers are grouped according to the following categories: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteoblastic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, lymphoma, leukemia, liver cancer, glioblastoma, melanoma, myeloma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and more. Adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, follicular lymphoma, Hodgkin's lymphoma, B-cell lymphoma, and any combination thereof.

[0058] In some respects, diseases or conditions include fibrosis. In some respects, diseases or conditions include fibrosis selected from the group consisting of: liver fibrosis (NASH), cirrhosis, pulmonary fibrosis, cystic fibrosis, chronic ulcerative colitis / IBD, bladder fibrosis, renal fibrosis, CAPS (Muller-Weir syndrome), atrial fibrosis, endocardial myocardial fibrosis, old myocardial infarction, glial scars, arteriosclerosis, joint fibrosis, Crohn's disease, Dupuytren contracture, keloid fibrosis, mediastinal fibrosis, myelofibrosis, Peroni disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, adhesive capsulitis, and any combination thereof.

[0059] Some aspects of this disclosure relate to a method for activating meningeal macrophages in a subject who requires this, the method comprising administering to the subject an extracellular vesicle disclosed herein, an ASO disclosed herein, a conjugate disclosed herein, or a pharmaceutical composition disclosed herein.

[0060] Some aspects of this disclosure relate to a method of treating a subject with a cancer of the central nervous system, the method comprising administering to the subject an effective amount of the extracellular vesicle disclosed herein, the ASO disclosed herein, the conjugate disclosed herein, or the pharmaceutical composition disclosed herein.

[0061] Some aspects of this disclosure relate to a method for inducing M1 polarization of meningeal macrophages in a subject with such need, the method comprising administering to the subject an effective amount of the extracellular vesicles disclosed herein, the ASO disclosed herein, the conjugates disclosed herein, or the pharmaceutical compositions disclosed herein.

[0062] Some aspects of this disclosure relate to a method for inducing meningeal macrophage infiltration of a tumor in a subject with this need, the method comprising administering to the subject an effective amount of the extracellular vesicles disclosed herein, the ASO disclosed herein, the conjugates disclosed herein, or the pharmaceutical compositions disclosed herein. Attached Figure Description

[0063] Figure 1A-1C This is a schematic diagram of an exemplary antisense oligonucleotide construct, including ASO-1838 (SEQ ID NO:218); Figure 1A ASO-1853 (SEQ ID NO:233) Figure 1B ) and ASO-995 (SEQ ID NO:234; Figure 1C ).

[0064] Figure 2A-2C This is an example of an ASO's IC50 (nM); Figure 2A ), CEBP / β mRNA expression (normalized relative to untreated cells); Figure 2B ) and CEBP / β protein levels (normalized relative to untreated cells; Figure 3C The graphical representation of ).

[0065] Figures 3A-3F It is compared with the positive control ASO ( Figure 3D-3F Compared to ), after increasing the dosage of various exemplary ASOs, IL6 ( Figure 3A and 3D ), TNFα ( Figure 3B and 3E ) and IL12p40 ( Figure 3C and 3F concentration of ) Figures 3A-3C A graphical representation of the relative changes in EC50 and EC60. Detailed Implementation

[0066] Some aspects of this disclosure relate to an antisense oligonucleotide (ASO) comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to a nucleic acid sequence within nucleotides 1438-2106 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. Some aspects of this disclosure relate to an extracellular vesicle (EV), such as an exosome, comprising an ASO comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to a nucleic acid sequence within nucleotides 1438-2106 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to nucleic acid sequences within nucleotides 1500-2106, 1600-2106, 1700-2106, 1800-2106, 1500-2000, 1500-1900, 1600-2100, 1700-2000, or 1800-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to nucleic acid sequences within nucleotides 1800-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to nucleic acid sequences within nucleotides 1838-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1838-1857 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1853-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0067] I. Definition

[0068] To facilitate understanding of this specification, some terms are defined first. Further definitions are provided throughout the detailed description.

[0069] It should be noted that the term "a / an" refers to one or more of that entity; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a / an", "one or more", and "at least one" are used interchangeably herein.

[0070] Furthermore, the term “and / or” as used herein should be interpreted as specifically disclosing that each of two specified features or components exists with or without the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: “A, B, and C”; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0071] It should be understood that wherever the term “comprising” is used to describe an aspect, other similar aspects described as “composed of” and / or “substantially composed of” are also provided.

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art relating to this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many of the terms used in this disclosure.

[0073] Units, prefixes, and symbols are represented in their International System of Units (SI) accepted form. Numerical ranges include the numbers defining the range. Unless otherwise specified, nucleotide sequences are written from left to right in a 5' to 3' orientation. Amino acid sequences are written from left to right in an amino-to-carboxyl orientation. The headings provided herein do not limit the various aspects of this disclosure, which can be derived by referring to the entire specification. Therefore, the terms that are defined immediately thereafter are defined more fully by referring to the entire specification.

[0074] The term “about” is used in this document to mean approximately, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the listed values. Generally, the term “about” can modify values ​​above or below the stated values ​​by, for example, a change of 10% up or down (increase or decrease). For example, if the statement “After administration of ASO, ASO reduced the expression of CEBP / β protein in cells by at least about 60%”, it implies a range of 50% to 70% reduction in CEBP / β levels.

[0075] The term "antisense oligonucleotide" (ASO) refers to an oligomer or polymer of nucleosides (such as naturally occurring nucleosides or their modified forms) covalently linked together by internucleotide bonds. The ASOs used in this disclosure comprise at least one non-naturally occurring nucleoside. The ASO is at least partially complementary to the target nucleic acid, such that the ASO hybridizes with the target nucleic acid sequence.

[0076] The terms "nucleic acid" or "nucleotide" are intended to encompass a variety of nucleic acids. In some aspects, the term "nucleic acid" or "nucleotide" refers to a target sequence, such as pre-mRNA, mRNA, or DNA, whether in vivo or in vitro. When the term refers to a nucleic acid or nucleotide in a target sequence, the nucleic acid or nucleotide can be a naturally occurring sequence within a cell. In other aspects, "nucleic acid" or "nucleotide" refers to a sequence in the ASO of this disclosure. When the term refers to a sequence in an ASO, the nucleic acid or nucleotide can be non-naturally occurring, i.e., chemically synthesized, enzymatically produced, recombinant-produced, or any combination thereof. In some aspects, the nucleic acid or nucleotide in the ASO is synthetically or recombinantly produced, but is not a naturally occurring sequence or fragment thereof. In some aspects, the nucleic acid or nucleotide in the ASO is not naturally occurring because it contains at least one nucleoside analogue that is not naturally occurring in nature.

[0077] As used herein, the term "nucleotide" refers to a glycoside comprising a sugar moiety, a base moiety, and a covalent linking group (linking group) such as an internucleotide linking group of a phosphate ester or thiophosphate ester, and encompasses two naturally occurring nucleotides such as DNA or RNA, as well as non-naturally occurring nucleotides comprising modified sugar and / or base moieties, which are also referred to herein as "nucleotide analogs." In this document, a single nucleotide may be referred to as a monomer or unit. In some respects, the term "nucleotide analog" refers to a nucleotide having a modified sugar moiety. Non-limiting examples of nucleotides having modified sugar moieties (e.g., LNA) are disclosed elsewhere herein. In other respects, the term "nucleotide analog" refers to a nucleotide having a modified nucleobase moiety. Nucleotides having modified nucleobase moieties include, but are not limited to, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine. In some respects, the terms “nucleotide,” “unit,” and “monomer” are used interchangeably. It will be recognized that when referring to the sequence of a nucleotide or monomer, the sequence of bases such as A, T, G, C, or U and their analogues is being referred to.

[0078] As used herein, the term "nucleoside" refers to a glycoside comprising a sugar moiety and a base moiety, and is therefore applicable when referring to nucleotide units covalently linked by internucleotide bonds between nucleotides via ASO. In the field of biotechnology, the term "nucleotide" is generally used to refer to a nucleic acid monomer or unit. In the context of ASO, the term "nucleotide" can refer to a single base, i.e., a nucleobase sequence comprising cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA), and uracil (RNA), where the presence of the sugar backbone and internucleotide bonds is implicit. Similarly, particularly in the case of oligonucleotides in which one or more of the internucleotide linking groups are modified, the term "nucleotide" can refer to "nucleoside." For example, the term "nucleotide" can be used, even when specifying the presence or nature of bonds between nucleosides.

[0079] As used herein, the term "nucleotide length" refers to the total number of nucleotides (monomers) in a given sequence. For example, the sequence ASO-CEBP / β-540 (SEQ ID NO:194) has 15 nucleotides; therefore, the nucleotide length of the sequence is 15. Thus, the term "nucleotide length" may be used interchangeably with "nucleotide number" herein.

[0080] As will be recognized by those skilled in the art, the 5' terminal nucleotide of an oligonucleotide does not contain a 5' internucleotide linking group, although it may contain a 5' terminal group.

[0081] The compounds described herein may contain several asymmetric centers and may exist as optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, mixtures of diastereomers, diastereomeric racemates, or mixtures of diastereomeric racemates. In some aspects, the asymmetric center may be an asymmetric carbon atom. The term "asymmetric carbon atom" means a carbon atom having four different substituents. According to the Cahn-Ingold-Prelog convention, the asymmetric carbon atom may have an "R" or "S" configuration.

[0082] As used herein, the term "bicyclic sugar" refers to a modified sugar moiety comprising a 4- to 7-membered ring containing a bridging link between two atoms of the 4- to 7-membered ring to form a second ring, thus producing a bicyclic structure. In some respects, this bridging links the C2' and C4' of the ribosomal ring of the nucleoside (i.e., a 2'-4' bridging), as observed in LNA nucleosides.

[0083] As used herein, a “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. While a “stop codon” (TAG, TGA, or TAA) is generally not translated into amino acids and can be considered part of a coding region, any winged sequence (e.g., promoters, ribosome binding sites, transcription terminators, introns, untranslated regions (“UTRs”), etc.) is not part of a coding region. The boundaries of a coding region are generally determined by the start codon at the 5' end encoding the amino terminus of the resulting polypeptide and the translation stop codon at the 3' end encoding the carboxyl terminus of the resulting polypeptide.

[0084] As used herein, the term "non-coding region" refers to a nucleotide sequence that is not a coding region. Examples of non-coding regions include, but are not limited to, promoters, ribosome binding sites, transcription terminators, introns, untranslated regions ("UTRs"), and non-coding exons. Some exons may be all or part of the 5' UTR or 3' UTR of each transcript. Untranslated regions are important for efficient translation of transcripts and for controlling the translation rate and half-life of transcripts.

[0085] When used in the context of nucleotide sequences, the term "region" refers to a segment of that sequence. For example, the phrases "region within a nucleotide sequence" or "region within the complementary sequence of a nucleotide sequence" refer to a sequence shorter than the nucleotide sequence but longer than at least 10 nucleotides located within a particular nucleotide sequence or its complementary sequence. The terms "sub-sequence" or "subsequence" can also refer to a region of a nucleotide sequence.

[0086] When referring to nucleotide sequences, the term "downstream" means that the nucleic acid or nucleotide sequence is located at the 3' end of a reference nucleotide sequence. In some respects, downstream nucleotide sequences involve sequences following the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0087] The term "downstream" refers to the nucleotide sequence located at the 5' end of the reference nucleotide sequence.

[0088] As used herein, the term "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding region that influences transcription, RNA processing, stability, or translation of the relevant coding region. Regulatory regions may include promoters, pretranslational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, UTRs, and stem-loop structures. If the coding region is intended for expression in eukaryotic cells, the polyadenylation signal and transcription termination sequence are typically located at the 3' end of the coding region.

[0089] As used herein, the term "transcription" can refer to the primary transcript synthesized through the transcription of DNA and processed into messenger RNA (mRNA) (i.e., pre-mRNA and the processed mRNA itself). The term "transcription" is used interchangeably with "pre-mRNA" and "mRNA." After the DNA strand is transcribed into primary transcripts, the newly synthesized primary transcripts are modified in several ways to transform into their mature functional forms, producing various proteins and RNAs, such as mRNA, tRNA, rRNA, lncRNA, miRNA, etc. Therefore, the term "transcription" can include exons, introns, 5'UTR, and 3'UTR.

[0090] As used herein, the term "expression" refers to the process by which polynucleotides produce gene products, such as RNA or polypeptides. It includes, but is not limited to, the transcription of polynucleotides into messenger RNA (mRNA) and the translation of mRNA into polypeptides. Expression produces "gene products." As used herein, gene products can be nucleic acids, such as messenger RNA produced by gene transcription, or polypeptides translated from transcripts. Gene products described herein further include nucleic acids with post-transcriptional modifications, such as polyadenylation or splicing; or polypeptides with post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0091] In the context of two or more nucleic acids, the term "identical" or "identity" percentage refers to the fact that two or more sequences are identical or have a specified percentage of identical nucleotide or amino acid residues when compared and aligned against the largest correspondence (introducing gaps if necessary), regardless of any conserved amino acid substitutions as part of sequence identity. The identity percentage can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments of amino acid or nucleotide sequences.

[0092] A non-limiting example of such sequence alignment algorithms is the one described in: Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877 and incorporated into the NBLAST and XBLAST procedures (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In some respects, gapped BLAST can be used as described in: Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR) are other publicly available software programs that can be used for sequence alignment. In some respects, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using the NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 90, and length weights of 1, 2, 3, 4, 5, or 6). In some alternatives, 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 percentage of identity between two amino acid sequences (e.g., using a BLOSUM 62 matrix or a PAM250 matrix with vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, or 5). Alternatively, in some aspects, the algorithm of Myers and Miller (CABIOS, 4: 11-17 (1989)) is used to determine the percentage of identity between nucleotide or amino acid sequences. For example, the ALIGN program (version 2.0) and PAM120 with a residue table, a vacancy length penalty of 12, and a vacancy penalty of 4 can be used to determine the percentage of identity. Those skilled in the art can determine the appropriate parameters for achieving maximum alignment using specific alignment software. In some aspects, the default parameters of the alignment software are used.

[0093] In some respects, the percentage of identity "X" between the first and second amino acid sequences is calculated as 100x(Y / Z), where Y is the number of amino acid residues that score as identical matches in the alignment of the first and second sequences (e.g., by visual inspection or a specific sequence alignment procedure), and Z is the total number of residues in the second sequence. If the first sequence is longer than the second sequence, the percentage of identity between the first and second sequences will be higher than the percentage of identity between the second and first sequences.

[0094] Different regions within a single polynucleotide target sequence aligned to a polynucleotide reference sequence can each have their own percentage of sequence identity. It should be noted that the percentage of sequence identity values ​​are 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 should also be noted that length values ​​will always be integers.

[0095] As used herein, the terms “homogeneous” and “homology” are interchangeable with the terms “identity” and “identical”.

[0096] The term “naturally occurring variants” refers to variants of the CEBP / β polypeptide or nucleic acid sequence (e.g., transcripts) that are naturally present in defined taxa, such as mammals, such as mice, monkeys, and humans. Generally, when referring to “naturally occurring variants” of polynucleotides, the term may also encompass any allelic variants of the genomic DNA encoding CEBP / β found via chromosomal translocation or replication at nucleotides 247,416,156–247,449,108 at chromosomal location 1q44 (i.e., nucleotides 247,416,156–247,449,108 in GenBank accession number NC_000001.11), as well as RNA such as mRNA derived therefrom. “Naturally occurring variants” may also include variants derived from alternative splicing of CEBP / β mRNA. When referring to a specific polypeptide sequence, the term also includes, for example, the naturally occurring form of the protein, which can therefore be processed, for example, through co-translation or post-translational modifications (such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc.).

[0097] When determining the degree of “complementarity” between the ASO (or its region) of this disclosure and a target region (such as those disclosed herein) encoding a nucleic acid of mammalian CEBP / β (e.g., the CEBP / β gene), the degree of “complementarity” (also referred to as “homology” or “identity”) is expressed as the percentage of identity (or homology percentage) between the sequence of the ASO (or its region) and the sequence of its best-aligned target region (or the reverse complementary sequence of the target region). This percentage is calculated by counting the number of identical aligned bases between the two sequences, dividing by the total number of adjacent monomers in the ASO, and multiplying by 100. In such comparisons, if vacancies are present, preferably, such vacancies are simply mismatches and not regions with different numbers of monomers within the vacancies between the ASO and the target region of this disclosure.

[0098] As used herein, the term "complementary sequence" refers to a sequence complementary to a reference sequence. Complementarity is a fundamental principle of DNA replication and transcription, as it is a property shared between two DNA or RNA sequences, meaning that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequence will be complementary, like looking in a mirror and seeing the opposite side of something. Therefore, for example, the complementary sequence of the 5'"ATGC"3' sequence can be written as 3'"TACG"5' or 5'"GCAT"3'. The terms "reverse complementary sequence," "reverse complementarity," and "reverse complementarity" as used herein are interchangeable with the terms "complementary sequence," "complementarity," and "complementarity." In some respects, the term "complementarity" refers to a 100% match or complementarity (i.e., complete complementarity) with adjacent nucleic acid sequences within the CEBP / β transcript. In some respects, the term “complementarity” refers to a match or complementarity with adjacent nucleic acid sequences within the CEBP / β transcript of 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%.

[0099] When referring to two separate nucleic acid or nucleotide sequences, the terms "corresponding to" and "corresponds to" are used to clarify sequence regions that correspond or are similar to each other based on homology and / or function, even though the nucleotides of the specific sequences may be numbered differently. For example, different isotypes of a gene transcript may have similar or conserved portions of the nucleotide sequence, and their numbering may differ within the respective isotype based on alternative splicing and / or other modifications. Furthermore, it should be recognized that different numbering systems may be used when characterizing nucleic acid or nucleotide sequences (e.g., gene transcripts and whether the sequence is numbered starting from the translation start codon or whether it includes a 5' UTR). Additionally, it should be recognized that the nucleic acid or nucleotide sequences of different variants of a gene or gene transcript may vary. However, as used herein, variant regions sharing nucleic acid or nucleotide sequence homology and / or function are considered to "correspond" to each other. For example, the nucleotide sequence of the CEBP / β transcript corresponding to nucleotides X to Y of SEQ ID NO:1 (“reference sequence”) refers to a CEBP / β transcript sequence (e.g., CEBP / β pre-mRNA or mRNA) having the same or similar sequence as nucleotides X to Y of SEQ ID NO:1, where X is the start site and Y is the stop site (as shown in Figure 1). Those skilled in the art can identify the corresponding X and Y residues in the CEBP / β transcript sequence by comparing the CEBP / β transcript sequence with SEQ ID NO:1.

[0100] The terms “corresponding nucleotide analog” and “corresponding nucleotide” are intended to indicate that the nucleobases in a nucleotide analog and a naturally occurring nucleotide have the same pairing or hybridization ability. For example, when the 2-deoxyribose unit of a nucleotide is linked to adenine, the “corresponding nucleotide analog” contains a pentose unit (different from the 2-deoxyribose) linked to adenine.

[0101] The ASO chemistry notes are as follows: β-D-oxyLNA nucleotides are designated by the oxygen group B, where B represents a nucleotide base such as thymine (T), uridine (U), cytosine (C), 5-methylcytosine (MC), adenine (A), or guanine (G), and therefore include oxygen A, oxygen T, oxygen MC, oxygen C, and oxygen G. DNA nucleotides are designated by DNAb, where lowercase b represents a nucleotide base such as thymine (T), uridine (U), cytosine (C), 5-methylcytosine (Mc), adenine (A), or guanine (G), and therefore include DNAa, DNAt, DNA, and DNAg. The letter M preceding C or c indicates 5-methylcytosine. The letter "s" indicates a phosphate thioester nucleotide bond.

[0102] As used herein, the term "ASO Number" or "ASO No." refers to a unique number assigned to a nucleotide sequence having a detailed chemical structure of components such as, for example, nucleosides (e.g., DNA), nucleoside analogs (e.g., β-D-oxy-LNA), nucleobases (e.g., A, T, G, C, U, or MC), and backbone structures (e.g., phosphate thioesters or phosphate diesters). For example, ASO-CEBP / β-1838 may refer to CEBP / β-1838 (SEQ ID NO: 218).

[0103] Unless otherwise stated, "potency" is usually expressed in IC50 units in μM, nM or pM. 50 or EC 50 Value. Efficacy can also be expressed as a percentage of inhibition. IC 50 This is the median inhibitory concentration of the therapeutic molecule. EC 50 It is the median effective concentration of the therapeutic molecule relative to the medium or control (e.g., saline). In functional assays, IC50 is... 50 This refers to the concentration of a therapeutic molecule that can reduce a biological response (e.g., mRNA transcription or protein expression) by 50% compared to responses induced by the therapeutic molecule. In functional assays, EC... 50 This is the concentration of a therapeutic molecule that produces a 50% biological response (e.g., mRNA transcription or protein expression). The IC can be calculated using any method known in the art. 50 or EC 50 .

[0104] As used herein, the term "inhibition," for example, the expression of CEBP / β gene transcripts and / or CEBP / β protein, refers to the reduction of CEBP / β gene transcript and / or CEBP / β protein expression in cells or tissues by ASO. In some respects, the term "inhibition" refers to complete inhibition (100% inhibition or undetectable level) of CEBP / β gene transcripts or CEBP / β protein. In other respects, the term "inhibition" refers to inhibition of CEBP / β gene transcript and / or CEBP / β protein expression in cells or tissues by 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%.

[0105] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle containing a membrane enclosing an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, nanovesicles) with a diameter smaller than that of the cell from which they originate. In some aspects, extracellular vesicles have a diameter ranging from 20 nm to 1000 nm and may contain various macromolecular payloads within an internal space (i.e., a lumen), displayed on the outer surface of the extracellular vesicle, and / or transmembrane. In some aspects, the payload may include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some aspects, the extracellular medium includes a scaffold portion. Extracellular vesicles include, for example and without limitation, apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), organelles containing sacs, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosome fusion with the plasma membrane). Extracellular vesicles can originate from living or dead organisms, explant tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. In some respects, extracellular vesicles are produced by cells expressing one or more transgenic products.

[0106] As used herein, the term "exosome" refers to an extracellular vesicle with a diameter between 20 and 300 nm (e.g., between 40 and 200 nm). Exosomes comprise a membrane enclosing an internal space (i.e., a lumen) and, in some respects, can be generated from cells (e.g., production cells) via direct plasma membrane budding or via late endosome fusion with the plasma membrane. In some respects, exosomes comprise a scaffold portion. As described below, exosomes can be derived from production cells and isolated from production cells based on their size, density, biochemical parameters, or combinations thereof. In some respects, the EVs (e.g., exosomes) of this disclosure are generated by cells expressing one or more transgenic products.

[0107] As used herein, the term "nanovesicle" refers to an extracellular vesicle with a diameter between 20 and 250 nm (e.g., between 30 and 150 nm) that is generated by a cell (e.g., a production cell) through direct or indirect manipulation such that the cell will not generate nanovesicles without manipulation. Suitable manipulation of the cell for the generation of nanovesicles includes, but is not limited to, continuous extrusion, treatment with an alkaline solution, sonication, or combinations thereof. In some aspects, the generation of nanovesicles may lead to the destruction of the production cell. In some aspects, the nanovesicle populations described herein are substantially free of vesicles derived from the cell by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. In some aspects, the nanovesicles include a scaffold portion. Once derived from the production cell, nanovesicles can be isolated from the production cell based on their size, density, biochemical parameters, or combinations thereof.

[0108] As used herein, the term "surface-engineered EV, such as exosome" (e.g., scaffold-engineered EV, such as exosome) refers to an EV (e.g., exosome) whose membrane or surface has been modified in terms of its composition such that the surface of the engineered EV (e.g., exosome) differs from the surface of the unmodified EV (e.g., exosome) or the surface of a naturally occurring EV (e.g., exosome). Engineering can be performed on the surface of the EV (e.g., exosome) or in the membrane of the EV (e.g., exosome) to alter the surface of the EV (e.g., exosome). For example, the membrane may be modified in terms of its protein, lipid, small molecule, carbohydrate, etc. The composition can be altered by chemical, physical, or biological methods or by production from cells previously or simultaneously modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering or by production from cells previously modified by genetic engineering. In some aspects, surface-engineered EVs (e.g., exosomes) contain exogenous proteins (i.e., proteins not naturally expressed by the EV (e.g., exosomes)) or fragments or variants thereof, which may be exposed on the surface of the EV (e.g., exosomes) or may serve as anchoring sites (connections) for portions exposed on the surface of the EV (e.g., exosomes). In other aspects, surface-engineered EVs (e.g., exosomes) contain higher expression (e.g., higher amounts) of native exosomal proteins (e.g., scaffold X) or fragments or variants thereof, which may be exposed on the surface of the EV (e.g., exosomes) or may serve as anchoring sites (connections) for portions exposed on the surface of the EV (e.g., exosomes).

[0109] As used herein, the term "cavity-engineered exosome" (e.g., scaffold Y-engineered exosome) refers to an EV (e.g., exosome) whose membrane or cavity has been modified in terms of its composition such that the cavity of the engineered EV (e.g., exosome) differs from the cavity of the unmodified EV (e.g., exosome) or the cavity of a naturally occurring EV (e.g., exosome). Engineering can be performed directly within the cavity or membrane of the EV (e.g., exosome) to alter its cavity. For example, the membrane can be modified in terms of its protein, lipid, small molecule, carbohydrate, etc., to modify the cavity of the EV (e.g., exosome). The composition can be altered by chemical, physical, or biological methods or by production from cells previously modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering or by production from cells previously modified by genetic engineering. In some aspects, the cavity-engineered exosome contains exogenous proteins (i.e., proteins not naturally expressed by the EV (e.g., exosome)) or fragments or variants thereof, which may be exposed within the lumen of the EV (e.g., exosome) or may serve as anchoring sites (connections) for portions exposed on the inner layer of the EV (e.g., exosome). In other aspects, the cavity-engineered EV (e.g., exosome) contains higher expression of native exosomal proteins (e.g., scaffold X or scaffold Y) or fragments or variants thereof, which may be exposed within the lumen of the exosome or may serve as anchoring sites (connections) for portions exposed within the lumen of the exosome.

[0110] When used in the context of EVs (e.g., exosomes) as described herein, the term "modified" refers to an alteration or engineering of an EV (e.g., exosome and / or its producing cell) such that the modified EV (e.g., exosome) differs from naturally occurring EVs (e.g., exosomes). In some aspects, the modified EVs (e.g., exosomes) described herein comprise membranes that differ from those of naturally occurring EVs (e.g., exosomes) in composition of proteins, lipids, small molecules, carbohydrates, etc. (e.g., membranes containing a higher density or number of native exosomal proteins and / or membranes containing proteins not naturally occurring in the exosome (e.g., ASO)). In some aspects, such modifications to the membrane alter the outer surface of the EV (e.g., exosome) (e.g., surface-engineered EVs, such as exosomes, as described herein). In some aspects, such modifications to the membrane alter the cavity of the EV (e.g., exosome) (e.g., cavity-engineered EVs, such as exosomes, as described herein).

