Extracellular vesicles comprising bioactive molecules and cleavable linkers

Abstract

The present disclosure relates to extracellular vesicles (e.g., exosomes) comprising a biologically active molecule covalently linked to the extracellular vesicles via a cleavable linker and an anchoring moiety. The extracellular vesicles can be used as agents for the prevention or treatment of cancer or other diseases. Also provided herein are methods for producing the extracellular vesicles and methods for treating a disease or condition using the extracellular vesicles.

Description

[0001] References to electronically submitted sequence listings via EFS-WEB

[0002] The contents of the electronically submitted sequence listing submitted in this application (Name: 0132-0307WO1_Seqlisting_ST26.xml, Size: 1,523,864 bytes; and Creation Date: August 16, 2022) are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure provides extracellular vesicles (EVs), such as exosomes, which can be used as agents for preventing or treating cancer and other diseases, the extracellular vesicles comprising at least one biologically active molecule attached to the extracellular vesicle, such as exosome, via a cleavable linker and an anchoring moiety. Background Art

[0004] Many bioactive compounds have effective biological activity, which is of therapeutic significance. However, these compounds generally show toxicity in non-target organs. One way to limit non-target tissue exposure is to chemically conjugate small molecules with affinity-based reagents (such as antibodies), which can guide therapeutic compounds to specific cell types (Dosio, F. et al., Toxins (Basel) 3 (7): 848-883 (2011)), but this approach is subject to the following limitations: the number of molecules of the target compound that can be attached to the antibody (usually 2 to 6 molecules per antibody), and the availability / presence of antibodies that specifically bind to targeted, relevant diseased / effector cells but not to non-target cells. These two problems limit the use of antibody-drug conjugates (ADCs) by reducing efficacy and increasing systemic toxicity, respectively. Therefore, there is a need for a delivery system with a higher payload than ADC that can selectively target specific tissues or organs while limiting overall systemic exposure to the therapeutic compound.

[0005] EVs, such as exosomes, are important mediators of intercellular communication. They are also important biomarkers for the diagnosis and prognosis of many diseases (such as cancer). As drug delivery vehicles, EVs, such as exosomes, have many advantages over traditional drug delivery methods (such as peptide immunity, DNA vaccines) as new treatment modalities in many therapeutic areas. However, despite their advantages, many EVs, such as exosomes, have limited clinical efficacy. For example, in a phase II clinical trial, dendritic cell-derived exosomes (DEX) were studied as maintenance immunotherapy after first-line chemotherapy for patients with inoperable non-small cell lung cancer (NSCLC). However, the trial was terminated because the primary endpoint (at least 50% of patients had progression-free survival (PFS) 4 months after chemotherapy cessation) was not reached. Besse, B., et al., Oncoimmunology 5(4):e1071008(2015).

[0006] Therefore, novel and more efficient engineered EVs, such as exosomes, are needed to better realize the therapeutic use and other applications of EV-based technologies. Summary of the Invention

[0007] The present disclosure provides an extracellular vesicle (EV) comprising a biologically active molecule (BAM) attached to the EV via an anchoring moiety (AM) according to Formula I:

[0008] AM-SP1-L1-SP2-BAM (Formula 1)

[0009] wherein L1 is a cleavable bond comprising: a polynucleotide group; a peptide group selected from alanine-alanine-asparagine, valine-glycine, glycine-glycine, glutamic acid-valine-citrulline, aspartic acid-valine-citrulline, serine-valine-citrulline, lysine-valine-citrulline, glycine-glycine-glycine-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate acyloxy group; or a silyl ether; and SP1 and SP2 are optional first and second spacers, respectively.

[0010] In some aspects, AM is covalently linked to BAM at the 5' position. In some aspects, AM is covalently linked to BAM at the 3' position.

[0011] In some aspects, L1 comprises a polynucleotide group that is a trinucleotide group or higher nucleotide group. In some aspects, the polynucleotide group is a tetranucleotide group comprising dTdTdTdT, wherein dT is deoxythymidine.

[0012] In some aspects, L1 comprises a peptidyl group selected from alanine-alanine-asparagine, valine-glycine, glycine-glycine, glutamic acid-valine-citrulline, aspartic acid-valine-citrulline, serine-valine-citrulline, lysine-valine-citrulline, glycine-glycine-glycine-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine.

[0013] In some aspects, L1 comprises a pyrophosphate group.

[0014] In some aspects, L1 comprises a silyl ether. In some aspects, the silyl ether comprises -OSiR 1 R 2 O-, where R 1 and R 2 The same or different and each is C 1-8 In some aspects, the silyl ether comprises -OSiR 1 R 2 O- and R 1 and R 2 Both are isopropyl.

[0015] In some aspects, the AM comprises a sterol, a lipid, a vitamin, a peptide, or a combination thereof.

[0016] In some aspects, the AM comprises a sterol comprising cholesterol, mercaptocholesterol, ergosterol, 7-dehydrocholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fucoxosterol, sargassum sterol, campesterol, β-sitosterol, sitostanol, coprostanol, avenasterol, stigmasterol, or a combination thereof. In some aspects, the sterol is cholesterol.

[0017] In some respects, AM comprises lipid, and this lipid comprises fatty acid or phospholipid.In some respects, fatty acid is straight chain fatty acid, branched chain fatty acid, saturated fatty acid, unsaturated fatty acid, hydroxy fatty acid, polycarboxylic acid or its arbitrary combination.In some respects, straight chain fatty acid is butyric acid, caproic acid, sad, capric acid, lauric acid, myristic acid, palmitic acid or stearic acid.In some respects, straight chain fatty acid is palmitic acid. In some aspects, the phospholipid comprises 16:01,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (16:0PDP PE), 16:01,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (16:0PE MCC), or 16:01,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cyanuric acid) (16:0 Cyanuric acid PE).

[0018] In some aspects, the AM comprises a vitamin comprising tocopherol, tocotrienol, vitamin D, vitamin K, riboflavin, niacin, or pyridoxine. In some aspects, the vitamin is tocopherol.

[0019] In some aspects, the AM is attached to the outer surface of the EV.

[0020] In some aspects, BAM includes peptides, polypeptide bases, polynucleotide bases, proteins, antibodies or their antigen-binding fragments, chemical compounds or their combinations in any. In some aspects, BAM includes antisense oligonucleotide bases (ASOs), siRNAs, miRNAs, shRNAs, nucleic acids or their combinations in any. In some aspects, BAM includes ASOs. In some aspects, ASO targets transcripts. In some aspects, transcripts are STAT6 transcripts, EGFP transcripts, CEBP / β transcripts, STAT3 transcripts, KRAS transcripts, NRAS transcripts, NLPR3 transcripts, FFLUC transcripts, RLUC transcripts, MYC transcripts or their combinations in any.

[0021] In some aspects, SP1 and SP2 are the same or different and each comprises an alkylene group, a polyoxyalkylene group, a succinimidyl group, a maleimido group, an aryl group, an ether, a carbonyl group, a carboxylate group, a carbamoyl group, a thioether group, a sulfo group, a thiocarbonyl group, a thiocarbamoyl group, a thiosuccinimidyl group, an amino group, an amide group, a hydrazido group, a thiophosphate group, a 1,2,3-triazolyl group, a dibenzoylcyclooctenyl group, a bicyclononenyl group, a p-aminobenzoyl group, a p-aminobenzylcarbamate group, or a combination thereof, and at least one of SP1 and SP2 is present in formula (I). In some aspects, at least one of SP1 and SP2 comprises C 2-8 alkylene, polyoxyalkenyl, maleimide, carbamoyl, thio, amide, 1,2,3-triazolyl, dibenzoylcyclooctenyl, bicyclononenyl, 1,2,3-triazolyldibenzoylcyclooctenyl, 1,2,3-triazolylbicyclononenyl, p-aminobenzoyl, p-aminobenzylcarbamate, or a combination thereof. In some aspects, at least one of SP1 and SP2 comprises a C3 alkylene, C6 alkylene, or C8 alkylene. In some aspects, at least one of SP1 and SP2 comprises a polyoxyalkylene containing 2 to 15 repeating –OCH2CH2- units. In some aspects, at least one of SP1 and SP2 further comprises a carbamoyl, amide, sulfosuccinimidyl, 1,2,3-triazolyldibenzoylcyclooctenyl, 1,2,3-triazolylbicyclononenyl, or a combination thereof.

[0022] In some aspects, SP1 is present in formula (I). In some aspects, SP2 is present in formula (I). In some aspects, both SP1 and SP2 are present in formula (I) and are the same. In some aspects, both SP1 and SP2 are present in formula (I) and are different.

[0023] In some aspects, Formula (I) is a construct selected from

[0024]

[0025] as well as

[0026]

[0027] The present disclosure provides a pharmaceutical composition comprising the EVs described herein and a pharmaceutically acceptable carrier.

[0028] The present disclosure also provides a kit comprising the EVs described herein or a pharmaceutical composition thereof and instructions for use.

[0029] The present disclosure provides a method for treating or preventing a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an EV as described herein or a pharmaceutical composition thereof. In some aspects, the disease or condition is cancer, graft-versus-host disease (GvHD), an autoimmune disease, an infectious disease, a fibrotic disease, an inflammatory disease, a neurodegenerative disorder, a central nervous system disease, a muscular dystrophy, or a metabolic disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagrams showing: the general structure of an exosome (left); an exemplary bioactive molecule (e.g., an oligonucleotide) linked to a ligand that allows attachment to the outer surface of the exosome via a linker (center); and how a bioactive molecule (e.g., an oligonucleotide) linked to an anchoring moiety (e.g., a lipid such as cholesterol) via a linker can be attached to the membrane of an exosome (right).

[0031] Figure 2 Sequences of exemplary ASOs are shown. FFLUC and RLUC are named after the luminescent reporter genes they target. MYC and STAT6 ASOs are named after the genes they target. Nb: LNA residue (including LNA-5MeC and LNAT / LNA-5MeU). Nm: 2′-O′ MOE residue (including MOE-5MeC and MOE-T / MOED-5MeU). dN: DNA residue. (5MdC): 5-methyl-dC. s: Phosphorothioate backbone modification.

[0032] Figure 3 is a chemical scheme for the multi-step synthesis of constructs of formula (I).

[0033] Figure 4 is a table showing some properties of lipid-linker-ASO(-CPP) stock solutions.

[0034] Figure 5 is a bar graph of the average diameter (nm) of reconstituted lipid-linker-ASO (-CPP) after filtration through a 0.2 μm filter.

[0035] Figure 6 This is a table showing some characteristics of the loaded ASO concentration.

[0036] Figure 7 is a bar graph of exemplary exoASO loading density, which represents the number of ASOs loaded per number of exosomes.

[0037] Figure 8 is an exemplary exoASO IC 50 Graph of values (nm).

[0038] Figure 9 is the IC of an exemplary exoASO normalized to loading density 50 “*” means that the ASO was loaded under non-optimized loading conditions, so the loading density has the potential to be further increased.

[0039] Figure 10 This is a table showing some characteristics of exoASO, including the loaded ASO concentration and IC 50 .

[0040] Figure 11 Graph showing the percentage of gene expression (hSTAT6) for various exoASOs numbered 1 to 11, normalized to the ASO concentration (nM).

[0041] Figure 12 Graph showing the percentage of gene expression (hSTAT6) normalized to the exosome concentration for various exoASOs numbered 1 to 11.

[0042] Figure 13 is the IC normalized to ASO concentration (nM) for ASO numbers 1 to 11 50 Comparison diagram.

[0043] Figure 14 is the IC normalized to exosome concentration (p / mL) for ASO numbers 1 to 11 50 Comparison diagram.

[0044] Figure 15Figure 2 is a graph of mSTAT6 knockdown (KD) in mouse liver using a single dose of exoASO (5 or 10 μg dose based on ASO weight) with various cleavable linkers and cell penetrating peptides (CPPs). DETAILED DESCRIPTION

[0045] The present disclosure relates to extracellular vesicles (EVs), such as exosomes, comprising at least one biologically active molecule covalently attached to the EVs (e.g., exosomes) via a cleavable linker and an anchoring moiety, and uses thereof. Non-limiting examples of various aspects are shown in the present disclosure.

[0046] Before describing the present disclosure in more detail, it should be understood that the present invention is not limited to the specific compositions or process steps described, as these specific compositions or process steps may of course vary. As will be clear to those skilled in the art after reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that can be readily separated or combined with features of any other several aspects without departing from the scope or spirit of the present invention. Any described method can be carried out in the order of events described or in any other order that is logically possible.

[0047] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting, as the scope of the disclosure will be limited only by the appended claims.

[0048] Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0049] definition

[0050] To facilitate understanding of this description, certain terms are first defined. Additional definitions are provided throughout the detailed description.

[0051] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" or "an," "one or more," and "at least one" are used interchangeably herein. It should be further noted that the claims can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a precondition for the use of exclusive terms such as "solely," "only," or the use of negative limitations in conjunction with the recitation of claim elements.

[0052] Furthermore, the term "and / or" as used herein is considered to be a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, 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).

[0053] It should be understood that whenever an aspect is described herein with the language "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0054] 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 to which this disclosure belongs. For example, 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 Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, 2000, Oxford University Press, provide a general dictionary for many of the terms used in this disclosure for those of skill in the art.

[0055] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include numerals defining the range. In the case of enumerated value ranges, it will be understood that each intermediate integer value and each fraction thereof, as well as each subrange between such values, is also specifically disclosed. The upper and lower limits of any range may be independently included in the range or excluded from the range, and each range including any one, not including any one or including two limits is also encompassed in the present disclosure. Therefore, the ranges enumerated herein are understood to be shorthand for all values within the range, including the endpoints enumerated. For example, a range of 1 to 10 is understood to include any number, combination of numbers or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0056] Where values are explicitly enumerated, it will be understood that values of approximately the same quantity or amount as the enumerated values are also within the scope of the present disclosure. Where combinations are disclosed, each subcombination of the elements of the combination is also specifically disclosed and within the scope of the present disclosure. Conversely, where different elements or groups of elements are disclosed separately, their combinations are also disclosed. Where any element of the present disclosure is disclosed as having multiple alternatives, examples of the disclosure in which each alternative is excluded individually or in any combination with other alternatives are also hereby disclosed; more than one element of the present disclosure may have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0057] Nucleotides are represented by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Nucleotides are represented herein by their commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0058] Amino acid sequences are written left to right in amino to carboxyl orientation.Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0059] The term "about" is used herein to mean approximately, roughly, around, or in the region of a certain range. 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 numerical values. Generally speaking, the term "about" can modify a numerical value above or below the stated value by, for example, a variance of 10%.

[0060] The term "administration" and grammatical variations thereof refer to the introduction of a composition such as an EV (e.g., exosome) of the present disclosure into a subject via a pharmaceutically acceptable route. The introduction of a composition such as an EV (e.g., exosome) of the present disclosure into a subject is performed by any suitable route, including intratumoral, oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, periocular, or topical. Administration includes self-administration and administration by another person. Suitable routes of administration allow the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the vein of the subject.

[0061] As used herein, the term "agonist" refers to a molecule that binds to a receptor and activates the receptor to produce a biological response. Receptors can be activated by endogenous or exogenous agonists. Non-limiting examples of endogenous agonists include hormones, neurotransmitters, and cyclic dinucleotides. Non-limiting examples of exogenous agonists include drugs, small molecules, and cyclic dinucleotides. Agonists can be full agonists, partial agonists, or inverse agonists.

[0062] The term "amino acid substitution" refers to replacing the amino acid residue present in a parent sequence or a reference sequence (e.g., wild-type sequence) with another amino acid residue. Amino acid can be substituted in a parent sequence or a reference sequence (e.g., wild-type polypeptide sequence), for example, via chemical peptide synthesis or by recombinant methods known in the art. Therefore, reference to "substitution at position X" refers to replacing the amino acid present at position X with an alternative amino acid residue. In some aspects, substitution pattern can be described according to scheme AnY, wherein A is the single-letter code corresponding to the amino acid that is natural or initially present at position n, and Y is the amino acid residue that replaces. In other aspects, substitution pattern can be described according to scheme An(YZ), wherein A is the single-letter code corresponding to the amino acid residue that replaces the amino acid that is natural or initially present at position n, and Y and Z are amino acid residues that alternately replace A.