[0111] As used herein, the term "scaffold portion" refers to a molecule that can be used to anchor a target payload or any other compound (e.g., ASO) to the luminal or lateral surface of an EV (e.g., exosome). In some aspects, a scaffold portion comprises a synthetic molecule. In some aspects, a scaffold portion comprises a non-peptide portion. In other aspects, a scaffold portion comprises lipids, carbohydrates, or proteins naturally present in EVs (e.g., exosomes). In some aspects, a scaffold portion comprises lipids, carbohydrates, or proteins not naturally present in EVs (e.g., exosomes). In some aspects, a scaffold portion is scaffold X. In some aspects, a scaffold portion is scaffold Y. In still other aspects, a scaffold portion comprises both scaffold X and scaffold Y. Non-limiting examples that can be used with other scaffold components of this disclosure include: aminopeptidase N (CD13); enkephalin, also known as membrane metalloendopeptidase (MME); exonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1); neuropilin-1 (NRP1); CD9, CD63, CD81, PDGFR, GPI ankyrin, lactoglucosin (MFGE8), LAMP2, and LAMP2B.

[0112] As used herein, the term "scaffold X" refers to an exosomal protein that has been identified on the surface of an exosome. See, for example, U.S. Patent No. 10,195,290, which is incorporated herein by reference in its entirety. Non-limiting examples of scaffold X proteins include: a negative regulator of the prostaglandin F2 receptor (“PTGFRN protein”); a basic glycoprotein (“BSG protein”); immunoglobulin superfamily member 2 (“IGSF2 protein”); immunoglobulin superfamily member 3 (“IGSF3 protein”); immunoglobulin superfamily member 8 (“IGSF8 protein”); integrin β-1 (“ITGB1 protein”); integrin α-4 (“ITGA4 protein”); 4F2 cell surface antigen heavy chain (“SLC3A2 protein”); a class of ATP transporters (“ATP1A1 protein”, “ATP1A2 protein”, “ATP1A3 protein”, “ATP1A4 protein”, “ATP1B3 protein”, “ATP2B1 protein”, “ATP2B2 protein”, “ATP2B3 protein”, “ATP2B protein”); and their functional fragments. In some respects, the scaffold X protein may be a complete protein or a fragment thereof (e.g., a functional fragment, such as the smallest fragment capable of anchoring another portion to the outer or luminal surface of an EV (e.g., an exosome). In some respects, scaffold X may anchor a portion (e.g., ASO) to the outer or luminal surface of an exosome.

[0113] As used herein, the term "scaffold Y" refers to an exosomal protein identified within the lumen of an exosome. See, for example, International Publication No. WO / 2019 / 099942, which is incorporated herein by reference in its entirety. Non-limiting examples of scaffold Y proteins include: myristylated alanine-rich protein kinase C substrate ("MARCKS protein"); myristylated alanine-rich protein kinase C substrate-like 1 ("MARCKSL1 protein"); and encephalolysin 1 ("BASP1 protein"). In some aspects, scaffold Y proteins may be complete proteins or fragments thereof (e.g., functional fragments, such as the smallest fragments capable of anchoring portions to the luminal surface of an exosome). In some aspects, scaffold Y may anchor portions (e.g., ASO) to the luminal surface of an EV (e.g., an exosome). In some aspects, scaffold Y may anchor portions (e.g., ASO) to the outer surface of an EV (e.g., an exosome).

[0114] As used herein, the term "fragment" of a protein (e.g., therapeutic protein, scaffold X, or scaffold Y) refers to an amino acid sequence of a protein that is shorter than the naturally occurring sequence and that lacks the N- and / or C-terminus or any portion thereof compared to the naturally occurring protein. As used herein, the term "functional fragment" refers to a protein fragment that retains the protein's function. Thus, in some aspects, a functional fragment of scaffold X protein retains the ability to partially anchor onto the luminal or outer surface of an EV (e.g., exosome). Similarly, in some aspects, a functional fragment of scaffold Y protein retains the ability to partially anchor onto the luminal or outer surface of an EV (e.g., exosome). Whether a fragment is a functional fragment can be assessed by any method known in the art for determining the protein content of an EV (e.g., exosome), including Western blotting, FACS analysis, and fusion of the fragment with an autofluorescent protein (e.g., GFP). In some aspects, the functional fragment of the 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 capabilities of the naturally occurring scaffold X protein, such as the ability to anchor to a specific region. In some aspects, the functional fragment of the 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 capabilities of the naturally occurring scaffold Y protein, such as the ability to anchor to another molecule.

[0115] As used herein, a “variant” of the term molecule (e.g., a functional molecule, an antigen, scaffold X, and / or scaffold Y) means a molecule that shares certain structural and functional properties with another molecule when compared by methods known in the art. For example, a variant of a protein may include substitutions, insertions, deletions, frameshifts, or rearrangements in another protein.

[0116] In some aspects, variants of scaffold X include variants that share at least about 70% identity with full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporter, or fragments (e.g., functional fragments) of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporter. In some aspects, variants of PTGFRN or variants of fragments 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 or its functional fragment according to SEQ ID NO:302. In some respects, variants or fragments of BSG 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 BSG or a functional fragment thereof according to SEQ ID NO:303. In some respects, variants or fragments of IGSF2 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 IGSF2 or a functional fragment thereof according to SEQ ID NO:308. In some aspects, variants or fragments of IGSF3 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 IGSF3 or a functional fragment thereof according to SEQ ID NO:309. In some aspects, variants or fragments of IGSF8 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 IGSF8 or a functional fragment thereof according to SEQ ID NO:304. In some aspects, variants or fragments of ITGB1 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 ITGB1 or a functional fragment thereof according to SEQ ID NO:305. In some aspects, variants or fragments of ITGA4 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 ITGA4 or a functional fragment thereof according to SEQ ID NO:306.In some respects, variants or fragments of SLC3A2 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 SLC3A2 or a functional fragment thereof according to SEQ ID NO:307. In some respects, variants or fragments of ATP1A1 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 ATP1A1 or a functional fragment thereof according to SEQ ID NO:310. In some respects, variants or fragments of ATP1A2 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 ATP1A2 or a functional fragment thereof according to SEQ ID NO:312. In some respects, variants or fragments of ATP1A3 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 ATP1A3 or a functional fragment thereof according to SEQ ID NO:312. In some respects, variants or fragments of ATP1A4 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 ATP1A4 or a functional fragment thereof according to SEQ ID NO:313. In some respects, variants or fragments of ATP1B3 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 ATP1B3 or a functional fragment thereof according to SEQ ID NO:314. In some respects, variants or fragments of ATP2B1 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 ATP2B1 or a functional fragment thereof according to SEQ ID NO:315. In some respects, variants or fragments of ATP2B2 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 ATP2B2 or a functional fragment thereof according to SEQ ID NO:316.In some aspects, variants or fragments of ATP2B3 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 ATP2B3 or a functional fragment thereof according to SEQ ID NO:317. In some aspects, variants or fragments of ATP2B4 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 ATP2B4 or a functional fragment thereof according to SEQ ID NO:318. In some aspects, variants or fragments of the scaffold X protein disclosed herein retain the ability to specifically target EVs (e.g., exosomes). In some aspects, scaffold X contains one or more mutations, such as conserved amino acid substitutions.

[0117] In some aspects, variants of stent Y include variants that share at least 70% identity with fragments of MARCKS, MARCKSL1, BASP1, or MARCKS, MARCKSL1, or BASP1. In some aspects, variants of MARCKS or its fragments 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 or its functional fragments according to SEQ ID NO:402. In some aspects, variants of MARCKSL1 or its fragments 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 MARCKSL1 or its functional fragments according to SEQ ID NO:402. In some aspects, variants or 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 or a functional fragment thereof according to SEQ ID NO:403. In some aspects, variants or fragments of the scaffold Y protein retain the ability to specifically target the luminal surface of EVs (e.g., exosomes). In some aspects, scaffold Y contains one or more mutations, such as conserved amino acid substitutions.

[0118] "Conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered conserved. Alternatively, amino acid strings can be conservatively substituted by structurally similar strings that differ in the sequence and / or composition of side chain family members.

[0119] The term "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location in which the same nucleotide or amino acid is presented in both the target and reference sequences. Vacancies presented in the target sequence are not counted because vacancies are not nucleotides or amino acids. Similarly, vacancies presented in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0120] The percentage of sequence identity is calculated by determining the number of positions in both sequences where the same amino acid residues or nucleic acid bases appear (resulting in the number of matching positions), dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using readily available software that is available online and for download. Suitable software programs are available from various sources and are used for the alignment of protein and nucleotide sequences. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the U.S. government's National Center for Biotechnology Information (BLAST) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute for Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0121] Different regions within a single polynucleotide or polypeptide target sequence aligned to a reference polynucleotide or polypeptide sequence may each have their own percentage of sequence identity. It should be noted that the percentage of sequence identity values ​​are 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 should also be noted that length values ​​will always be integers.

[0122] Those skilled in the art will understand that the generation of sequence alignments used to calculate the percentage of sequence identity is not limited to binary sequence-sequence comparisons driven solely by primary sequence data. Sequence alignments can originate from multiple sequence alignments. A suitable program for generating multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com / muscle / . ClustalW2 and MUSCLE can alternatively be obtained, for example, from EBI.

[0123] It should also be understood that sequence alignment 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., mutation locations), or phylogenetic data. A suitable procedure for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, available at www.tcoffee.org and alternatively, for example, from EBI. It should also be understood that the final alignment used to calculate the percentage of sequence identity can be verified automatically or manually.

[0124] Polynucleotide variants can contain alterations in coding regions, non-coding regions, or both. In one respect, polynucleotide variants contain changes that produce silent substitutions, additions, or deletions, but do not alter the properties or activity of the encoded polypeptide. In another respect, nucleotide variants are produced through silent substitutions due to the degeneracy of the genetic code. In still other respects, variants include those that substitute, delete, or add 5-10, 1-5, or 1-2 amino acids in any combination. Polynucleotide variants can be generated for a variety of reasons, such as to optimize codon expression for a specific host (changing codons in human mRNA to other codons, for example, in bacterial hosts such as Escherichia coli).

[0125] Naturally occurring variants are called “allele variants” and refer to one of several alternative forms of a gene occupying a given locus on an organism’s chromosome (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allele variants can vary at the polynucleotide and / or polypeptide level and are included in this disclosure. Alternatively, non-naturally occurring variants may be produced by mutagenesis or direct synthesis.

[0126] Known methods using protein engineering and recombinant DNA techniques can produce variants to improve or alter the characteristics of peptides. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secretory protein without substantially losing its biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993) (incorporated herein by reference in its entirety) reported variants of KGF proteins that retained heparin-binding activity even after the deletion of 3, 8, or 27 amino acid residues from the N-terminus. Similarly, interferon-γ exhibits up to a tenfold increase in activity after the deletion of 8-10 amino acid residues from the C-terminus of such proteins. (Dobeli et al., J. Biotechnology 7:199-216 (1988), in its entirety (incorporated herein by reference).

[0127] Furthermore, substantial evidence suggests that variants often retain biological activities similar to those of naturally occurring proteins. For example, Gayle and colleagues (J. Biol. Chem 268:22105-22111 (1993), incorporated herein by reference in its entirety) conducted an extensive mutational analysis of the human cytokine IL-1a. They generated over 3,500 individual IL-1a mutants using random mutagenesis, each variant exhibiting an average of 2.5 amino acid changes across the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The researchers found that "[most] molecules can be altered with little effect on [binding or biological activity]" (see abstract). In fact, of the more than 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins with significantly different activities from the wild-type.

[0128] As described above, peptide variants include, for example, modified peptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent linkage of flavin, covalent linkage of heme moieties, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, covalent linkage of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cysteine ​​residues, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristylation, oxidation, polyethylene glycolation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, isopreneation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as argininoylation and ubiquitous proteination. In some respects, bracket X and / or bracket Y can be modified in any convenient location.

[0129] As used herein, the terms “connected to” or “conjugated to” are used interchangeably and refer to a covalent or non-covalent bond formed between the first part and the second part (e.g., scaffold X and ASO, respectively, e.g., scaffold portion expressed in or on extracellular vesicles and ASO, e.g., scaffold X (e.g., PTGFRN protein) expressed in or on the luminal or outer surface of extracellular vesicles).

[0130] The term “encapsulated,” or its grammatically different forms (e.g., encapsulation or encapsulating),” refers to a state or process in which a first part (e.g., ASO) is contained within a second part (e.g., EV, e.g., exosome) and the two parts are not chemically or physically connected. In some respects, the term “encapsulated” may be used interchangeably with “in the cavity of.” Non-limiting examples of encapsulating a first part (e.g., ASO) into a second part (e.g., EV, e.g., exosome) are disclosed elsewhere herein.

[0131] As used herein, the term "productive cell" refers to a cell used to produce EVs (e.g., exosomes). Productive cells can be cells cultured in vitro or cells in vivo. Productive cells include, but are not limited to, cells known to be effective at producing EVs (e.g., exosomes), such as HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, etc. Neuronal precursor cells, Amniotic fluid cells, adipose-derived mesenchymal stem cells, RPTEC / TERT1 cells. In some respects, the producing cells are not antigen-presenting cells. In some respects, the producing cells are not dendritic cells, B cells, mast cells, macrophages, neutrophils, Kupffer-Browicz cells, cells derived from any of these cells, or any combination thereof. In some respects, the EVs (e.g., exosomes) used in this disclosure do not carry antigens on MHC class I or II molecules exposed on the surface of the EV (e.g., exosome), but rather carry antigens in the lumen of the EV (e.g., exosome) or on the surface of the EV (e.g., exosome) by means of attachment to scaffold X and / or scaffold Y.

[0132] As used herein, the terms “isolate,” “isolated,” and “isolating,” or “purify,” “purified,” and “purifying,” and “extracted” and “extracting” are used interchangeably and refer to the formulation state (e.g., multiple known or unknown amounts and / or concentrations) of a desired EV that has undergone one or more purification processes (e.g., selection or enrichment of the desired EV formulation). In some respects, isolation or purification, as used herein, is the process of removing, partially removing (e.g., a portion), EVs from a sample containing production cells. In some respects, the isolated EV composition does not have detectable undesirable activity, or alternatively, the level or amount of undesirable activity is at or below an acceptable level or amount. In other respects, the amount and / or concentration of the desired EV in the isolated EV composition is at or above an acceptable amount and / or concentration. In other respects, the isolated EV composition is enriched compared to the starting material from which the composition was obtained (e.g., the production cell formulation). This enrichment can be 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% compared to the starting material. In some respects, the isolated EV formulation is substantially free of residual biological products. In some respects, the isolated EV formulation is 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 material. Residual biological products can include non-biological substances (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites. Being substantially free of residual biological products can also mean that the EV composition does not contain detectable production cells, and only EVs are detectable.

[0133] As used herein, the term "payload" refers to an agent that acts on a target (e.g., a target cell) in contact with an EV. A non-limiting example of a payload that may be contained on an EV (e.g., an exosome) is an ASO. Payloads that may be introduced into EVs (e.g., exosomes) and / or producing cells include agents such as nucleotides (e.g., nucleotides containing partial or complete detection of toxins or disruptive transcription), nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides such as enzymes, or RNA molecules with regulatory functions such as miRNA, dsDNA, lncRNA, and siRNA), amino acids (e.g., amino acids containing partial or complete detection of toxins or disruptive translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins). In some respects, payloads contain ASOs. As used herein, the term "antibody" covers immunoglobulins (whether naturally occurring or partially or completely synthetically produced) and fragments thereof. The term also covers any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" further includes polypeptides containing a framework region from an immunoglobulin gene or a fragment thereof that specifically binds to and recognizes an antigen. As used herein, the term "antigen" refers to any agent that, when introduced into a subject, elicits an immune response (cellular or humoral) against itself. The term "antibody" is intended to include complete antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and further include single-chain antibodies, humanized antibodies, mouse antibodies, chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments (such as, for example, scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments), bifunctional antibodies, and antibody-associated peptides. Antibodies include both bispecific and multispecific antibodies, provided they exhibit the desired biological activity or function.

[0134] As used herein, the terms “individual,” “subject,” “host,” and “patient” are used interchangeably and refer to any mammalian subject, particularly a human, in the presence of a diagnostic, therapeutic, or therapeutic agent. The compositions and methods described herein are intended for therapeutic and veterinary applications in humans. In some respects, the subject is a mammal, and in others, the subject is a human. As used herein, “mammal subject” includes all mammals, including but not limited to humans, livestock (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.).

[0135] The term "pharmaceutical composition" refers to a formulation in which the biological activity of the active ingredient is permitted and which does not contain any additional components that would have unacceptable toxicity to a subject to whom the composition will be administered. Such a composition may be sterile.

[0136] As used herein, the term “substantially free” means that a sample containing EVs (e.g., exosomes) contains less than 10% macromolecules by mass / volume (m / v) percentage concentration. Some fractions may contain less than 0.001%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% (m / v) of macromolecules.

[0137] As used in this article, the term "macromolecule" means nucleic acid, contaminant protein, lipid, carbohydrate, metabolite, or a combination thereof.

[0138] As used herein, the term “common exosomal protein” means proteins previously known to be enriched in exosomes, including but not limited to CD9, CD63, CD81, PDGFR, GPI ankyrin, lactoglucosin (MFGE8), LAMP2 and LAMP2B, fragments thereof, or peptides bound to them.

[0139] As used herein, “administration” means giving a subject the composition disclosed herein containing EVs (e.g., exosomes) via a pharmaceutically acceptable route. Routes of administration may be intravenous, such as intravenous injection and intravenous infusion. Other routes of administration include, for example, subcutaneous, intramuscular, oral, nasal, and pulmonary administration. EVs (e.g., exosomes) may be administered as part of a pharmaceutical composition comprising at least one excipient.

[0140] The “effective amount” of, for example, ASO or extracellular vesicles disclosed herein is an amount sufficient to perform the stated purpose. The “effective amount” may be determined empirically and in a conventional manner regarding the stated purpose.

[0141] As used herein, “treat,” “treatment,” or “treating” means, for example, a reduction in the severity of a disease or ailment; a shortening of the duration of a disease; an improvement or elimination of one or more symptoms associated with a disease or ailment; or providing a beneficial effect to a subject suffering from a disease or ailment, but not necessarily curing the disease or ailment. The term also includes prevention or avoidance of a disease or ailment or its symptoms. In one aspect, “treating” or “treatment” includes inducing hematopoiesis in a subject in need of it. In some aspects, the disease or ailment is associated with hematopoiesis or a deficiency thereof. In some aspects, the disease or ailment is cancer. In some aspects, treatment enhances hematopoiesis in a subject suffering from cancer, wherein enhanced hematopoiesis includes increased proliferation and / or differentiation of one or more immune cells in the subject.

[0142] As used herein, the terms “prevent” or “preventing” refer to reducing or mitigating the occurrence or severity of a particular outcome. In some respects, preventative treatment achieves preventative outcomes. In some respects, the use of EVs (e.g., exosomes) containing ASOs as described herein is preventatively administered to subjects. In some respects, subjects are at risk of developing cancer. In some respects, subjects are at risk of developing hematopoietic disorders.

[0143] II. Antisense Oligonucleotides (ASO)

[0144] This disclosure employs antisense oligonucleotides (ASOs) for regulating the function of nucleic acid molecules encoding mammalian CEBP / β (such as CEBP / β nucleic acids, for example CEBP / β transcripts, including CEBP / β pre-mRNA and CEBP / β mRNA) or natural variants of such nucleic acid molecules encoding mammalian CEBP / β. In the context of this disclosure, the term "ASO" refers to a molecule (i.e., an oligonucleotide) formed by the covalent bonding of two or more nucleotides.

[0145] An ASO comprises a continuous nucleotide sequence of about 10 to about 30, such as 10-20, 14-20, 16-20, or 15-25 nucleotides in length. In some aspects, the ASO is 20 nucleotides long. In some aspects, the ASO is 18 nucleotides long. In some aspects, the ASO is 19 nucleotides long. In some aspects, the ASO is 17 nucleotides long. In some aspects, the ASO is 16 nucleotides long. In some aspects, the ASO is 15 nucleotides long. As used herein, the terms “antisense ASO,” “antisense oligonucleotide,” and “oligomer” are used interchangeably with the term “ASO.” The ASOs used in this disclosure are not naturally occurring and cannot be found in nature. In some aspects, the ASOs are chemically modified.

[0146] References to SEQ ID numbers include a specific nucleobase sequence but not any design or complete chemical structure. Furthermore, the ASOs disclosed in the figures herein illustrate representative designs but are not limited to the specific designs shown in the figures unless otherwise stated. For example, when a claim (or this specification) refers to SEQ ID NO:101, it contains only the nucleotide sequence of SEQ ID NO:101. Any ASO design disclosed herein may be written as SEQ ID NO:XX, wherein each of the first, second, third, first, second, and Nth nucleotides from the 5' end is a modified nucleotide, such as LNA, and each of the other nucleotides is an unmodified nucleotide (e.g., DNA).

[0147] In various aspects, the ASO of this disclosure does not contain RNA (units). In some aspects, the ASO contains one or more DNA units. In one aspect, the ASO according to this disclosure is a linear molecule or is synthesized as a linear molecule. In some aspects, the ASO is a single-stranded molecule and does not contain, for example, short regions of at least 3, 4, or 5 adjacent nucleotides that are complementary to an equivalent region (i.e., a double strand) within the same ASO—in this respect, the ASO is (substantially) not double-stranded. In some aspects, the ASO is not substantially double-stranded. In some aspects, the ASO is not siRNA. In various aspects, the ASO of this disclosure may consist entirely of adjacent nucleotide regions. Therefore, in some aspects, the ASO is not substantially self-complementary.

[0148] In other respects, this disclosure includes fragments of ASO. For example, this disclosure includes at least one nucleotide, at least two adjacent nucleotides, at least three adjacent nucleotides, at least four adjacent nucleotides, at least five adjacent nucleotides, at least six adjacent nucleotides, at least seven adjacent nucleotides, at least eight adjacent nucleotides, or at least nine adjacent nucleotides of the ASO disclosed herein. Fragments of any sequence disclosed herein are considered part of this disclosure.

[0149] In some aspects, the ASO used in this disclosure includes diaminophosphate morpholine oligomers (PMO) or peptide-conjugated diaminophosphate morpholine oligomers (PPMO).

[0150] II.A. Target

[0151] Suitablely, the ASO of this disclosure is capable of downregulating (e.g., reducing or eliminating) the expression of CEBP / β mRNA or CEBP / β protein. In this respect, the ASO of this disclosure can promote the differentiation of M2 macrophages and / or reduce the differentiation of M1 macrophages. In particular, this disclosure relates to an ASO that targets one or more of nucleotides 1438-2106 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11.

[0152] Unless otherwise stated, the term “CEBP / β” as used herein may refer to CEBP / β derived from one or more species (e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).

[0153] CEBP / β (CEBP / β) is also known as CCAAT / enhancer-binding protein β. Synonyms for CEBP / β are known and include C / EBPβ; liver activator protein; LAP; liver-enriched repressor protein; LIP; nuclear factor NF-IL6; transcription factor 5; TCF-5; CEBPB; CEBPb; CEBPβ; CEBP / B; and TCF5. The sequence of the human CEBP / β gene can be found in the publicly available GenBank accession number NC_000020.11 (50190583..50192690). The human CEBP / β gene is located at chromosome 20q13.13, 50190583-50192690.

[0154] The sequence of the human CEBP / β pre-mRNA transcript (SEQ ID NO: 11) corresponds to the inverse complementary sequence of residues 50190583-50192690 on chromosome 20q13.13. The CEBP / β mRNA sequence is publicly available in GenBank under accession number NM_001285878.1. The sequence of the human CEBP / β protein is available under publicly available accession numbers: P17676 (canonical sequence, SEQ ID NO: 12), P17676-2, and P17676-3, each of which is incorporated herein by reference in its entirety.

[0155] Table 1. CEBP / β mRNA and protein sequences

[0156]

[0157]

[0158] Natural variants of the human CEBP / β gene product are known. For example, natural variants of the human CEBP / β protein may contain one or more amino acid substitutions selected from A241P, A253G, G195S, and any combination thereof. Other variants of the human CEBP / β protein generated by alternative splicing are also known in the art. CEBP / β isotype 2 (identifier for UniProt: P17676-2) differs from the canonical sequence in that residues 1-23 are deleted. CEBP / β isotype 3 (sequence identifier: P17676-3) differs from the standard sequence (deletion of residues 1-198). Therefore, the ASO of this disclosure can be designed to reduce or suppress the expression of natural variants of the protein.

[0159] An example of a target nucleic acid sequence for ASO is CEBP / β premRNA. SEQ ID NO:11 represents the human CEBP / β genome sequence (i.e., the reverse complementary sequence of nucleotides 50190583-50192690 on chromosome 20q13.13). SEQ ID NO:11 is identical to the CEBP / β premRNA sequence, except that the nucleotide “t” in SEQ ID NO:11 is shown as “u” in the premRNA. In some respects, the “target nucleic acid” comprises a nucleic acid encoding the CEBP / β protein or a naturally occurring variant thereof, and an intron of the RNA nucleic acid (e.g., premRNA) derived therefrom. In other respects, the target nucleic acid comprises a nucleic acid encoding the CEBP / β protein or a naturally occurring variant thereof, and an exon region of the RNA nucleic acid (e.g., premRNA) derived therefrom. In still other respects, the target nucleic acid comprises an exon-intron junction of a nucleic acid encoding the CEBP / β protein or a naturally occurring variant thereof, and an exon-intron junction of the RNA nucleic acid (e.g., premRNA) derived therefrom. In some applications, such as research or diagnostics, the “target nucleic acid” can be cDNA or synthetic oligonucleotides derived from the aforementioned DNA or RNA nucleic acid targets. The sequence of the human CEBP / β protein encoded by the CEBP / β pre-mRNA is shown as SEQ ID NO:13.

[0160] In some aspects, the ASO of this disclosure comprises a continuous nucleotide sequence complementary to a nucleic acid sequence within nucleotides 1438-2106 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1500-2106, 1600-2106, 1700-2106, 1800-2106, 1500-2000, 1500-1900, 1600-2100, 1700-2000, or 1800-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1500-2106 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1600-2106 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1700-2106 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1800-2106 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1500-2000 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1500-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1600-2100 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some respects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1700-2000 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0161] In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1800-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1810-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1820-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1825-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1830-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1835-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1840-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1845-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1850-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0162] In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1800-1890 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1800-1880 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1800-1875 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1800-1870 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1800-1865 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some respects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1800-1860 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0163] In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1810-1890 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1820-1880 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1830-1875 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1838-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1838-1857 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some respects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1853-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0164] In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1830-1840 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1831-1841 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1832-1842 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1833-1843 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1834-1844 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1835-1845 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1836-1846 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1837-1847 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1838-1848 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1839-1849 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0165] In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1840-1850 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1841-1851 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1842-1852 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1843-1853 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1844-1854 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1845-1855 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1846-1856 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1847-1857 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1848-1858 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1849-1859 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0166] In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1850-1860 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1851-1861 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1852-1862 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1853-1863 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1854-1864 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1855-1865 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1856-1866 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1857-1867 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1858-1868 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1859-1869 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0167] In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1860-1870 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1861-1871 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1862-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1863-1873 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1864-1874 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1865-1875 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1866-1876 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1867-1877 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1868-1878 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1869-1879 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0168] In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1870-1880 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1871-1881 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1872-1882 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1873-1883 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1874-1884 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1875-1885 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1876-1886 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1877-1887 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1878-1888 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11. In some aspects, the continuous nucleotide sequence is complementary to the nucleic acid sequence within nucleotides 1879-1889 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:11.