[0063] As used herein, the term "antagonist" refers to a molecule that, when bound to a receptor, blocks or inhibits an agonist-mediated response rather than stimulating its own biological response. Many antagonists achieve their effectiveness by competing with endogenous ligands or substrates at a structurally defined binding site on the receptor. Non-limiting examples of antagonists include alpha blockers, beta blockers, and calcium channel blockers. Antagonists can be competitive antagonists, noncompetitive antagonists, or uncompetitive antagonists.

[0064] As used herein, the term "antibody" encompasses natural or partially or completely synthetically produced immunoglobulins and fragments thereof. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" further includes polypeptides containing framework regions from immunoglobulin genes or fragments thereof that specifically bind to and recognize antigens. The use of the term antibody is intended to include complete antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, mouse-human antibodies, mouse-primate antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments, such as, for example, scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, diabodies, and antibody-related polypeptides. Antibodies include bispecific antibodies and multispecific antibodies, as long as they exhibit the desired biological activity or function. In some aspects of the present disclosure, the bioactive molecule is an antibody or a molecule comprising an antigen-binding fragment thereof.

[0065] The terms "antibody-drug conjugate" and "ADC" are used interchangeably and refer to an antibody linked (e.g., covalently linked) to a therapeutic agent (sometimes referred to herein as a pharmaceutical agent, drug, or active pharmaceutical ingredient) or pharmaceutical agent. In some aspects of the present disclosure, the biologically active molecule is an antibody-drug conjugate.

[0066] As used herein, the term "approximately" when applied to one or more target values refers to a value that is similar to a stated reference value. In certain aspects, the term "approximately" refers to a range of values that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) a stated reference value, unless otherwise specified or otherwise obvious from the context (except where such number exceeds 100% of the possible value).

[0067] As used herein, the term "bioactive molecule" refers to any molecule that can be attached to an EV (e.g., an exosome) via an anchoring portion, wherein the molecule can have a therapeutic or prophylactic effect in a subject in need thereof, or for diagnostic purposes. Thus, for example, the term bioactive molecule includes proteins (e.g., antibodies, proteins, polypeptides, and derivatives, fragments, and variants thereof), lipids and derivatives thereof, carbohydrates (e.g., glycan moieties in glycoproteins), or small molecules (e.g., molecules with a molecular weight of 1000 g / mol or less). In some aspects, the bioactive molecule includes a radioisotope. In some aspects, the bioactive molecule is a detectable moiety, such as a radionuclide, a fluorescent molecule, or a contrast agent. In some aspects, the bioactive molecule can be or can include a targeting moiety or a tropism moiety. In some aspects, the bioactive molecule can be or can include, for example, an affinity ligand (e.g., biotin, digoxin, or dinitrophenol). In some aspects, the bioactive molecule can be or can include a moiety that can improve pharmacokinetic or pharmacodynamic properties, such as a moiety that can increase plasma half-life, such as a PEG moiety.

[0068] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a substitution is considered conservative if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family. In another aspect, an amino acid string can be conservatively replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0069] As used herein, the term "conserved" refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that do not vary at the same position in two or more sequences being compared. Relatively conserved nucleotides or amino acids are those that are more conserved among more related sequences than nucleotides or amino acids that occur elsewhere in the sequence.

[0070] In some aspects, if two or more sequences are 100% identical to each other, they are said to be "fully conserved" or "identical". In some aspects, if two or more sequences are about 70% identical to each other or higher, such as about 80% identical, about 90% identical, about 95%, about 98% or about 99% identical, they are said to be "highly conserved". In some aspects, if two or more sequences are at most about 70% identical to each other, including about 30% identical, about 40% identical, about 50% identical, about 60% identical or about 65% identical, they are said to be "conserved". Sequence conservation can be applied to the full length of a polynucleotide or polypeptide or to a portion, region or feature thereof.

[0071] As used herein, the term "conventional EV protein" refers to proteins that were previously known to be enriched in EVs.

[0072] As used herein, the term "conventional exosomal proteins" refers to proteins previously known to be enriched in exosomes, including but not limited to CD9, CD63, CD81, PDGFR, GPI-anchored proteins, lactadherins LAMP2 and LAMP2B, fragments thereof, or peptides bound thereto.

[0073] As used herein, the term "derivative" refers to an EV (e.g., an exosome), a component (e.g., a protein such as Scaffold X, a lipid, or a carbohydrate), or a biologically active molecule (e.g., a polypeptide, a polynucleotide, a lipid, a carbohydrate, an antibody or fragment thereof, a PROTAC, etc.) that has been chemically modified to introduce at least one reactive moiety (e.g., a phosphoramidite moiety).

[0074] The terms “excipient” and “carrier” are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound or EV.

[0075] As used herein, the terms "extracellular vesicle," "EV," and grammatical variants thereof are used interchangeably and refer to cell-derived vesicles comprising a membrane surrounding an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, microvesicles, nanovesicles, extracellular granules, cancer bodies, or apoptotic bodies) having a diameter smaller than that of the cell from which they are derived. In some aspects, the diameter of the extracellular vesicle ranges from 20 nm to 1000 nm (e.g., 50 nm to 1000 nm, 50 nm to 200 nm, or 200 nm to 1000 nm), and may comprise various macromolecular payloads located within the internal space (i.e., lumen), displayed on the external surface of the extracellular vesicle, and / or across the membrane. In some aspects, the payload may comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In certain aspects, the extracellular carrier comprises a scaffold portion. By way of example and not limitation, extracellular vesicles include apoptotic bodies, cell debris, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicles of vesicled organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. In some aspects, extracellular vesicles are produced by cells expressing one or more transgenic products.

[0076] As used herein, the term "exosome" refers to an extracellular vesicle with a diameter between 20 and 300 nm (e.g., 40 to 200 nm, 50 to 200 nm). Exosomes comprise a membrane surrounding an internal space (i.e., an inner cavity), and in some aspects, can be produced from cells (e.g., production cells) by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. In certain aspects, exosomes comprise a scaffold portion. As described below, exosomes can be derived from production cells and separated from production cells based on their size, density, biochemical parameters, or a combination thereof. In some aspects, the exosomes of the present disclosure are produced by cells expressing one or more transgenic products.

[0077] In some aspects, the EVs, e.g., exosomes, e.g., nanovesicles, of the present disclosure are engineered by covalently linking at least one bioactive molecule (e.g., a protein such as an antibody or antibody drug conjugate (ADC), ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) such as an antisense oligonucleotide, a small molecule drug, or a small molecule toxin) to the EV, e.g., exosome or nanovesicle, via an anchoring moiety.

[0078] In some aspects, EVs of the present disclosure, such as exosomes or nanovesicles, may comprise various macromolecular payloads located within the internal space (i.e., lumen), displayed on the external (external) surface or internal (lumen) surface of EV, and / or across the membrane. In some aspects, the payload may comprise, for example, nucleic acids, proteins, carbohydrates, lipids, small molecules, and combinations thereof. In certain aspects, EVs, such as exosomes, comprise a scaffold portion (e.g., scaffold X). EVs, such as exosomes, may be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. In some aspects, EVs, such as exosomes, are produced by cells expressing one or more transgenic products. In other aspects, EVs of the present disclosure are not limited to nanovesicles, microsomes, microvesicles, extracellular bodies, or apoptotic bodies.

[0079] The following diagram is shown in Figure 1 Middle: General structure of exosomes; exemplary bioactive molecules (e.g., oligonucleotides) linked to ligands (e.g., anchoring moieties) that allow attachment to the outer surface of exosomes via linkers; and how bioactive molecules (e.g., oligonucleotides) linked to anchoring moieties (e.g., lipids such as cholesterol) via linkers can be attached to the exosome membrane. In some aspects, AMs are attached to the outer surface of EVs.

[0080] As used herein, the term "fragment" of a protein (e.g., a biologically active molecule such as a therapeutic protein, or a scaffold protein such as Scaffold X) refers to an amino acid sequence of a protein that is shorter than the naturally occurring N- and / or C-terminus deleted sequence or any portion of the protein deleted, compared to the naturally occurring protein.

[0081] As used herein, the term "functional fragment" refers to a protein fragment that retains protein function. Thus, in some aspects, a functional fragment of a scaffold protein (e.g., Scaffold X protein) retains the ability to anchor a bioactive molecule to the lumen or exterior surface of an EV (e.g., an exosome).

[0082] Whether a fragment is a functional fragment can be assessed by any method known in the art to determine the protein content of EVs (e.g., exosomes), including Western blot, fluorescence activated cell sorting (FACS) analysis, and fusion of the fragment to an autofluorescent protein, such as, for example, green fluorescent protein (GFP). In certain aspects, the functional fragment of the scaffold X protein retains, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100% of the ability of the naturally occurring scaffold X protein to anchor a biologically active molecule to the lumen or external surface of an EV (e.g., exosome).

[0083] As used herein, "anchoring" a bioactive molecule to the lumen or external surface of an EV (e.g., exosome) of the present disclosure via a scaffold protein refers to covalently or non-covalently attaching the bioactive molecule to a portion of the scaffold molecule located on the lumen or external surface of the EV (e.g., exosome), respectively.

[0084] As used herein, the term "homology" refers to the overall relatedness between polymer molecules, such as nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In general, the term "homology" means the evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present disclosure, the term homology encompasses both identity and similarity.

[0085] In some aspects, polymer molecules are considered "homologous" to each other if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the monomers in the molecule are identical (exactly identical monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0086] In the context of this disclosure, substitutions (even when they are referred to as amino acid substitutions) are made at the nucleic acid level, i.e., replacement of an amino acid residue with an alternative amino acid residue is made by substituting the codon encoding the first amino acid with the codon encoding the second amino acid.

[0087] As used herein, the term "identity" refers to the overall monomer conservation between polymeric molecules, such as polypeptide molecules or polynucleotide molecules (e.g., DNA molecules and / or RNA molecules). The term "identical" without any additional qualifiers, e.g., protein A is identical to protein B, means that the sequences are 100% identical (100% sequence identity). Describing two sequences as being "70% identical" is equivalent to describing them as having, for example, "70% sequence identity."

[0088] For example, the calculation of percent identity between two polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second polypeptide sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In some aspects, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The amino acids at corresponding amino acid positions are then compared.

[0089] When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of intervals and the length of each interval, which needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be achieved using a mathematical algorithm.

[0090] Suitable software programs are available from various sources and are used for the comparison of both protein and nucleotide sequences. A suitable program for determining sequence identity percentage is bl2seq, which is a part of the BLAST (Basic Local Alignment Search Tool) program suite, available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, which are a part of the EMBOSS bioinformatics program suite and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0091] Sequence alignment can be performed using methods known in the art, such as MAFFT (Multiple Alignment using Fast Fourier Transform), Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE (Multiple Sequence Comparison by Logarithmic Expectation), and the like.

[0092] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Note that percent 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. Also note that length values will always be integers.

[0093] In certain aspects, the percent identity (%ID) of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID=100x(Y / Z), where Y is the number of amino acid residues (or nucleobases) scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0094] It will be appreciated by those skilled in the art that the generation of the sequence alignment for calculating sequence identity percentage ratio is not limited to the binary sequence-sequence comparison driven only by primary sequence data.It will also be appreciated that sequence alignment can be produced by integrating sequence data with the data from heterogeneous sources, the data from heterogeneous sources such as structural data (for example crystal protein structure), functional data (for example position of mutation) or phylogeny data.Integrating heterogeneous data to generate the appropriate program for multiple sequence alignment is T-Coffee, which can be obtained on www.tcoffee.org and alternatively for example, obtained from EBI.It will also be appreciated that the final comparison for calculating sequence identity percentage ratio can be automatic or manually planned.

[0095] As used herein, the terms "isolated," "purified," "extracted," and grammatical variations thereof are used interchangeably and refer to a state of preparation of a desired EV (e.g., a plurality of EVs of known or unknown amount and / or concentration) that has undergone one or more purification processes, such as selection or enrichment, preparation of a desired EV (e.g., exosomes). In some aspects, isolation or purification as used herein is a process of removing, partially removing (e.g., a portion) EVs (e.g., exosomes) from a sample containing producer cells. In some aspects, the isolated EV (e.g., exosome) composition has no detectable undesirable activity, or alternatively, the level or amount of the undesirable activity is at or below an acceptable level or amount. In other aspects, the isolated EV (e.g., exosome) composition has an amount and / or concentration of the desired EV (e.g., exosomes) that is at or above an acceptable amount and / or concentration. In other aspects, the isolated EV (e.g., exosome) composition is enriched compared to the starting material (e.g., producer cell preparation) from which the composition is obtained. Compared to the starting material, the enrichment can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, at least 99.99%, at least 99.999%, at least 99.9999% or greater than 99.9999%. In some aspects, the isolated EV (e.g., exosome) preparation is substantially free of residual biological products. In some aspects, the isolated EV (e.g., exosome) preparation is 100% free, at least 99% free, at least 98% free, at least 97% free, at least 96% free, at least 95% free, at least 94% free, at least 93% free, at least 92% free, at least 91% free or at least 90% free of any contaminating biological matter. Residual biological products may include non-biological materials (including chemicals) or unnecessary nucleic acids, proteins, lipids and / or metabolites. Substantially free of residual biological products can also mean that the EV (e.g., exosome) composition does not contain detectable producer cells and only EVs (e.g., exosomes) are detectable.

[0096] The terms "linked," "fused," and their grammatical variations are used interchangeably and refer to a first moiety, such as a first amino acid sequence or nucleotide sequence, being covalently or non-covalently linked to a second moiety, such as a second amino acid sequence, nucleotide sequence, and / or lipid (e.g., cholesterol), respectively. The first moiety can be directly linked or juxtaposed to the second moiety, or alternatively, an intervening moiety can covalently link the first moiety to the second moiety. The term "linked" not only refers to the fusion of the first moiety to the second moiety at the C-terminus or N-terminus, but also includes the insertion of the entire first moiety (or second moiety) into any two points in the second moiety (or first moiety, respectively), such as amino acids. In one aspect, the first moiety is linked to the second moiety via a peptide bond or a linker. The first moiety can be linked to the second moiety via a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (for a polypeptide chain) or a nucleotide or nucleotide chain (for a nucleotide chain), or any chemical moiety (for a polypeptide or polynucleotide chain or any chemical molecule). The term "linked" is also represented by a hyphen (-). In some aspects, the scaffold X protein on an EV (e.g., an exosome) can be linked to or fused to a biologically active molecule via a linker, a spacer, or both a linker and a spacer.

[0097] The term "modified" as described herein, when used in the context of EVs (e.g., exosomes), refers to changes or engineering of EVs (e.g., exosomes) and / or their producing cells such that the modified EVs (e.g., exosomes) are different from naturally occurring EVs (e.g., exosomes). In some aspects, the modified EVs (e.g., exosomes) described herein comprise a membrane that is different in composition from the membrane of naturally occurring EVs (e.g., exosomes) in terms of proteins, lipids, small molecules, carbohydrates, etc. In one aspect, the membrane comprises a higher density or number of natural EVs (e.g., exosomes), proteins, and / or the membrane comprises proteins that are not naturally found in EVs (e.g., exosomes). In certain aspects, such modifications to the membrane alter the outer surface of EVs, such as exosomes (e.g., surface-engineered EVs and exosomes described herein).

[0098] As used herein, the term "modified protein" or "protein modification" refers to a protein that has at least 15% identity to the non-mutated amino acid sequence of the protein. Modifications of the protein include fragments or variants of the protein. Modifications of the protein may further include chemical or physical modifications to the fragments or variants of the protein.

[0099] As used herein, the terms "modulate," "modify," and grammatical variations thereof generally refer to the ability to alter, such as by increasing or decreasing, for example, directly or indirectly promoting / stimulating / upregulating or interfering / inhibiting / downregulating, a specific concentration, level, expression, function, or behavior, when applied to a specific concentration, level, expression, function, or behavior, such as, for example, acting as an antagonist or agonist. In some cases, a modulator can increase and / or decrease a certain concentration, level, activity, or function relative to a control, or relative to a generally expected average activity level, or relative to a control activity level.

[0100] 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) and is produced by a cell (e.g., a production cell) by direct or indirect manipulation so that the nanovesicle is not produced by the cell in the absence of manipulation. Suitable manipulations for producing nanovesicles in cells include, but are not limited to, continuous extrusion, treatment with an alkaline solution, ultrasonic treatment, or a combination thereof. In some aspects, the production of nanovesicles can result in the destruction of production cells. In some aspects, the nanovesicle colony described herein is substantially free of vesicles derived from cells by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. In some aspects, the nanovesicle includes a scaffold portion, such as scaffold X. Once derived from production cells, nanovesicles can be separated from production cells based on their size, density, biochemical parameters, or a combination thereof.

[0101] As used herein, the term "payload" refers to a bioactive molecule (e.g., a therapeutic agent) that acts on a target (e.g., a target cell) in contact with the EV (e.g., exosome) of the present disclosure. Non-limiting examples of payloads that can be introduced into EVs (e.g., exosomes) include therapeutic agents such as nucleotides (e.g., nucleotides containing a detectable moiety or toxin or nucleotides that disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides (e.g., enzymes), or RNA molecules with regulatory functions, such as miRNA, dsDNA, lncRNA, and siRNA), amino acids (e.g., amino acids containing a detectable moiety or toxin or amino acids that disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins). In certain aspects, the payload comprises an antigen. As used herein, the term "antigen" refers to any agent that, when introduced into a subject, elicits an immune response (cellular or bodily fluid) to itself. In some aspects, the payload molecule is covalently linked to an EV, such as an exosome, via a linker, a spacer, or both a linker and a spacer, as disclosed herein. In other aspects, the payload comprises an adjuvant.

[0102] The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," and grammatical variations thereof encompass any pharmaceutical agent approved by a U.S. federal regulatory agency or listed in the U.S. Pharmacopoeia for use in animals (including humans), as well as any carrier or diluent that does not cause undesirable physiological effects to an extent that would prohibit administration of the composition to a subject and does not abrogate the biological activity and properties of the administered compound. Excipients and carriers that are useful in preparing pharmaceutical compositions and that are generally safe, nontoxic, and desirable are encompassed.

[0103] As used herein, the term "pharmaceutical composition" refers to one or more compounds described herein, such as, for example, EVs, such as the exosomes of the present disclosure, mixed or doped with, or suspended in, one or more other chemical components, such as pharmaceutically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate the administration of a formulation of EVs (e.g., exosomes) to a subject.

[0104] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The term refers to the primary structure of a molecule. Therefore, the term includes triple-stranded, double-stranded, and single-stranded DNA, as well as triple-stranded, double-stranded, and single-stranded RNA. The term also includes modified, for example, by alkylation and / or by end-capping, and unmodified polynucleotides. More particularly, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide of the N- or C-glycosides of purine or pyrimidine bases, and other polymers containing positive nucleotide backbones, such as polyamides (e.g., peptide nucleic acids "PNA") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains a core base in a configuration that allows base pairing and base stacking, such as those found in DNA and RNA. In some aspects of the present disclosure, as disclosed herein, the bioactive molecule attached to EVs (e.g., exosomes) via a linker, a spacer, or both a linker and a spacer is a polynucleotide, such as an antisense oligonucleotide. In specific aspects, the polynucleotide comprises mRNA. In other aspects, the mRNA is a synthetic mRNA. In some aspects, the synthetic mRNA comprises at least one non-natural nucleobase. In some aspects, all nucleobases of a certain class have been replaced with non-natural nucleobases (e.g., all uridines in the polynucleotides disclosed herein can be replaced with non-natural nucleobases, such as 5-methoxyuridine). In some aspects of the present disclosure, the bioactive molecule is a polynucleotide (e.g., an antisense oligonucleotide, ASO).

[0105] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may comprise modified amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. In some aspects of the present disclosure, as disclosed herein, the biologically active molecule attached to an EV (e.g., an exosome) via a linker, a spacer, or both a linker and a spacer is a polypeptide, such as an antibody or derivative thereof such as an ADC, a proteolytic targeting chimera (PROTAC), a toxin, a fusion protein, or an enzyme.

[0106] As used herein, the term "polypeptide" refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the aforementioned. Polypeptides can be single polypeptides, or can be multimolecular complexes, such as dimers, trimers, or tetramers. They can also include single-chain or multi-chain polypeptides. The most common disulfide bonds are found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some aspects, a "peptide" can be less than or equal to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0107] As used herein, the terms "prevent" and "preventing" and variations thereof refer to partially or completely delaying the onset of a disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or clinical manifestations of a particular disease, disorder, and / or condition; partially or completely delaying progression from a particular disease, disorder, and / or condition; and / or reducing the risk of developing pathology associated with a disease, disorder, and / or condition. In some aspects, prevention of a certain outcome is achieved through prophylactic treatment.

[0108] As used herein, the term "producer cell" refers to a cell used to produce EVs (e.g., exosomes). Producer cells can be cells cultured in vitro or in vivo. Producer cells include, but are not limited to, cells known to effectively produce EVs (e.g., exosomes), such as HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, AGE.HNTM Neuronal precursor cells, CAP TM In some aspects, the production cell is not an antigen presenting cell. In some aspects, the production cell is not a dendritic cell, a B cell, a mast cell, a macrophage, a neutrophil, a Kupffer-Browicz cell, a cell derived from any of these cells, or any combination thereof.

[0109] As used herein, "prevention" or "prophylaxis" refers to a treatment or regimen of action intended to prevent the onset of a disease or condition, or to prevent or delay the symptoms associated with a disease or condition.

[0110] As used herein, "prevention" refers to measures taken to maintain health and prevent or delay bleeding episodes, or to prevent or delay symptoms associated with a disease or condition.

[0111] A "recombinant" polypeptide or protein refers to a polypeptide or protein produced via recombinant DNA technology. For the purposes of this disclosure, recombinantly produced polypeptides and proteins expressed in engineered host cells are considered isolated, as are natural or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique. The polypeptides disclosed herein can be recombinantly produced using methods known in the art. Alternatively, the proteins and peptides disclosed herein can be chemically synthesized. In some aspects of the present disclosure, the scaffold X protein present in EVs (e.g., exosomes) is recombinantly produced by overexpressing the scaffold protein in producer cells, such that the level of the scaffold protein in the resulting EVs (e.g., exosomes) is increased relative to the level of the scaffold protein present in EVs (e.g., exosomes) from producer cells that do not overexpress such scaffold protein.

[0112] As used herein, the term "scaffold moiety" refers to a molecule, such as a protein (such as Scaffold X), that can be used to anchor a payload, such as a bioactive molecule, to an EV (e.g., an exosome), for example, on the exterior surface of an EV. In certain aspects, the scaffold moiety comprises a synthetic molecule. In certain aspects, the scaffold moiety comprises a non-polypeptide moiety. In other aspects, the scaffold moiety comprises, for example, a lipid, carbohydrate, protein, or combination thereof (e.g., a glycoprotein or proteolipid) that naturally occurs in EVs (e.g., exosomes). In certain aspects, the scaffold moiety comprises a lipid, carbohydrate, or protein that does not naturally occur in EVs (e.g., exosomes). In certain aspects, the scaffold moiety comprises a lipid or carbohydrate that naturally occurs in EVs (e.g., exosomes), but is enriched in EVs (e.g., exosomes) relative to basal / natural / wild-type levels. In certain aspects, the scaffold moiety comprises a protein that naturally occurs in EVs (e.g., exosomes), but is enriched in EVs (e.g., exosomes) relative to basal / natural / wild-type levels, for example, by recombinant overexpression in a producer cell. In certain aspects, the scaffold moiety is Scaffold X.

[0113] As used herein, the term "scaffold X" refers to an EV (e.g., exosome) protein that has been identified on the surface of an EV (e.g., 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: prostaglandin F2 receptor negative regulator ("PTGFRN"); basophil activating factor ("BSG"); immunoglobulin superfamily member 2 ("IGSF2"); immunoglobulin superfamily member 3 ("IGSF3"); immunoglobulin superfamily member 8 ("IGSF8"); integrin beta-1 ("ITGB1"); integrin alpha-4 ("ITGA4"); 4F2 cell surface antigen heavy chain ("SLC3A2"); and a class of ATP transporters ("ATP1A1," "ATP1A2," "ATP1A3," "ATP1A4," "ATP1B3," "ATP2B1," "ATP2B2," "ATP2B3," "ATP2B"). In some aspects, the scaffold X protein can be an entire protein or a fragment thereof (e.g., a functional fragment, such as the smallest fragment capable of anchoring another moiety to the external surface or lumen of an EV (e.g., an exosome). In some aspects, scaffold X can anchor a bioactive molecule to the external surface or lumen of an EV (e.g., an exosome). In some aspects of the present disclosure, the bioactive molecule can be covalently attached to scaffold X, for example, via a linker, a spacer, or both a linker and a spacer, as disclosed herein. Non-limiting examples of other scaffold moieties that can be used with the present disclosure include: aminopeptidase N (CD13); neprilysin, also known as membrane metalloendopeptidase (MME); ectonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1); neuropilin-1 (NRP1); CD9, CD63, CD81, PDGFR, GPI-anchored proteins, lactadherin, LAMP2, and LAMP2B.

[0114] As used herein, the term "similarity" refers to the overall correlation between polymer molecules, such as between polynucleotide molecules (such as DNA molecules and / or RNA molecules) and / or between polypeptide molecules. The calculation of the similarity percentages of polymer molecules to each other can be performed in the same manner as the calculation of the identity percentage, except that the calculation of the similarity percentages takes into account conservative substitutions as understood in the art. It should be understood that the percentage of similarity depends on the comparative scale used, i.e., whether, for example, amino acids are compared according to their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or a combination thereof.

[0115] As used herein, the term "small molecule" refers to an organic compound that can pass through a cell membrane and has a molecular weight of about 1000 g / mol or less (e.g., about 900 g / mol or less). The organic compound can be, for example, a biomolecule, a drug, or a toxin. Suitable biomolecules include, for example, cyclic dinucleotides, fatty acids, sugars (e.g., glucose), amino acids, lipids (e.g., cholesterol), phenolic compounds, and alkaloids. Suitable drugs include, for example, analgesics, antibacterials, antivirals, anticonvulsants, antipsychotics, antitumor agents, anti-inflammatory agents, antiobesity agents, antiparasitics, contraceptives, ear agents, ophthalmic agents, skeletal muscle relaxants, sleep disorder agents, central nervous system agents, cardiovascular agents, blood glucose regulators, immune agents, infertility agents, respiratory agents, tyrosine kinase inhibitors, and mTOR inhibitors. Specific examples include, but are not limited to, aspirin, naproxen, celecoxib, diclofenac, ketorolac, oxycodone, insulin, methotrexate, sulfasalazine, imiquimod, cyclophosphamide, mycophenolate mofetil, marmastat, dimercaprol, adriamycin, paclitaxel and paclitaxel. Suitable toxins include, for example, bacterial toxins, including hemotoxins, phototoxins, hepatotoxins and neurotoxins, mycotoxins, aflatoxins, ochratoxins, citrinin and ergot alkaloids. Specific examples include monomethyl riocetine E (MMAE) botulinum toxin A, tetanus toxin A, edema toxin, exotoxin A, cholera toxin, pertussis toxin, diphtheria toxin, dioxins, muscarines and toad toxins, or synthetic toxins, such as bisphenol A, perchlorate, tetrachloroethylene, 2-butoxyethanol and formaldehyde.

[0116] Unless otherwise stated, reference to compounds having one or more stereocenters is intended to include each stereoisomer and all combinations of stereoisomers thereof.

[0117] The terms "subject," "patient," "individual," and "host," and variations thereof, are used interchangeably herein and refer to any mammalian subject, including but not limited to humans, domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.), and experimental animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.), for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications.

[0118] As used herein, the term "substantially free" means that a sample comprising EVs (e.g., exosomes) contains less than 10% of macromolecules, such as contaminants, by mass / volume (m / v) 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.

[0119] As used herein, the term “surface-engineered EVs” (e.g., scaffold-X engineered exosomes) refers to EVs whose membrane or surface is modified in its composition such that the surface of the engineered EV is different from the surface of the EV before modification or the surface of naturally occurring EVs.

[0120] As used herein, the term "surface-engineered exosomes" (e.g., scaffold-X engineered exosomes) refers to exosomes whose membrane or surface (external surface or luminal surface) is modified in its composition such that the surface of the engineered exosome is different from the surface of the exosome before modification or the surface of naturally occurring exosomes.

[0121] Engineering can be on the surface of EV (e.g., exosomes), or on the membrane of EV (e.g., exosomes), so that the surface of EV (e.g., exosomes) is changed. For example, the composition of the membrane, such as proteins, lipids, small molecules, carbohydrates, or a combination thereof, can be modified. The composition can be changed by chemical, physical, or biological methods, or by producing cells that have been previously or simultaneously modified by chemical, physical, or biological methods. Specifically, the composition can be changed by genetic engineering, or by producing cells that have been previously modified by genetic engineering. In some aspects, surface-engineered EVs (e.g., exosomes) contain exogenous proteins (i.e., proteins that are not naturally expressed by EVs, such as exosomes) or fragments or variants thereof that can be exposed to the surface of EVs (e.g., exosomes), or can be anchor points (attachments) of portions exposed to the surface of EVs (e.g., exosomes). In other aspects, the surface engineered EVs (e.g., exosomes) comprise native EVs, such as higher expression (e.g., higher numbers) of exosomal proteins (e.g., scaffold X) or fragments or variants thereof that can be exposed on the surface of EVs (e.g., exosomes), or anchor points (attachments) of portions that can be exposed on the surface of EVs (e.g., exosomes). In specific aspects, the surface engineered EVs, such as exosomes, comprise modifications to one or more membrane components, such as proteins (such as scaffold X), lipids, small molecules, carbohydrates, or combinations thereof, wherein at least one of the components is covalently attached to a bioactive molecule via a linker, a spacer, or both a linker and a spacer, as disclosed herein.

[0122] As used herein, the term "therapeutically effective amount" is an amount of an agent or pharmaceutical compound comprising the EVs or exosomes of the present disclosure that is sufficient to produce a desired therapeutic, pharmacological, and / or physiological effect on a subject in need thereof. A therapeutically effective amount can be a "prophylactically effective amount" because prevention can be considered therapy.

[0123] As used herein, the terms "treat" or "treatment" refer to, for example, reducing the severity of a disease or condition; shortening the duration of the course of the disease; ameliorating or eliminating one or more symptoms associated with the disease or condition; providing any appropriate degree of beneficial effect to a subject suffering from the disease or condition, but not necessarily curing the disease or condition. The terms also include prophylaxis or prevention of a disease or condition or its symptoms. In one aspect, the terms "treating" or "treatment" means inducing an immune response to an antigen in a subject.

[0124] As used herein, the term "variant" of a molecule (e.g., a functional molecule, antigen, or scaffold X) refers to a molecule that shares certain structural and functional identities with another molecule after comparison by methods known in the art. For example, a variant of a protein may include a substitution, insertion, deletion, frameshift, or rearrangement in another protein.

[0125] In some aspects, a variant of Scaffold X or a derivative comprises a variant of Scaffold X that is at least 70% identical to full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or an ATP transporter, or a fragment (e.g., a functional fragment) of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or an ATP transporter.

[0126] In some aspects, variants or fragments of the Scaffold X protein disclosed herein, or derivatives thereof, retain the ability to specifically target EVs (e.g., exosomes). In some aspects, Scaffold X or Scaffold X derivatives include one or more mutations, such as conservative amino acid substitutions.

[0127] Naturally occurring variants are called "allelic variants" and refer to one of several alternative forms of a gene occupying a given locus on a chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at the polynucleotide and / or polypeptide level and are encompassed by the present disclosure. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.