[0169] Some aspects of this disclosure relate to an ASO comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, the continuous nucleotide sequence being complementary to a nucleic acid sequence within nucleotides 995-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, the continuous nucleotide sequence being complementary to a nucleic acid sequence within nucleotides 996-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, the continuous nucleotide sequence being complementary to a nucleic acid sequence within nucleotides 997-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, the continuous nucleotide sequence being complementary to a nucleic acid sequence within nucleotides 998-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 999-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 1000-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 1001-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 1003-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 1000-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO: 11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 1004-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO: 11.In some respects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to the nucleic acid sequence within nucleotides 1005-1014 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11.

[0170] In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 995-1013 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 995-1012 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 995-1011 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 995-1010 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 995-1009 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 995-1008 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 995-1007 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 995-1006 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 995-1005 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11. In some aspects, the ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to the nucleic acid sequence within nucleotides 995-1004 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11.

[0171] In some aspects, the ASO of this disclosure is capable of hybridizing with target nucleic acids (e.g., CEBP / β transcripts) under physiological conditions (i.e., in vivo conditions). In some aspects, the ASO of this disclosure is capable of hybridizing with target nucleic acids (e.g., CEBP / β transcripts) in vitro. In some aspects, the ASO of this disclosure is capable of hybridizing with target nucleic acids (e.g., CEBP / β transcripts) in vitro under stringent conditions. The stringent conditions used for in vitro hybridization depend in particular on productive cell uptake, RNA accessibility, temperature, association free energy, salt concentration, and time (see, for example, Stanley T. Crooke, Antisense Drug Technology: Principles, Strategies and Applications, 2nd edition, CRC Press (2007)). Generally, high to moderate stringency conditions are used for in vitro specific hybridization so that hybridization occurs between substantially similar nucleic acids, but not between dissimilar nucleic acids. Examples of stringent hybridization conditions include hybridization for 1 hour at 40°C in 5X saline citrate (SSC) buffer (0.75M sodium chloride / 0.075M sodium citrate), followed by washing the sample 10 times in 1X SSC at 40°C and 5 times in 1X SSC buffer at room temperature. In vivo hybridization conditions consist of intracellular conditions (e.g., physiological pH and intracellular ionic conditions) that govern the hybridization of antisense oligonucleotides with the target sequence. In vivo conditions can be simulated in vitro with relatively less stringent conditions. For example, hybridization can be performed in vitro at 37°C in 2X SSC (0.3M sodium chloride / 0.03M sodium citrate) and 0.1% SDS. A wash solution containing 4X SSC and 0.1% SDS can be used at 37°C, followed by washing in 1X SSC at 45°C.

[0172] In some aspects, the ASOs disclosed herein are capable of targeting CEBP / β transcripts from one or more species (e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears). In some aspects, the ASOs disclosed herein are capable of targeting CEBP / β transcripts in humans and rodents (e.g., mice or rats). Therefore, in some aspects, the ASOs are capable of downregulating (e.g., reducing or eliminating) the expression of CEBP / β mRNA or protein in both humans and rodents (e.g., mice or rats). In some aspects, any ASO described herein is part of a conjugate comprising an ASO covalently linked to at least one nonnucleotide or nonpolynucleotide.

[0173] Some aspects of this disclosure relate to a conjugate comprising the ASO described herein. In some aspects, the conjugate comprises an ASO covalently linked to at least one nonnucleotide. In some aspects, the conjugate comprises an ASO covalently linked to at least one non-polynucleotide molecule. In some aspects, the nonnucleotide or non-polynucleotide portion comprises a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or any combination thereof.

[0174] II.B.ASO sequence

[0175] The ASO disclosed herein comprises a continuous nucleotide sequence corresponding to a complementary sequence to the CEBP / β transcript region, for example, a nucleic acid sequence within nucleotides 995-1014 or 1438-2106 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11.

[0176] In some aspects, this disclosure provides an ASO of length 10-30, such as 10-15 nucleotides, 10-20 nucleotides, 10-25 nucleotides, or about 20 nucleotides, wherein the continuous 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 with the nucleic acid sequence within nucleotides 995-1014 or 1438-2106 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO: 11. Thus, for example, the ASO hybridizes with a single-stranded nucleic acid molecule having a sequence having SEQ ID NO: 12 or a portion thereof.

[0177] The ASO may comprise a continuous nucleotide sequence that is fully complementary to the equivalent region of the nucleic acid encoding the mammalian CEBP / β protein (e.g., the nucleic acid sequence within nucleotides 995-1014 or 1438-2106 of the CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:11). The ASO may also comprise a continuous nucleotide sequence that is fully complementary to the nucleic acid sequence corresponding to nucleotide XY of SEQ ID NO:11 or a region within that sequence, where X and Y are the start and stop sites, respectively.

[0178] In some aspects, the nucleotide sequence or continuous nucleotide sequence of the ASO disclosed herein has at least about 80% sequence identity with sequences selected from SEQ ID NO:101-233 (Table 2), 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%, at least about 97%, at least about 98%, at least about 99%, such as about 100% sequence identity (homologous). In some aspects, the ASO has the design described elsewhere herein or the chemical structure shown elsewhere herein.

[0179] Table 2. ASO Sequences

[0180]

[0181]

[0182] In some respects, the ASO (or its adjacent nucleotide portion) is selected from or comprises a sequence selected from the group consisting of: SEQ ID NO: 102 to 233 or a region of at least 10 adjacent nucleotides thereof, wherein the ASO (or its adjacent nucleotide portion) may optionally contain one, two, three or four mismatches when compared with the corresponding CEBP / B transcript.

[0183] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:218. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:233.

[0184] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:101. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:102. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:103. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:104. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:105. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:106. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:107. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:108. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:109. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:110.

[0185] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:111. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:112. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:113. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:114. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:115. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:116. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:117. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:118. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:119. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:120.

[0186] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:121. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:122. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:123. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:124. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:125. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:126. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:127. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:128. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:129. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:130.

[0187] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:131. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:132. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:133. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:134. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:135. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:136. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:137. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:138. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:139. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:140.

[0188] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:141. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:142. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:143. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:144. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:145. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:146. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:147. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:148. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:149. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:150.

[0189] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:151. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:152. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:153. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:154. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:155. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:156. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:157. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:158. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:159. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:160.

[0190] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:161. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:162. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:163. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:164. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:165. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:166. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:167. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:168. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:169. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:170.

[0191] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:171. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:172. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:173. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:174. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:175. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:176. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:177. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:178. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:179. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:180.

[0192] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:181. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:182. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:183. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:184. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:185. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:186. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:187. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:188. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:189. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:190.

[0193] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:191. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:192. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:193. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:194. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:195. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:196. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:197. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:198. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:199. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:200.

[0194] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:201. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:202. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:203. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:204. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:205. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:206. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:207. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:208. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:209. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:210.

[0195] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:211. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:212. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:213. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:214. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:215. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:216. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:217. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:218. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:219. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:220.

[0196] In some aspects, the ASO comprises the sequence shown in SEQ ID NO:231. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:232. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:233.

[0197] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:101. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:102. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:103. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:104. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:105. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:106. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:107. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:108. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:109. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:110.

[0198] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:111. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:112. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:113. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:114. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:115. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:116. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:117. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:118. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:119. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:120.

[0199] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:121. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:122. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:123. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:124. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:125. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:126. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:127. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:128. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:129. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:130.

[0200] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:131. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:132. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:133. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:134. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:135. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:136. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:137. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:138. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:139. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:140.

[0201] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:141. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:142. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:143. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:144. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:145. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:146. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:147. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:148. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:149. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:150.

[0202] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:151. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:152. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:153. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:154. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:155. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:156. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:157. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:158. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:159. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:160.

[0203] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:161. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:162. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:163. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:164. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:165. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:166. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:167. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:168. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:169. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:170.

[0204] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:171. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:172. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:173. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:174. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:175. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:176. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:177. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:178. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:179. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:180.

[0205] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:181. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:182. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:183. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:184. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:185. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:186. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:187. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:188. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:189. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:190.

[0206] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:191. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:192. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:193. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:194. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:195. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:196. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:197. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:198. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:199. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:200.

[0207] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:201. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:202. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:203. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:204. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:205. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:206. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:207. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:208. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:209. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:210.

[0208] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:211. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:212. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:213. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:214. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:215. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:216. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:217. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:218. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:219. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:220.

[0209] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:231. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:232. In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:233.

[0210] In some aspects, the ASO comprises a sequence having at least about 80%, at least about 85%, or at least about 95% of the sequence shown in SEQ ID NO:234. In some aspects, the ASO comprises the sequence shown in SEQ ID NO:233.

[0211] In some aspects, the ASO of this disclosure binds to a target nucleic acid sequence (e.g., CEBP / B transcript) and is able to inhibit or reduce the expression of CEBP / B transcript by at least 10% or 20% compared to normal (i.e., control) expression levels in cells, for example, by 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 normal expression levels (e.g., expression levels in cells not exposed to ASO).

[0212] In some respects, compared with cells not exposed to ASO (e.g., exposed to saline), the ASO of this disclosure is able to reduce the expression of in vitro CEBP / B mRNA in target cells 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%.

[0213] In some respects, when hybridizing with the target sequence, ASO can tolerate 1, 2, 3, or 4 (or more) mismatches and still bind sufficiently to the target to exhibit the desired effect, namely downregulation of the target mRNA and / or protein. Mismatches can be compensated, for example, by increasing the length of the ASO nucleotide sequence and / or increasing the number of nucleotide analogs, as disclosed elsewhere herein.

[0214] In some respects, the ASO of this disclosure contains no more than three mismatches when hybridizing with the target sequence. In other respects, the consecutive nucleotide sequences contain no more than two mismatches when hybridizing with the target sequence. In still other respects, the consecutive nucleotide sequences contain no more than one mismatch when hybridizing with the target sequence.

[0215] II.C.ASO Length

[0216] An ASO may consist of a continuous nucleotide sequence with a total length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 adjacent nucleotides. It should be understood that when a range of ASO or continuous nucleotide sequence length is given, the range includes the lower and upper limits provided within that range, such as 10-30 (or in between), including both 10 and 30.

[0217] In some aspects, the ASO comprises a continuous nucleotide sequence of a total length of about 14-20, 14, 15, 16, 17, 18, 19, or 20 adjacent nucleotides. In some aspects, the ASO comprises a continuous nucleotide sequence of a total length of about 20 adjacent nucleotides. In some aspects, the ASO of this disclosure is 14 nucleotides in length. In some aspects, the ASO of this disclosure is 15 nucleotides in length. In some aspects, the ASO of this disclosure is 16 nucleotides in length. In some aspects, the ASO of this disclosure is 17 nucleotides in length. In some aspects, the ASO of this disclosure is 18 nucleotides in length. In some aspects, the ASO of this disclosure is 19 nucleotides in length.

[0218] II.D. Nucleosides and Nucleoside Analogs

[0219] In one aspect of this disclosure, the ASO comprises one or more non-naturally occurring nucleoside analogs. As used herein, a “nucleoside analog” is a variant of a natural nucleoside (such as a DNA or RNA nucleoside) obtained through modification of its sugar and / or base moieties. In principle, in the case of oligonucleotides, the analog may simply be “silencing” or “equivalent” to the natural nucleoside, i.e., acting in a manner that inhibits the expression of the target gene without having a functional effect. However, such “equivalent” analogs may still be useful if, for example, they are easier or cheaper to manufacture, or more stable under storage or manufacturing conditions, or they represent a tag or label. However, in some aspects, the analog will have a functional effect in a manner that inhibits the expression of the ASO; 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. Specific examples of nucleoside analogues are described, for example, by Freier and 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 ASO of this disclosure may 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 ten, more than eleven, more than twelve, more than thirteen, more than fourteen, more than fifteen, more than sixteen, more than eighteen, more than nineteen, or more than twenty nucleoside analogues. In some respects, the nucleoside analogues in the ASO are identical. In other respects, the nucleoside analogues in the ASO are different. The nucleotide analogues in the ASO may be any one or a combination of the following nucleoside analogues.

[0220] In some aspects, nucleoside analogs include 2'-O-alkyl-RNA; 2'-O-methylRNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; Arabica nucleic acid (ANA); 2'-fluoro-ANA; bicyclic nucleoside analogs; or any combination thereof. In some aspects, nucleoside analogs include nucleosides comprising sugar modifications. In some aspects, nucleoside analogs include nucleosides comprising bicyclic sugars. In some aspects, nucleoside analogs include LNA.

[0221] In some respects, nucleoside analogs are selected from the group consisting of: restricted ethyl nucleosides (cEt), 2',4'-restricted 2'-O-methoxyethyl (cMOE), α-L-LNA, β-D-LNA, 2'-O,4'-C-ethylene-bridged nucleic acids (ENA), amino-LNA, oxy-LNA, thio-LNA, and any combination thereof. In some respects, ASOs contain one or more 5'-methyl-cytosine nucleobases.

[0222] II.D.1 nucleobase

[0223] The term nucleobase includes purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds during nucleic acid hybridization. In the context of this disclosure, the term nucleobase also encompasses modified nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In some aspects, nucleobase moieties are modified by modifying or substituting nucleobases. In this context, “nucleobase” refers to naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-natural variants. Such variants are described, for example, in Hirao et al., (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry, Supplement 371.4.1.

[0224] In some respects, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl-cytosine, 5-thiazo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazo-uracil, 2-thiouracil, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0225] The nucleobase moiety may be indicated by a letter code for each corresponding nucleobase, such as A, T, G, C, or U, wherein each letter may optionally include a modified nucleobase with an equivalent function. For example, in an exemplary oligonucleotide, the nucleobase moiety is selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA interstitial polymers, 5-methylcytosine LNA nucleosides may be used.

[0226] II.D.2. Sugar Modification

[0227] The ASO disclosed herein may comprise one or more nucleosides having a modified sugar moiety (i.e., a modification of the sugar moiety when compared to the ribosome found in DNA and RNA). Many nucleosides modified with a ribosome have been prepared, primarily with the aim of improving certain properties of the oligonucleotide, such as affinity and / or nuclease resistance.

[0228] Such modifications include those in which the ribocyclic structure is modified, for example, by replacing it with a hexose ring (HNA) or a bicyclic ring (LNA) that typically has a bis-molecular bridge between the C2' and C4' carbons on the ribocyclic ring, or an unlinked ribocyclic ring that typically lacks a bond between the C2' and C3' carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclic hexosinic nucleic acids (WO2011 / 017521) or tricyclic nucleic acids (WO2013 / 154798). Modified nucleosides also include nucleosides in which the sugar portion is replaced by a non-sugar portion, such as in the case of peptide nucleic acids (PNA) or morpholino nucleic acids.

[0229] Sugar modification also includes modifications by changing the substituents on the ribose ring to groups other than hydrogen or by altering the naturally occurring 2'-OH group in RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' positions. Nucleosides with modified sugar moieties also include 2'-modified nucleosides, such as 2'-substituted nucleosides. In fact, much attention has been paid to the development of 2'-substituted nucleosides, and many 2'-substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity.

[0230] II.D.2.a.2' modified nucleosides

[0231] 2'-sugar modified nucleosides are nucleosides having a substituent other than H or –OH at the 2' position (2'-substituted nucleosides) or containing a 2'-linked bimolecular group, and include 2'-substituted nucleosides and LNA (2'-4' bimolecular bridging) nucleosides. For example, 2'-modified sugars can provide enhanced binding affinity (e.g., affinity-enhancing 2'-sugar modified nucleosides) and / or increased nuclease resistance to oligonucleotides. Examples of 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'-fluoro-DNA, arabinonucleotide (ANA), and 2'-fluoro-ANA nucleosides. For further examples, see, for instance, Freier and Altmann; Nucl. Acid Res., 1997, 25, 4429-4443; Uhlmann, Curr. Opinion in Drug Development, 2000, 3(2), 293-213; and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are some descriptions of 2'-substituted modified nucleosides.

[0232]

[0233] II.D.2.b Locked nucleoside (LNA).

[0234] LNA nucleotides are modified nucleotides containing a linker group (called a binucleotide or bridge) between the C2' and C4' ends of the ribose ring (i.e., a 2'-4' bridge) that restricts or locks the conformation of the ribose ring. These nucleotides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). When LNAs are incorporated into oligonucleotides of complementary RNA or DNA molecules, the locking of the ribose conformation is associated with enhanced hybridization affinity (double-strand stabilization). This can be routinely determined by measuring the melting temperature of the oligonucleotide / complement double strand.

[0235] Non-limiting exemplary LNA nucleosides are described 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 Published in 2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al., J. Org. Chem. 2010, Vol. 75(5), pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238.

[0236] In some respects, the modified nucleosides or LNA nucleosides of the ASO disclosed herein have the general formula structure of formula I or II:

[0237]

[0238] in

[0239] W is selected from -O-, -S-, -N(R) a )-、-C(R a R b )-, especially -O-;

[0240] B is a nucleobase or a modified nucleobase portion;

[0241] Z is a nucleoside bond with an adjacent nucleoside or a 5'-terminal group;

[0242] Z* is a nucleoside linker to an adjacent nucleoside or a 3'-terminal group;

[0243] R 1 R 2 R 3 R 5 and R 5* Independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, hydroxyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, azide, heterocycle, and aryl; and

[0244] X, Y, R a and R b As defined in this article.

[0245] In some aspects of –XY-, Ra It is hydrogen or alkyl, particularly hydrogen or methyl. In some aspects of –XY-, R b It is hydrogen or alkyl, particularly hydrogen or methyl. In other aspects of –XY-, R a and R b One or both of them are hydrogen. In the other aspect of –XY-, R a and R b Only one of them is hydrogen. In some aspects of -XY-, R a and R b One of them is a methyl group and the other is hydrogen. In some aspects of -XY-, R a and R b Both are methyl groups.

[0246] In some aspects of –X-, R a It is hydrogen or alkyl, particularly hydrogen or methyl. In some aspects of –X-, R b It is hydrogen or alkyl, particularly hydrogen or methyl. In other aspects of –X-, R a and R b One or both of them are hydrogen. In some aspects of –X-, R a and R b Only one of them is hydrogen. In some aspects of –X-, R a and R b One of them is methyl and the other is hydrogen. In other aspects of –X-, R a and R b Both are methyl groups.

[0247] In some aspects of –Y-, R a It is hydrogen or alkyl, particularly hydrogen or methyl. In some aspects of –Y-, R b It is hydrogen or alkyl, particularly hydrogen or methyl. In other aspects of –Y-, R a and R b One or both of them are hydrogen. In some aspects of –Y-, R a and R b Only one of them is hydrogen. In the other aspects of –Y-, R a and R b One of them is a methyl group and the other is hydrogen. In some aspects of –Y-, R a and R b Both are methyl groups.

[0248] In some respects, R 1 R 2 R 3 R 5 and R 5* It is independently selected from hydrogen and alkyl groups, especially hydrogen and methyl groups.

[0249] In some respects, R 1 R 2 R 3 R 5 and R 5* Both are hydrogen.

[0250] In some respects, R 1 R 2 R 3 Both are hydrogen, R 5 and R 5* One of them is hydrogen and the other, as defined above, is particularly alkyl, and more particularly methyl.

[0251] In some respects, R 1 R 2 R 3 Both are hydrogen, R 5 and R 5* One of them is hydrogen and the other is an azide.

[0252] In some respects, -XY- is -O-CH2-, W is oxygen and R is oxygen. 1 R 2 R 3 R 5 and R 5* All of these are hydrogen. Such LNA nucleosides are disclosed in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352 and WO 2004 / 046160, all of which are hereby incorporated by reference, and include β-D-oxyLNA and α-L-oxyLNA nucleosides known in the art.

[0253] In some respects, -XY- is -S-CH2-, W is oxygen and R is oxygen. 1 R 2 R 3 R 5 and R 5* Both are hydrogen. Such thio-LNA nucleosides are disclosed in WO 99 / 014226 and WO 2004 / 046160, which are hereby incorporated by reference.

[0254] In some respects, -XY- is -NH-CH2-, W is oxygen and R is oxygen. 1 R 2 R 3 R 5 and R 5* Both are hydrogen. Such amino-LNA nucleosides are disclosed in WO 99 / 014226 and WO 2004 / 046160, which are hereby incorporated by reference.

[0255] In some respects, -XY- is -O-CH2CH2- or -OCH2CH2CH2-, W is oxygen and R is oxygen. 1 R 2 R 3 R 5 and R 5* Both are hydrogen. Such LNA nucleosides are disclosed in WO 00 / 047599 and Morita et al., Bioorganic & Med. Chem. Lett. 12, 73-76, which are hereby incorporated by reference, and include 2'-O-4'C-ethylene-bridged nucleic acids (ENA) known in the art.

[0256] In some respects, -XY- is -O-CH2-, W is oxygen, and R is... 1 R 2 R 3 Both are hydrogen, R 5 and R 5* One of them is hydrogen and the other is not hydrogen, such as an alkyl group, for example a methyl group. Such 5'-substituted LNA nucleosides are disclosed in WO 2007 / 134181, which is hereby incorporated by reference.

[0257] In some respects, -XY- is -O-CR a R b -, where R a and R b One or both of them are not hydrogen, especially alkyl groups such as methyl, W is oxygen, R 1 R 2 R 3 Both are hydrogen, R 5 and R 5* One of them is hydrogen and the other is not hydrogen, particularly alkyl, such as methyl. Such dual-modified LNA nucleosides are disclosed in WO 2010 / 077578, which is hereby incorporated by reference.

[0258] In some respects, -XY- is -O-CH(CH2-O-CH3)- ("2'O-methoxyethyl bicyclic nucleic acid", Seth et al., J.Org.Chem. 2010, Vol. 75(5), pp. 1569-81).

[0259] In some respects, -XY- is -O-CHR a -, W is oxygen and R 1 R 2 R 3 R 5 and R 5*Both are hydrogen. Such 6'-substituted LNA nucleosides are disclosed in WO 2010 / 036698 and WO 2007 / 090071, which are hereby incorporated by reference. In such 6'-substituted LNA nucleosides, R a In particular, C1-C6 alkyl groups, such as methyl.

[0260] In some respects, -XY- is -O-CH(CH2-O-CH3)-, W is oxygen and R is oxygen. 1 R 2 R 3 R 5 and R 5* Both are hydrogen atoms. This type of LNA nucleoside is also known in the art as cyclic MOE (cMOE) and is disclosed in WO 2007 / 090071.

[0261] In some respects, -XY- is -O-CH(CH3)-.

[0262] In some respects, -XY- is -O-CH 2- O-CH2- (Seth et al., J.Org.Chem 2010, see above).

[0263] In some respects, -XY- is -O-CH(CH3)-, W is oxygen and R is oxygen. 1 R 2 R 3 R 5 and R 5* Both are hydrogen. Such 6'-methyl LNA nucleosides are also known in the art as cET nucleosides, and can be (S)-cET or (R)-cET diastereomers, as disclosed in WO 2007 / 090071 (β-D) and WO 2010 / 036698 (α-L), both of which are hereby incorporated by reference.

[0264] In some respects, XY- is -O-CR a R b -, where R a and R b Neither is hydrogen, W is oxygen and R 1 R 2 R 3 R 5 and R 5* Both are hydrogen. In some respects, R a and R b All of them are alkyl groups, and in particular, all of them are methyl groups. Such 6'-disubstituted LNA nucleosides are disclosed in WO 2009 / 006478, which is hereby incorporated by reference.

[0265] In some respects, -XY- is -S-CHR a -, W is oxygen and R 1 R 2 R 3 R 5 and R 5* Both are hydrogen. Such 6'-substituted thio-LNA nucleosides are disclosed in WO 2011 / 156202, which is hereby incorporated by reference. In certain aspects of such 6'-substituted thio-LNAs, R a It is an alkyl group, especially a methyl group.

[0266] In some respects, -XY- is -C(=CH2)C(R) a R b )-, such as W is oxygen and R 1 R 2 R 3 R 5 and R 5* Both are hydrogen. Such vinyl carbon LNA nucleosides are disclosed in WO 2008 / 154401 and WO 2009 / 067647, both of which are hereby incorporated by reference.

[0267] In some respects, -XY- is -N (OR) a )-CH2-, W is oxygen and R 1 R 2 R 3 R 5 and R 5* Both are hydrogen. In some respects, R a It is an alkyl group, such as methyl. Such LNA nucleosides are also called N-substituted LNAs and are disclosed in WO 2008 / 150729, which is hereby incorporated by reference.

[0268] In some respects, -XY- is -O-NCH3- (Seth et al., J.Org.Chem 2010, see above).

[0269] In some respects, -XY- is ON(R a )-–N(R a -O-、-NR a -CR a R b -CR a R b -or–NR a -CR a R b -, W is oxygen and R 1 R 2 R 3 R 5 and R5* Both are hydrogen. In some respects, R a It is an alkyl group, such as methyl. (Seth et al., J.Org.Chem 2010, see above).

[0270] In some respects, R 5 and R 5* Both are hydrogen. In other respects, R... 5 and R 5* One of them is hydrogen and the other is an alkyl group, such as a methyl group. In this respect, R 1 R 2 and R 3 Specifically, hydrogen and -XY- can be particularly -O-CH2- or -O-CHC(R) a )3-, such as -O-CH(CH3)-.

[0271] In some respects, -XY- is -CR a R b -O-CR a R b -, such as -CH2-O-CH2-, where W is oxygen and R is... 1 R 2 R 3 R 5 and R 5* Both are hydrogen. In this respect, R a In particular, alkyl groups, such as methyl groups, may be used. Such LNA nucleotides are also known as conformation-restricted nucleotides (CRNs) and are disclosed in WO 2013 / 036868, which is hereby incorporated by reference.

[0272] In some respects, -XY- is -O-CR a R b -O-CR a R b -, such as -O-CH2-O-CH2-, where W is oxygen and R is... 1 R 2 R 3 R 5 and R 5* Both are hydrogen. In some respects, R a In particular, alkyl groups, such as methyl groups, may be used. Such LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, which are hereby incorporated by reference.

[0273] Unless otherwise stated, it will be recognized that LNA nucleosides can be β-D or α-L stereoisoforms.

[0274] Scheme 1 presents some examples of LNA nucleotides.

[0275] Option 1

[0276]

[0277] As shown elsewhere, in some aspects of this disclosure, the LNA nucleotide in the oligonucleotide is β-D-oxy-LNA nucleotide.

[0278] III.E. Nuclease-mediated degradation

[0279] Nuclease-mediated degradation refers to oligonucleotides that can mediate the degradation of a sequence when it forms a double helix with a complementary nucleotide sequence.