[0128] Using known methods of protein engineering and recombinant DNA technology, variants can be generated to improve or alter the characteristics of a polypeptide. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without significant loss of biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993), which is incorporated herein by reference in its entirety, reported variant KGF proteins that had heparin binding activity even after deletion of 3, 8, or 27 amino-terminal amino acid residues. Similarly, after deletion of 8 to 10 amino acid residues from the carboxyl terminus of this protein, interferon gamma exhibited up to ten times higher activity. (Dobeli et al., J. Biotechnology 7:199-216 (1988), which is incorporated herein by reference in its entirety.)

[0129] In addition, there is ample evidence that variants generally retain biological activities similar to naturally occurring proteins. For example, Gayle and colleagues (J.Biol.Chem 268:22105-22111 (1993), which is incorporated herein by reference in its entirety) conducted extensive mutational analysis of the human cytokine IL-1a. They used random mutagenesis to generate more than 3,500 individual IL-1a mutants, with an average of 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at each possible amino acid position. The researchers found that "most molecules can be changed with minimal effect on [binding or biological activity]. " (See abstract.) In fact, of the more than 3,500 nucleotide sequences tested, only 23 unique amino acid sequences produced proteins whose activity was significantly different from that of the wild type.

[0130] As described above, variants or derivatives include, for example, modified polypeptides. In some aspects, variants or derivatives of, for example, polypeptides, polynucleotides, lipids, glycoproteins are the result of chemical modification and / or endogenous modification. In some aspects, variants or derivatives are the result of in vivo modification. In some aspects, variants or derivatives are the result of in vitro modification. In yet other aspects, variants or derivatives are the result of intracellular modification in a production cell.

[0131] Modifications present in variants and derivatives include, for example, acetylation, acylation, adenosine diphosphate ribose (ADP) ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginylation, and ubiquitination.

[0132] In some aspects, Scaffold X can be modified at any convenient position. In some aspects, bioactive molecules can be modified at any convenient position. In certain aspects of the present disclosure, EV (e.g., exosome) components (e.g., proteins such as Scaffold X, lipids, or polysaccharides) and / or bioactive molecules (e.g., antibodies or ADCs, PROTACs, small molecules (such as cyclic dinucleotides), toxins (such as MMAE), STING (stimulator of interferon genes) agonists (e.g., CAS Nos. 702662-50-8 and 849214-04-6), tolerogens (e.g., agents disclosed in U.S. Patent No. 7,910,113 and U.S. Patent Application Publication No. 2009 / 0169578), or antisense oligonucleotides) can be modified to produce derivatives comprising at least one linker, spacer, or both a linker and a spacer, as disclosed herein.

[0133] The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated, a alkyl radical having the specified number of carbon atoms (e.g., C1-C 10 The term "alkyl" refers to a straight or branched hydrocarbon group having 1 to 10 carbon atoms. Typically, an alkyl group will have 1 to 15 carbon atoms, for example, 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms. A "low alkyl" group is an alkyl group having 1 to 4 carbon atoms (for example, 1 to 3 carbon atoms or 1 to 2 carbon atoms). The term "alkyl" includes monovalent, divalent, and polyvalent groups. For example, where appropriate, the term "alkyl" includes "alkylidene" when the formula indicates that the alkyl group is divalent or when the substituents are connected to form a ring. 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.

[0134] The term "alkylene" by itself or as part of another substituent means a divalent (diradical) alkyl group, where alkyl is defined herein. "Alkylene" is exemplified by, but not limited to, -CH2CH2CH2CH2-. Typically, an "alkylene" group will have from 1 to 15 carbon atoms, for example, 10 or fewer carbon atoms (e.g., 1 to 8 or 1 to 6 carbon atoms). A "lower alkylene" group is an alkylene group having from 1 to 4 carbon atoms (e.g., 1 to 3 carbon atoms or 1 to 2 carbon atoms).

[0135] The term "alkenyl" itself or as part of another substituent refers to a straight or branched hydrocarbon radical having 2 to 15 carbon atoms and at least one double bond. Typical alkenyl groups have 2 to 10 carbon atoms and at least one double bond. In one aspect, the alkenyl group has 2 to 8 carbon atoms or 2 to 6 carbon atoms and 1 to 3 double bonds. Exemplary alkenyl groups include vinyl, 2-propenyl, 1-but-3-enyl, crotyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), 2-isopentenyl, 1-pent-3-enyl, 1-hex-5-enyl, etc.

[0136] The term "alkynyl" by itself or as part of another substituent refers to a straight or branched, unsaturated or polyunsaturated hydrocarbon group having 2 to 15 carbon atoms and at least one triple bond. Typical "alkynyl" groups have 2 to 10 carbon atoms and at least one triple bond. In one aspect of the present disclosure, alkynyl groups have 2 to 6 carbon atoms and at least one triple bond. Exemplary alkynyl groups include prop-1-ynyl, prop-2-ynyl (i.e., propargyl), ethynyl, and 3-butynyl.

[0137] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to an alkyl group attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0138] The term "heteroalkyl" by itself or in combination with another term means a alkyl radical consisting of the specified number of carbon atoms (e.g., C2-C 10 or C2-C8) and at least one stable straight or branched hydrocarbon radical consisting of a heteroatom selected from, for example, N, O, S, Si, B, and P (in one aspect, N, O, S), wherein the nitrogen, sulfur, and phosphorus atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The heteroatom is located at any interior 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. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0139] Similarly, the term "heteroalkylene" by itself or as part of another substituent refers to a divalent radical 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 (carbon and heteroatoms, excluding hydrogen) (3-24 membered heteroalkyl). In another example, a heteroalkyl group has a total of 3 to 10 atoms (3-10 membered heteroalkyl) or 3 to 8 atoms (3-8 membered heteroalkyl). Where appropriate, such as when the formula indicates that the heteroalkyl group is divalent or when substituents are linked to form a ring, the term "heteroalkyl" includes "heteroalkylene".

[0140] As used herein, the term "C 1-8 "Alkyl" refers to a straight or branched chain saturated hydrocarbon having 1 to 8 (e.g., 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) carbon atoms. Representative "C 1-8 “Alkyl” groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and 2-methylbutyl.

[0141] The term "C 1-10 "Alkylene" refers to a group of the formula -(CH2) 1-10 -saturated straight chain hydrocarbon group. 1-10 Examples of the alkylene group include a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, a heptylene group, an octylene group, a nonylene group, and a decylene group.

[0142] The term "cycloalkyl" by itself or in combination with other terms means a saturated or unsaturated non-aromatic carbocyclic group having 3 to 24 carbon atoms, for example, a C3-C8 cycloalkyl or C3-C6 cycloalkyl group having 3 to 12 carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl and cyclooctadienyl. The term "cycloalkyl" also includes bridged polycyclic (e.g., bicyclic) structures such as norbornyl, adamantyl and bicyclo[2.2.1]heptyl. A "cycloalkyl" group can be fused to at least one (e.g., 1 to 3) ring 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, then the "cycloalkyl" group is attached to the remainder of the molecule through the carbocyclic ring.

[0143] The terms "heterocycloalkyl," "heterocyclic," "heterocycle," or "heterocyclyl," by themselves or in combination with other terms, mean a carbocyclic, non-aromatic ring (e.g., a 3- to 8-membered ring and, for example, a 4-, 5-, 6-, or 7-membered ring) containing at least one and up to 5 heteroatoms selected from, for example, N, O, S, Si, B, and P (in one aspect, N, O, and S), wherein the nitrogen, sulfur, and phosphorus atoms are optionally oxidized, and the nitrogen atom is optionally quaternized (e.g., 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur), or a fused ring system of a 4- to 8-membered ring containing at least one and up to 10 heteroatoms (e.g., 1 to 5 heteroatoms selected from N, O, and S) in stable combinations known to those skilled in the art. Exemplary heterocycloalkyl groups include fused benzene rings. When the "heterocycle" group includes a fused aryl, heteroaryl, or cycloalkyl ring, then the "heterocycle" group is attached to the remainder of the molecule via the heterocycle. A heteroatom may occupy the position at which the heterocycle is attached to the remainder of the molecule.

[0144] Exemplary heterocycloalkyl or heterocyclic groups of the present disclosure include morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S,S-dioxide, piperazinyl, homopiperazinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, tetrahydropyranyl, piperidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, homopiperidinyl, homomorpholinyl, homothiomorpholinyl, homothiomorpholinyl S,S-dioxide, oxazolidinone, dihydropyrazolyl, dihydropyrrolyl, dihydropyrazolyl , dihydropyridinyl, dihydropyrimidinyl, dihydrofuranyl, dihydropyranyl, tetrahydrothiophene S-oxide, tetrahydrothiophene S,S-dioxide, high-thiomorpholinyl S-oxide, 1-(1,2,5,6-tetrahydropyridinyl), 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.

[0145] "Aryl" means a 5, 6 or 7-membered aromatic carbocyclic group having a monocycle (e.g., phenyl) or fused to other aromatic or non-aromatic rings (e.g., 1 to 3 other rings). When an "aryl" group includes a non-aromatic ring (such as 1,2,3,4-tetrahydronaphthyl) or a heteroaryl group, the "aryl" group is bonded to the rest of the molecule via an aryl ring (e.g., a phenyl ring). The aryl group is optionally substituted (e.g., with 1 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, anthracenyl, quinoline, indanyl, indenyl, dihydronaphthyl, fluorenyl, tetrahydronaphthyl, benzo [d] [1,3] dioxolyl or 6,7,8,9-tetrahydro-5H-benzo [a] cycloheptenyl. In one aspect, the aryl group is selected from phenyl, benzo[d][1,3]dioxolyl, and naphthyl. In yet another aspect, the aryl group is phenyl.

[0146] The term "arylene" refers to an aryl group having two open valences and which can be in the ortho, meta, or para configuration, as shown in the following structure:

[0147]

[0148] The arylene group may be unsubstituted or substituted with up to four (e.g., 1, 2, 3, or 4) groups including, but not limited to, C 1-8 Alkyl, -O-(C 1-8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-, -NHC(O)R', -S(O)2R', -S(O)R', -OH, -halo, -N3, -NH2, -NH(R'), -N(R')2-NO2, and -CN, wherein each R' is independently H, -C 1-8 Alkyl or aryl.

[0149] The term "arylalkyl" or "aralkyl" is intended to include those groups in which an aryl group or a heteroaryl group is attached to an alkyl group to form the groups -alkyl-aryl and -alkyl-heteroaryl, wherein alkyl, aryl, and heteroaryl are defined herein. Exemplary "arylalkyl" or "aralkyl" groups include benzyl, phenethyl, pyridylmethyl, and the like.

[0150] "Aryloxy" means the group -O-aryl, wherein aryl is as defined herein. In one example, the aryl portion of the aryloxy group is phenyl or naphthyl. In one aspect, the aryl portion of the aryloxy group is phenyl.

[0151] The term "heteroaryl" or "heteroaromatic" refers to a polyunsaturated 5-, 6-, or 7-membered aromatic moiety containing at least one heteroatom (e.g., 1 to 5 heteroatoms, such as 1 to 3 heteroatoms) selected from N, O, S, Si, and B (in one aspect, N, O, and S), wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. A "heteroaryl" group can be a monocyclic ring or fused to other aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings (e.g., 1 to 3 other rings). When a "heteroaryl" group includes a fused aryl, cycloalkyl, or heterocycloalkyl ring, the "heteroaryl" group is attached to the rest of the molecule via the heteroaryl ring. A heteroaryl group can be attached to the rest of the molecule through a carbon or heteroatom.

[0152] In one example, the heteroaryl group has 4 to 10 carbon atoms and 1 to 5 heteroatoms selected from O, S, and N. Non-limiting examples of heteroaryl groups include pyridyl, pyrimidinyl, quinolinyl, benzothiophenyl, indolyl, indolinyl, pyridazinyl, pyrazinyl, isoindolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, imidazolyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, furanyl, thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, isothiazolyl, naphthyridinyl, isobenzodihydropyrrolyl, pyranyl, chromanyl, tetrahydroisoquinolyl, isoindolyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothiophenyl, isobenzothiophenyl, benzoxazolyl, pyridopyridinyl, benzotetrahydrofuranyl, benzotetrahydrothiophenyl, purinyl, benzodioxolyl, triazinyl, pteridinyl, benzothiazolyl, imidazopyridinyl, imidazothiazolyl, dihydrobenzisoxazinyl, benzoisoxazinyl, benzoxazinyl, dihydrobenzisothiazinyl, benzopyranyl, benzothiopyranyl, chromonyl , chromanyl, pyridyl-N-oxide, tetrahydroquinolinyl, dihydroquinolinyl, dihydroquinolinyl, dihydroisoquinolinyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindolyl, benzodioxanyl, benzoxazolinyl, pyrrolyl N-oxide, pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinyl N-oxide, quinolinyl N-oxide, indolyl N-oxide, indolinyl N-oxide, isoquinolinyl N-oxide, quinazolinyl N-oxide, quinoxalinyl In some embodiments, the present invention includes benzothiazolyl N-oxide, benzothiazolyl N-oxide, benzoimidazolyl N-oxide, pyrrolyl N-oxide, oxadiazolyl N-oxide, thiadiazolyl N-oxide, triazolyl N-oxide, tetrazolyl N-oxide, benzothiopyranyl S-oxide, benzothiopyranyl S, S-dioxide. Exemplary heteroaryl groups include imidazolyl, pyrazolyl, thiadiazolyl, triazolyl, isoxazolyl, isothiazolyl, imidazolyl, thiazolyl, oxadiazolyl and pyridyl. Other exemplary heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, pyridin-4-yl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[0153] Any organic residue described herein (e.g., alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, etc.) may be unsubstituted or substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) groups, as appropriate. Typical substituents include, but are not limited to, C 1-8 Alkyl, -O-(C 1-8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-, -NHC(O)R', -S(O)2R', -S(O)R', -OH, -halo, -N3, -NH2, -NH(R'), -N(R')2, and -CN, wherein each R' is independently H, -C 1-8 Alkyl or aryl.

[0154] Extracellular vesicles comprising the construct of formula (I)

[0155] The present disclosure provides an extracellular vesicle (EV) comprising a biologically active molecule (BAM) attached to the EV via an anchoring moiety (AM) according to Formula I:

[0156] AM-SP1-L1-SP2-BAM (Formula 1)

[0157] in

[0158] L1 is a cleavable bond comprising: a polynucleotide group; a peptidyl group selected from alanine-alanine-asparagine, valine-glycine, glycine-glycine, glutamic acid-valine-citrulline, aspartic acid-valine-citrulline, serine-valine-citrulline, lysine-valine-citrulline, glycine-glycine-glycine-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate acyloxy group; or a silyl ether; and

[0159] SP1 and SP2 are optional first and second spacers, respectively.

[0160] As used herein, the term "linker" refers to a combination of structural elements comprising, for example, a "bond" (both cleavable and non-cleavable) and a "spacer," which connects the anchoring moiety AM and the bioactive molecule BAM. In the absence of the cleavable bonds of the present disclosure, these linkers allow for more efficient and increased loading of bioactive molecules (e.g., ASOs) onto the surface of EVs (e.g., exosomes) compared to corresponding constructs comprising the same anchoring moiety (AM) and bioactive molecule (BAM) in the absence of the presently disclosed cleavable bonds. In other words, the constructs disclosed herein, such as those of formula IAM-SP1-L1-SP2-BAM, result in: (i) higher EV loading efficiency, (ii) higher numbers of BAMs per EV, (iii) higher BAM density per EV, (iv) higher BAM potency, or (v) any combination thereof.

[0161] As used herein, the term "bond" refers to any bond or chemical group that connects, for example, an anchoring moiety AM and a spacer SP, a spacer SP and a bioactive molecule BAM, or an anchoring moiety AM and a bioactive molecule BAM. In constructs in which there is more than one anchoring moiety AM or bioactive molecule BAM, a bond can connect two anchoring moieties or two bioactive moieties. In some aspects, a "bond" can be cleavable, non-cleavable, or both cleavable and non-cleavable. In some aspects, a bond can comprise multiple linkers and bonds that can respond to different stimuli, such as pH, temperature, enzymes, and the like.