[0280] In some respects, oligonucleotides can function via nuclease-mediated target nucleic acid degradation, wherein the oligonucleotides of this disclosure are capable of recruiting nucleases, particularly endonucleases, preferably ribonucleases (RNases), such as RNase H. Examples of oligonucleotide designs functioning via nuclease-mediated mechanisms are oligonucleotides that typically contain a region of at least 5 or 6 DNA nucleotides and are side-joined with nucleotides (e.g., interstitial polymers) that enhance affinity on one or both sides.

[0281] II.F. RNase H activity and recruitment

[0282] The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H and induce the degradation of the complementary RNA molecule when forming a double strand with it. WO01 / 23613 provides in vitro methods for determining RNase H activity, which can be used to determine the ability to recruit RNase H. Generally, an oligonucleotide is considered capable of recruiting RNase H if it has an initial rate of at least 5%, such as at least 10% or more, or more than 20%, when a complementary target nucleic acid sequence is provided. This initial rate is determined using an oligonucleotide having the same base sequence as the modified oligonucleotide being tested but containing only DNA monomers, with phosphate thioester bonds between all monomers in the oligonucleotide, and using the methods provided in Examples 91-95 of WO01 / 23613.

[0283] In some respects, if the initial rate of RNase H (e.g., measured in pmol / L / min) when the complementary target nucleic acid is provided is less than 20%, such as less than 10%, such as less than 5%, then the oligonucleotide is considered to be substantially unable to recruit RNase H. This initial rate is determined using an oligonucleotide with the same base sequence as the oligonucleotide being tested but containing only DNA monomers, without 2' substitutions, and with phosphothioester bonds between all monomers in the oligonucleotide, and using the method provided in Examples 91-95 of WO01 / 23613. In some respects, ASO is fully phosphothioesterified.

[0284] II.G.ASO Design

[0285] The ASO disclosed herein may comprise a nucleotide sequence comprising both a nucleoside and a nucleoside analogue, and may be in the form of an interstitial polymer. Examples of configurations of interstitial polymers that may be used with the ASO disclosed herein are described in U.S. Patent Application Publication No. 2012 / 0322851.

[0286] As used herein, the term "interstitial aggregate" refers to an antisense oligonucleotide containing a region of an RNase H-recruiting oligonucleotide (interstitial aggregate) flanked by one or more affinity-enhancing modified nucleosides (flanks) at the 5' and 3' sides. The term "LNA interstitial aggregate" is an interstitial oligonucleotide in which at least one of the affinity-enhancing modified nucleosides is an LNA nucleoside. The term "mixed-wing interstitial aggregate" refers to an LNA interstitial aggregate in which the flanking region contains at least one LNA nucleoside and at least one DNA nucleoside or a 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'-fluoro-DNA, arabinonucleotide (ANA), and 2'-fluoro-ANA nucleoside.

[0287] In some aspects, the ASO of this disclosure may be in the form of a hybrid polymer. In some aspects, the ASO of this disclosure may be in the form of a holopolymer. In some aspects, in addition to enhancing the affinity of the ASO for the target region, some nucleoside analogs also mediate the binding and cleavage of RNases (e.g., RNase H). Since α-L-LNA monomers recruit RNase H activity to some extent, in some aspects, the interstitial region (e.g., region B as mentioned herein) of the ASO containing α-L-LNA monomers consists of fewer monomers that can be recognized and cleaved by RNase H, and introduces more flexibility in the construction of hybrid polymers.

[0288] In some aspects, the ASO contains at least one, at least two, or at least three LNA portions at the 5' end of the ASO sequence. In some aspects, the ASO contains three LNA portions at the 5' end of the ASO sequence. In some aspects, the ASO contains at least one, at least two, or at least three LNA portions at the 3' end of the ASO sequence. In some aspects, the ASO contains three LNA portions at the 3' end of the ASO sequence. In some aspects, the ASO contains at least one, at least two, or at least three LNA portions at the 5' end of the ASO sequence and at the 3' end of the ASO sequence. In some aspects, the ASO contains three LNA portions at the 5' end of the ASO sequence and at the 3' end of the ASO sequence.

[0289] II.G.1. Interstitial polymer design

[0290] In some aspects, the ASO of this disclosure is a spacer polymer and comprises adjacent nucleotide segments (e.g., one or more DNA molecules) capable of recruiting RNases (such as RNase H), referred herein as region B (B), wherein region B is flanked by regions of nucleoside analogs at the 5' and 3' sides, the regions of which are located at the 5' and 3' of the adjacent nucleotide segments of region B—these regions are referred to as region A (A) and region C (C), respectively. In some aspects, the nucleoside analogs are sugar-modified nucleosides (e.g., high-affinity sugar-modified nucleosides). In some aspects, the sugar-modified nucleosides of regions A and C enhance the affinity of the ASO for target nucleic acids (i.e., 2' sugar-modified nucleosides that enhance affinity). In some aspects, the sugar-modified nucleosides are 2' sugar-modified nucleosides, such as high-affinity 2' sugar modifications, such as LNA and / or 2'-MOE.

[0291] In the interstitial polymer, the 5' and 3' nucleotides of region B are DNA nucleotides and are located near the nucleotide analogs (e.g., high-affinity sugar-modified nucleotides) of regions A and C, respectively. In some respects, regions A and C can be further defined by having nucleotide analogs at the ends furthest from region B (i.e., at the 5' end of region A and the 3' end of region C).

[0292] In some aspects, the ASO disclosed herein comprises a nucleotide sequence of formula (5' to 3')ABC, wherein: (A) (5' region or 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) capable of recruiting RNases (when forming a double strand with complementary RNA molecules such as pre-mRNA or mRNA targets); and (C) (3' region or second wing sequence) comprises at least one nucleoside analog (e.g., 3-5 LNA units).

[0293] In some aspects, region A contains 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, and region C consists of 3 or 4 nucleoside analogs such as LNA. Such designs include (ABC) 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. In some aspects, ASO has LLLD. n LLL, LLLLD n LLLL or LLLLLD n The design is LLLLL, where L is a nucleoside analog, D is DNA, and n can be any integer between 4 and 24. In some aspects, n can be any integer between 6 and 14. In some aspects, n can be any integer between 8 and 12. In some aspects, ASO has designs of LLLLMMDnMMLLL, LLLMDnMLLL, LLLMMDnMMLLLL, LLLMDnMLLLL, LLLLLMMDnMMLLLLL, or LLLLLMDnMLLLLL, where D is DNA, n can be any integer between 3 and 15, L is LNA, and M is 2' MOE.

[0294] Other interstitial polymer designs are disclosed in WO2004 / 046160, WO 2007 / 146511 and WO2008 / 113832, which are hereby incorporated herein by reference in their entirety.

[0295] II.H. Nucleotide linkages

[0296] The monomers of the ASO described herein are coupled together via a linking group. Suitablely, each monomer is linked to its 3' adjacent monomer via a linking group.

[0297] Those skilled in the art will understand that, in the context of this disclosure, the 5' monomer at the ASO terminus does not contain a 5' linking group, although it may or may not contain a 5' terminal group.

[0298] In some respects, a continuous nucleotide sequence contains one or more modified internucleotide links. The term "linking group" or "internucleotide link" is intended to refer to a group capable of covalently coupling two nucleosides together. Non-limiting examples include phosphate groups and thiophosphate groups.

[0299] The nucleosides or their adjacent nucleoside sequences of the ASO disclosed herein are coupled together via a linking group. Suitably, each nucleoside is linked to its 3' adjacent nucleoside via a linking group.

[0300] In some respects, the nucleoside-to-nucleotide linkages are modified from their normal phosphodiester form to one that is more resistant to nuclease attack, such as a thiophosphate that can be cleaved by RNase H and also allows the antisense repression pathway to reduce the expression of the target gene. In some respects, 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 the nucleoside-to-nucleotide linkages are modified.

[0301] III. Extracellular vesicles, such as exosomes

[0302] This article discloses EVs containing ASOs, such as exosomes. An ASO can be any ASO described herein or a functional fragment thereof. In some respects, ASOs reduce the levels of CEBP / β mRNA or CEBP / β protein in target cells.

[0303] In some aspects, an EV (e.g., an exosome) contains at least one ASO. In some aspects, an EV (e.g., an exosome) contains at least two ASOs, for example, a first ASO containing a first nucleotide sequence and a second ASO containing a second nucleotide sequence. In some aspects, an EV (e.g., an exosome) contains at least three ASOs, at least four ASOs, at least five ASOs, at least six ASOs, or more than six ASOs. In some aspects, each of the first ASO, second ASO, third ASO, fourth ASO, fifth ASO, sixth ASO, and / or ninth ASO is distinct.

[0304] In some aspects, the EV (e.g., exosome) comprises a first ASO and a second ASO, wherein the first ASO comprises a first nucleotide sequence complementary to a first target sequence in the first transcript, and wherein the second ASO comprises a second nucleotide sequence complementary to a second target sequence in the first transcript. In some aspects, the first target sequence does not overlap with the second target sequence.

[0305] In some respects, the EV (e.g., exosome) comprises a first ASO and a second ASO, wherein the first ASO comprises a nucleotide sequence complementary to a target sequence in the CEBP / β transcript (i.e., the ASO disclosed herein), and wherein the second ASO comprises a second nucleotide sequence complementary to a second target sequence in a second transcript, wherein the second transcript is not a CEBP / β transcript.

[0306] In some respects, EVs (e.g., exosomes) target tumor cells, dendritic cells, T cells, B cells, macrophages, monocytes, neurons, hepatocytes, alveolar macrophages, interstitial macrophages, Kupffer cells, myeloid cells (e.g., neutrophils, myeloid-derived suppressor cells (MDSCs, e.g., monocytic MDSCs or granulocytic MDSCs), monocytes, macrophages, hematopoietic stem cells, basophils, neutrophils, or eosinophils) or any combination thereof. In some respects, EVs (e.g., exosomes) target cells selected from the group consisting of macrophages, myeloid-derived suppressor cells (MDSCs), monocytes, basophils, neutrophils, eosinophils, and any combination thereof. In some respects, EVs (e.g., exosomes) target myeloid cells. In some respects, EVs (e.g., exosomes) target macrophages. In some respects, EVs (e.g., exosomes) target the liver, heart, lungs, brain, kidneys, central nervous system, peripheral nervous system, muscles, bones, joints, skin, intestines, bladder, pancreas, lymph nodes, spleen, blood, bone marrow, or any combination thereof.

[0307] In some respects, EVs (e.g., exosomes) reduce the expression of one or more genes upregulated by CEBP / β. In some respects, EVs (e.g., exosomes) promote the differentiation of M2 macrophages. In some respects, EVs (e.g., exosomes) reduce the differentiation of M1 macrophages. In some respects, EVs (e.g., exosomes) promote the differentiation of myeloid-derived suppressor cells (MDSCs) into pro-inflammatory phenotypes.

[0308] In some respects, EVs (e.g., exosomes) are used to treat cancers in subjects who require this treatment. In some respects, the cancers are selected from the group consisting of: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteoblastic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, lymphoma, leukemia, liver cancer, glioblastoma, melanoma, myeloma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma. Adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, and any combination thereof. In some respects, EVs (e.g., exosomes) increase the infiltration of immune cells (e.g., macrophages) into the tumor.

[0309] In some aspects, EVs (e.g., exosomes) treat central nervous system tumors in subjects. In some aspects, EVs (e.g., exosomes) treat brain tumors in subjects. In some aspects, EVs (e.g., exosomes) treat glioblastomas in subjects. In some aspects, the glioblastoma is glioblastoma multiforme (GBM). In some aspects, EVs (e.g., exosomes) treat meningeal carcinomatosis in subjects. In some aspects, EVs (e.g., exosomes) containing ASO activate macrophages within the central nervous system. In some aspects, EVs (e.g., exosomes) containing ASO induce M1 polarization of macrophages within the central nervous system. In some aspects, EVs (e.g., exosomes) containing ASO activate meningeal macrophages. In some aspects, EVs (e.g., exosomes) containing ASO induce M1 polarization of meningeal macrophages. In some aspects, EVs (e.g., exosomes) containing ASO induce tumor infiltration of meningeal macrophages.

[0310] In some respects, EV (e.g., exosomes) can treat fibrosis in subjects who require this treatment. Excessive M2 macrophage activation leads to the sustained production of TGFβ and growth factors, which promote myofibroblast proliferation, EMT / EndoMT activation, and extracellular matrix deposition. M2 macrophages represent a turning point between wound healing and the worsening of the pro-fibrotic process. In some contexts, fibrosis is selected from liver fibrosis (NASH), cirrhosis, pulmonary fibrosis, cystic fibrosis, chronic ulcerative colitis / IBD, bladder fibrosis, renal fibrosis, CAPS (Muckle-Wells syndrome), atrial fibrosis, endocardial myocardial fibrosis, old myocardial infarction, glial scars, arterial stiffness, joint fibrosis, Crohn's disease, Dupuytren's contracture, keloid fibrosis, mediastinal fibrosis, myelofibrosis, Peyronie's disease, renal systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, adhesive capsulitis, and any combination thereof. In some contexts, EVs (e.g., exosomes) are used to treat liver fibrosis (NASH). In some contexts, EVs (e.g., exosomes) are used to treat CAPS (Muckle-Wells syndrome).

[0311] In some respects, EVs (e.g., exosomes) are used to treat neurodegenerative diseases. In some respects, neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, prions, motor neuron disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, and any combination thereof.

[0312] In some respects, EVs (e.g., exosomes) are used to treat metabolic disorders / CVD. In some respects, metabolic disorders / CVD are selected from acid-base imbalances, metabolic encephalopathy, calcium metabolism disorders, DNA repair defects, glucose metabolism disorders, hyperlactatemia, iron metabolism disorders, lipid metabolism disorders, malabsorption syndrome, metabolic syndrome X, congenital metabolic defects, mitochondrial diseases, phosphorus metabolism disorders, porphyria, protein homeostasis deficiencies, metabolic skin diseases, wasting syndrome, electrolyte imbalances, and any combination thereof.

[0313] As described above, the EVs (e.g., exosomes) discussed herein are extracellular vesicles with a diameter between approximately 20 and 300 nm. The size of the EVs (e.g., exosomes) described herein can be measured according to the methods described below.

[0314] In some aspects, the EVs (e.g., exosomes) of this disclosure comprise a double lipid membrane (“EV (e.g., exosome) membrane”) comprising an inner (luminal) surface and an outer surface. In some aspects, the inner (luminal) surface faces the core (i.e., the lumen) of the EV (e.g., exosome). In some aspects, the outer surface may be in contact with the membrane / cytoplasm of an endosome, a multivesicular body, or a producing cell or target cell.

[0315] In some respects, EV (e.g., exosome) membranes contain lipids and fatty acids. In other respects, EV (e.g., exosome) membranes contain phospholipids, glycolipids, fatty acids, sphingolipids, glycerol phosphates, sterols, cholesterol, and phosphatidylserine.

[0316] In some aspects, the EV (e.g., exosome) membrane comprises an inner lobule and an outer lobule. The composition of the inner and outer lobules can be determined by transbilayer distribution determinations known in the art, see, for example, Kuypers et al., Biohim Biophys Acta 1985 819:170. In some aspects, the outer lobule is composed of about 70%–90% choline phospholipids, about 0%–15% acidic phospholipids, and about 5%–30% phosphatidylethanolamine. In some aspects, the inner lobule is composed of about 15%–40% choline phospholipids, about 10%–50% acidic phospholipids, and about 30%–60% phosphatidylethanolamine.

[0317] In some respects, EV (e.g., exosome) membranes contain one or more polysaccharides, such as glycans.

[0318] In some aspects, the EV (e.g., exosome) disclosed herein includes an ASO, wherein the ASO is connected to the EV via a support portion, on the outer surface of the EV or on the cavity surface of the EV.

[0319] In some aspects, EVs containing ASO (e.g., exosomes) include an anchoring portion between the ASO and the exosome membrane, which optionally includes a connector. Non-limiting examples of connectors are disclosed elsewhere herein.

[0320] III.A. Anchoring Section (AM)

[0321] One or more anchoring moieties (AMs) can be used to anchor ASO to the EV of this disclosure. In some aspects, the ASO is directly attached to the anchoring moieties or via a connector. In some aspects, the ASO can be attached to the anchoring moieties or connector combinations via a reaction between a reactive group (RG; e.g., amine, thiol, hydroxyl, carboxylic acid, or azide) and a reactive moiety (RM; e.g., maleimide, succinate, NHS). Several potential synthetic routes are envisioned, such as:

[0322] [AM]- / reactive moiety / + / reactive group / -[ASO]

[0323] [AM]-[connector]n- / reactive moiety / + / reactive group / -[ASO]

[0324] [AM]- / reactive moiety / + / reactive group / -[connector]n-[ASO]

[0325] [AM]-[connector]n- / reactive moiety / + / reactive group / -[connector]n-[ASO]

[0326] The anchoring portion can be inserted into the lipid bilayer of EVs (e.g., exosomes), thereby allowing exosomes to load ASO. Currently, a major obstacle to the commercialization of exosomes as delivery mediators for polar ASO is the highly inefficient loading process. This obstacle can be overcome by modifying exosomes before loading polar ASO. Therefore, as described herein, modification of ASO facilitates their loading into exosomes.

[0327] Compared to the previously reported loading efficiencies of introducing unmodified ASO into exosomes via methods such as electroporation or cationic lipid transfection, the method described in this paper for loading exosomes with modified polar ASO significantly improves loading efficiency.

[0328] In some respects, the modification increases 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 times compared to natural (unmodified) ASO. In some respects, the modification increases 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 compared to natural (unmodified) ASO.

[0329] In some respects, the modification increases 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 the natural (unmodified) ASO (e.g., the corresponding unmodified ASO).

[0330] In some aspects, the anchoring moiety may be chemically conjugated to the ASO to enhance its hydrophobic properties. In exemplary aspects, the anchoring moiety is a sterol (e.g., cholesterol), GM1, a lipid, a vitamin, a small molecule, a peptide, or a combination thereof. In some aspects, the moiety is a lipid. In some aspects, the anchoring moiety is a sterol, such as cholesterol. Other hydrophobic moieties include, for example, phospholipids, lysophospholipids, fatty acids, or vitamins (e.g., vitamin D or vitamin E).

[0331] In some respects, the anchoring portion is directly or via one or more joints attached to the end of the ASO (i.e., "end finishing"). In other respects, the anchoring portion is attached to other parts of the ASO.

[0332] In some aspects, the ASO may contain a detectable marker. Exemplary markers include fluorescent and / or radioactive markers. In some aspects, where the ASO is fluorescently labeled, the detectable marker may be, for example, Cy3. Adding a detectable marker to the ASO can be used as a way to label exosomes and track their biodistribution. In other aspects, the detectable marker may be directly attached to exosomes, for example, by labeling exosomal lipids and / or exosomal peptides.

[0333] The different components of ASO (i.e., anchoring parts, connectors and connector combinations, and ASO) can be linked by amides, esters, ethers, thioethers, disulfides, aminophosphates, triphosphates, dithiophosphates, methyl phosphonate, phosphate diesters or thiophosphates, or alternatively any or other linkages.

[0334] In some respects, different components of ASO can be connectors using bifunctional connectors (i.e., connectors containing two functional groups), such as N-succinimide-3-(2-pyridyl dithio)propionate, N-4-maleimide butyric acid, S-(2-pyridyl dithio)cysteine, iodoacetoxysuccinimide, N-(4-maleimide butoxy)succinimide, N-[5-(3'-maleimide propylamide)-1-carboxypentyl]iminodiacetic acid, N-(5-aminopentyl)-iminodiacetic acid, etc.

[0335] III.A.1. Anchoring Part

[0336] As described in detail below, suitable anchoring portions capable of anchoring ASO to the surface of EVs (e.g., exosomes) include, for example, sterols (e.g., cholesterol), lipids, lysophospholipids, fatty acids, or fat-soluble vitamins.

[0337] In some aspects, the anchoring moiety can be a lipid. The lipid anchoring moiety can be any lipid known in the art, such as palmitic acid or glycosylphosphatidylinositol. In some aspects, the lipid is a fatty acid, phosphatide, phospholipid (e.g., phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine) or its analogues (e.g., phosphatidylcholine, lecithin, phosphatidylethanolamine, cephalin, or phosphatidylserine or its analogues or portions, such as partially hydrolyzed portions).

[0338] Typically, anchoring moieties are chemically linked. However, anchoring moieties can be enzymatically linked to ASOs. In some respects, it is possible to link anchoring moieties to ASOs by altering cell culture conditions. For example, by using a medium that restricts myristic acid, some other fatty acids, including short-chain and unsaturated fatty acids, can be linked to N-terminal glycine. For instance, it has been reported that in BK channels, myristic acid is linked posttranslatively to internal serine / threonine or tyrosine residues via a hydroxyl ester bond.

[0339] The anchoring portion can be attached to the ASO directly or indirectly via a joint assembly at any chemically feasible location, such as at the 5' and / or 3' end of the ASO. In one aspect, the anchoring portion is attached only to the 3' end of the ASO. In another aspect, the anchoring portion is attached only to the 5' end of the ASO. In another aspect, the anchoring portion is attached at a location that is not the 3' or 5' end of the ASO.

[0340] Some types of membrane anchors that can be used to practice the methods of this disclosure are presented in the following table:

[0341]

[0342] In some aspects, the anchoring portion of this disclosure comprises two or more types of anchoring portions disclosed herein. For example, in some aspects, the anchoring portion comprises two lipids (e.g., phospholipids and fatty acids), or two phospholipids, or two fatty acids, or lipids and vitamins, or cholesterol and vitamins, etc., having together 6-80 carbon atoms (i.e., 6-80 equivalent carbons (ECN)).

[0343] In some aspects, the combination of anchoring components, such as lipids (e.g., fatty acids), has the following combinations: 6-80, 8-80, 10-80, 12-80, 14-80, 16-80, 18-80, 20-80, 22-80, 24-80, 26-80, 28-80, 30-80, 4-76, 6-76, 8-76, 10-76, 12-76, 14-76, 16-76, 18-76, 20-76, 22-76, 24-76, 26-76, 28-76, 30-76, 6-72, 8-72, 10-72, 12-72, 14-72, 16-72, 18-72, 20-72, 22-7 2, 24-72, 26-72, 28-72, 30-72, 6-68, 8-68, 10-68, 12-68, 14-68, 16-68, 18-68, 20-68, 22-68, 24-68, 26-68, 28-68, 30-68, 6-64, 8-64, 10-64, 12-64, 14-64, 16-64, 18-64, 20-64, 22-64, 24-64, 26-64, 28-64, 30-64, 6-60, 8-60, 10-60, 12-56, 14-56, 16-56, 18-56, 20-56, 22-56, 24-56, 26 -56, 28-56, 30-56, 6-52, 8-52, 10-52, 12-52, 14-52, 16-52, 18-52, 20-52, 22-52, 24-52, 26-52, 28-52, 30-52, 6-48, 8-48, 10-48, 12-48, 14-48, 16-48, 18-48, 20-48, 22-48, 24-48, 26-48, 28-48, 30-48, 6-44, 8-44, 10-44, 12-44, 14-44, 16-44, 18-44, 20-44, 22-44, 24-44, 26-44, 28-44 ECNs for 30-44, 6-40, 8-40, 10-40, 12-40, 14-40, 16-40, 18-40, 20-40, 22-40, 24-40, 26-40, 28-40, 30-40, 6-36, 8-36, 10-36, 12-36, 14-36, 16-36, 18-36, 20-36, 22-36, 24-36, 26-36, 28-36, 30-36, 6-32, 8-32, 10-32, 12-32, 14-32, 16-32, 18-32, 20-32, 22-32, 24-32, 26-32, 28-32, or 30-32.

[0344] III.A.1.a. Cholesterol and other sterols

[0345] In some aspects, the anchoring moiety comprises sterols, steroids, hopanes, hydroxysteroids, open-ring steroids, or their lipophilic analogues. In some aspects, the anchoring moiety comprises sterols, such as phytosterols, mycosterols, or animal sterols. Exemplary animal sterols include cholesterol and 24S-hydroxycholesterol; exemplary phytosterols include ergosterol (mycosterol), campesterol, sitosterol, and stigmasterol. In some aspects, the sterol is selected from ergosterol, 7-dehydrocholesterol, cholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fucosterol, saringosterol, campesterol, β-sitosterol, sitosterol, coprosterol, oat sterol, or stigmasterol. Sterols can exist as free sterols, acylated (sterol esters), alkylated (sterol alkyl ethers), sulfated (sterol sulfates), or linked to a glycoside moiety (sterol glycosides), and can themselves be acylated (acylated sterol glycosides).

[0346] In some respects, the anchoring component contains steroids. In some respects, the steroids are selected from dihydrotestosterone, arbutin, agave saponins, diosgenin saponins, progesterone, or cortisol.

[0347] For example, sterols can be conjugated to ASO directly or via a linker at a available -OH group of the sterol. Exemplary sterols have the following general skeleton:

[0348]

[0349] As another example, ergosterol has the following structure:

[0350]

[0351] Cholesterol has the following structure:

[0352]

[0353] Therefore, in some embodiments, the free -OH group of a sterol or steroid is used to conjugate ASO directly or via a linker combination to a sterol (e.g., cholesterol) or steroid.

[0354] III.A.1.b Fatty Acids

[0355] In some respects, the anchoring component is a fatty acid. In some respects, fatty acids are short-chain, medium-chain, or long-chain fatty acids. In some respects, fatty acids are saturated fatty acids. In some respects, fatty acids are unsaturated fatty acids. In some respects, fatty acids are monounsaturated fatty acids. In some respects, fatty acids are polyunsaturated fatty acids, such as ω-3 (ω-3) or ω-6 (ω-6) fatty acids.