[0162] As used herein, the term "spacer" refers to a chemical moiety that is capable of covalently linking two spacer moieties (e.g., a biologically active molecule and an anchoring moiety) together to form a generally stable two-part molecule. Generally, spacers are non-cleavable. For example, a spacer can be an alkyl chain or a polyalkoxy chain, as described herein.

[0163] In some aspects, the length of the linker connecting the anchoring moiety AM and the biologically active molecule BAM is between about 2 nm and about 30 nm. In some aspects, the length of the linker connecting the anchoring moiety AM and the biologically active molecule BAM is about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, about 25 nm, about 26 nm, about 27 nm, about 28 nm, about 29 nm, or about 30 nm. In some aspects, the length of the optimized linker is at least 2 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 6 nm, at least 7 nm, at least 8 nm, at least 9 nm, at least 10 nm, at least 11 nm, at least 12 nm, at least 13 nm, at least 14 nm, at least 15 nm, at least 16 nm, at least 17 nm, at least 18 nm, at least 19 nm, at least 20 nm, at least 21 nm, at least 22 nm, at least 23 nm, at least 24 nm, at least 25 nm, at least 26 nm, at least 27 nm, at least 28 nm, at least 29 nm, or at least 30 nm. In some aspects, the length of the optimized linker is less than about 2 nm, less than about 3 nm, less than about 4 nm, less than about 5 nm, less than about 6 nm, less than about 7 nm, less than about 8 nm, less than about 9 nm, less than about 10 nm, less than about 11 nm, less than about 12 nm, less than about 13 nm, less than about 14 nm, less than about 15 nm, less than about 16 nm, less than about 17 nm, less than about 18 nm, less than about 19 nm, less than about 20 nm, less than about 21 nm, less than about 22 nm, less than about 23 nm, less than about 24 nm, less than about 25 nm, less than about 26 nm, less than about 27 nm, less than about 28 nm, less than about 29 nm, or less than about 30 nm.

[0164] In some aspects, the length of the linker connecting the anchoring moiety AM and the biologically active molecule BAM is about 2 nm to about 4 nm, about 3 nm to about 5 nm, about 4 nm to about 6 nm, about 5 nm to about 7 nm, about 6 nm to about 8 nm, about 7 nm to about 9 nm, about 8 nm to about 10 nm, about 9 nm to about 11 nm, about 10 nm to about 12 nm, about 11 nm to about 13 nm, about 12 nm to about 14 nm, about 13 nm to about 15 nm, about 14 nm to about 16 nm, about 15 nm to about 17 nm, about 16 nm to about 18 nm, about 17 nm to about 19 nm, or about 20 nm to about 21 nm. m to about 19 nm, about 18 nm to about 20 nm, about 19 nm to about 21 nm, about 20 nm to about 22 nm, about 21 nm to about 23 nm, about 22 nm to about 24 nm, about 23 nm to about 25 nm, about 24 nm to about 26 nm, about 25 nm to about 27 nm, about 26 nm to about 28 nm, about 27 nm to about 29 nm, about 28 nm to about 30 nm, about 2 nm to about 6 nm, about 4 nm to about 8 nm, about 6 nm to about 10 nm, about 8 nm to about 12 nm, about 10 nm to about 14 nm, about 12 nm to about 16 nm, about 14 nm to about 18 nm, about 16 nm to about 20 nm, about 18 nm to about 22 nm, about 20 nm to about 24 nm, about 22 nm to about 26 nm, about 24 nm to about 28 nm, about 26 nm to about 30 nm, about 2 nm to about 10 nm, about 4 nm to about 12 nm, about 6 nm to about 14 nm, about 8 nm to about 16 nm, about 10 nm to about 18 nm, about 12 nm to about 20 nm, about 14 nm to about 22 nm, about 16 nm to about 24 nm, about 18 nm to about 26 nm, about 20 nm to about 2 8 nm, about 22 nm to about 30 nm, about 2 nm to about 12 nm, about 4 nm to about 14 nm, about 6 nm to about 16 nm, about 8 nm to about 18 nm, about 10 nm to about 20 nm, about 12 nm to about 22 nm, about 14 nm to about 24 nm, about 16 nm to about 26 nm, about 18 nm to about 28 nm, about 20 nm to about 30 nm, about 2 nm to about 5 nm, about 5 nm to about 10 nm, about 10 nm to about 15 nm, about 15 nm to about 20 nm, about 20 nm to about 25 nm, or about 25 nm to about 30 nm.

[0165] Extracellular vesicles

[0166] Extracellular vesicles (EVs) typically have a diameter of 20 nm to 1000 nm. Exosomes are small extracellular vesicles with a diameter typically of about 50 to 200 nm (e.g., 100 to 200 nm). EVs, such as exosomes, consist of a restricted lipid bilayer and a variety of protein and nucleic acid groups (Maas, SLN, et al., Trends. Cell Biol. 27 (3): 172-188 (2017)). EVs, such as exosomes, exhibit preferential uptake in discrete cell types and tissues, and can be directed to their tropism by adding proteins to their surface that interact with receptors on the surface of target cells (Alvarez-Erviti, L., et al., Nat. Biotechnol. 29 (4): 341-345 (2011)).

[0167] The EVs (e.g., exosomes) of the present disclosure may have a diameter between about 20 and about 300 nm. In certain aspects, the EVs (e.g., exosomes) of the present disclosure have a diameter of about 20 to about 290 nm, about 20 to about 280 nm, about 20 to about 270 nm, about 20 to about 260 nm, about 20 to about 250 nm, about 20 to about 240 nm, about 20 to about 230 nm, about 20 to about 220 nm, about 20 to about 210 nm, about 20 to about 200 nm, about 20 to about 190 nm, about 20 to about 180 nm, about 20 to about The size of EVs (e.g., exosomes) described herein can be measured according to methods known in the art.

[0168] Unlike antibodies, EVs (e.g., exosomes) can accommodate a large number of molecules attached to their surface, on the order of thousands to tens of thousands of molecules per EV (e.g., exosome). Therefore, EV (e.g., exosome)-drug conjugates represent a platform for delivering high concentrations of therapeutic compounds to discrete cell types while limiting overall systemic exposure to the compound, thereby reducing off-target toxicity. The accommodation of a large number of molecules on the surface of EVs (e.g., exosomes) may be affected by, for example, the type of bioactive molecule used (e.g., antibodies are bulkier than antisense oligonucleotides), the type of membrane anchor used (e.g., protein anchors are bulkier than lipid or lipid plus spacer anchors), and the combination of linkers and spacers connecting the bioactive molecule and the membrane anchor. In this regard, the present disclosure provides specific combinations of linkers and spacers connecting bioactive molecules (e.g., ASOs) and membrane anchors (e.g., lipids), wherein the membrane anchor attaches the bioactive molecule to the surface of the EV (e.g., exosome).

[0169] In some aspects, the present disclosure provides "biologically active molecules" (BAMs), such as ASOs, that are directly or indirectly attached (e.g., covalently bonded), for example, via one or more linker combinations, to one or more anchoring moieties (AMs). The anchoring moieties can be inserted into the lipid bilayer of EVs (e.g., exosomes), allowing the exosomes to be loaded with BAMs (e.g., ASOs). Currently, the main obstacle to the commercialization of exosomes as delivery vehicles for polar BAMs (e.g., ASOs) is the very inefficient loading. As shown herein, this obstacle can be overcome by connecting the BAM to the AM using a specific linker / spacer combination (i.e., "linker") before loading the BAM into the EV (e.g., exosome). Thus, as described herein, the use of optimized linkers facilitates the loading of BAMs (e.g., ASOs) onto EVs (e.g., exosomes).

[0170] The compositions and methods of loading EVs (e.g., exosomes) with constructs comprising BAMs (e.g., ASOs) linked to AMs (e.g., lipids such as sterols) via optimized linkers described herein improve loading efficiency and BAM density compared to previously reported loading efficiencies and BAM densities of unmodified BAMs introduced into EVs (e.g., exosomes) by, for example, electroporation or cationic lipid transfection. The compositions and methods disclosed herein also significantly improve the efficacy of EVs (e.g., exosomes) by, for example, electroporation or cationic lipid transfection compared to previously reported efficiencies when unmodified BAMs are introduced into EVs (e.g., exosomes).

[0171] The EVs (e.g., exosomes) disclosed herein comprise a bilipid membrane ("exosome membrane" or "EV membrane") comprising an inner surface (luminal surface) and an outer surface. The inner surface faces the inner core of the EV (e.g., exosome), i.e., the lumen of the EV. The EV or exosome membrane comprises lipids and fatty acids. Exemplary lipids include phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterol, and phosphatidylserine. The EV or exosome membrane comprises an inner leaflet and an outer leaflet. The composition of the inner leaflet and the outer leaflet can be determined by trans-bilayer distribution assays known in the art, see, for example, Kuypers et al., Biohim Biophys Acta 1985 819:170.

[0172] In some aspects, the outer leaflet is composed of between about 70% and about 90% choline phospholipids, between about 0% and about 15% acidic phospholipids, and between about 5% and about 30% phosphatidylethanolamine. In some aspects, the inner leaflet is composed of between about 15% and about 40% choline phospholipids, between about 10% and about 50% acidic phospholipids, and between about 30% and about 60% phosphatidylethanolamine. In some aspects, the EV or exosome membrane comprises one or more polysaccharides, such as glycans. Glycans on the surface of EVs or exosomes can serve as attachment sites for maleimide moieties or linkers connecting glycans and maleimide moieties. Glycans can be present on one or more proteins on the surface of EVs (e.g., exosomes), such as Scaffold X, such as the PTGFRN polypeptide, or on the lipid membrane of EVs (e.g., exosomes). Glycans can be modified to have sulfofucose, which can serve as functional groups for attaching maleimide moieties to glycans. In some aspects, Scaffold X can be modified to express a large number of glycans to allow additional attachment to EVs (e.g., exosomes).

[0173] Anchoring part

[0174] In some aspects, the anchoring moiety AM of Formula I comprises a sterol, a lipid (e.g., a phospholipid), a vitamin, a peptide, or a combination thereof, and an optional spacer. In general, AM may comprise any hydrophobic moiety or combination thereof that can be inserted into the lipid bilayer of an EV (e.g., an exosome), electrostatically interact with the surface of an EV (e.g., an exosome), or a combination thereof. Suitable anchoring moieties AM capable of anchoring the bioactive molecule BAM to the surface of an EV (e.g., an exosome) include, for example, sterols (e.g., cholesterol), lipids, phospholipids, lysophospholipids, fatty acids, vitamins (e.g., fat-soluble vitamins), scaffold moieties (e.g., protein X), or a combination thereof as described herein.

[0175] In some aspects, the anchoring moiety AM comprises a sterol, a steroid, a hopane steroid, a hydroxysteroid, a secosteroid or its analogue having lipophilic properties. In some aspects, the anchoring moiety comprises a sterol, such as a plant sterol, a fungus sterol or a zoosterol. Exemplary zoosterols include cholesterol and 24S-hydroxycholesterol; exemplary plant sterols include ergosterol (fungosterol), campesterol, sitosterol and stigmasterol. In some aspects, the sterol is selected from ergosterol, 7-dehydrocholesterol, cholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fuccasterol, sargassum sterol, campesterol, β-sitosterol, sitostanol, coprostanol, avenasterol, stigmasterol or a combination thereof. Sterols may be present as free sterols, acylated (sterol esters), alkylated (alkyl sterol ethers), sulfated (sterol sulfates), or attached to a glycosidic moiety (sterol glycosides), which may itself be acylated (acylated sterol glycosides).

[0176] The sterol can be attached (via solid phase synthesis or conjugation) to the spacer SP, for example, via an available —OH group of the sterol. Exemplary sterols have the general skeleton shown below:

[0177]

[0178] For example, ergosterol has the following structure:

[0179]

[0180] In another example, cholesterol has the following structure:

[0181]

[0182] In some aspects, the anchoring moiety AM comprises, consists of, or consists essentially of a sterol, cholesterol, mercaptocholesterol, ergosterol, 7-dehydrocholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fuccasterol, sargassum sterol, campesterol, β-sitosterol, sitostanol, coprostanol, avenasterol, stigmasterol, or a combination thereof. In some specific aspects, the anchoring moiety AM comprises cholesterol.

[0183] In some aspects, the anchoring moiety AM comprises a steroid. In some aspects, the steroid is selected from dihydrotestosterone, linalool, safflower oil, diosgenin, progesterone, or cortisol.

[0184] In some aspects, the anchoring moiety AM comprises or is composed of a lipid. The lipid anchoring moiety AM may comprise any lipid known in the art, such as palmitic acid or glycosylphosphatidylinositol. In some aspects, AM comprises a lipid, and the lipid comprises a fatty acid or a phospholipid. In some aspects, the lipid is a fatty acid, a phospholipid (phosphatide), a phospholipid (phospholipid) (such as phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine) or its analog (such as phosphatidylcholine, lecithin, phosphatidylethanolamine, cephalin or phosphatidylserine or its analog or part, such as a part of its partial hydrolysis).

[0185] In some aspects, the anchoring moiety AM comprises, consists of, or consists essentially of a fatty acid, such as a straight-chain fatty acid. In some aspects, the fatty acid is a straight-chain fatty acid, a branched-chain fatty acid, a saturated fatty acid, an unsaturated fatty acid, a hydroxy fatty acid, a polycarboxylic acid, or any combination thereof. In some aspects, the fatty acid is a short-chain, medium-chain, or long-chain fatty acid. In some aspects, the fatty acid is a saturated fatty acid. In some aspects, the fatty acid is an unsaturated fatty acid. In some aspects, the fatty acid is a monounsaturated fatty acid. In some aspects, the fatty acid is a polyunsaturated fatty acid, such as an ω-3 (omega-3) or ω-6 (omega-6) fatty acid. In some aspects, the anchoring moiety AM comprises, consists of, or consists essentially of a straight-chain fatty acid, a branched-chain fatty acid, an unsaturated fatty acid, a monounsaturated fatty acid, a polyunsaturated fatty acid, a hydroxy fatty acid, a polycarboxylic acid, or any combination thereof.

[0186] In some aspects, lipids, such as fatty acids, have C2-C 18 In some aspects, lipids, such as fatty acids, have C2, C3, C4, C5, C6, C7, C8, C9, C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 or C 18 In some aspects, the fatty acids have C2 chains. In some aspects, the fatty acids have C3 chains. In some aspects, the fatty acids have C4 chains. In some aspects, the fatty acids have C5 chains. In some aspects, the fatty acids have C6 chains. In some aspects, the fatty acids have C7 chains. In some aspects, the fatty acids have C8 chains. In some aspects, the fatty acids have C9 chains. In some aspects, the fatty acids have C 10 In some aspects, the fatty acid has C 11 In some aspects, the fatty acid has C 12 In some aspects, the fatty acid has C 13In some aspects, the fatty acid has C 14 In some aspects, the fatty acid has C 15 In some aspects, the fatty acid has C 16 In some aspects, the fatty acid has C 17 In some aspects, the fatty acid has C 18 chain.

[0187] In some aspects, the fatty acid has a C4-C 18 In some aspects, the fatty acids have C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C2-C9, C2-C 10 、C2-C 11 、C2-C 12 、C2-C 13 、C2-C 14 、C2-C 15 、C2-C 16 、C2-C 17 、C2-C 18 ,C3-C4,C3-C5,C3-C6,C3-C7,C3-C8,C3-C9,C3-C 10 、C3-C 11 、C3-C 12 、C3-C 13 、C3-C 14 、C3-C 15 、C3-C 16 、C3-C 17 、C3-C 18 ,C4-C5,C4-C6,C4-C7,C4-C8,C4-C9,C4-C 10 、C4-C 11 、C4-C 12 、C4-C 13 、C4-C 14 、C4-C 15 、C4-C 16 、C4-C 17 、C4-C 18 ,C5-C6,C5-C7,C5-C8,C5-C9,C5-C 10 、C5-C 11 、C5-C 12 、C5-C 13 、C5-C 14 、C5-C 15 、C5-C 16 、C5-C 17 、C5-C 18、C6-C7、C6-C8、C6-C9、C6-C 10 、C6-C 11 、C6-C 12 、C6-C 13 、C6-C 14 、C6-C 15 、C6-C 16 、C6-C 17 、C6-C 18 、C7-C8、C7-C9、C7-C 10 、C7-C 11 、C7-C 12 、C7-C 13 、C7-C 14 、C7-C 15 、C7-C 16 、C7-C 17 、C7-C 18 、C8-C9、C8-C 10 、C8-C 11 、C8-C 12 、C8-C 13 、C8-C 14 、C8-C 15 、C8-C 16 、C8-C 17 、C8-C 18 、C9-C 10 、C9-C 11 、C9-C 12 、C9-C 13 、C9-C 14 、C9-C 15 、C9-C 16 、C9-C 17 、C9-C 18 、C 10 -C 11 、C 10 -C 12 、C 10 -C 13 、C 10 -C 14 、C 10 -C 15 、C 10 -C 16 、C 10 -C 17 、C 10 -C 18 、C 11 -C 12 、C 11 -C 13 、C 11 -C14 、C 11 -C 15 、C 11 -C 16 、C 11 -C 17 、C 11 -C 18 、C 12 -C 13 、C 12 -C 14 、C 12 -C 15 、C 12 -C 16 、C 12 -C 17 、C 12 -C 18 、C 13 -C 14 、C 13 -C 15 、C 13 -C 16 、C 13 -C 17 、C 13 -C 18 、C 14 -C 15 、C 14 -C 16 、C 14 -C 17 、C 14 -C 18 、C 15 -C 16 、C 15 -C 17 、C 15 -C 18 、C 16 -C 17 、C 16 -C 18 or C 17 -C 18 chain.

[0188] In some aspects, the anchoring moiety AM comprises two fatty acids, each of which is independently selected from fatty acids having a chain with any of the aforementioned ranges or numbers of carbon atoms. In some aspects, one of the fatty acids is independently C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C2-C9, C2-C 10 、C2-C 11 、C2-C 12 、C2-C 13 、C2-C 14、C2-C 15 、C2-C 16 、C2-C 17 、C2-C 18 、C3-C4、C3-C5、C3-C6、C3-C7、C3-C8、C3-C9、C3-C 10 、C3-C 11 、C3-C 12 、C3-C 13 、C3-C 14 、C3-C 15 、C3-C 16 、C3-C 17 、C3-C 18 、C4-C5、C4-C6、C4-C7、C4-C8、C4-C9、C4-C 10 、C4-C 11 、C4-C 12 、C4-C 13 、C4-C 14 、C4-C 15 、C4-C 16 、C4-C 17 、C4-C 18 、C5-C6、C5-C7、C5-C8、C5-C9、C5-C 10 、C5-C 11 、C5-C 12 、C5-C 13 、C5-C 14 、C5-C 15 、C5-C 16 、C5-C 17 、C5-C 18 、C6-C7、C6-C8、C6-C9、C6-C 10 、C6-C 11 、C6-C 12 、C6-C 13 、C6-C 14 、C6-C 15 、C6-C 16 、C6-C 17 、C6-C 18 、C7-C8、C7-C9、C7-C 10 、C7-C 11 、C7-C 12 、C7-C 13 、C7-C 14 、C7-C 15 、C7-C 16 、C7-C 17 、C7-C 18、C8-C9、C8-C 10 、C8-C 11 、C8-C 12 、C8-C 13 、C8-C 14 、C8-C 15 、C8-C 16 、C8-C 17 、C8-C 18 、C9-C 10 、C9-C 11 、C9-C 12 、C9-C 13 、C9-C 14 、C9-C 15 、C9-C 16 、C9-C 17 、C9-C 18 、C 10 -C 11 、C 10 -C 12 、C 10 -C 13 、C 10 -C 14 、C 10 -C 15 、C 10 -C 16 、C 10 -C 17 、C 10 -C 18 、C 11 -C 12 、C 11 -C 13 、C 11 -C 14 、C 11 -C 15 、C 11 -C 16 、C 11 -C 17 、C 11 -C 18 、C 12 -C 13 、C 12 -C 14 、C 12 -C 15 、C 12 -C 16 、C 12 -C 17 、C 12 -C 18 、C 13 -C 14 、C13 -C 15 、C 13 -C 16 、C 13 -C 17 、C 13 -C 18 、C 14 -C 15 、C 14 -C 16 、C 14 -C 17 、C 14 -C 18 、C 15 -C 16 、C 15 -C 17 、C 15 -C 18 、C 16 -C 17 、C 16 -C 18 or C 17 -C 18 chain fatty acids and the other independently is a fatty acid having C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C2-C9, C2-C 10 、C2-C 11 、C2-C 12 、C2-C 13 、C2-C 14 、C2-C 15 、C2-C 16 、C2-C 17 、C2-C 18 ,C3-C4,C3-C5,C3-C6,C3-C7,C3-C8,C3-C9,C3-C 10 、C3-C 11 、C3-C 12 、C3-C 13 、C3-C 14 、C3-C 15 、C3-C 16 、C3-C 17 、C3-C 18 ,C4-C5,C4-C6,C4-C7,C4-C8,C4-C9,C4-C 10 、C4-C 11 、C4-C 12 、C4-C 13 、C4-C 14 、C4-C 15 、C4-C 16 、C4-C17 、C4-C 18 、C5-C6、C5-C7、C5-C8、C5-C9、C5-C 10 、C5-C 11 、C5-C 12 、C5-C 13 、C5-C 14 、C5-C 15 、C5-C 16 、C5-C 17 、C5-C 18 、C6-C7、C6-C8、C6-C9、C6-C 10 、C6-C 11 、C6-C 12 、C6-C 13 、C6-C 14 、C6-C 15 、C6-C 16 、C6-C 17 、C6-C 18 、C7-C8、C7-C9、C7-C 10 、C7-C 11 、C7-C 12 、C7-C 13 、C7-C 14 、C7-C 15 、C7-C 16 、C7-C 17 、C7-C 18 、C8-C9、C8-C 10 、C8-C 11 、C8-C 12 、C8-C 13 、C8-C 14 、C8-C 15 、C8-C 16 、C8-C 17 、C8-C 18 、C9-C 10 、C9-C 11 、C9-C 12 、C9-C 13 、C9-C 14 、C9-C 15 、C9-C 16 、C9-C 17 、C9-C 18 、C 10 -C 11 、C 10 -C 12 、C 10 -C 13 、C10 -C 14 、C 10 -C 15 、C 10 -C 16 、C 10 -C 17 、C 10 -C 18 、C 11 -C 12 、C 11 -C 13 、C 11 -C 14 、C 11 -C 15 、C 11 -C 16 、C 11 -C 17 、C 11 -C 18 、C 12 -C 13 、C 12 -C 14 、C 12 -C 15 、C 12 -C 16 、C 12 -C 17 、C 12 -C 18 、C 13 -C 14 、C 13 -C 15 、C 13 -C 16 、C 13 -C 17 、C 13 -C 18 、C 14 -C 15 、C 14 -C 16 、C 14 -C 17 、C 14 -C 18 、C 15 -C 16 、C 15 -C 17 、C 15 -C 18 、C 16 -C 17 、C 16 -C 18 or C 17 -C 18In some aspects, each fatty acid independently has a chain of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms.

[0189] Examples of useful saturated straight-chain fatty acids include those with an even number of carbon atoms, such as butyric acid (C4), hexanoic acid (C6), caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), or stearic acid (C18), and those with an odd number of carbon atoms, such as propionic acid (C3), n-pentanoic acid (C5), heptanoic acid (C7), nonanoic acid (C9), undecanoic acid (C11), tridecanoic acid (C13), pentadecanoic acid (C15), or heptadecanoic acid (C17).

[0190] Examples of suitable saturated branched-chain fatty acids include isobutyric acid, isohexanoic acid, isooctanoic acid, isodecanoic acid, isolauric acid, 11-methyldodecanoic acid, isomyristic acid, 13-methyl-tetradecanoic acid, isopalmitic acid, 15-methyl-hexadecanoic acid, or isostearic acid. Suitable saturated odd-carbon branched-chain fatty acids include anteiso fatty acids terminated with an isobutyl group, such as 6-methyl-octanoic acid, 8-methyl-decanoic acid, 10-methyl-dodecanoic acid, 12-methyl-tetradecanoic acid, or 14-methyl-hexadecanoic acid.

[0191] Examples of suitable unsaturated fatty acids include 4-decenoic acid, 9-decenoic acid (caproleic acid), 4-dodecenoic acid, 5-dodecenoic acid, lauroleic acid, 4-tetradecenoic acid, 5-tetradecenoic acid, 9-tetradecenoic acid, palmitoleic acid, 6-octadecenoic acid, oleic acid, and the like.

[0192] 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, 9-hydroxy-trans-10,12-octadecadienoic acid, 9,10-dihydroxystearic acid, 12-hydroxystearic acid, and the like.

[0193] Examples of suitable polycarboxylic acids include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, D,L-malic acid, and the like.

[0194] In some aspects, 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, heptadecanoic acid, or stearic acid.

[0195] In some aspects, each fatty acid is independently selected from alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, gamma-linoleic acid, dihomo-gamma-linoleic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, paullinic acid, oleic acid, elaidic acid, bosseopentaenoic acid, sardinic acid, or another monounsaturated or polyunsaturated fatty acid.

[0196] In some aspects, one or two of the fatty acids are essential fatty acids. Given the beneficial health effects of certain essential fatty acids, the therapeutic benefits of the disclosed therapeutic agent-loaded exosomes can be increased by including such fatty acids in the therapeutic agent. In some aspects, the essential fatty acid is an n-6 or n-3 essential fatty acid selected from the group consisting of linolenic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, adrenic acid, docosapentaenoic acid n-6, alpha-linolenic acid, or stearic acid.

[0197] Fatty acid chains vary widely in their chain lengths and can be classified according to chain length, for example, from short to very long. Short-chain fatty acids (SCFA) are fatty acids (such as butyric acid) with a chain of about five or less carbons. In some respects, fatty acid is a SCFA. Medium-chain fatty acids (MCFA) include fatty acids with a chain of about 6 to 12 carbons, which can form medium-chain triglycerides. In some respects, fatty acid is a MCFA. Long-chain fatty acids (LCFA) include fatty acids with a chain of 13 to 21 carbons. In some respects, fatty acid is a LCFA. In some respects, fatty acid is a LCFA.

[0198] In some aspects, the anchoring moiety AM is formed by a straight-chain fat comprising, consisting of, or consisting essentially of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, or a combination thereof. In some specific aspects, the anchoring moiety AM is formed by palmitic acid.

[0199] In some aspects, the anchoring moiety AM comprises, consists of, or consists essentially of a phospholipid. The structure of a phospholipid molecule generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. For example, a phospholipid can be a lipid according to the formula:

[0200]

[0201] Wherein Rp is the phospholipid moiety, and R 1 and R 2The fatty acid moieties may be the same or different and each may be a fatty acid moiety with or without unsaturation. The fatty acid moiety may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid or linoleic acid.

[0202] The phospholipid moiety can be, for example, phosphatidylcholine, phosphatidylcholine (e.g., 2-lysophosphatidylcholine), phosphoinositol, phospho-sphingolipid, phosphoethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and sphingomyelin, or any combination thereof.

[0203] The phospholipids used as anchoring moieties AM in the present disclosure can be natural or non-natural phospholipids. Non-natural phospholipid substances are also encompassed, including natural substances with modifications and substitutions including branching, oxidation, cyclization, and alkynes. For example, phospholipids can be functionalized or cross-linked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, alkynyl groups can undergo copper-catalyzed cycloadditions when exposed to azides.

[0204] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Examples of phospholipids that can be used in the anchoring moieties disclosed herein include phosphatidylethanolamines (e.g., dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-oleoyl-2-palmitoylphosphatidylethanolamine, and dieucoylphosphatidylethanolamine), phosphatidylglycerols (e.g., dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, 1-palmitoyl-2-oleoyl-phosphatidylglycerol, 1-oleoyl-2-palmitoyl-phosphatidylglycerol, and dieucoylphosphatidylglycerol); phosphatidylserines (e.g., dilauroylphosphatidylserine, dimyristoylphosphatidylserine, Dipalmitoylphosphatidylserine, distearoylphosphatidylserine, dioleoylphosphatidylserine, 1-palmitoyl-2-oleoyl-phosphatidylserine, 1-oleoyl-2-palmitoyl-phosphatidylserine, and dieruoylphosphatidylserine); phosphatidic acids (e.g., dilauroylphosphatidic acid, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid, dioleoylphosphatidic acid, 1-palmitoyl-2-oleoyl-phosphatidylserine, and dieruoylphosphatidylserine); -oleoylphosphatidic acid, 1-oleoyl-2-palmitoyl-phosphatidic acid, and dieruoylphosphatidic acid); and phosphatidylinositols (e.g., dilauroylphosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol, distearoylphosphatidylinositol, dioleoylphosphatidylinositol, 1-palmitoyl-2-oleoyl-phosphatidylinositol, 1-oleoyl-2-palmitoyl-phosphatidylinositol, and dieruoylphosphatidylinositol).

[0205] Phospholipids can be of symmetrical or asymmetrical type. As used herein, the term "symmetrical phospholipids" includes glycerophospholipids with matching fatty acid moieties and sphingolipids in which the hydrocarbon chains of the variable fatty acid moieties and the sphingosine backbone include a considerable number of carbon atoms. As used herein, the term "asymmetrical phospholipids" includes lysolipids; glycerophospholipids with different fatty acid moieties (e.g., fatty acid moieties with different carbon atom numbers and / or degrees of unsaturation (e.g., double bonds); and sphingolipids in which the hydrocarbon chains of the variable fatty acid moieties and the sphingosine backbone include dissimilar carbon atom numbers (e.g., the variable fatty acid moieties include at least two more carbon atoms than the hydrocarbon chain or at least two less carbon atoms than the hydrocarbon chain).

[0206] In some aspects, the anchoring moiety AM comprises a phospholipid, such as a symmetrical phospholipid having 10 carbons (C10), 12 carbons (C12), 14 carbons (C14), 16 carbons (C16), or 18 carbons (C18). In some aspects, the anchoring moiety AM comprises a symmetrical phospholipid having 14 carbons (C14). In some aspects, the anchoring moiety AM comprises a symmetrical phospholipid having 16 carbons (C16). In some aspects, the anchoring moiety AM comprises a symmetrical phospholipid having 18 carbons (C18). In some aspects, the phospholipid is phosphatidylethanolamine (PE). Thus, in some aspects, the anchoring moiety AM comprises C14 PE. In some aspects, the anchoring moiety AM comprises C16 PE. In some aspects, the anchoring moiety AM comprises C18 PE. In some aspects, the acyl chain of PE does not contain unsaturation. Thus, in some aspects, the anchoring moiety AM comprises C14:0PE, C16:0PE, or C18:0PE. In some aspects, the anchoring moiety AM comprises 16:01,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (16:0PDPPE).

[0207] In some aspects, the anchoring moiety AM comprises a phospholipid containing a cyanuric acid (cyanuric acid (cyanur)) group. In some aspects, the phospholipid comprising a cyanuric acid group is PE. In some aspects, the phospholipid comprising a cyanuric acid group is C14:0PE, C16:0PE, or C18:0PE. In some aspects, the anchoring moiety AM comprises 16:01,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (16:0PE MCC). In some aspects, the anchoring moiety AM comprises 16:01,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cyanuric acid) (16:0 cyanuric acid PE).