[0356] In some respects, lipids (such as fatty acids) have a C2-C ratio. 60 Chain. In some embodiments, lipids (e.g., fatty acids) have a C2-C chain. 28 Chain. In some respects, fatty acids have a C2-C chain. 40 Chain. In some respects, fatty acids have a C2-C chain. 12 Or C4-C 12 Chain. In some respects, fatty acids have a C4-C chain. 40 Chain. In some respects, fatty acids have a C4-C chain. 40 C2-C 38 C2-C 36 C2-C 34 C2-C 32 C2-C 30 C4-C 30 C2-C 28 C4-C 28 C2-C 26 C4-C 26 C2-C 24 C4-C 24 C6-C 24 C8-C 24 C 10 -C 24 C2-C 22 C4-C 22 C6-C 22 C8-C 22 C 10 -C 22 C2-C 20 C4-C 20 C6-C 20 C8-C 20 C 10 -C 20 C2-C 18 C4-C 18 C6-C 18 C8-C 18 C 10 -C 18 C 12 -C 18 C 14 -C 18 C 16 -C 18 C2-C 16 C4-C 16 C6-C 16 C8-C 16 C 10 -C 16、C 12 -C 16 、C 14 -C 16 、C2-C 15 、C4-C 15 、C6-C 15 、C8-C 15 、C9-C 15 、C 10 -C 15 、C 11 -C 15 、C 12 -C 15 、C 13 -C 15 、C2-C 14 、C4-C 14 、C6-C 14 、C8-C 14 、C9-C 14 、C 10 -C 14 、C 11 -C 14 、C 12 -C 14 、C2-C 13 、C4-C 13 、C6-C 13 、C7-C 13 、C8-C 13 、C9-C 13 、C 10 -C 13 、C 10 -C 13 、C 11 -C 13 、C2-C 12 、C4-C 12 、C6-C 12 、C7-C 12 、C8-C 12 、C9-C 12 、C 10 -C 12 、C2-C 11 、C4-C 11 、C6-C 11 、C7-C 11 、C8-C 11 、C9-C 11 、C2-C 10 、C4-C 10Fatty acids can have C2-C9, C4-C9, C2-C8, C2-C7, C4-C7, C2-C6, or C4-C6 chains. In some respects, fatty acids have C2, C3, C4, C5, C6, C7, C8, C9, C1, C2 ... 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22 C 23 C 24 C 25 C 26 C 27 C 28 C 29 C 30 C 31 C 32 C 33 C 34 C 35 C 36 C 37 C 38 C 39 C 40 C 41 C 42 C 43 C 44 C 45 C 46 C 47 C 48 C 49 C 50 C 51 C 52 C 53 C 54 C 55 C 56 C 57 C 58 C 59 Or C 60 chain.

[0357] In some aspects, the anchoring portion comprises two fatty acids, each independently selected from fatty acids having a chain having any of the aforementioned ranges or numbers of carbon atoms. In some aspects, one fatty acid is independently a fatty acid having a C6-C21 chain, and the other fatty acid is independently a fatty acid having a C12-C36 chain. In some embodiments, each fatty acid independently has a chain with 11, 12, 13, 14, 15, 16, or 17 carbon atoms.

[0358] Suitable fatty acids include saturated straight-chain fatty acids, saturated branched-chain fatty acids, unsaturated fatty acids, hydroxy fatty acids, and polycarboxylic acids. In some respects, these fatty acids have up to 32 carbon atoms.

[0359] Examples of useful saturated straight-chain fatty acids include those with an even number of carbon atoms, such as butyric acid, hexanoic acid, octanoic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, tetracosanoic acid, hexacosanoic acid, octacosanoic acid, triacontanic acid, and trichomeoic acid, and those with an odd number of carbon atoms, such as propionic acid, valeric acid, heptanoic acid, nonanoic acid, undecanoic acid, tridecanoic acid, pentadecanoic acid, heptadecanic acid, nonadecanic acid, dodecanoic acid, trisanoic acid, pentadecanoic acid, and heptadecanic acid.

[0360] Examples of suitable saturated branched fatty acids include isobutyric acid, isohexanoic acid, isooctanoic acid, isodecanic acid, isolaric acid, 11-methyldodecanoic acid, isomearic acid, 13-methyltetradecanoic acid, isopalmitic acid, 15-methylhexadecanoic acid, isostearic acid, 17-methyloctadecanoic acid, isarachidic acid, 19-methyleicosanoic acid, α-ethylhexanoic acid, α-hexyldecanoic acid, α-heptylundecanoic acid, 2-decyltetradecanoic acid, 2-undecyltetradecanoic acid, 2-decylpentadecananoic acid, 2-undecylpentadecananoic acid, and Fine oxocol 1800 acid (a product of Nissan Chemical Industries, Ltd.). Suitable saturated odd-chain branched fatty acids include trans-iso-fatty acids terminated with isobutyl groups, such as 6-methyl-octanoic acid, 8-methyl-decanoic acid, 10-methyl-dodecanoic acid, 12-methyl-tetradecanoic acid, 14-methyl-hexadecanoic acid, 16-methyl-octadecanoic acid, 18-methyl-eicosanoic acid, 20-methyl-docosahexadecanoic acid, 22-methyl-tetracosanoic acid, 24-methyl-hexacosanoic acid, and 26-methyl-octadecanoic acid.

[0361] Examples of suitable unsaturated fatty acids include 4-decenoic acid, decenoic acid, 4-dodecenoic acid, 5-dodecenoic acid, myrcenoic acid, 4-tetradecenoic acid, 5-tetradecenoic acid, 9-tetradecenoic acid, palmitoleic acid, 6-octadecenoic acid, oleic acid, 9-octadecenoic acid, 11-octadecenoic acid, 9-eicosenoic acid, cis-11-eicosenoic acid, cetyl oleate, 13-docosaenoic acid, 15-docosaenoic acid, 17- Hexadecanoic acid, 6,9,12,15-hexadecanoic acid, linoleic acid, linolenic acid, α-tungstic acid, β-tungstic acid, punicic acid, 6,9,12,15-octadecanoic acid, perilla acid, 5,8,11,14-eicosatetraenoic acid, 5,8,11,14,17-eicosatepentanoic acid, 7,10,13,16,19-docosapentaenoic acid, 4,7,10,13,16,19-docosahexaenoic acid, etc.

[0362] Examples of suitable hydroxy fatty acids include α-hydroxylauric acid, α-hydroxymyristic acid, α-hydroxypalmitic acid, α-hydroxystearic acid, ω-hydroxylauric acid, α-hydroxyarachidic acid, 9-hydroxy-12-octadecenoic acid, ricinoleic acid, α-hydroxybehenic acid, 9-hydroxy-trans-10,12-octadecadienoic acid, saffron acid, safranin, 9,10-dihydroxystearic acid, 12-hydroxystearic acid, etc.

[0363] Examples of suitable polycarboxylic acids include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, octanoic acid, azelaic acid, sebacic acid, D,L-malic acid, etc.

[0364] In some respects, each fatty acid is independently selected from propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanic acid, stearic acid, nonadecanic acid, arachidic acid, timocyanic acid, behenic acid, tridecanoic acid, limonic acid, timocyanic acid, succinic acid, lac ceric acid, lecithinic acid, geraniic acid, hexadecanoic acid, heptadecanic acid, and octadecanoic acid.

[0365] In some respects, each fatty acid is independently selected from alpha-linolenic acid, octadecanoic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, gamma-linoleic acid, dihomo-gamma-linoleic acid, arachidonic acid, docosahexaenoic acid, palmitoleic acid, isoleic acid, paullinic acid, oleic acid, transoleic acid, gondoic acid, erucic acid, nervonic acid, mead acid, adrenal acid, bosseopentaenoic acid, ozubondo acid, sardine acid, herring acid, docosahexaenoic acid, or docosapentaenoic acid, or another monounsaturated or polyunsaturated fatty acid.

[0366] In some respects, one or both of the fatty acids are essential fatty acids. Given the health benefits of certain essential fatty acids, the therapeutic benefits of the exosomes carrying the therapeutic agent can be increased by including such fatty acids in the therapeutic agent. In some respects, essential fatty acids are n-6 or n-3 essential fatty acids selected from the group consisting of: linolenic acid, gamma-linolenic acid, dihoxy-gamma-linolenic acid, arachidonic acid, adrenaline, docosapentaenoic acid (DNA), alpha-linolenic acid, octadecanoic acid, 20:4 DNA, eicosapentaenoic acid, docosapentaenoic acid (DNA), or docosahexaenoic acid (DNA).

[0367] In some respects, each fatty acid is independently selected from all-cis-7,10,13-hexadecanoic acid, alpha-linolenic acid, octadecanoic acid, eicosatrienoic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid, docosahexaenoic acid (DHA), docosapentaenoic acid, docosahexaenoic acid, or lipoic acid. In other respects, the fatty acid is selected from eicosapentaenoic acid, docosahexaenoic acid, or lipoic acid. Other examples of fatty acids include all-cis-7,10,13-hexadecanoic acid, alpha-linolenic acid (ALA or all-cis-9,12,15-octadecanoic acid), octadecanoic acid (STD or all-cis-6,9,12,15-octadecanoic acid), eicosaterienoic acid (ETE or all-cis-11,14,17-eicosaterienoic acid), and eicosatetraenoic acid (ETA or all-cis-8,11,14,17-eicosatetraenoic acid). Docosapentaenoic acid (EPA), docosapentaenoic acid (DPA, squalene or cis-7,10,13,16,19-docosapentaenoic acid), docosahexaenoic acid (DHA or cis-4,7,10,13,16,19-docosahexaenoic acid), docosapentaenoic acid (cis-9,12,15,18,21-docosahexaenoic acid), or docosahexaenoic acid (nisinic acid or cis-6,9,12,15,18,21-docosahexaenoic acid). In some respects, fatty acids are medium-chain fatty acids, such as lipoic acid.

[0368] Fatty acid chains vary greatly in their chain length and can be classified according to chain length, such as short to very long. Short-chain fatty acids (SCFAs) are fatty acids with chains of about five or fewer carbons (e.g., butyric acid). In some respects, fatty acids are SCFAs. Medium-chain fatty acids (MCFAs) include fatty acids with chains of about 6-12 carbons, which can form medium-chain triglycerides. In some respects, fatty acids are MCFAs. Long-chain fatty acids (LCFAs) include fatty acids with chains of 13-21 carbons. In some respects, fatty acids are LCFAs. In some respects, fatty acids are LCFAs. Very long-chain fatty acids (VLCFAs) include fatty acids with chains of 22 or more carbons, such as 22-60, 22-50, or 22-40 carbons. In some respects, fatty acids are VLCFAs.

[0369] III.A.1.c. Phospholipids

[0370] In some respects, the anchoring portion contains phospholipids. Phospholipids are a class of lipids that are major components of all cell membranes. Due to their amphiphilic properties, they can form lipid bilayers. The structure of a phospholipid molecule typically consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" composed of phosphate ester groups. For example, phospholipids can be lipids according to the following formula:

[0371]

[0372] Where R p R1 and R2 represent the phospholipid portion, and R1 and R2 represent the fatty acid portions, which may be the same or different and may or may not have unsaturated fatty acids.

[0373] The phospholipid portion may be selected from, for example, the non-restricted group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, lysophosphatidylcholine, and sphingomyelin.

[0374] Certain phospholipids can promote fusion with lipid bilayers, such as those of exosome membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in the membrane. Phospholipid fusion with the membrane allows one or more elements of a lipid-containing composition to bind to or pass through the membrane.

[0375] The fatty acid portion may be selected from, for example, the non-restricted group consisting of: lauric acid, myristic acid, myristone acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0376] The phospholipids used as anchoring moieties in this disclosure can be natural or non-natural phospholipids. Non-natural phospholipid substances are also considered, including natural substances with modifications and substitutions (including branching, oxidation, cyclization, and alkynes). For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., one or more alkenyl groups with double bonds replaced by triple bonds). Under appropriate reaction conditions, the alkyne group can undergo copper-catalyzed cycloaddition upon exposure to azides.

[0377] Phospholipids include, but are not limited to, glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid.

[0378] Examples of phospholipids that can be used for the anchoring portions disclosed herein include

[0379] Phosphatidylethanolamine: For example, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearate phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, 1-palmitoyl-2-oleylenyl phosphatidylethanolamine, 1-oleylenyl-2-palmitoyl phosphatidylethanolamine and disqualyl phosphatidylethanolamine;

[0380] Phosphatidylglycerols: for example, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, 1-palmitoyl-2-oleylenyl-phosphatidylglycerol, 1-oleylenyl-2-palmitoylenyl-phosphatidylglycerol and disorhoyl phosphatidylglycerol;

[0381] Phosphatidylserine: For example, such as dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, distearate phosphatidylserine, dioleoyl phosphatidylserine, 1-palmitoyl-2-oleylenyl-phosphatidylserine, 1-oleylenyl-2-palmitoylenyl-phosphatidylserine and disqualyl phosphatidylserine;

[0382] Phosphatidic acids: for example, dilauroyl phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearyloyl phosphatidic acid, dioleoyl phosphatidic acid, 1-palmitoyl-2-oleylenyl phosphatidic acid, 1-oleylenyl-2-palmitoyl-phosphatidic acid, and disqualyl phosphatidic acid; and,

[0383] Phosphatidylinositol: For example, dilauroyl phosphatidylinositol, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, distearoyl phosphatidylinositol, dioleoyl phosphatidylinositol, 1-palmitoyl-2-oleylenyl-phosphatidylinositol, 1-oleylenyl-2-palmitoylenyl-phosphatidylinositol and disqualyl phosphatidylinositol.

[0384] Phospholipids can be symmetrical or asymmetrical. As used herein, the term "symmetrical phospholipid" includes glycerophospholipids having matched fatty acid moieties and sphingolipids, wherein the hydrocarbon chain of the variable fatty acid moieties and the sphingosine backbone contains a considerable number of carbon atoms. As used herein, the term "asymmetrical phospholipid" includes lysolipin, glycerophospholipids with different fatty acid moieties (e.g., fatty acid moieties with different numbers of carbon atoms and / or degrees of unsaturation (e.g., double bonds), and sphingolipids wherein the hydrocarbon chain of the variable fatty acid moieties and the sphingosine backbone contains different numbers of carbon atoms (e.g., the variable fatty acid moieties contain at least two more carbon atoms than the hydrocarbon chain or at least two fewer carbon atoms than the hydrocarbon chain).

[0385] In some aspects, the anchoring portion comprises at least one symmetrical phospholipid. The symmetrical phospholipid may be selected from the non-limiting group consisting of:

[0386] 1,2-Dipropionyl-sn-glycero-3-phosphocholine (03:0 PC)

[0387] 1,2-Dibutyryl-sn-glycero-3-phosphocholine (04:0 PC)

[0388] 1,2-Divaleryl-sn-glycero-3-phosphocholine (05:0 PC)

[0389] 1,2-Dihexanoyl-sn-glycero-3-phosphocholine (06:0 PC)

[0390] 1,2-Diheptanyl-sn-glycero-3-phosphocholine (07:0 PC)

[0391] 1,2-Dioctanoyl-sn-glycero-3-phosphocholine (08:0 PC)

[0392] 1,2-Dinonanoyl-sn-glycero-3-phosphocholine (09:0 PC)

[0393] 1,2-Didecanoyl-sn-glycero-3-phosphocholine (10:0 PC)

[0394] 1,2-Diundecanoyl-sn-glycero-3-phosphocholine (11:0 PC, DUPC)

[0395] 1,2-Dilauroyl-sn-glycero-3-phosphocholine (12:0 PC)

[0396] 1,2-Didecanoyl-sn-glycero-3-phosphocholine (13:0 PC)

[0397] 1,2-Dimyristicoyl-sn-glycero-3-phosphocholine (14:0 PC, DMPC)

[0398] 1,2-Dipentadecanyl-sn-glycero-3-phosphocholine (15:0 PC)

[0399] 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (16:0 PC, DPPC)

[0400] 1,2-Diphyranoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC)

[0401] 1,2-Diheptadecanoyl-sn-glycero-3-phosphocholine (17:0 PC)

[0402] 1,2-Distearyl-sn-glycero-3-phosphocholine (18:0 PC, DSPC)

[0403] 1,2-Dinonadecanoyl-sn-glycero-3-phosphocholine (19:0 PC)

[0404] 1,2-Diicosanoyl-sn-glycero-3-phosphocholine (20:0 PC)

[0405] 1,2-Diicosanoyl-sn-glycero-3-phosphocholine (21:0 PC)

[0406] 1,2-Dibenzyl-sn-glycero-3-phosphocholine (22:0 PC)

[0407] 1,2-Diicosanoyl-sn-glycero-3-phosphocholine (23:0 PC)

[0408] 1,2-Diicosicoyl-sn-glycero-3-phosphocholine (24:0 PC)

[0409] 1,2-Dimyristyl-sn-glycero-3-phosphocholine (14:1(Δ9-cis)PC),

[0410] 1,2-Ditransmyristyl-sn-glycero-3-phosphocholine (14:1(Δ9-trans)PC),

[0411] 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (16:1(Δ9-cis)PC),

[0412] 1,2-Ditranspalmitoyl-sn-glycero-3-phosphocholine (16:1(Δ9-trans)PC),

[0413] 1,2-Diisocyanoyl-sn-glycero-3-phosphocholine (18:1(Δ6-cis)PC),

[0414] 1,2-Dioleoyl-sn-glycero-3-phosphocholine (18:1(Δ9-cis)PC, DOPC),

[0415] 1,2-Ditransoleoyl-sn-glycero-3-phosphocholine (18:1(Δ9-trans)PC),

[0416] 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (18:2(cis)PC, DLPC),

[0417] 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (18:3(cis)PC, DLnPC),

[0418] 1,2-Diicosenoyl-sn-glycero-3-phosphocholine (20:1 (cis)PC),

[0419] 1,2-Diarachidonico-sn-glycero-3-phosphocholine (20:4(cis)PC, DAPC),

[0420] 1,2-Dipyridyl-sn-glycero-3-phosphocholine (22:1 (cis)PC),

[0421] 1,2-Bisdocosahexaenoyl-sn-glycero-3-phosphocholine (22:6(cis)PC, DHAPC),

[0422] 1,2-Diceryl-sn-glycero-3-phosphocholine (24:1 (cis)PC),

[0423] 1,2-Dihexanoyl-sn-glycero-3-phosphoethanolamine (06:0 PE)

[0424] 1,2-Dioctanoyl-sn-glycero-3-phosphoethanolamine (08:0 PE)

[0425] 1,2-Didecanoyl-sn-glycero-3-phosphate ethanolamine (10:0 PE)

[0426] 1,2-Dilauroyl-sn-glycero-3-phosphate ethanolamine (12:0 PE)

[0427] 1,2-Dimyristicoyl-sn-glycero-3-phosphoethanolamine (14:0 PE)

[0428] 1,2-Dipentadecanoyl-sn-glycero-3-phosphate ethanolamine (15:0 PE)

[0429] 1,2-Dipalmitoyl-sn-glycero-3-phosphate ethanolamine (16:0 PE)

[0430] 1,2-Diphyranoyl-sn-glycero-3-phosphoethanolamine (4ME 16:0 PE)

[0431] 1,2-Diheptadecanoyl-sn-glycero-3-phosphate ethanolamine (17:0 PE)

[0432] 1,2-Distearyl-sn-glycero-3-phosphoethanolamine (18:0 PE, DSPE)

[0433] 1,2-Dipalmitoyl-sn-glycero-3-phosphate ethanolamine (16:1 PE)

[0434] 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (18:1(Δ9-cis)PE, DOPE),

[0435] 1,2-Ditransoleoyl-sn-glycero-3-phosphate ethanolamine (18:1(Δ9-trans)PE),

[0436] 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine (18:2 PE, DLPE)

[0437] 1,2-Dilinoleoyl-sn-glycero-3-phosphate ethanolamine (18:3 PE, DLnPE)

[0438] 1,2-Diarachidonico-sn-glycero-3-phosphate ethanolamine (20:4 PE, DAPE)

[0439] 1,2-Bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6PE, DHAPE)

[0440] 1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC),

[0441] Sodium 1,2-dioleoyl-sn-glycero-3-phosphate-racemic-(1-glycerol) (DOPG) and any combination thereof.

[0442] In some aspects, the anchoring portion comprises at least one symmetrical phospholipid selected from the non-restrictive group consisting of: DLPC, DMPC, DOPC, DPPC, DSPC, DUPC, 18:0 diether PC, DLnPC, DAPC, DHAPC, DOPE, 4ME 16:0PE, DSPE, DLPE, DLnPE, DAPE, DHAPE, DOPG, and any combination thereof.

[0443] In some aspects, the anchoring portion comprises at least one asymmetric phospholipid. The asymmetric phospholipid may be selected from the non-limiting group consisting of:

[0444] 1-Myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0 PC, MPPC)

[0445] 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0 PC, MSPC)

[0446] 1-Palmitoyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0 PC)

[0447] 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0 PC, PMPC)

[0448] 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC, PSPC)

[0449] 1-Palmyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 PC, POPC)

[0450] 1-Palmyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC, PLPC)

[0451] 1-Palmitoyl-2-arachidonico-sn-glycero-3-phosphocholine (16:0-20:4 PC)

[0452] 1-Palmitoyl-2-docosahenoyl-sn-glycero-3-phosphocholine (14:0-22:6PC)

[0453] 1-Stearyl-2-myristoyl-sn-glycero-3-phosphocholine (18:0-14:0 PC, SMPC)

[0454] 1-Stearyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:0-16:0 PC, SPPC)

[0455] 1-Stearyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1 PC, SOPC)

[0456] 1-Stearyl-2-linoleyl-sn-glycero-3-phosphocholine (18:0-18:2 PC)

[0457] 1-Stearyl-2-arachidonico-sn-glycero-3-phosphocholine (18:0-20:4 PC)

[0458] 1-Stearyl-2-docosahenoyl-sn-glycero-3-phosphocholine (18:0-22:6PC)

[0459] 1-Oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:1-14:0 PC, OMPC)

[0460] 1-Oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0 PC, OPPC)

[0461] 1-Oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0 PC, OSPC)

[0462] 1-Palmyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE, POPE)

[0463] 1-Palmyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:2 PE)

[0464] 1-Palmyl-2-arachidonico-sn-glycero-3-phosphoethanolamine (16:0-20:4 PE)

[0465] 1-Palmitoyl-2-docoshexaenoyl-sn-glycero-3-phosphate ethanolamine (16:0-22:6PE)

[0466] 1-Stearyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:1 PE)

[0467] 1-Stearyl-2-linoleyl-sn-glycero-3-phosphoethanolamine (18:0-18:2 PE)

[0468] 1-Stearyl-2-arachidonico-sn-glycero-3-phosphoethanolamine (18:0-20:4 PE)

[0469] 1-Stearyl-2-docoshexaenoyl-sn-glycero-3-phosphate ethanolamine (18:0-22:6PE)

[0470] 1-Oleoyl-2-cholesterolylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC) and any combination thereof.

[0471] To provide more significant nuclease resistance, cellular uptake efficiency, and more significant RNA interference effects, phosphatidylethanolamine can be used as the anchoring moiety, such as dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, 1-palmitoyl-2-oleylenyl-phosphatidylethanolamine, and dioleoyl phosphatidylethanolamine.

[0472] The binding sites for lipid (e.g., phospholipids) and adapter combinations or BAMs (e.g., ASOs) can be appropriately selected based on the type of lipid and adapter or ASO. Any site other than the hydrophobic group of the lipid can be chemically bonded to the adapter or ASO. For example, when using phosphatidylethanolamine, bonding can be achieved by forming an amide bond between the amino group of the phosphatidylethanolamine and the adapter or ASO. When using phosphatidylglycerol, bonding can be achieved by forming an ester bond, ether bond, etc., between the hydroxyl group of the glycerol residue and the adapter or ASO. When using phosphatidylserine, bonding can be achieved by forming an amide bond or ester bond, etc., between the amino or carboxyl group of the serine residue and the adapter or ASO. When using phosphatidic acid, bonding can be achieved by forming a phosphate ester bond, etc., between the phosphate ester residue and the adapter or ASO. When using phosphatidylinositol, bonding can be achieved by forming an ester bond, ether bond, etc., between the hydroxyl group of the inositol residue and the adapter or ASO.

[0473] III.A.1.d. Lysolic lipids (e.g., lysophospholipids)

[0474] In some cases, the anchoring moiety comprises lysolipin, such as lysophospholipid. Lysolipin is a lipid derivative in which one or two fatty acyl chains have been removed (usually by hydrolysis). Lysophospholipid is a phospholipid derivative in which one or two fatty acyl chains have been removed by hydrolysis.

[0475] In some respects, the anchoring portion comprises any of the phospholipids disclosed above, one or both of which have been hydrolyzed, and the resulting lysophospholipid may or may not contain a fatty acid acyl chain.

[0476] In some respects, the anchoring moiety includes lysophosphatidylinosides, lysophosphoglycolipids, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, or lysophosphatidylserine.

[0477] In some respects, the anchoring portion comprises hemolyzed lipids selected from the non-restricted group consisting of:

[0478] 1-Hexanoyl-2-hydroxy-sn-glycero-3-phosphocholine (06:0 Lyso PC)

[0479] 1-Heptanoyl-2-hydroxy-sn-glycero-3-phosphocholine (07:0 Lyso PC)

[0480] 1-Octayl-2-hydroxy-sn-glycero-3-phosphocholine (08:0 Lyso PC)

[0481] 1-Nonanoyl-2-hydroxy-sn-glycero-3-phosphocholine (09:0 Lyso PC)

[0482] 1-Decanyl-2-hydroxy-sn-glycero-3-phosphocholine (10:0 Lyso PC)

[0483] 1-Undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (11:0 Lyso PC)

[0484] 1-Lauroyl-2-hydroxy-sn-glycero-3-phosphocholine (12:0 Lyso PC)

[0485] 1-Trigedecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (13:0 Lyso PC)

[0486] 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0 Lyso PC)

[0487] 1-Pentadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (15:0 Lyso PC)

[0488] 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 Lyso PC)

[0489] 1-Heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (17:0 Lyso PC)

[0490] 1-Stearyl-2-hydroxy-sn-glycero-3-phosphocholine (18:0 Lyso PC)

[0491] 1-Oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:1 Lyso PC)

[0492] 1-Noctadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (19:0 Lyso PC)

[0493] 1-Eicosanoyl-2-hydroxy-sn-glycero-3-phosphocholine (20:0 Lyso PC)

[0494] 1-Behenyl-2-hydroxy-sn-glycero-3-phosphocholine (22:0 Lyso PC)

[0495] 1-Tecosanoyl-2-hydroxy-sn-glycero-3-phosphocholine (24:0 Lyso PC)

[0496] 1-Hexacosanoyl-2-hydroxy-sn-glycero-3-phosphocholine (26:0 Lyso PC)

[0497] 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (14:0 Lyso PE)

[0498] 1-Palmyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (16:0 Lyso PE)

[0499] 1-Stearyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:0 Lyso PE)

[0500] 1-Oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 Lyso PE)

[0501] 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and any combination thereof.

[0502] III.A.I.e. Vitamins

[0503] In some respects, the anchoring component contains lipophilic vitamins, such as folic acid, vitamin A, vitamin E, or vitamin K.

[0504] In some respects, the anchoring portion contains vitamin A. Vitamin A is a group of unsaturated nutrient organic compounds that include retinol, retinal, retinoic acid, and several provitamin A carotenoids (most notably beta-carotene). In some respects, the anchoring portion contains retinol. In some respects, the anchoring portion contains retinoids. Retinoids are a class of vitamins that are equivalent to vitamin A or chemically related to it. In some respects, the anchoring portion contains first-generation retinoids (e.g., retinol, retinoic acid, isotretinoin, or avitamin A), second-generation retinoids (e.g., etretinate or acitretin), third-generation retinoids (e.g., adapalene, bexarotene, or tazarotene), or any combination thereof.

[0505]

[0506] In some respects, the anchoring moiety contains vitamin E. Tocopherols are a class of methylated phenols, many of which have vitamin E activity. Therefore, in some respects, the anchoring moiety contains α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, or combinations thereof.

[0507]

[0508] Tocotrienols also possess vitamin E activity. The key chemical structural difference between tocotrienols and tocopherols lies in the fact that, compared to the saturated side chains of tocopherols, tocotrienols have unsaturated isoprene-like side chains containing three carbon-carbon double bonds. In some aspects, the anchoring moiety comprises α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, or combinations thereof. Tocotrienols can be represented by the following formula:

[0509]

[0510] α(α)-Tocotrienols: R1 = Me, R2 = Me, R3 = Me;

[0511] β(β)-Tocotrienol: R1=Me, R2=H, R3=Me;

[0512] γ(γ)-Tocotrienol: R1=H, R2=Me, R3=Me;

[0513] δ(δ)-Tocotrienols: R1=H, R2=H, R3=Me.

[0514] In some respects, the anchoring moiety contains vitamin K. Chemically, the vitamin K family includes 2-methyl-1,4-naphthoquinone (3-) derivatives. Vitamin K comprises two natural vitamins: vitamin K1 and vitamin K2. The structure of vitamin K1 (also known as phytonaphthoquinone, phylloquinone, or (E)-phytonaphthoquinone) is characterized by the presence of a phytoyl group. The structure of vitamin K2 (methylnaphthoquinone) is characterized by the presence of a polyisoprene side chain in the molecule, which may contain six to 13 isoprene units. Thus, vitamin K2 consists of many related chemical isoforms with carbon side chains of varying lengths composed of isoprene-like atom groups. MK-4 is the most common form of vitamin K2. Longer-chain forms such as MK-7, MK-8, and MK-9 are predominant in fermented foods. Longer-chain forms of vitamin K2, such as MK-10 to MK-13, are synthesized by bacteria, but they are not well absorbed and have little biological function. In addition to the natural form of vitamin K, there are many synthetic forms of vitamin K, such as vitamin K3 (menaquinone; 2-methylnaphthyl-1,4-dione), vitamin K4, and vitamin K5.

[0515] Therefore, in some respects, the anchoring portion contains vitamins K1, K2 (e.g., MK-4, MK-5, MK-6, MK-7, MK-8, MK-9, MK-10, MK-11, MK-12 or MK-13), K3, K4, K5 or any combination thereof.

[0516]

[0517] III.A.2. Connector Assembly

[0518] In some respects, the ASO is attached to the hydrophobic membrane anchoring portion disclosed herein via a adapter assembly, which may comprise any combination of cleavable and / or non-cleavable adapters. The primary function of the adapter assembly is to provide optimal spacing between one or more anchoring portions and the BAM target. For example, in the case of the ASO, the adapter assembly should reduce steric hindrance and position the ASO so that it can interact with the target nucleic acid (e.g., mRNA or miRNA).

[0519] Linkers are readily cleavable (“cleavable linkers”), thereby facilitating the release of bioactive molecules. Therefore, in some aspects, the linker assemblies disclosed herein may comprise cleavable linkers. Such cleavable linkers may be susceptible to, for example, acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, while the bioactive molecules remain active. Alternatively, linkers may be substantially resistant to cleavage (“uncleavable linkers”). In some aspects, cleavable linkers comprise spacers. In some aspects, the spacers are PEG.

[0520] In some aspects, the connector assembly comprises at least 2, at least 3, at least 4, at least 5, or at least 6 or more different connectors disclosed herein. In some aspects, the connectors in the connector assembly may be linked by ester bonds (e.g., phosphate diesters or thiophosphate esters).

[0521] In some respects, the joint is a direct key between the anchoring portion and the BAM (e.g., ASO).

[0522] III.A.2.a. Non-disintegrable joint

[0523] In some respects, the linker assembly comprises a “non-cleavable linker.” A non-cleavable linker is a chemical part that can stably covalently link two or more components of the modified bioactive molecule of this disclosure (e.g., the bioactive molecule and the anchoring moiety; the bioactive molecule and the cleavable linker; the anchoring moiety and the cleavable linker) and does not belong to any of the categories listed above for cleavable linkers. Thus, a non-cleavable linker is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.

[0524] Furthermore, non-cleavage refers to the ability of the chemical bonds in or adjacent to the linker to undergo cleavage induced by acids, light-labile cleaving agents, peptidases, esterases, or chemical or physiological compounds that cleave disulfide bonds, without the cyclic dinucleotide and / or antibody losing its activity. In some respects, bioactive molecules are attached to the linker via another linker (e.g., a self-degrading linker).

[0525] In some aspects, the linker assembly includes a non-cleavable linker comprising, for example, tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), succinimide, or any combination thereof. In some aspects, the non-cleavable linker includes spacer subunits to attach bioactive molecules to the non-cleavable linker.

[0526] In some aspects, one or more non-cleavable linkers comprise smaller units linked together (e.g., HEG, TEG, glycerol, C2 to C12 alkyl groups, etc.). In another aspect, the linking is an ester linking (e.g., a phosphate diester or a thiophosphate) or other linking.

[0527] III.A.2.b. Ethylene glycol (HEG, TEG, PEG)

[0528] In some aspects, the joint assembly includes a non-split joint, wherein the non-split joint comprises a feature of formula R 3 -(O-CH2-CH2) n -or R 3 -(0-CH2-CH2) n -O- polyethylene glycol (PEG), where R 3 It is hydrogen, methyl, or ethyl, and n has a value from 2 to 200. In some aspects, the connector contains spacers, where the spacers are PEG.

[0529] In some respects, PEG connectors are oligoethylene glycol connectors, such as diethylene glycol, triethylene glycol, tetraethylene glycol (TEG), pentaethylene glycol, or hexaethylene glycol (HEG).

[0530] In some respects, n has the values ​​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, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 15 The values ​​are 7, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 189, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200.

[0531] In some respects, 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.

[0532] In some specific aspects, n has values ​​from 3 to 200, 3 to 20, 10 to 30, or 9 to 45.

[0533] In some respects, PEG is branched PEG. Branched PEG has three to ten PEG chains derived from a central core group.

[0534] In some respects, the PEG moiety is monodisperse polyethylene glycol. In the context of this disclosure, monodisperse polyethylene glycol (mdPEG) is a PEG having a single, defined chain length and molecular weight. mdPEG is typically produced by chromatographic separation from polymer mixtures. In some formulas, the monodisperse PEG moiety is designated as the abbreviation mdPEG.

[0535] In some respects, PEG is star-shaped PEG. Star-shaped PEG has 10 to 100 PEG chains derived from a central core group.

[0536] In some respects, PEG is comb-shaped PEG. Comb-shaped PEG has multiple PEG chains that are typically grafted onto the polymer backbone.

[0537] In some respects, PEG has a molar mass between 100 g / mol and 3000 g / mol, particularly between 100 g / mol and 2500 g / mol, and more particularly between about 100 g / mol and 2000 g / mol. In some respects, PEG has a molar mass between 200 g / mol and 3000 g / mol, particularly between 300 g / mol and 2500 g / mol, and more particularly between about 400 g / mol and 2000 g / mol.

[0538] In some respects, PEG is PEG 100 PEG 200 PEG 300 PEG 400 PEG 500 PEG 600 PEG 700 PEG 800 PEG 900 PEG 1000 PEG 1100 PEG 1200 PEG 1300 PEG 1400 PEG 1500 PEG 1600 PEG 1700 PEG 1800 PEG 1900 PEG 2000 PEG 2100 PEG 2200 PEG2300 PEG 2400 PEG 2500 PEG 1600 PEG 1700 PEG 1800 PEG 1900 PEG 2000 PEG 2100 PEG 2200 PEG 2300 PEG 2400 PEG 2500 PEG 2600 PEG 2700 PEG 2800 PEG 2900或 PEG 3000 In one particular aspect, PEG is PEG. 400 In another specific aspect, PEG is PEG. 2000 .

[0539] In some respects, the connector assemblies disclosed herein may include several PEG connectors, such as pyrolytic connectors with side-connected PEG, HEG or TEG connectors.

[0540] In some respects, the connector assembly comprises (HEG)n and / or (TEG)n, where n is an integer between 1 and 50, and each unit is connected, for example, via a phosphate ester connector, a thiophosphate ester bond, or a combination thereof.

[0541] III.A.2.c. Glycerin and Polyglycerol (PG)

[0542] In some aspects, the joint assembly includes a non-disintegrable joint comprising a component of the formula ((R3—O—(CH2—CHOH—CH2O)). n —) describes a glycerol unit or polyglycerol (PG), wherein R3 is hydrogen, methyl, or ethyl, and n has a value of 3 to 200. In some aspects, n has a value of 3 to 20. In some aspects, n has a value of 10 to 30.

[0543] In some respects, the PG connector is a diglyceride, triglyceride, tetraglyceride (TG), pentaglyceride, or hexaglyceride (HG) connector.

[0544] In some respects, n has the values ​​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, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 15 The values ​​are 7, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 189, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200.

[0545] In some respects, 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.

[0546] In some alternatives to these embodiments, n has values ​​from 9 to 45. In some aspects, the heterogeneous portion is derived from formula (R) 3 —O—(CH2—CHOR 5 —CH2—O) n —) describes branched polyglycerol, where R 5 It is hydrogen, or by formula (R) 3 —O—(CH2—CHOH—CH2—O) n —) describes a straight-chain glycerol chain, and R 3 It is hydrogen, methyl, or ethyl. In some respects, the heterosource portion is composed of formula (R 3 —O—(CH2—CHOR 5 —CH2—O) n —) describes hyperbranched polyglycerol, where R 5 It is hydrogen; or it is derived from formula (R) 3 —O—(CH2—CHOR 6 —CH2—O) n —) describes a glycerol chain, where R 6 It is hydrogen; or it is derived from formula (R) 3 —O—(CH2—CHOR 7 —CH2—O) n —) describes a glycerol chain, where R 7 It is hydrogen; or it is derived from formula (R) 3 —O—(CH2—CHOH—CH2—O) n —) describes a straight-chain glycerol chain, and R 3 It is hydrogen, methyl, or ethyl. Hyperbranched glycerol and its synthetic methods are described in Oudshorn et al. (2006) Biomaterials 27:5471-5479; Wilms et al. (20100 Acc. Chem. Res. 43, 129-41 and the references cited therein).

[0547] In some respects, PG has a molar mass between 100 g / mol and 3000 g / mol, particularly between 100 g / mol and 2500 g / mol, and even more particularly between about 100 g / mol and 2000 g / mol. In some respects, PG has a molar mass between 200 g / mol and 3000 g / mol, particularly between 300 g / mol and 2500 g / mol, and even more particularly between about 400 g / mol and 2000 g / mol.

[0548] In some respects, PG is PG 100 PG 200 PG 300 PG 400 PG500 PG 600 PG 700 PG 800 PG 900 PG 1000 PG 1100 PG 1200 PG 1300 PG 1400 PG 1500 PG 1600 PG 1700 PG 1800 PG 1900 PG 2000 PG 2100 PG 2200 PG 2300 PG 2400 PG 2500 PG 1600 PG 1700 PG 1800 PG 1900 PG 2000 PG 2100 PG 2200 PG 2300 PG 2400 PG 2500 PG 2600 PG 2700 PG 2800 PG 2900 or PG 3000 In one particular aspect, PG is PG 400 In another specific aspect, PG is PG. 2000 .

[0549] In some respects, the connector assembly comprises (glycerol)n, and / or (HG)n and / or (TG)n, where n is an integer between 1 and 50, and each unit is connected, for example, via a phosphate ester connector, a thiophosphate ester bond, or a combination thereof.

[0550] III.A.2.d. Aliphatic (alkyl) joints

[0551] In some respects, the connector assembly contains at least one aliphatic (alkyl) connector, such as propyl, butyl, hexyl, or C2-C12 alkyl, such as C2-C10 alkyl or C2-C6 alkyl.

[0552] In some aspects, the linker assembly comprises an alkyl chain, such as an unsubstituted alkyl group. In some aspects, the linker assembly comprises a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, an arylalkyl group, an arylalenyl group, an arylalkynyl group, a heteroarylalkyl group, a heteroarylalenyl group, a heteroarylalkynyl group, a heterocyclic alkyl group, a heterocyclic alkenyl group, a heterocyclic alkenyl group, an aryl group, a heterocyclic group, a cycloalkyl group, a cycloalkenyl group, an alkylarylalkyl group, an alkylarylalenyl group, an alkylarylalkynyl group, an alkenylarylalkyl group, an alkenylreylalkenyl group, an alkenylarylalkynyl group, an alkenylreylalkenyl group, an alkenylarylalkynyl group, an alkenylarylalkynyl group, an alkenylreylalkenyl group, an alkenylarylalkynyl group, an alkenylarylalkynyl group, an alkenylreylalkenyl group, an alkenylarylalkynyl group, an alkenylarylalkyl ...alkyl group, an alkenylarylalkyl group, an alkenylreylalkenyl group, an alkenylarylalkyl group, an alkenylarylalkyl group, an alkenylreylalkenyl group, an alkenylarylalkylal Alkynyl arylalkyl, alkynyl aryl alkenyl, alkynyl aryl alkynyl, alkyl heteroarylalkyl, alkyl heteroarylalkyl, alkyl heteroaryl alkenyl, alkyl heteroaryl alkynyl, alkenyl heteroarylalkyl, alkenyl heteroaryl alkenyl, alkenyl heteroaryl alkynyl, alkynyl heteroarylalkyl, alkynyl heteroaryl alkenyl, alkynyl heteroaryl alkynyl, alkyl heterocyclic alkyl, alkyl heterocyclic alkenyl, alkyl heterocyclic alkynyl, alkenyl heterocyclic alkyl, alkenyl heterocyclic alkenyl or alkenyl heterocyclic alkynyl.

[0553] Optionally, these components are substituted. Substituents include alcohols, alkoxy groups (such as methoxy, ethoxy, and propoxy), straight-chain or branched alkyl groups (such as C1-C12 alkyl), amines, aminoalkyl groups (such as aminoC1-C12 alkyl), phosphoramides, phosphate esters, aminophosphate esters, dithiophosphate esters, thiophosphate esters, acyl hydrazides, hydrazines, halogens (such as F, Cl, Br, or I), amides, alkylamides (such as amide C1-C12 alkyl), carboxylic acids, carboxylic acid esters, carboxylic anhydrides, carboxylic acid halides, ethers, sulfonyl halides, imine esters, isocyanates, isothiocyanates, haloformates, carbodiimide adducts, aldehydes, ketones, mercapto groups, haloacetyl groups, alkyl halides, alkyl sulfonates, C(═O)CH═CHC(═O) (maleimide), thioethers, cyano groups, sugars (such as mannose, galactose, and glucose), α,β-unsaturated carbonyl groups, alkylmercuric groups, or α,β-unsaturated sulfones.

[0554] Unless otherwise stated, the term "alkyl" on its own or as part of another substituent means a straight-chain or branched hydrocarbon group having a specified number of carbon atoms (e.g., C1-C1). 10 (This refers to one to ten carbon atoms). Typically, alkyl groups will have 2 to 24 carbon atoms, for example, 2 to 10 carbon atoms, 2 to 8 carbon atoms, or 2 to 6 carbon atoms. A “lower alkyl” group is an alkyl group having 2 to 4 carbon atoms. The term “alkyl” includes both divalent and polyvalent groups. For example, where appropriate, such as when the formula indicates that the alkyl group is divalent or when the substituents are linked together to form a ring, the term “alkyl” includes “alkylene.” Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, and homologues and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, and n-octyl.

[0555] The term "alkylene" itself, or as part of another substituent, refers to a divalent (dimethyl) alkyl group, where alkyl is defined herein. "alkylene" is, for example, but not limited to –CH2CH2CH2CH2-. Typically, an "alkylene" will have 2 to 24 carbon atoms, for example, 10 or fewer carbon atoms (e.g., 2 to 8 or 2 to 6 carbon atoms). "Lower alkylene" is an alkylene group having 2 to 4 carbon atoms.

[0556] The term "alkenyl" itself, or as part of another substituent, refers to a straight-chain or branched hydrocarbon group having 2 to 24 carbon atoms and at least one double bond. A typical alkenyl group has 2 to 10 carbon atoms and at least one double bond. In one embodiment, the alkenyl group has 2 to 8 carbon atoms or 2 to 6 carbon atoms and 2 to 3 double bonds. Exemplary alkenyl groups include vinyl, 2-propenyl, 1-but-3-enyl, crotonyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), 2-isopentenyl, 1-pent-3-enyl, 1-hex-5-enyl, etc.

[0557] The term "alkynyl" itself, or as part of another substituent, refers to a straight-chain or branched, unsaturated or polyunsaturated hydrocarbon group having 2 to 24 carbon atoms and at least one triple bond. A typical "alkynyl" group has 2 to 10 carbon atoms and at least one triple bond. In one aspect of the invention, the alkynyl group has 2 to 6 carbon atoms and at least one triple bond. Exemplary alkynyl groups include prop-1-alkynyl, prop-2-alkynyl (i.e., propynyl), ethynyl, and 3-butynyl.

[0558] The terms “alkoxy,” “alkylamino,” and “alkoxythio” (or thioalkoxy) are used in their conventional sense and refer to an alkyl group that is attached to the rest of the molecule by an oxygen atom, an amino group, or a sulfur atom, respectively.

[0559] The term "heteroalkyl" on its own or in combination with another term means consisting of a specified number of carbon atoms (e.g., C2-C). 10A stable straight-chain or branched hydrocarbon group consisting of at least one heteroatom selected from, for example, N, O, S, Si, B, and P (N, O, and S in one embodiment), wherein nitrogen, sulfur, and phosphorus atoms are optionally oxidized, and one or more nitrogen atoms are optionally quaternized. One or more heteroatoms are positioned at any internal position of the heteroalkyl group. Examples of heteroalkyl groups include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -CH2-Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. At most two heteroatoms can be consecutive, such as -CH2-NH-OCH3 and –CH2-O-Si(CH3)3, for example.

[0560] Similarly, the term "heteroalkylene" itself, or as part of another substituent, refers to a divalent group derived from a heteroalkyl group, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and –CH2-S-CH2-CH2-NH-CH2-. Typically, a heteroalkyl group will have 3 to 24 atoms (carbons and heteroatoms, excluding hydrogen) (3 to 24-membered heteroalkyl). In another instance, the heteroalkyl group has a total of 3 to 10 atoms (3 to 10-membered heteroalkyl) or 3 to 8 atoms (3 to 8-membered heteroalkyl). Where appropriate, for example, when the formula indicates that the heteroalkyl group is divalent or when the substituents are linked together to form a ring, the term "heteroalkyl" includes "heteroalkylene".

[0561] The term "cycloalkyl" on its own or in combination with other terms refers to a saturated or unsaturated non-aromatic carbocyclic group having 3 to 24 carbon atoms, for example, having 3 to 12 carbon atoms (e.g., C3-C8 cycloalkyl or C3-C6 cycloalkyl). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. The term "cycloalkyl" also includes bridged polycyclic (e.g., bicyclic) structures such as norbornyl, adamantyl, and bicyclic [2.2.1]heptyl. A "cycloalkyl" group may be fused with at least one (e.g., 2 to 3) other rings selected from aryl (e.g., phenyl), heteroaryl (e.g., pyridyl), and non-aromatic (e.g., carbocyclic or heterocyclic) rings. When a "cycloalkyl" group includes a fused aryl, heteroaryl, or heterocyclic ring, the "cycloalkyl" group is connected to the remainder of the molecule via a carbocyclic ring.

[0562] The terms “heterocyclic alkyl,” “heterocyclic,” “heterocyclic,” or “heterocyclic group,” on their own or in combination with other terms, refer to stable combinations known to those skilled in the art of a fused ring system containing at least one and at most five heteroatoms selected from, for example, N, O, S, Si, B, and P (e.g., N, O, and S), of a carbocyclic nonaromatic ring (e.g., 3- to 8-membered rings and, for example, 4, 5, 6, or 7-membered rings), wherein nitrogen, sulfur, and phosphorus atoms are optionally oxidized, and one or more nitrogen atoms are optionally quaternized (e.g., 2 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur), or of a 4- to 8-membered ring containing at least one and at most 10 heteroatoms (e.g., 2 to 5 heteroatoms selected from N, O, and S). Exemplary heterocyclic alkyl groups include fused benzene rings. When a “heterocyclic” group includes a fused aryl, heteroaryl, or cycloalkyl ring, the “heterocyclic” group is connected to the remainder of the molecule via the heterocycle. Heteroatoms may occupy positions where the heterocycle is connected to the remainder of the molecule.

[0563] Exemplary heterocyclic alkyl or heterocyclic groups disclosed herein include morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S,S-dioxide, piperazine, homopiperazine, pyrrolyl, pyrrololinyl, imidazoalkyl, tetrahydropyranyl, piperidinyl, tetrahydrofuranyl, tetrahydrothiopheneyl, piperidinyl, homopiperidinyl, homomorpholinyl, homothiomorpholinyl, homothiomorpholinyl S,S-dioxide, oxazolidinone, dihydropyrazolyl, and dihydropyrazolyl. Hydropyrroleyl, dihydropyrazolyl, dihydropyridyl, dihydropyrimidinyl, dihydrofuranyl, dihydropyranyl, tetrahydrothiopheneyl S-oxide, tetrahydrothiopheneyl S,S-dioxide, high-thiomorpholinyl S-oxide, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, 1-piperazinyl, 2-piperazinyl, etc.

[0564] “Aryl” means a 5, 6, or 7-membered aromatic carbon ring group having a monocyclic ring (e.g., phenyl) or fused to other aromatic or non-aromatic rings (e.g., 2 to 3 other rings). When the “aryl” group includes a non-aromatic ring (such as in 1,2,3,4-tetrahydronaphthyl) or a heteroaryl group, the “aryl” group is connected to the rest of the molecule via an aryl ring (e.g., a benzene ring). The aryl group is optionally substituted (e.g., having 2 to 5 substituents as described herein). In one example, the aryl group has 6 to 10 carbon atoms. Non-limiting examples of aryl groups include phenyl, 1-naphthyl, 2-naphthyl, quinoline, indanyl, indenyl, dihydronaphthyl, fluorenyl, tetrahydronaphthyl, benzo[d][1,3]dioxacyclopentenyl, or 6,7,8,9-tetrahydro-5H-benzo[a]cycloheptenyl. In one embodiment, the aryl group is selected from phenyl, benzo[d][1,3]dioxanepentenyl, and naphthyl. In another embodiment, the aryl group is phenyl.

[0565] The term "arylalkyl" or "arylalkyl" is intended to include those groups in which an aryl group or a heteroaryl group is attached to an alkyl group to form alkyl-aryl and -alkyl-heteroaryl groups (wherein alkyl, aryl, and heteroaryl are defined herein). Exemplary "arylalkyl" or "arylalkyl" groups include benzyl, phenethyl, pyridylmethyl, etc.

[0566] "Aryloxy group" means -O-aryl group, where aryl is as defined herein. In one example, the aryl portion of the aryloxy group is phenyl or naphthyl. In one embodiment, the aryl portion of the aryloxy group is phenyl.

[0567] The term "heteroaryl" or "heteroaromatic" refers to a polyunsaturated 5, 6, or 7-membered aromatic moiety containing at least one heteroatom (e.g., 2 to 5 heteroatoms, such as 1 to 3 heteroatoms) selected from N, O, S, Si, and B (e.g., N, O, and S), wherein nitrogen and sulfur atoms are optionally oxidized, and one or more nitrogen atoms are optionally quaternized. The "heteroaryl" group can be monocyclic or fused with other aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings (e.g., 2 to 3 other rings). When the "heteroaryl" group includes a fused aryl, cycloalkyl, or heterocycloalkyl ring, the "heteroaryl" group is attached to the remainder of the molecule via a heteroaryl ring. The heteroaryl group can be attached to the remainder of the molecule via carbon or heteroatom.

[0568] In one example, the heteroaryl group has 4 to 10 carbon atoms and 2 to 5 heteroatoms selected from O, S, and N. Non-limiting examples of heteroaryl groups include pyridinyl, pyrimidinyl, quinolinyl, benzothiopheneyl, indolyl, indololinyl, pyridazinyl, pyrazinyl, isoindolyl, isoquinolinyl, quinazolinyl, quinoxalolinyl, phthalazinyl, imidazolyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, indolazinyl, inzozolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, furanyl, thiopheneyl, pyrroleyl, oxadiazolyl, thiazolyl, triazolyl, tetrazolyl, isothiazolyl, naphridinyl, isochromyl, chromanyl, tetrahydroisoquinolinyl, isoindololinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrofuranyl Thiopheneyl, isobenzothiopheneyl, benzoxazolyl, pyridopyridyl, benzotetrahydrofuranyl, benzotetrahydrothiopheneyl, purineyl, benzodioxolyl, triazineyl, pteridinyl, benzothiazolyl, imidazopyridyl, imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl, dihydrobenzisothiazinyl, benzopyran alkyl, benzothiaranyl, chromanonyl, pyridyl-N-oxide, tetrahydroquinolinyl, dihydroquinolinyl, dihydroquinolinone, dihydroisoquinolinone, dihydrocoumarinyl, dihydroisocoumarinyl, isoindolinonyl, benzodioxane, benzoxazolinonyl, pyrroleyl N-oxide, pyrimidinyl N-oxide, pyrazinyl N-oxide, pyrazinyl-N-oxide, quinolinyl-N-oxide, indoleyl N-oxide, indole Phinyl N-oxide, isoquinolinyl N-oxide, quinazolinyl N-oxide, quinoxalolinyl N-oxide, phthalazinyl N-oxide, imidazoleyl N-oxide, isoxazolyl N-oxide, oxazolyl N-oxide, thiazolyl N-oxide, indoleazinyl N-oxide, indazoleyl N-oxide, benzothiazolyl N-oxide, benzimidazolyl N-oxide, pyrroleyl N-oxide, oxadiazolyl N-oxide, thiazolyl N-oxide, triazolyl N-oxide, tetrazolyl N-oxide, benzothiaranyl S-oxide, benzothiaranyl S,S-dioxide. Exemplary heteroaryl groups include imidazole, pyrazolyl, thiazolyl, triazolyl, isoxazolyl, isothiazolyl, imidazole, thiazolyl, oxadiazolyl, and pyridinyl.Other exemplary heteroaryl groups include 1-pyrrole, 2-pyrrole, 3-pyrrole, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isooxazolyl, 4-isooxazolyl, 5-isooxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, pyridin-4-yl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinel, 2-benzimidazolyl, 5-indolyl, 1-isoquinolinyl, 5-isoquinolinyl, 2-quinoxolinyl, 5-quinoxolinyl, 3-quinolinyl, and 6-quinolinyl. The substituents in each of the aryl ring and heteroaryl ring systems mentioned above are selected from the group consisting of the acceptable aryl group substituents described below.