[0208] In some aspects, the anchoring moiety AM comprises at least one symmetrical phospholipid. The symmetrical phospholipid can be selected from the non-limiting group consisting of: 1,2-dipropionyl sn-glycero3-phosphocholine (03:0PC), 1,2-dibutyryl sn-glycero3-phosphocholine (04:0PC), 1,2-divaleranoyl sn-glycero3-phosphocholine (05:0PC), 1,2-dihexanoyl sn-glycero3-phosphocholine (06:0PC), 1,2-diheptanoyl sn-glycero3-phosphocholine (07:0PC), 1,2-dioctanoyl sn-glycero3-phosphocholine (08:0PC), 1,2-dinonanoyl sn-glycero3-phosphocholine (09:0PC), 1,2-didecanoyl sn-glycero3-phosphocholine (010:0PC), 1,2-dioctanoyl sn-glycero3-phosphocholine (011:0PC), 1,2-didecanoyl sn-glycero3-phosphocholine (012:0PC), 1,2-dioctanoyl sn-glycero3-phosphocholine (013:0PC), 1,2-didecanoyl sn-glycero3-phosphocholine (014:0PC), 1,2-dioctanoyl sn-glycero3-phosphocholine (015:0PC), 1,2-dioctanoyl sn-glycero3-phosphocholine (016:0PC), 1,2-didecanoyl sn-glycero3-phosphocholine (017:0PC), 1,2-dioctanoyl sn-glycero3-phosphocholine (018:0PC), 1,2-dioctanoyl sn-glycero3-phos 3-phosphocholine (10:0PC), 1,2-dicondecanoyl sn-glycerol 3-phosphocholine (11:0PC, DUPC), 1,2-dilauroyl sn-glycerol 3-phosphocholine (12:0PC), 1,2-tridecanoyl sn-glycerol 3-phosphocholine (13:0PC), 1,2-dimyristoyl sn-glycerol 3-phosphocholine (14:0PC, DMPC), 1,2-dipentadecanoyl sn-glycerol 3-phosphocholine (15:0PC), 1,2-dipalmitoyl sn-glycerol 3-phosphocholine (16:0PC, DPPC), 1,2-diphytoyl sn-glycerol 3-phosphocholine (4ME 16:0PC), 1,2-dipeptadecanoyl sn-glycerol 3-phosphocholine (17:0PC), 1,2-distearoyl sn-glycerol 3-phosphocholine (18:0PC, DSPC), 1,2-dimyristoyl sn-glycerol 3-phosphocholine (14:1(Δ9-trans)PC), 1,2-dipalmitoleoyl sn-glycerol 3-phosphocholine (16:1(Δ9-cis)PC), 1,2-dipalmitoleoyl sn-glycerol 3-phosphocholine (16:1(Δ9-trans)PC), 1,2-dioctadecanoyl sn-glycerol 3-phosphocholine (18:1(Δ6-cis)PC), 1,2-dioleoyl sn-glycerol 3-phosphocholine (18:1(Δ9-cis)PC, DOPC), 1,2-dioleoyl sn-glycerol 3-phosphocholine (18:1(Δ9-trans)PC), 1,2- Linoleoyl sn glycerol 3 phosphocholine (18:2 (cis) PC, DLPC), 1,2-dilinoleoyl sn glycerol 3 phosphocholine (18:3 (cis) PC, DLnPC), 1,2-dihexanoyl sn glycerol 3 phosphoethanolamine (06:0PE), 1,2-dioctanoyl sn glycerol 3 phosphoethanolamine (08:0PE), 1,2-didecanoyl sn glycerol 3 phosphoethanolamine (10:0PE), 1,2-dilauroyl sn glycerol 3 phosphoethanolamine (12:0PE), 1,2-dimyristoyl sn glycerol 3 phosphoethanolamine (14:0PE), 1,2-dipentadecanoyl sn glycerol 3 phosphoethanolamine (15:0PE), 1,2-dipalmitoyl sn glycerol 3 phosphoethanolamine (16:0PE), 1,2-diphytoyl sn glycerol 3 phosphoethanolamine (4ME 16:0PE), 1,2-diheptadecanoyl sn-glycerol-3-phosphoethanolamine (17:0PE), 1,2-distearoyl sn-glycerol-3-phosphoethanolamine (18:0PE, DSPE), 1,2-dipalmitoleoyl sn-glycerol-3-phosphoethanolamine (16:1PE), 1,2-dioleoyl sn-glycerol-3-phosphoethanolamine (18:1(Δ9-cis)PE, DOPE), 1,2-dioleoyl sn-glycerol-3-phosphoethanolamine (18:1(Δ9-trans)PE), 1,2-dilinoleoyl sn-glycerol-3-phosphoethanolamine (18:2PE, DLPE), 1,2-dilinoleoyl sn-glycerol-3-phosphoethanolamine (18:3PE, DLnPE), 1,2-dioctadecenyl sn-glycerol-3-phosphocholine (18:0 diether PC), 1,2-dioleoyl sn-glycerol-3-phosphorac(1 glycerol) sodium salt (DOPG), and any combination thereof.

[0209] In some aspects, the anchoring moiety AM comprises at least one symmetric phospholipid selected from the non-limiting 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.

[0210] In some aspects, the anchoring moiety AM comprises at least one asymmetric phospholipid. The asymmetric phospholipid can be selected from the non-limiting group consisting of: 1 myristoyl 2 palmitoyl sn glycerol 3 phosphocholine (14:0-16:0PC, MPPC), 1 myristoyl 2 stearoyl sn glycerol 3 phosphocholine (14:0-18:0PC, MSPC), 1 palmitoyl 2 acetyl sn glycerol 3 phosphocholine (16:0-02:0PC), 1 palmitoyl 2 myristoyl sn glycerol 3 phosphocholine (16:0-14:0PC, PMPC), 1 palmitoyl 2 stearo ... sn-glycerol 3 phosphocholine (16:0-18:0PC, PSPC), 1 palmitoyl 2 oleoyl sn-glycerol 3 phosphocholine (16:0-18:1PC, POPC), 1 palmitoyl 2 linoleoyl sn-glycerol 3 phosphocholine (16:0-18:2PC, PLPC), 1 palmitoyl 2 arachidonoyl sn-glycerol 3 phosphocholine (16:0-20:4PC), 1 palmitoyl 2 docosahexaenoyl sn-glycerol 3 phosphocholine (14:0-22:6PC), 1 stearoyl 1 stearoyl 2 palmitoyl sn glycerol 3 phosphocholine (18:0-14:0PC, SMPC), 1 stearoyl 2 palmitoyl sn glycerol 3 phosphocholine (18:0-16:0PC, SPPC), 1 stearoyl 2 oleoyl sn glycerol 3 phosphocholine (18:0-18:1PC, SOPC), 1 stearoyl 2 linoleoyl sn glycerol 3 phosphocholine (18:0-18:2PC), 1 stearoyl 2 arachidonoyl sn glycerol 3 phosphocholine (18:0-20:4PC), 1 Stearoyl 2 docosahexaenoyl sn glycerol 3 phosphocholine (18:0-22:6PC), 1 oleoyl 2 myristoyl sn glycerol 3 phosphocholine (18:1-14:0PC, OMPC), 1 oleoyl 2 palmitoyl sn glycerol 3 phosphocholine (18:1-16:0PC, OPPC), 1 oleoyl 2 stearoyl sn glycerol 3 phosphocholine (18:1-18:0PC, OSPC), 1 palmitoyl 2 oleoyl sn glycerol 3 phosphoethanolamine (16:0-18:1PE,POPE), 1 palmitoyl 2 linoleoyl sn glycerol 3 phosphoethanolamine (16:0-18:2PE), 1 palmitoyl 2 arachidonoyl sn glycerol 3 phosphoethanolamine (16:0-20:4PE), 1 palmitoyl 2 docosahexaenoyl sn glycerol 3 phosphoethanolamine (16:0-22:6PE), 1 stearoyl 2 oleoyl sn glycerol 3 phosphoethanolamine (18:0-18:1PE), 1 stearoyl 2 linoleoyl sn glycerol 3 phosphoethanolamine (18:0-18:2PE), 1 stearoyl 2 arachidonoyl sn glycerol 3 phosphoethanolamine (18:0-20:4PE), 1 stearoyl 2 docosahexaenoyl sn glycerol 3 phosphoethanolamine (18:0-22:6PE), 1 oleoyl 2 cholesteryl hemisuccinyl sn glycerol 3 phosphocholine (OChemsPC), and any combination thereof.

[0211] In order to provide improved nuclease resistance, cellular uptake efficiency and more significant RNA interference effect, phosphatidylethanolamine (e.g., dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine and dioleoylphosphatidylethanolamine) can be used as the anchoring moiety AM.

[0212] In some aspects, the anchoring moiety AM comprises or consists of a lysolipid, such as a lysophospholipid. A lysolipid is a derivative of a lipid in which one or two fatty acyl chains have been removed, typically by hydrolysis. A lysophospholipid is a derivative of a phospholipid in which one or two fatty acyl chains have been removed by hydrolysis.

[0213] In some aspects, the anchoring moiety comprises any phospholipid disclosed herein in which one or both acyl chains have been removed by hydrolysis, and thus the resulting lysophospholipid comprises one or no fatty acid acyl chains.

[0214] In some aspects, the anchoring moiety comprises a lysoglycerophospholipid, a lysoglycosphingolipid, a lysophosphatidylcholine, a lysophosphatidylethanolamine, a lysophosphatidylinositol, or a lysophosphatidylserine.

[0215] In some aspects, the anchoring moiety AM comprises a lysolipid selected from the non-limiting group consisting of: 1 hexanoyl 2 hydroxy sn glycerol 3 phosphocholine (06:0 Lyso PC), 1 heptanoyl 2 hydroxy sn glycerol 3 phosphocholine (07:0 Lyso PC), 1 octanoyl 2 hydroxy sn glycerol 3 phosphocholine (08:0 Lyso PC), 1 nonanoyl 2 hydroxy sn glycerol 3 phosphocholine (09:0 Lyso PC), 1 decanoyl 2 hydroxy sn glycerol 3 phosphocholine (10:0 Lyso PC), 1 undecanoyl 2 hydroxy sn glycerol 3 phosphocholine (11:0 Lyso PC), 1 lauroyl 2 hydroxy sn glycerol 3 phosphocholine (12:0 Lyso PC), 1 tridecanoyl 2 hydroxy sn glycerol 3 phosphocholine (13:0 Lyso PC), 1 myristoyl 2 hydroxy sn glycerol 3 phosphocholine (14:0 Lyso PC), PC), 1 pentadecanoyl 2 hydroxy sn glycerol 3 phosphocholine (15:0Lyso PC), 1 palmitoyl 2 hydroxy sn glycerol 3 phosphocholine (16:0Lyso PC), 1 heptadecanoyl 2 hydroxy sn glycerol 3 phosphocholine (17:0Lyso PC), 1 stearoyl 2 hydroxy sn glycerol 3 phosphocholine (18:0Lyso PC) or 1 oleoyl 2 hydroxy sn glycerol 3 phosphocholine (18:1Lyso PC), 1 myristoyl 2 hydroxy sn glycerol 3 phosphoethanolamine (14:0Lyso PE), 1 palmitoyl 2 hydroxy sn glycerol 3 phosphoethanolamine (16:0Lyso PE), 1 stearoyl 2 hydroxy sn glycerol 3 phosphoethanolamine (18:0Lyso PE), 1 oleoyl 2 hydroxy sn glycerol 3 phosphoethanolamine (18:1LysoPE), 1 hexadecyl sn glycerol 3 phosphocholine (C16 Lyso PC), and any combination thereof.

[0216] In some aspects, the anchoring moiety AM comprises, consists of, or consists essentially of a vitamin, such as a lipophilic vitamin. Suitable vitamins include, for example, vitamin A, vitamin B (e.g., vitamin B3 (niacin), vitamin B6 (pyridoxine), vitamin B9 (folic acid), or vitamin B12 (riboflavin)), vitamin E (tocopherol or tocotrienol), vitamin D (e.g., vitamin D2 or ergocalciferol, vitamin D3 or cholecalciferol, or a combination thereof), vitamin K, or a combination thereof. In some aspects, the vitamin is tocopherol, tocotrienol, vitamin D, vitamin K, riboflavin, niacin, or pyridoxine. In some aspects, the vitamin is tocopherol.

[0217] In some aspects, the anchoring moiety AM comprises or consists of vitamin D. Vitamin D is a group of fat-soluble, open-ring steroids responsible for increasing intestinal absorption of calcium, magnesium, and phosphate, as well as many other biological effects. In humans, the most important compounds in this group are vitamin D3 (also known as cholecalciferol) and vitamin D2 (ergocalciferol).

[0218]

[0219] In some aspects, the anchoring moiety AM comprises or consists of vitamin B9 (folic acid).

[0220]

[0221] In some aspects, the anchoring moiety AM comprises or consists of vitamin B2 (riboflavin).

[0222]

[0223] In some aspects, the anchoring moiety AM comprises or consists of vitamin B3 (niacin)

[0224]

[0225] In some aspects, the anchoring moiety AM comprises or consists of vitamin B6 (pyridoxine).

[0226]

[0227] In some aspects, the anchoring moiety AM comprises or consists of vitamin A. Vitamin A is a group of unsaturated nutritional organic compounds that includes retinol, retinal, retinoic acid, and several provitamin A carotenoids (most notably beta-carotene). In some aspects, the anchoring moiety comprises retinol. In some aspects, the anchoring moiety comprises a retinoid. Retinoids are a class of chemical compounds that are vitamin equivalents to or chemically related to vitamin A. In some aspects, the anchoring moiety comprises a first generation retinoid (e.g., retinol, tretinoin, isotretinoin, or alitretinoin), a second generation retinoid (e.g., acetylcholine or acitretin), a third generation retinoid (e.g., adapalene, bexarotene, or tazarotene), or any combination thereof.

[0228]

[0229] In some aspects, the anchoring moiety AM comprises or consists of vitamin E. Tocopherols are a class of methylated phenols, many of which have vitamin E activity. Thus, in some aspects, the anchoring moiety comprises α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, or a combination thereof.

[0230]

[0231] Tocotrienols also have vitamin E activity. The structural difference between tocotrienols and tocopherols is that tocotrienols have unsaturated isoprenoid side chains with three carbon-carbon double bonds, while tocopherols have saturated side chains. In some aspects, the anchoring moiety comprises α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, or a combination thereof. Tocotrienols can be represented by the formula

[0232]

[0233] α(α)-Tocotrienol: R 1 =Me, R 2 =Me, R 3 =Me;

[0234] β(β)-Tocotrienol: R 1 =Me, R 2 =H, R 3 =Me;

[0235] γ(γ)-Tocotrienol: R 1 =H, R 2 =Me, R 3 =Me;

[0236] δ(δ)-Tocotrienol: R 1 =H, R 2 =H, R 3 =Me.

[0237] In some aspects, the anchoring moiety AM comprises or consists of vitamin K. Chemically, the vitamin K family includes 2-methyl-1,4-naphthoquinone(3-) derivatives. Vitamin K includes two naturally occurring equivalents: vitamin K1 and vitamin K2. The structure of vitamin K1 (also known as phytomenaquinone, phylloquinone, or (E)-phytomenaquinone) is characterized by the presence of a phytyl group. The structure of vitamin K2 (methyl menaquinones) is characterized by the presence of a polyisoprenyl side chain in the molecule, which can contain six to 13 isoprenyl units. Therefore, vitamin K2 consists of many related chemical subtypes with carbon side chains of varying lengths composed of isoprenoid atom groups. MK-4 is the most common form of vitamin K2. Long-chain forms, such as MK-7, MK-8, and MK-9, predominate in fermented foods. Long-chain forms of vitamin K2 (such as MK-10 to MK-13) are synthesized by bacteria, but they are poorly absorbed and have limited biological function. In addition to natural forms of vitamin K, there are many synthetic forms of vitamin K, such as vitamin K3 (menadione; 2-methylnaphthalene-1,4-dione), vitamin K4, and vitamin K5.

[0238] Thus, in some aspects, the anchoring moiety comprises vitamin 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.

[0239]

[0240] Chemically, the vitamin K family includes 2-methyl-1,4-naphthoquinone(3-) derivatives. Vitamin K includes two naturally occurring equivalents: vitamin K1 (phylloquinone) and vitamin K2 (menaquinone). Vitamin K2, in turn, consists of many related chemical isoforms, with carbon side chains of varying lengths composed of isoprenoid atom groups. The two most studied are menaquinone-4 (MK-4) and menaquinone-7 (MK-7). Thus, in some aspects, vitamin K is MK-4, MK-5, or a combination thereof.