[0569] Examples of aliphatic connectors include the following structures:

[0570] —O—CO—O—

[0571] —NH—CO—O—

[0572] —NH—CO—NH—

[0573] —NH—(CH2) n1 —

[0574] —S—(CH2) n1 —

[0575] —CO—(CH2) n1 —CO—

[0576] —CO—(CH2) n1 —NH—

[0577] —NH—(CH2) n1 —NH—

[0578] —CO—NH—(CH2) n1 —NH—CO—

[0579] —C(═S)—NH—(CH2) n1 —NH—CO—

[0580] —C(═S)—NH—(CH2) n1 —NH—C—(═S)—

[0581] —CO—O—(CH2) n1 —O—CO—

[0582] —C(═S)—O—(CH2) n1 —O—CO—

[0583] —C(═S)—O—(CH2) n1 —O—C—(═S)—

[0584] —CO—NH—(CH2) n1 —O—CO—

[0585] —C(═S)—NH—(CH2) n1 —O—CO—

[0586] —C(═S)—NH—(CH2) n1 —O—C—(═S)—

[0587] —CO—NH—(CH2) n1 —O—CO—

[0588] —C(═S)—NH—(CH2) n1 —CO—

[0589] —C(═S)—O—(CH2) n1 —NH—CO—

[0590] —C(═S)—NH—(CH2) n1 —O—C—(═S)—

[0591] —NH—(CH2CH2O) n2 —CH(CH2OH)—

[0592] —NH—(CH2CH2O) n2 —CH2—

[0593] —NH—(CH2CH2O) n2 —CH2—CO—

[0594] —O—(CH2) n3 —S—S—(CH2) n4 —O—P(═O)2—

[0595] —CO—(CH2) n3 —O—CO—NH—(CH2) n4 —

[0596] —CO—(CH2) n3 —CO—NH—(CH2) n4 —

[0597] —(CH2) n1 NH—

[0598] —C(O)(CH2) n1 NH—

[0599] —C(O)—(CH2) n1 -C(O)—

[0600] —C(O)—(CH2) n1 -C(O)O—

[0601] —C(O)—O—

[0602] —C(O)—(CH2) n1 -NH—C(O)—

[0603] —C(O)—(CH2) n1 —

[0604] —C(O)—NH—

[0605] —C(O)—

[0606] —(CH2) n1 -C(O)—

[0607] —(CH2) n1 -C(O)O—

[0608] —(CH2) n1 —

[0609] —(CH2) n1 -NH—C(O)—

[0610] n1 is an integer between 1 and 40 (e.g., 2 to 20 or 2 to 12); n2 is an integer between 1 and 20 (e.g., 2 to 10 or 2 to 6); n3 and n4 may be the same or different, and are integers between 1 and 20 (e.g., 2 to 10 or 2 to 6).

[0611] In some respects, the connector assembly comprises (C3)n, (C4)n, (C5)n, (C6)n, (C7)n, or (C8)n or combinations thereof, where n is an integer between 1 and 50, and each unit is connected, for example, via a phosphate ester connector, a thiophosphate ester bond, or a combination thereof.

[0612] III.A.3. Disintegrable Connector

[0613] In some respects, the different components of the ASO disclosed herein can be connectors of cleavable connectors. The term cleavable connector refers to a connector containing at least one breakable or cleavable bond or chemical bond. As used herein, the term cleavage refers to the breaking of one or more chemical bonds in a relatively large molecule in a manner that produces two or more relatively small molecules. Cleavage can be mediated, for example, by nucleases, peptidases, proteases, phosphatases, oxidases, or reductases, or by specific physicochemical conditions, such as redox environments, pH, the presence of reactive oxygen species, or light of specific wavelengths.

[0614] In some respects, the term “cleavable” as used herein refers to, for example, rapidly degradable linkers, such as, for example, phosphate diesters and disulfides, while the term “non-cleavable” refers to, for example, more stable links, such as, for example, nuclease-resistant thiophosphates.

[0615] In some respects, a cleavable linker is a dinucleotide or trinucleotide linker, a disulfide, an imine, a thioacetate, a val-cit dipeptide, or any combination thereof.

[0616] In some respects, the cleavable linker comprises valine-alanine-carbamate p-aminobenzyl ester or valine-citrulline-carbamate p-aminobenzyl ester.

[0617] III.A.3.a. Redox-reducible pyrolytic junction

[0618] In some respects, the linker assembly includes a redox-cleavable linker. As a non-limiting example, one type of cleavable linker is a redox-cleavable linking group that cleaves upon reduction or oxidation.

[0619] In some respects, redox-degradable joints contain disulfide bonds, that is, they are disulfide-degradable joints.

[0620] The redox-reductant linker can be reduced, for example, by intracellular thiols, oxidases, or reductases.

[0621] III.A.3.b. Reactive Oxygen Species (ROS) Degradable Connectors

[0622] In some aspects, the adapter assembly may include a cleavable adapter that can be cleaved by reactive oxygen species (ROS), such as superoxide (O3) or hydrogen peroxide (H2O2) produced by inflammatory processes such as activated neutrophils. In some aspects, the ROS-cleavable adapter is a thioacetate-cleavable adapter. See, for example, U.S. Patent 8,354,455B2, which is incorporated herein by reference in its entirety.

[0623] III.A.3.c. pH-dependent pyrolytic linkers

[0624] In some respects, a connector is an “acid-instable connector” containing an acid-cleavable linker group that selectively cleaves under acidic conditions (pH < 7).

[0625] As a non-limiting example, acid-cleavable linkers cleave in acidic environments, such as about 6.0, 5.5, 5.0, or lower. In some respects, the pH is about 6.5 or lower. In some respects, the linker is cleaved by enzymes such as peptidases (which can be substrate-specific) or phosphatases that can act as generalized acids. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid-cleavable linkers. While the pH of human serum is 7.4, the average pH in cells is slightly lower, ranging from about 7.2 to 7.3. Endosomes also have acidic pH, ranging from 5.5 to 6.0, and lysosomes are at even more acidic pH, around 5.0. Therefore, pH-dependent cleavable linkers are sometimes referred to in the art as endosome-instantaneous linkers.

[0626] The acid-cleavable linker may have the general formula -C=NN-, C(O)O, or -OC(O). In another non-limiting example, for instance, when the carbon atom linked to the ester oxygen (alkoxy group) is linked to an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or tert-butyl. Examples of acid-cleavable linking groups include, but are not limited to, amines, imines, amino esters, imine benzoates, dicarboxylate esters, polyphosphate esters, polyphosphazenes, acetals, vinyl ethers, hydrazones, cis-aconitate esters, acylhydrazides, thiocarbamoyl groups, imizine, azidomethyl-methylmaleic anhydride, thiopropionate esters, masked endogenous solubilizers, citraconyl groups, or any combination thereof. Disulfide linkage is also pH sensitive.

[0627] In some respects, the linker contains low-pH unstable hydrazone bonds. Such acid-labile bonds have been widely used in the field of conjugates, such as antibody-drug conjugates. See, for example, Zhou et al., Biomacromolecules 2011, 12, 1460-7; Yuan et al., Acta Biomater. 2008, 4, 1024-37; Zhang et al., Acta Biomater. 2007, 6, 838-50; Yang et al., J. Pharmacol. Exp. Ther. 2007, 321, 462-8; Reddy et al., Cancer Chemother. Pharmacol. 2006, 58, 229-36; Doronina et al., Nature Biotechnol. 2003, 21, 778-84.

[0628] In some embodiments, the connector comprises a low-pH unstable bond selected from: ketals that are unstable in acidic environments (e.g., pH less than 7 and greater than about 4) to form diols and ketones; acetals that are unstable in acidic environments (e.g., pH less than 7 and greater than about 4) to form diols and aldehydes; imines or iminiums that are unstable in acidic environments (e.g., pH less than 7 and greater than about 4) to form amines and aldehydes or ketones; silicon-oxygen-carbon bonds that are unstable under acidic conditions; silicon-nitrogen (silazane) bonds; silicon-carbon bonds (e.g., arylsilanes, vinylsilanes, and allylsilanes); maleamic acids (amide bonds synthesized from maleic anhydride derivatives and amines); orthoesters; hydrazones; activated carboxylic acid derivatives (e.g., esters, amides) designed to undergo acid-catalyzed hydrolysis; or vinyl ethers.

[0629] Further examples can be found in U.S. Patent Nos. 9,790,494B2 and 8,137,695B2, the contents of which are incorporated herein by reference in their entirety.

[0630] III.A.3d. Enzyme-cleavable linker

[0631] In some respects, a linker assembly may contain linkers that can be cleaved by intracellular or extracellular enzymes, such as proteases, esterases, nucleases, and amidases. The range of enzymes capable of cleaving a particular linker in a linker assembly depends on the specific bonds and chemical structure of the linker. Thus, peptide linkers can be cleaved, for example, by peptidases; linkers containing ester bonds can be cleaved, for example, by esterases; linkers containing amide bonds can be cleaved, for example, by amidases; and so on.

[0632] III.A.3.e. Protease-cleavable linkers

[0633] In some respects, linker assemblies contain protease-cleavable linkers, i.e., linkers that can be cleaved by endogenous proteases. Only certain peptides are readily cleaved, either intracellularly or extracellularly. See, for example, Trout et al., 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al., 43 Int. J. Cancer, 677-684 (1989). Cleavable linkers may contain a cleavable site consisting of an α-amino acid unit and a peptide bond, chemically an amide bond between the carboxyl group of one amino acid and the amino group of a second amino acid. Other amide bonds, such as the bond between a carboxyl group and the α-amino group of lysine, are not considered peptide bonds and are considered non-cleavable.

[0634] In some respects, protease cleavable linkers contain cleavage sites of proteases.This protease includes, for example, enkephalinase (CALLA or CD10), phorate oligopeptidase (TOP), leukotriene A4 hydrolase, endothelial angiotensin-converting peptide convertase, ste24 protease, neurolysin, mitochondrial intermediate peptidase, interstitial collagenase, collagenase, lysosome, macrophage elastase, matrix dissolving factor, gelatinase, membrane-penetrating peptidase (meprin), procollagen C-endopeptidase, procollagen N-endopeptidase, ADAM and ADAMT metalloproteinases, myelin-associated metalloproteinases, enamelysin, tumor necrosis factor α-convertase, insulin lysozyme, nardilysin, mitochondrial processing peptidase, and magnolia. Magnolysin, dactylysin-like metalloproteinases, neutrophil collagenase, matrix metalloproteinases, membrane-type matrix metalloproteinases, SP2 endopeptidase, prostate-specific antigen (PSA), plasmin, urokinase, human fibroblast activation protein (FAPα), trypsin, chymotrypsin, caldecrin, pancreatic elastase, pancreatic endopeptidase, intestinal peptidase, leukocyte elastase, myelocytes, chymase-like enzymes, granzymes, keratinocyte chymotrypsin, acrosomal protease, kallikrein, complement components and factors, alternative complement pathway C3 / C5 convertase, mannose-binding protein-associated serine. Proteases, coagulation factors, thrombin, protein C, U and T plasminogen activators, cathepsin G, serine transmembrane proteases (hepsin), prostasin, hepatocyte growth factor-activated endopeptidases, subtilisin / kexin-type proprotein convertase, furin proteases, proprotein convertase, prolyl peptidase, acylaminoacyl peptidase, peptidyl-glycaminase, signal peptidase, N-terminal nucleophilic aminohydrolase, 20S proteasome, gamma-glutamyl transpeptidase, mitochondrial endopeptidase, mitochondrial endopeptidase Ia, htra2 peptidase, matriptase, site 1 protease, asparagine endopeptidase (leguma) (in), cathepsins, cysteine ​​cathepsins, calpapsins, ubiquitin isopeptidase T, caspase, glycosylphosphatidylinositol protein transaminases, cancer procoagulants, hormone prothiol proteases, gamma-glutamyl hydrolases, bleomycin hydrolases, fibroblast activating protein (seprase), cathepsin B, cathepsin D, cathepsin L, cathepsin M, proteinase K, pepsin, chymosyn, gastric subprotease, renin, yeast asparagine and / or mapsin, prostate-specific antigen (PSA) or generally any Asp-N, Glu-C, Lys-C, or Arg-C proteases. See, for example,Cancer Res. 77(24):7027-7037 (2017), which is incorporated herein by reference in its entirety.

[0635] In some respects, the cleavable linker component comprises a peptide containing one to ten amino acid residues. In these respects, the peptide allows proteases to cleave the linker, thereby facilitating the release of the bioactive molecule upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Exemplary peptides include, but are not limited to, dipeptides, tripeptides, tetrapeptides, pentapeptides, and hexapeptides.

[0636] Peptides may contain naturally occurring and / or non-natural amino acid residues. The term "naturally occurring amino acid" refers to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. "Non-natural amino acids" (i.e., amino acids that are not naturally occurring) include, as non-limiting examples, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, leucine ("Nle"), valine ("Nva"), β-alanine, L-naphthylalanine or D-naphthylalanine, ornithine ("Orn"), etc. Peptides may be engineered and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasminogen activator proteases.

[0637] Amino acids also include the D-forms of both natural and non-natural amino acids. "D-" indicates an amino acid with a "D" (dextral) configuration, the opposite of the configuration in naturally occurring ("L-") amino acids. Natural or non-natural amino acids are commercially available (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0638] Exemplary dipeptides include, but are not limited to, valine-alanine, valine-citrulline, phenylalanine-lysine, N-methyl-valine-citrulline, cyclohexylalanine-lysine, and β-alanine-lysine. Exemplary tripeptides include, but are not limited to, gly-valine-citrulline (gly-val-cit) and gly-gly-gly (gly-gly-gly).

[0639] III.A.3.f. Esterase-cleavable linkers

[0640] Some linkers are cleaved by esterases (“esterase-cleavable linkers”). Only certain esters can be cleaved by esterases and amidases present both inside and outside the cell. Esters are formed through the condensation between a carboxylic acid and an alcohol. Simple esters are esters produced from simple alcohols, such as aliphatic alcohols and small cyclic alcohols, as well as small aromatic alcohols. Examples of ester-based cleavable linkers include, but are not limited to, esters with alkylene, alkenyl, and ynylene groups. Ester-cleavable linkers have the general formula -C(O)O- or -OC(O)-.

[0641] III.A.3g phosphatase can cleave the linker.

[0642] In some respects, the linker assembly may comprise a phosphate-ester-based cleavable linker group, which is cleaved by an agent that degrades or hydrolyzes the phosphate ester group. An example of an agent that cleaves intracellular phosphate ester groups is an enzyme, such as an intracellular phosphatase. Examples of phosphate-ester-based linkers are —O—P(O)(ORk)—O—, —O—P(S)(ORk)—O—, —O—P(S)(SRk)—O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(R k )-O-、-OP(S)(R k )-O-、-SP(O)(R k )-O-、-SP(S)(R k )-O-、-SP(O)(R k )-S- or -OP(S)(Rk)-S-.

[0643] In all aspects, R k It is any of the following: NH2, BH3, CH3, C 1-6 Alkyl, C 6-10 Aryl, C 1-6 Alkoxy and C 6-10 Aryl-oxygen group. In some respects, C 1-6 Alkyl and C 6-10The aryl group is unsubstituted. Other non-limiting examples are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-S-, -OP(S)(H)-S-, or -OP(O)(OH)-O-.

[0644] III.A.3.h. Photoactivated pyrolytic connector

[0645] In some respects, the combination connector includes a photoactivated cleavable connector, such as a nitrobenzyl connector or a connector containing a nitrobenzyl reactive group.

[0646] III.A.3.i. Self-disintegrating connector

[0647] In some aspects, the adapter assembly comprises a self-degrading adapter. In some aspects, the self-degrading adapter in the EV (e.g., exosome) of this disclosure undergoes 1,4-elimination following enzymatic cleavage of the protease-cleavable adapter. In some aspects, the self-degrading adapter in the EV (e.g., exosome) of this disclosure undergoes 1,6-elimination following enzymatic cleavage of the protease-cleavable adapter. In some aspects, the self-degrading adapter is, for example, a p-aminobenzyl (pAB) derivative, such as p-aminobenzyl carbamate (pABC), p-aminobenzyl ether (PABE), p-aminobenzyl carbonate, or combinations thereof.

[0648] In some respects, the self-decomposing linker contains an aromatic group. In some respects, the aromatic group is selected from the group consisting of benzyl, cinnamyl, naphthyl, and biphenyl. In some respects, the aromatic group is heterocyclic. In other respects, the aromatic group contains at least one substituent. In some respects, at least one substituent is selected from the group consisting of F, Cl, I, Br, OH, methyl, methoxy, NO2, NH2, NO 3+ NHCOCH3, N(CH3)2, NHCOCF3, alkyl, haloalkyl, C1-C8 alkyl halides, carboxylic acid esters, sulfate esters, aminosulfonates, and sulfonates. In other respects, at least one C in the aromatic group is substituted with N, O, or CR*, wherein R* is independently selected from H, F, Cl, I, Br, OH, methyl, methoxy, NO2, NH2, NO 3+ NHCOCH3, N(CH3)2, NHCOCF3, alkyl, haloalkyl, C1-C8 alkyl halides, carboxylic acid esters, sulfate esters, aminosulfonates and sulfonates.

[0649] In some respects, the self-degrading linker comprises an aminobenzyl carbamate group (e.g., p-aminobenzyl carbamate), an aminobenzyl ether group, or an aminobenzyl carbonate group. In one respect, the self-degrading linker is p-aminobenzyl carbamate (pABC).

[0650] pABC is the most efficient and widely used connector for site-specific prodrug activation of autodegradable sites (see, for example, Carl et al. J. Med. Chem. 24:479-480 (1981); WO1981 / 001145; Rautio et al., Nature Reviews Drug Discovery 7:255-270 (2008); Simplicio et al., Molecules 13:519-547 (2008)).

[0651] In some respects, the self-degrading linker connects a bioactive molecule (e.g., ASO) to a protease-cleavable substrate (e.g., Val-Cit). In specific respects, the carbamate group of the pABC self-degrading linker is linked to the amino group of the bioactive molecule (e.g., ASO), and the amino group of the pABC self-degrading linker is linked to the protease-cleavable substrate.

[0652] The aromatic ring of the aminobenzyl group may optionally be substituted by one or more (e.g., R1 and / or R2) substituents on the aromatic ring, which replace hydrogen atoms otherwise connected to one of the four unsubstituted carbons forming the ring. As used herein, the symbol "R" is used in conjunction with the aromatic ring. x "(For example, R1, R2, R3, R4) are general abbreviations for substituents as described herein."

[0653] Substituents can enhance the self-decomposition ability of p-aminobenzyl groups (Hay et al., J. Chem. Soc., Perkin Trans. 1: 2759-2770 (1999); see also Sykes et al., J. Chem. Soc., Perkin Trans. 1: 1601-1608 (2000)).

[0654] Self-decomposing elimination can occur, for example, via 1,4-elimination, 1,6-elimination (e.g., pABC), 1,8-elimination (e.g., p-aminocinnamyl alcohol), -elimination, cyclization elimination (e.g., 4-aminobutanol ester and ethylenediamine), cyclization / lactoneation, cyclization / lactoneation, etc. See, for example, Singh et al. Curr. Med. Chem. 15:1802-1826 (2008); Greenwald et al. J. Med. Chem. 43:475-487 (2000).

[0655] In some aspects, the self-degrading linker may contain, for example, a cinnamyl, naphthyl, or biphenyl group (see, for example, Blencowe et al., Polym. Chem. 2:773-790 (2011)). In some aspects, the self-degrading linker contains a heterocycle (see, for example, U.S. Patent Nos. 7,375,078; 7,754,681). Many homologous aromatics (see, for example, Carl et al., J. Med. Chem. 24:479 (1981); Senter et al., J. Org. Chem. 55:2975 (1990); Taylor et al., J. Org. Chem. 43:1197 (1978); Andrianomenjanahary et al., Bioorg. Med. Chem. Lett. 2:1903 (1992)) and coumarin-based compounds that are self-degradable under aqueous and physiological conditions (see, for example, Weinstein et al., Chem. ... The heteroaromatic groups of furan, thiophene, thiazole, oxazole, isoxazole, pyrrole, pyrazole (see, for example, Hay et al. J.Med.Chem.46:5533(2003)), pyridine (see, for example, Perry-Feigenbaum et al. Org.Biomol.Chem.7:4825(2009)), imidazolide (see, for example, Nailor et al. Bioorg.Med.Chem.Lett.Z:1267(1999); Hay and Denny, Tetrahedron Lett.38:8425(1997)) and triazole (see, for example, Bertrand and Gesson, J.Org.Chem.72:3596(2007)) are known in the art. See also U.S. Patent Nos. 7,691,962; 7,091,186; U.S. Patent Publication Nos. US2006 / 0269480; US2010 / 0092496; US2010 / 0145036; US2003 / 0130189; US2005 / 0256030).

[0656] In some aspects, the adapter assemblies disclosed herein comprise more than one self-decomposing adapter in series, such as two or more pABC units. See, for example, de Groot et al., J. Org. Chem. 66:8815-8830 (2001). In some aspects, the adapter assemblies disclosed herein may comprise a self-decomposing adapter (e.g., a hemithioaminal derivative of p-aminobenzyl alcohol or p-carboxybenzaldehyde or glyoxylic acid) attached to a fluorescent probe (see, for example, Meyer et al., Org. Biomol. Chem. 8:1777-1780 (2010)).

[0657] When the substituents in self-decomposing linkers are specified by their conventional chemical formulas written from left to right, they also encompass chemically identical substituents that would be obtained from a structure written from right to left. For example, "-CH2O-" is also intended to represent "-OCH2-".

[0658] Self-decomposing property, for example, in the self-decomposing linker of p-aminobenzyl as described above, the substituents in R1 and / or R2 may include, for example, alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, aryloxy, heteroaryl, etc. When the compounds of this disclosure contain more than one substituent, each of the substituents is selected independently.

[0659] In some specific aspects, the self-disintegrating linker is connected to a cleavable peptide linker having the following formula, combined with the following formula:

[0660] -A a -Y y -

[0661] Each –A- is independently an amino acid unit, a is independently an integer from 2 to 12; and –Y- is a self-decomposing spacer, and y is 1 or 2. In some respects, –A a - It can be a dipeptide, tripeptide, tetrapeptide, pentapeptide, or hexapeptide. In some respects, –A a -Select the group consisting of the following: valine-alanine, valine-citrulline, phenylalanine-lysine, N-methylvaline-citrulline, cyclohexylalanine-lysine, and β-alanine-lysine. In some respects, –A a - It is either valine-alanine or valine-citrulline.

[0662] In some respects, self-disintegrating joints – Y y -Has the following formula:

[0663]

[0664] Each R 2 C is independent 1-8 Alkyl, -O-(C 1-8 Alkyl, halogen, nitro, or cyano; and m is an integer from 0 to 4. In some respects, m is 0, 1, or 2. In some respects, m is 0.

[0665] In some respects, the cleavable linker is valine-alanine-carbamate p-aminobenzyl ester or valine-citrulline-carbamate p-aminobenzyl ester.

[0666] III.A.4. Reactive Component (RM)

[0667] The ASOs of this disclosure are produced via chemical synthesis or via chemical reactions between their components. For example, in some aspects, an anchoring portion containing a reactive group (e.g., maleimide) may react with an ASO containing a maleimide reactive group to produce a hydrophobically modified ASO of this disclosure, wherein the anchoring portion may be inserted into the lipid bilayer of the exosome membrane, thereby attaching the ASO to the surface of the exosome.

[0668] Any component or grouping of components in the hydrophobically modified ASO of this disclosure may contain at least RG and / or RM, which will allow the components to be linked by a single reaction or a series of reactions to produce the hydrophobically modified ASO of this disclosure. Exemplary synthetic modes for producing the hydrophobically modified ASO include:

[0669] [AM]- / RG / + / RM / -[ASO]→[AM]-[ASO]

[0670] [AM]- / RM / + / RG / -[ASO]→[AM]-[ASO]

[0671] [AM]-[L]- / RM / + / RG / -[ASO]→[AM]-[L]-[ASO]

[0672] [AM]-[L]- / RG / + / RM / -[ASO]→[AM]-[L]-[ASO]

[0673] [AM]- / RM / + / RG / -[L]-[ASO]→[AM]-[L]-[ASO]

[0674] [AM]- / RG / + / RM / -[L]-[ASO]→[AM]-[L]-[ASO]

[0675] [AM]-[L]- / RM / + / RG / -[L]-[ASO]→[AM]-[L]-[L]-[ASO]

[0676] [AM]-[L]- / RG / + / RM / -[L]-[ASO]→[AM]-[L]-[L]-[ASO]

[0677] Where [AM] is the anchoring moiety, [ASO] is the antisense oligonucleotide, [L] is the linker or linker combination, / RM / is the reactive moiety, and / RG / is the reactive group. In any of the provided schematic diagrams, ASO may be attached, for example, via its 5' or 3' end.

[0678] Exemplary synthesis patterns for generating intermediates in the synthesis of ASO include:

[0679] [AM]- / RM / + / RG / -[L]→[AM]-[L]

[0680] [AM]- / RG / + / RM / -[L]→[AM]-[L]

[0681] [L]- / RM / + / RG / -[L]→[L]-[L]

[0682] [L]- / RG / + / RM / -[L]→[L]-[L]

[0683] [L]- / RM / + / RG / -[ASO]→[L]-[ASO]

[0684] [L]- / RG / + / RM / -[ASO]→[L]-[ASO]

[0685] Where [AM] is the anchoring moiety, [ASO] is the antisense oligonucleotide, [L] is the linker or linker combination, / RM / is the reactive moiety, and / RG / is the reactive group. In any of the provided schematic diagrams, ASO may be attached, for example, via its 5' or 3' end.

[0686] In some respects, the reactive group " / RG / " can be, for example, an amino group, a thiol group, a hydroxyl group, a carboxylic acid group, or an azide group. Specific reactive portions " / RM / " that can react with these reactive groups are described in more detail below.

[0687] [AM]-( / RM / )n+( / RG / -[L]-[ASO])n→[AM]-[L]-[ASO]

[0688] The anchoring portion, connector, or connector combination disclosed herein, or any of the ASOs, may be conjugated to a reactive portion, such as an amino reactive portion (e.g., NHS-ester, p-nitrophenol, isothiocyanate, isocyanate, or aldehyde), a thiol reactive portion (e.g., acrylate, maleimide, or pyridyl disulfide), a hydroxyl reactive portion (e.g., isothiocyanate or isocyanate), a carboxylic acid reactive portion (e.g., epoxide), or an azide reactive portion (e.g., alkyne).

[0689] Exemplary reactive portions that can be used to covalently bond two components disclosed herein (e.g., anchor portion and ASO, or anchor portion and connector, or anchor portion and connector, or both connectors, or connector and ASO, or both anchor portions) include, for example, N-succinimide-3-(2-pyridyldithio)propionate, N-4-maleimidebutyric acid, S-(2-pyridyldithio)cysteine, iodoacetoxysuccinimide, N-(4-maleimidebutyryloxy)succinimide, N-[5(3'-maleimidepropylamide)-1-carboxypentyl]iminodiacetic acid, N-(5-aminopentyl)iminodiacetic acid, and 1'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide). Bifunctional connectors (connectors containing two functional groups) are also available.

[0690] In some aspects, the anchoring portion, connector, or ASO may contain a terminal oxyamino group, such as -ONH2, hydrazine, -NHNH2, mercapto (i.e., SH or thiol), or olefin (e.g., CH═CH2). In some aspects, the anchoring portion, connector, or ASO may, for example, contain an electrophilic portion at the terminal position, such as an aldehyde, alkyl halide, methanesulfonate, toluenesulfonate, m-nitrobenzenesulfonic acid, or p-bromobenzenesulfonic acid, or an activated carboxylic acid ester, such as an NHS ester, phosphorous acid, or pentafluorophenyl ester. In some aspects, a covalent bond can be formed by coupling a nucleophilic group (e.g., hydroxyl, thiol, or amino group) of the ligand to an electrophilic group.

[0691] This invention applies to all types of reactive groups and reactive moieties, including but not limited to those known in the art.

[0692] As used herein, the term "protecting group" refers to an unstable chemical moiety known in the art that protects reactive groups (including, but not limited to, hydroxyl, amino, and thiol groups) from unwanted reactions during a synthetic procedure. 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 for use in further reactions. Protecting groups known in the art are typically described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York (1999).

[0693] Furthermore, various synthetic steps can be performed alternately or sequentially to obtain the desired compound. Synthetic chemical transformation and protecting group methodologies (protection and deprotection) that can be used to synthesize the compounds described herein are known in the art and include, for example, those described in the following literature: R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G. M. Watts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette (ed.), Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0694] S...

Claims

1. An extracellular vesicle comprising an antisense oligonucleotide (ASO), the antisense oligonucleotide comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length, the contiguous nucleotide sequence being complementary to a nucleic acid sequence within nucleotides 1800-1900 of a CEBP / β transcript corresponding to the nucleotide sequence as shown in SEQ ID NO: 11; wherein the extracellular vesicles selectively deliver the ASO to myeloid-derived suppressor cells (MDSCs) and other myeloid cells; wherein the ASO is capable of downregulating the expression of CEBP / β mRNA or CEBP / β protein; and Among them, downregulating CEBP / β expression can promote the immune regulation of MDSCs and other myeloid cells to a proinflammatory phenotype.

2. An extracellular vesicle comprising an antisense oligonucleotide (ASO), wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to a nucleic acid sequence within nucleotides 1500-2106, nucleotides 1600-2106, nucleotides 1700-2106, nucleotides 1800-2106, nucleotides 1500-2000, nucleotides 1500-1900, nucleotides 1600-2100, nucleotides 1700-2000 or nucleotides 1800-1900 of a CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

3. The extracellular vesicle of claim 2, wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1800-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

4. The extracellular vesicle according to any one of claims 2 to 3, wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1838-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

5. The extracellular vesicle according to any one of claims 2 to 4, wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1838-1857 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

6. The extracellular vesicle according to any one of claims 2 to 4, wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1853-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

7. The extracellular vesicle of any one of claims 2 to 6, wherein 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 CEBP / β transcript.

8. The extracellular vesicle according to any one of claims 2 to 7, wherein the contiguous nucleotide sequence is completely complementary to a nucleotide sequence within the CEBP / β transcript.

9. The extracellular vesicle according to any one of claims 2 to 7, wherein the ASO comprises (i) a nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 101-233, or (ii) a nucleotide sequence having one or two mismatches selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 101-233.

10. The extracellular vesicle according to any one of claims 2 to 8, wherein the ASO comprises the nucleotide sequence shown in SEQ ID NO:

218.

11. The extracellular vesicle according to any one of claims 2 to 8, wherein the ASO comprises the nucleotide sequence shown in SEQ ID NO:

233.

12. An extracellular vesicle comprising an antisense oligonucleotide (ASO), wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length, which is complementary to a nucleic acid sequence within nucleotides 995-1014 of a CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:

11.

13. The extracellular vesicle of claim 12, wherein 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 nucleotides 995-1014 of the CEBP / β transcript.

14. The extracellular vesicle of claim 12 or 13, wherein the contiguous nucleotide sequence is fully complementary to the nucleotide sequence within nucleotides 995-1014 of the CEBP / β transcript.

15. The extracellular vesicle according to claim 12 or 13, wherein the ASO comprises the nucleotide sequence shown in SEQ ID NO: 234 with one or two mismatches.

16. The extracellular vesicle according to any one of claims 12 to 14, wherein the ASO comprises the nucleotide sequence shown in SEQ ID NO:

234.

17. The extracellular vesicle according to any one of claims 2 to 16, which targets cells selected from the group consisting of macrophages, myeloid-derived suppressor cells (MDSCs), monocytes, basophils, neutrophils, eosinophils, and any combination thereof.

18. The extracellular vesicle of any one of claims 2 to 17, wherein the ASO is capable of reducing CEBP / β protein expression in human cells (eg, immune cells), wherein the human cells express the CEBP / β protein.

19. The extracellular vesicle of claim 18, wherein the CEBP / β 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 the CEBP / β protein expression in human cells not exposed to the ASO.

20. The extracellular vesicle of any one of claims 2 to 19, wherein the ASO is capable of reducing the level of CEBP / β mRNA in human cells (eg, immune cells), wherein the human cells express the CEBP / β mRNA.

21. The extracellular vesicle of claim 20, wherein the level of CEBP / β 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 CEBP / β mRNA in human cells not exposed to the ASO.

22. The extracellular vesicle of any one of claims 2 to 21, wherein the ASO is a gapmer, a mixedmer or a fullmer.

23. The extracellular vesicle of any one of claims 2 to 22, wherein the ASO comprises one or more nucleoside analogs.

24. The extracellular vesicle of claim 23, wherein one or more of the nucleoside analogs comprises 2'-O-alkyl-RNA; 2'-O-methyl RNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; arabinoic acid (ANA); 2'-fluoro-ANA; or a bicyclic nucleoside analog.

25. The extracellular vesicle of claim 23 or 24, wherein one or more of the nucleoside analogs is a sugar-modified nucleoside.

26. The extracellular vesicle of claim 25, wherein the sugar-modified nucleoside is an affinity-enhancing 2' sugar-modified nucleoside.

27. The extracellular vesicle of any one of claims 23 to 26, wherein one or more of the nucleoside analogs comprises a nucleoside comprising a bicyclic sugar.

28. The extracellular vesicle of any one of claims 23 to 26, wherein one or more of the nucleoside analogs comprises LNA.

29. The extracellular vesicle of any one of claims 23 to 28, wherein one or more of the nucleotide analogs is selected from the group consisting of constrained ethyl nucleosides (cEt), 2',4'-constrained 2'-O-methoxyethyl (cMOE), α-L-LNA, β-D-LNA, 2'-0,4'-C-ethylene-bridged nucleic acid (ENA), amino-LNA, oxy-LNA, thio-LNA, and any combination thereof.

30. The extracellular vesicle of any one of claims 2 to 29, wherein the ASO comprises one or more 5'-methyl-cytosine nucleobases.

31. The extracellular vesicle of any one of claims 2 to 30, wherein the ASO has a design selected from the group consisting of the designs in Figure 1, wherein the uppercase letters are sugar-modified nucleosides and the lowercase letters are DNA.

32. The extracellular vesicle of any one of claims 2 to 31, wherein the ASO is 14 to 20 nucleotides in length.

33. The extracellular vesicle of any one of claims 2 to 32, wherein the contiguous nucleotide sequence comprises one or more modified internucleoside linkages.

34. The extracellular vesicle of claim 33, wherein the one or more modified internucleoside linkages are phosphorothioate linkages.

35. The extracellular vesicle of claim 33 or 34, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the internucleoside linkages are modified.

36. The extracellular vesicle of claim 35, wherein each of the internucleoside linkages in the ASO is a phosphorothioate linkage.

37. The extracellular vesicle of any one of claims 2 to 36, further comprising an anchoring moiety.

38. The extracellular vesicle of claim 37, wherein the ASO is linked to the anchoring moiety.

39. The extracellular vesicle of any one of claims 2 to 38, further comprising an exogenous targeting moiety.

40. The extracellular vesicle of claim 39, wherein the exogenous targeting moiety comprises a peptide, an antibody or antigen-binding fragment thereof, a chemical compound, an RNA aptamer, or any combination thereof.

41. The extracellular vesicle of claim 39 or 40, wherein the exogenous targeting moiety comprises a peptide.

42. The extracellular vesicle of any one of claims 39 to 41, wherein the exogenous targeting moiety comprises a microprotein, a designed ankyrin repeat protein (darpin), a lipocalin, an antibody-mimicking protein, an aptamer, a peptide mimetic molecule, a natural ligand of a receptor, a camelid nanobody, or any combination thereof.

43. The extracellular vesicle of any one of claims 39 to 42, wherein 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), scFv, Fv, Fab, Fab', F(ab')2, or any combination thereof.

44. The extracellular vesicle of claim 43, wherein the antibody is a single chain antibody.

45. The extracellular vesicle of any one of claims 39 to 44, wherein the exogenous targeting moiety targets the exosomes to the liver, heart, lung, brain, kidney, central nervous system, peripheral nervous system, muscle, bone, joints, skin, intestine, bladder, pancreas, lymph nodes, spleen, blood, bone marrow, or any combination thereof.

46. ​​The extracellular vesicle of any one of claims 39 to 45, wherein the exogenous targeting moiety targets the exosomes to tumor cells, dendritic cells, T cells, B cells, macrophages, neurons, hepatocytes, Kupffer cells, myeloid cells (e.g., neutrophils, monocytes, macrophages, hematopoietic stem cells, MDSCs (e.g., monocytic MDSCs or granulocytic MDSCs)), or any combination thereof.

47. An extracellular vesicle according to any one of claims 39 to 46, wherein the EV comprises a scaffold portion that connects the exogenous targeting portion to the EV.

48. The extracellular vesicle of any one of claims 39 to 47, wherein the anchoring portion and / or the scaffold portion is Scaffold X.

49. The extracellular vesicle of any one of claims 39 to 47, wherein the anchoring portion and / or the scaffold portion is scaffold Y.

50. The extracellular vesicle of claim 48, wherein the scaffold X is a scaffold protein capable of anchoring the ASO on the luminal surface of the EV and / or on the outer surface of the EV.

51. The extracellular vesicle of claim 48 or 50, wherein the scaffold X is selected from the group consisting of: prostaglandin F2 receptor negative regulator (PTGFRN protein); basic glycoprotein (BSG protein); immunoglobulin superfamily member 2 (IGSF2 protein); immunoglobulin superfamily member 3 (IGSF3 protein); immunoglobulin superfamily member 8 (IGSF8 protein); integrin beta-1 (ITGB1 protein); integrin alpha-4 (ITGA4 protein); 4F2 cell surface antigen heavy chain (SLC3A2 protein); a class of ATP transporters (ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4 proteins); their functional fragments; and any combination thereof.

52. The extracellular vesicle according to any one of claims 37 to 51, wherein the anchoring portion and / or the scaffold portion is a PTGFRN protein or a functional fragment thereof.

53. The extracellular vesicle of any one of claims 37 to 52, wherein the anchoring portion and / or the scaffold portion comprises the amino acid sequence shown in SEQ ID NO:

302.

54. The extracellular vesicle of any one of claims 37 to 53, wherein the anchoring portion and / or the scaffold portion comprises an amino acid sequence that is 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:

301.

55. The extracellular vesicle of claim 49, wherein the scaffold Y is a scaffold protein capable of anchoring the ASO on the luminal surface of the EV and / or on the outer surface of the EV.

56. The extracellular vesicle of claim 49 or 55, wherein the scaffold Y is selected from the group consisting of: myristoylated alanine-rich protein kinase C substrate (MARCKS protein), myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1 protein), brain acid-soluble protein 1 (BASP1 protein), functional fragments thereof, and any combination thereof.

57. The extracellular vesicle according to any one of claims 49, 55 and 56, wherein the scaffold Y is BASP1 protein or a functional fragment thereof.

58. The extracellular vesicle of any one of claims 49 and 55 to 57, wherein the scaffold Y comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND and / or the ED associates with the luminal surface of the EV.

59. The extracellular vesicle of claim 58, wherein the ND is associated with the luminal surface of the exosome via myristoylation.

60. The extracellular vesicle of claim 58 or 59, wherein the ED is associated with the luminal surface of the exosome through ionic interactions.

61. The method of any one of claims 58 to 60, wherein the ND comprises an amino acid sequence selected from the group consisting of: (i) GGKLSKK (SEQ ID NO:411), (ii) GAKLSKK (SEQ ID NO:412), (iii) GGKQSKK (SEQ ID NO:413), (iv) GGKLAKK (SEQ ID NO:414), (v) GGKLSK (SEQ ID NO:415), or (vi) any combination thereof.

62. The extracellular vesicle of any one of claims 58 to 61, wherein the ND comprises the amino acid sequence GGKLSKK (SEQ ID NO: 411).

63. The extracellular vesicle of any one of claims 37 to 62, wherein the ASO is attached to the anchoring portion and / or the scaffold portion on the outer surface of the EV.

64. The extracellular vesicle of any one of claims 37 to 63, wherein the ASO is attached to the anchoring portion and / or the scaffold portion on the luminal surface of the EV.

65. The extracellular vesicle of any one of claims 37 to 64, wherein the anchoring moiety comprises a sterol, GM1, a lipid, a vitamin, a small molecule, a peptide, or a combination thereof.

66. The extracellular vesicle of any one of claims 37 to 64, wherein the anchoring moiety comprises cholesterol.

67. The extracellular vesicle of any one of claims 37 to 66, wherein 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.

68. The extracellular vesicle of any one of claims 37 to 67, wherein the ASO is linked to the anchoring portion and / or the scaffold portion via a linker.

69. The extracellular vesicle of any one of claims 2 to 68, wherein the ASO is linked to the EV via a linker.

70. The extracellular vesicle of claim 68 or 69, wherein the linker is a polypeptide.

71. The extracellular vesicle of claim 68 or 69, wherein the linker is a non-polypeptide moiety.

72. The extracellular vesicle of claim 68 or 69, wherein the linker comprises ethylene glycol.

73. The extracellular vesicle of claim 72, wherein the linker comprises HEG, TEG, PEG, or any combination thereof.

74. The extracellular vesicle of claim 68 or 69, wherein the linker comprises an acrylic acid phosphoramidite (e.g., ACRYDITE TM ), adenylation, azide (NHS ester), digoxin (NHS ester), cholesterol-TEG, I-LINKER TM , amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, or Uni-Link TM amino modifiers), 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 SS, dithiol, or thiol modifier C6S-S), or any combination thereof.

75. The extracellular vesicle of any one of claims 68 to 74, wherein the linker is a cleavable linker.

76. The extracellular vesicle of claim 75, wherein the linker comprises valine-alanine-p-aminobenzylcarbamate or valine-citrulline-p-aminobenzylcarbamate.

77. The extracellular vesicle of any one of claims 74 to 76, wherein the linker comprises (i) a maleimide moiety and (ii) valine-alanine-p-aminobenzylcarbamate or valine-citrulline-p-aminobenzylcarbamate.

78. The extracellular vesicle of any one of claims 2 to 77, wherein the EV is an exosome.

79. 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 1438-2106 of a CEBP / β transcript corresponding to the nucleotide sequence shown in SEQ ID NO:

11.

80. The ASO of claim 79, wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1500-2106, nucleotides 1600-2106, nucleotides 1700-2106, nucleotides 1800-2106, nucleotides 1500-2000, nucleotides 1500-1900, nucleotides 1600-2100, nucleotides 1700-2000, or nucleotides 1800-1900 of a CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

81. The ASO of claim 79 or 80, wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1800-1900 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

82. The ASO of any one of claims 79 to 81, wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1838-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

83. The ASO of any one of claims 79 to 82, wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1838-1857 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

84. The ASO of any one of claims 79 to 82, wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1853-1872 of the CEBP / β transcript corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

85. The ASO of any one of claims 79 to 84, wherein 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 CEBP / β transcript.

86. The ASO of any one of claims 79 to 85, wherein the contiguous nucleotide sequence is fully complementary to a nucleotide sequence within the CEBP / β transcript.

87. The ASO according to any one of claims 79 to 85, wherein the ASO comprises (i) a nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 101-223, or (ii) a nucleotide sequence having one or two mismatches selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 101-223.

88. The ASO of any one of claims 79 to 86, wherein the ASO comprises the nucleotide sequence shown in SEQ ID NO:

218.

89. The ASO of any one of claims 79 to 86, wherein the ASO comprises the nucleotide sequence shown in SEQ ID NO:

233.

90. An ASO comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within nucleotides 995-1014 of a CEBP / β transcript corresponding to the nucleotide sequence as shown in SEQ ID NO:

11.

91. The ASO of claim 90, wherein 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 nucleotides 995-1014 of the CEBP / β transcript.

92. The ASO of claim 90 or 91, wherein the contiguous nucleotide sequence is fully complementary to the nucleotide sequence within nucleotides 995-1014 of the CEBP / β transcript.

93. The ASO of claim 90 or 91, wherein the ASO comprises the nucleotide sequence shown in SEQ ID NO: 234 with one or two mismatches.

94. The ASO of any one of claims 90 to 92, wherein the ASO comprises the nucleotide sequence shown in SEQ ID NO:

234.

95. The ASO according to any one of claims 90 to 94, which is capable of reducing CEBP / β protein expression in human cells (eg, immune cells), wherein the human cells express the CEBP / β protein.

96. The ASO of claim 95, wherein the CEBP / β 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 the CEBP / β protein expression in human cells not exposed to the ASO.

97. The ASO according to any one of claims 79 to 96, which is capable of reducing the level of CEBP / β mRNA in a human cell (eg, an immune cell), wherein the human cell expresses the CEBP / β mRNA.

98. The ASO of claim 97, wherein the level of CEBP / β 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 CEBP / β mRNA in human cells not exposed to the ASO.

99. The ASO of any one of claims 79 to 98, which is a gapmer, a mixedmer or a fullmer.

100. The ASO of any one of claims 79 to 99, comprising one or more nucleoside analogs.

101. The ASO of claim 100, wherein one or more of the nucleoside analogs comprises 2'-O-alkyl-RNA; 2'-O-methyl RNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; arabinoic acid (ANA); 2'-fluoro-ANA; or a bicyclic nucleoside analog.

102. The ASO of claim 100 or 101, wherein one or more of the nucleoside analogs is a sugar-modified nucleoside.

103. The ASO of claim 102, wherein the sugar-modified nucleoside is an affinity-enhancing 2' sugar-modified nucleoside.

104. The ASO of any one of claims 100 to 103, wherein one or more of the nucleoside analogs comprises a nucleoside comprising a bicyclic sugar.

105. The ASO of any one of claims 100 to 104, wherein one or more of the nucleoside analogs comprises LNA.

106. The ASO of any one of claims 100 to 105, wherein one or more of the nucleotide analogs is selected from the group consisting of constrained ethyl nucleosides (cEt), 2',4'-constrained 2'-O-methoxyethyl (cMOE), α-L-LNA, β-D-LNA, 2'-0,4'-C-ethylene-bridged nucleic acid (ENA), amino-LNA, oxy-LNA, thio-LNA, and any combination thereof.

107. The ASO of any one of claims 79 to 106, wherein the ASO comprises one or more 5'-methyl-cytosine nucleobases.

108. The ASO of any one of claims 79 to 107, wherein the ASO has a design selected from the group consisting of the design in Figure 1, wherein the uppercase letters are sugar-modified nucleosides and the lowercase letters are DNA.

109. The ASO of any one of claims 79 to 108, wherein the ASO is 14 to 20 nucleotides in length.

110. The ASO of any one of claims 79 to 109, wherein the contiguous nucleotide sequence comprises one or more modified internucleoside linkages.

111. The ASO of claim 110, wherein the one or more modified internucleoside linkages are phosphorothioate linkages.

112. The ASO of claim 110 or 111, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the internucleoside linkages are modified.

113. The ASO of claim 112, wherein each of the internucleoside linkages in the ASO is a phosphorothioate linkage.

114. A conjugate comprising the ASO according to any one of claims 79 to 113, wherein the ASO is covalently attached to at least one non-nucleotide or non-polynucleotide moiety.

115. The conjugate of claim 114, wherein the non-nucleotide or non-polynucleotide moiety comprises a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or any combination thereof.

116. An extracellular vesicle comprising the ASO according to any one of claims 79 to 113 or the conjugate according to claim 114 or 115.

117. A pharmaceutical composition comprising the extracellular vesicle according to any one of claims 2 to 94 and 132, the ASO according to any one of claims 79 to 113 or the conjugate according to claim 114 or 115, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

118. The pharmaceutical composition of claim 117, wherein the pharmaceutically acceptable salt comprises a sodium salt, a potassium salt, an ammonium salt, or any combination thereof.

119. The pharmaceutical composition of claim 117 or 118, further comprising at least one additional therapeutic agent.

120. The pharmaceutical composition of claim 119, wherein the additional therapeutic agent is a CEBP / beta antagonist.

121. The pharmaceutical composition of claim 120, wherein the CEBP / β antagonist is a chemical compound, siRNA, shRNA, antisense oligonucleotide, protein, or any combination thereof.

122. The pharmaceutical composition of claim 120 or 121, wherein the CEBP / β antagonist is an anti-CEBP / β antibody or a fragment thereof.

123. The pharmaceutical composition of claim 120 or 121, wherein the CEBP / β antagonist comprises an antisense oligonucleotide (ASO).

124. A kit comprising an extracellular vesicle according to any one of claims 2 to 78 and 116, an ASO according to any one of claims 79 to 113, a conjugate according to claim 114 or 115, or a pharmaceutical composition according to any one of claims 117 to 123, and instructions for use.

125. A diagnostic kit comprising an extracellular vesicle according to any one of claims 2 to 78 and 116, an ASO according to any one of claims 79 to 113, a conjugate according to claim 114 or 115, or a pharmaceutical composition according to any one of claims 116 to 123, and instructions for use.

126. A method for inhibiting or reducing CEBP / β protein expression in a cell, the method comprising administering to the cell expressing CEBP / β protein an extracellular vesicle according to any one of claims 2 to 78 and 116, an ASO according to any one of claims 79 to 113, a conjugate according to claim 114 or 115, or a pharmaceutical composition according to any one of claims 117 to 123, wherein the CEBP / β protein expression in the cell is inhibited or reduced after the administration.

127. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an extracellular vesicle according to any one of claims 2 to 78 and 116, an ASO according to any one of claims 79 to 113, a conjugate according to claim 114 or 115, or a pharmaceutical composition according to any one of claims 117 to 123.

128. Use of the extracellular vesicle of any one of claims 2 to 78 and 116, the ASO of any one of claims 79 to 113, the conjugate of claim 114 or 115, or the pharmaceutical composition of any one of claims 117 to 123 in the manufacture of a medicament for treating cancer in a subject in need thereof.

129. The extracellular vesicle of any one of claims 2 to 78 and 116, the ASO of any one of claims 79 to 113, the conjugate of claim 114 or 115, or the pharmaceutical composition of any one of claims 117 to 123, for use in treating cancer in a subject in need thereof.

130. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an extracellular vesicle according to any one of claims 2 to 78 and 116, an ASO according to any one of claims 79 to 113, a conjugate according to claim 114 or 115, or a pharmaceutical composition according to any one of claims 117 to 123, wherein the disease or condition is selected from fibrosis, inflammation, neurodegenerative diseases, metabolic disorders / CVD, and any combination thereof.

131. Use of the extracellular vesicle of any one of claims 2 to 78 and 116, the ASO of any one of claims 79 to 113, the conjugate of claim 114 or 115, or the pharmaceutical composition of any one of claims 117 to 123 in the manufacture of a medicament for treating a disease or disorder in a subject in need thereof, wherein the disease or disorder is selected from fibrosis, inflammation, neurodegenerative diseases, metabolic disorders / CVD, and any combination thereof.

132. The extracellular vesicle of any one of claims 2 to 78 and 116, the ASO of any one of claims 79 to 113, the conjugate of claim 114 or 115, or the pharmaceutical composition of any one of claims 117 to 123, for use in treating a disease or disorder in a subject in need thereof, wherein the disease or disorder is selected from fibrosis, inflammation, neurodegenerative diseases, metabolic disorders / CVD, and any combination thereof.

133. The method of any one of claims 126, 127 and 130, the use of claim 128 or 131, or the composition for use of claim 129 or 132, wherein said ASO inhibits or reduces expression of CEBP / β mRNA in said cell after said administration.

134. The method, use or composition for use of claim 133, wherein the level of CEBP / β 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 said administration compared to the level of CEBP / β mRNA in cells not exposed to said ASO.

135. The method of any one of claims 126, 127 and 130, the use of claim 128 or 131, or the composition for use of claim 129 or 132, wherein the expression of CEBP / β 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 said administration compared to the expression of CEBP / β protein in cells not exposed to said ASO.

136. The method of any one of claims 126, 127 and 130, the use of claim 128 or 131, or the composition for use of claim 129 or 132, wherein the extracellular vesicle, the ASO, the conjugate or the pharmaceutical composition is administered intracardiacally, orally, parenterally, intrathecally, intracerebroventricularly, intrapulmonaryly, topically or intraventricularly.

137. The method of claim 127, the use of claim 128, or the composition for use of claim 128, wherein the cancer is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, squamous cell carcinoma of the head and neck, colorectal cancer, lymphoma, leukemia, hepatocellular carcinoma, glioblastoma, melanoma, myeloma-based Basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, follicular lymphoma, Hodgkin's lymphoma, B-cell lymphoma, and any combination thereof.

138. The method of claim 130, the use of claim 131, or the composition for use of claim 131, wherein the disease or disorder comprises fibrosis.

139. The method of claim 130, the use of claim 131, or the composition for use of claim 131, wherein the disease or disorder comprises fibrosis selected from the group consisting of liver fibrosis (NASH), liver cirrhosis, pulmonary fibrosis, cystic fibrosis, chronic ulcerative colitis / IBD, bladder fibrosis, kidney fibrosis, CAPS (Muller-Weid Syndrome), atrial fibrosis, endomyocardial fibrosis, old myocardial infarction, glial scar, arteriosclerosis, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloid fibrosis, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, adhesive capsulitis, and any combination thereof.

140. A method of activating meningeal macrophages in a subject in need thereof, the method comprising administering to the subject an extracellular vesicle according to any one of claims 2 to 78 and 116, an ASO according to any one of claims 79 to 113, a conjugate according to claim 114 or 115, or a pharmaceutical composition according to any one of claims 117 to 123.

141. A method of treating cancer of the central nervous system of a subject in need thereof, the method comprising administering to the subject an effective amount of an extracellular vesicle according to any one of claims 2 to 78 and 116, an ASO according to any one of claims 79 to 113, a conjugate according to claim 114 or 115, or a pharmaceutical composition according to any one of claims 117 to 123.

142. A method of inducing M1 polarization of meningeal macrophages in a subject in need thereof, the method comprising administering to the subject an effective amount of an extracellular vesicle according to any one of claims 2 to 78 and 116, an ASO according to any one of claims 79 to 113, a conjugate according to claim 114 or 115, or a pharmaceutical composition according to any one of claims 117 to 123.

143. A method of inducing meningeal macrophage infiltration of a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of an extracellular vesicle according to any one of claims 2 to 78 and 116, an ASO according to any one of claims 79 to 113, a conjugate according to claim 114 or 115, or a pharmaceutical composition according to any one of claims 117 to 123.

Images