[0241] In some aspects, the anchoring portion AM can include a scaffold protein (e.g., a scaffold X protein, such as PTGFRN or a fragment thereof), or a binding molecule that can bind to a scaffold protein present in the EV (e.g., exosome) membrane, for example, an antibody or a binding portion thereof that can specifically bind to naturally or recombinantly expressed PTGRN on the surface of an EV (e.g., exosome). Thus, in some aspects, the anchoring portion AM and / or the scaffold portion is scaffold X.

[0242] In some aspects, one or more scaffold moieties can be CD47, CD55, CD49, CD40, CD133, CD59, Glypican-1, CD9, CD63, CD81, integrins, selectins, lectins, cadherins, other similar polypeptides known to those skilled in the art, or any combination thereof. Non-limiting examples of other scaffold moieties that can be used with the present disclosure include: aminopeptidase N (CD13); neprilysin, also known as membrane metalloendopeptidase (MME); ectonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1); neuropilin-1 (NRP1); or any combination thereof.

[0243] In other aspects, one or more scaffold moieties are expressed in the membrane of EVs (e.g., exosomes) by recombinantly expressing the scaffold moieties in production cells. EVs (e.g., exosomes) obtained from production cells can be further modified to conjugate to maleimide moieties or linkers. In other aspects, the scaffold moieties, e.g., Scaffold X, are deglycosylated. In some aspects, the scaffold moieties, e.g., Scaffold X, are highly glycosylated, e.g., more than naturally occurring Scaffold X under the same conditions.

[0244] In some aspects, scaffold X is selected from the group consisting of prostaglandin F2 receptor negative regulator (PTGFRN protein); basophil activating factor (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); functional fragments thereof; and any combination thereof.

[0245] In some aspects, Scaffold X comprises a prostaglandin F2 receptor negative regulator (PTGFRN polypeptide). PTGFRN polypeptide may also be referred to as CD9 chaperone 1 (CD9P-1), protein F containing a Glu-Trp-Ile EWI motif (EWI-F), prostaglandin F2-alpha receptor regulatory protein, prostaglandin F2-alpha receptor-associated protein, or CD315.

[0246] In some aspects, Scaffold X is a PTGFRN protein or a functional fragment thereof. In some aspects, Scaffold X comprises an amino acid sequence as set forth in SEQ ID NO: 302. In some aspects, Scaffold X comprises an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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: 302.

[0247] Non-limiting examples of other Scaffold X proteins can be found in U.S. Patent No. US10195290B1, issued February 5, 2019, which is incorporated by reference in its entirety.

[0248] In the context of the present disclosure, the combination of lipid moieties in the anchoring moiety AM, such as different fatty acids, different sterols, different vitamins, or combinations thereof, means that some constructs disclosed herein in the construct population, such as the Formula I construct, may have different anchoring moieties AM and / or linkers. For example, some constructs of Formula I will comprise fatty acids, while other constructs may comprise vitamins or sterols. In another example, the anchoring moiety AM may comprise two lipids, such as a phospholipid and a fatty acid, or two phospholipids, or two fatty acids, or a lipid and a vitamin, or cholesterol and a vitamin, etc., which together have 6 to 30 carbon atoms (ie, an equivalent carbon number (ECN) of 6 to 30). Selecting a combination of constructs with different anchoring moieties AM and / or linkers generally results in better packaging of lipids in the membrane on EVs (e.g., exosomes), which can result in improved loading efficiency and BAM density.

[0249] In general, the anchoring portion is chemically attached, for example, via solid phase synthesis. However, the anchoring portion AM can be enzymatically attached to the bioactive molecule BAM. The anchoring portion AM can be combined via a linker or a spacer, as described herein, at any chemically feasible position, for example, at the 5' and / or 3' end of a nucleotide sequence (e.g., ASO), directly or indirectly conjugated to the bioactive molecule BAM. In some aspects, the anchoring portion AM is directly or indirectly conjugated to the 3' end of the bioactive molecule BAM via a cleavable linker disclosed herein. In some aspects, the anchoring portion AM is only conjugated to the 5' end of a nucleotide sequence (e.g., ASO). In one aspect, the anchoring portion AM is conjugated at a position of a non-3' end or 5' end of a nucleotide sequence (e.g., ASO).

[0250] In some aspects, the anchoring moiety AM of the present disclosure may include any of the hydrophobic group modifications disclosed below:

[0251]

[0252] In some aspects, the anchoring moiety AM can include a spacer SP (eg, SP1 or SP2) that enables attachment to BAM and includes a cleavable bond, as described herein (eg, L1). Suitable spacers are described herein.

[0253] Cleavable bond

[0254] The present disclosure provides a construct of Formula I comprising a cleavable bond: L1. The term "cleavable linker" refers to a linker or spacer comprising at least one bond or chemical bond that can be broken or cleaved under certain physiological conditions. 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 a nuclease, peptidase, protease, phosphatase, oxidase, or reductase, or by specific physicochemical conditions such as a redox environment, pH, the presence of reactive oxygen species, or light of a specific wavelength.

[0255] In some aspects, L1 is a cleavable bond comprising: a polynucleotide group; a peptidyl group selected from alanine-alanine-asparagine, valine-glycine, glycine-glycine, glutamic acid-valine-citrulline, aspartic acid-valine-citrulline, serine-valine-citrulline, lysine-valine-citrulline, glycine-glycine-glycine-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate group; or a silyl ether.

[0256] In some respects, L1 comprises a polynucleotide base. In some respects, the polynucleotide base is a trinucleotide base or a higher nucleotide base, such as a tetranucleotide base or a higher nucleotide base, a pentanucleotide base or a higher nucleotide base, a hexanucleotide base or a higher nucleotide base, a heptanucleotide base or a higher nucleotide base, an octanucleotide base or a higher nucleotide base, a nonanucleotide base or a higher nucleotide base or a decanucleotide base or a higher nucleotide base. Usually, the length of the polynucleotide base is not longer than 50 nucleotides (for example 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, 25 nucleotides or less, 20 nucleotides or less, 15 nucleotides or less, 14 nucleotides or less, 13 nucleotides or less, 12 nucleotides or less, 11 nucleotides or less, 10 nucleotides or less, 9 nucleotides or less, 8 nucleotides or less, 7 nucleotides or less, 6 nucleotides or less, 5 nucleotides or less, 4 nucleotides or less or 3 nucleotides). In some respects, L1 comprises a tetranucleotide base.

[0257] In general, the polynucleotide base will comprise adenosine (AMP, dAMP), guanylate (GMP, dGMP), cytidylate (CMP, dCMP), thymidylate (dTMP), uridine (UMP), or any combination thereof. In one example, the polynucleotide base is a tetranucleotide base comprising dTdTdTdT, wherein dT is thymidylate.

[0258] In some aspects, L1 is a peptidyl group comprising a cleavable bond selected from the group consisting of alanine-alanine-asparagine, valine-glycine, glycine-glycine, glutamic acid-valine-citrulline, aspartic acid-valine-citrulline, serine-valine-citrulline, lysine-valine-citrulline, glycine-glycine-glycine-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine. In some aspects, the peptidyl group comprises alanine-alanine-asparagine. In some aspects, the peptidyl group comprises valine-glycine. In some aspects, the peptidyl group comprises glycine-glycine. In some aspects, the peptidyl group comprises glutamic acid-valine-citrulline. In some aspects, the peptidyl group comprises aspartic acid-valine-citrulline. In some aspects, the peptidyl group comprises serine-valine-citrulline. In some aspects, the peptidyl group comprises lysine-valine-citrulline. In some aspects, the peptidyl group comprises glycine-glycine-glycine-valine-citrulline. In some aspects, the peptidyl group comprises cyclobutane-1,1-dicarboxamide-citrulline (cBu-Cit). In some aspects, the peptidyl group comprises alanine-phenylalanine-lysine.

[0259] In some aspects, L1 is a cleavable bond comprising a pyrophosphate acyloxy bond. In some aspects, the pyrophosphate acyloxy bond has the formula

[0260]

[0261] Where X+ is a monovalent cation such as a proton (H + ) or Group I cations (e.g. Li + 、Na + , K + or Rb + )and Indicates linkage to AM, BAM or a spacer.

[0262] In some aspects, L1 is a cleavable bond comprising a silyl ether. In some aspects, the silyl ether comprises -OSiR 1 R 2 O-, where R 1 and R 2 The same or different, and each is C 1-8 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl) or aryl (e.g., phenyl). 1 and R 2 Both are isopropyl.

[0263] In some aspects, L1 is a cleavable bond comprising a cell penetrating peptide (CPP). As used herein, a "cell penetrating peptide" is a short peptide (e.g., 30 amino acids or less, such as 29 amino acids or less, 28 amino acids or less, 27 amino acids or less, 26 amino acids or less, 25 amino acids or less, 24 amino acids or less, 23 amino acids or less, 22 amino acids or less, 21 amino acids or less, 20 amino acids or less, 19 amino acids or less, 18 amino acids or less, 17 amino acids or less, 16 amino acids or less, 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, or 2 amino acids) that can penetrate cells to promote the transfer of BAM across the plasma membrane. In one aspect, the cell penetrating peptide may comprise 3 to 30 amino acid residues (e.g., 3 to 25 amino acid residues, 3 to 20 amino acid residues, 3 to 15 amino acid residues, 3 to 10 amino acid residues, 3 to 9 amino acid residues, 3 to 6 amino acid residues, 5 to 25 amino acid residues, 5 to 20 amino acid residues, 5 to 15 amino acid residues, 5 to 10 amino acid residues, or 5 to 9 amino acid residues).

[0264] In some aspects, the cell penetrating peptide can be linear or cyclic. In some aspects, the cell penetrating peptide is linear.

[0265] In some aspects, cell penetrating peptides can be protein-derived, synthetic or chimeric. In some aspects, cell penetrating peptides can be classified as cationic, amphipathic (e.g., primary or secondary), non-amphipathic, hydrophilic and / or hydrophobic. In some aspects, cell penetrating peptides can be classified as cationic and hydrophobic. In some aspects, cell penetrating peptides can be classified as cationic and hydrophilic.

[0266] On the one hand, cell penetrating peptide is cationic, including polycationic.The charge on cationic cell penetrating peptide can be +1 or more (for example +2 or more, +3 or more, +4 or more, +5 or more, +6 or more, +7 or more, +8 or more, +9 or more, +10 or more, +11 or more, +12 or more, +14 or more, +16 or more, +18 or more, +20 or more, +22 or more, +24 or more, +26 or more or +28 or more).In general, charge will be +30 or less (for example +28 or less, +26 or less, +24 or less, +22 or less, +20 or less, +18 or less, +16 or less, +14 or less, +12 or less, +11 or less, +10 or less, +9 or less, +8 or less, +7 or less, +6 or less, +5 or less, +4 or less or +2 or less). In some aspects, the charge on the cell penetrating peptide will be in the range of 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 5, 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 5, 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 8, 3 to 5, 4 to 30, 4 to 25, 4 to 15, 4 to 10, 4 to 8, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, or 5 to 8. In one aspect, the cationic cell penetrating peptide can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) residues of arginine, lysine, histidine, glutamate, or a combination thereof. In one aspect, the cationic cell penetrating peptide comprises Tat or Arg9.

[0267] Cell penetrating peptides may comprise naturally occurring and / or non-natural amino acid residues. The term "naturally occurring amino acids" refers to alanine (A or Ala), arginine (R or Arg), asparagine (N or Asn), aspartic acid (D or Asp), cysteine (C or Cys), glutamic acid (E or Glu), glutamine (Q or Gln), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), leucine (L or Leu), lysine (K or Lys), methionine (M or Met), phenylalanine (F or Phe), proline (P or Pro), serine (S or Ser), threonine (T or Thr), tryptophan (W or Trp), tyrosine (Y or Tyr) and valine (V or Val). "Unnatural amino acids" (i.e., non-naturally occurring amino acids) include, by way of non-limiting example, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, norleucine ("Nle"), norvaline ("Nva"), β-alanine, L- or D-naphthylamine, ornithine ("Orn"), etc. Peptides can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0268] Amino acids also include the D- forms of natural and unnatural amino acids. "D-" denotes an amino acid having a "D" (dextrorotatory) configuration, as opposed to the configuration in naturally occurring ("L-") amino acids. Natural and unnatural amino acids can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0269] In some aspects, the cell penetrating peptide or fragment thereof can comprise low sequence diversity, e.g., comprising 50% or more of a single amino acid, such as arginyl, cysteinyl, prolyl, or lysyl. In one aspect, the cell penetrating peptide or fragment thereof can be arginyl-rich, cysteinyl-rich, prolyl-rich, or lysyl-rich, wherein the sequence or fragment comprises 50% or more of that particular amino acid.

[0270] In some aspects, cell penetrating peptides, fragments thereof, or side chains thereof can be classified as hydrophobic. In one aspect, hydrophobicity can be introduced by including one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) residues of tryptophan, phenylalanine, tyrosine, leucine, or a combination thereof. For example, the amino acid sequences WWWWW (SEQ ID NO: 1094) and FFLIPKG (SEQ ID NO: 1095) and the cell penetrating peptide gH 625 (sequence HGLASTLTRWAHYNALIRAF (SEQ ID NO: 1113) peptide group) are considered to be hydrophobic.

[0271] In some aspects, the cell penetrating peptide may comprise three or more arginyl moieties (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 arginyl moieties). In one aspect, the cell penetrating peptide may comprise three (Arg3), six (Arg6), eight (Arg8), or nine (Arg9) arginyl moieties. In some aspects, the cell penetrating peptide further comprises at least one amino acid other than arginyl, such as cysteinyl, glycyl, or a combination thereof. In some aspects, at least one amino acid other than arginyl is cysteinyl, glycyl, lysyl, glutaminyl, isoleucyl, tryptophanyl, phenylalanyl, valyl, threonyl, seryl, or a combination thereof.

[0272] In some aspects, the cell penetrating peptide comprises a cyclic peptide, TAT (peptide group of the sequence YGRKKRRQRRR (SEQ ID NO: 1096)), Antp (Antennapedia; peptide group of the sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 1097)), DPV3 (peptide group of the sequence RKKRRRESRKKRRRES (SEQ ID NO: 1098)), DPV6 (peptide group of the sequence GRPRESGKKRKRKRLKP (SEQ ID NO: 1099)), Penetratin (peptide group of the sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 1100)), R9-TAT (peptide group of the sequence GRRRRRRRRRPPQ (SEQ ID NO: 1101)), pVEC (peptide group of the sequence LLIILRRRIRKQAHAHSK (SEQ ID NO: 1102)), ARF(19-31) (peptide group of the sequence RVRVFVVHIPRLT (SEQ ID NO: 1103)), or a peptide comprising a peptide having a cyclic peptide. NO: 1103)), MPG (peptide group of the sequence GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 1104)), MAP (peptide group of the sequence KLALKLALKALKAALKLA (SEQ ID NO: 1105)), transportan (peptide group of the sequence GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 1106)), Bip4 (peptide group of the sequence VSALK (SEQ ID NO: 1107)), C105Y (peptide group of the sequence CSIPPEVKFNPFVYLI (SEQ ID NO: 1108)), melittin (peptide group of the sequence GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 1109)), gH625 (peptide group of the sequence HGLASTLTRWAHYNALIRAF (SEQ ID NO: 1110)), NO: 1110)), Pep-1 (peptide group of sequence KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 1111)), SAP (sweet arrow peptide;(peptide group of the sequence (VRLPPP) 3 (SEQ ID NO: 1112)), gH 625 (peptide group of the sequence HGLASTLTRWAHYNALIRAF (SEQ ID NO: 1113)), crot (27-29) (peptide group of the sequence KMDCRWRWKCCKK (SEQ ID NO: 1114)), R6 / W3 (peptide group of the sequence RRWWRWRR (SEQ ID NO: 1115)), TP10 (peptide group of the sequence AGYLLGKINLKALAALAKKIL (SEQ ID NO: 1116)), CADY (peptide group of the sequence Ac-GLWRALWRLLRSLWRLLWRA-cysteamide (SEQ ID NO: 1117)), sC18 (peptide group of the sequence GLRKRLRKFRNKIKEK (SEQ ID NO: 1118)), YTA4 (peptide group of the sequence IAWVKAFIRKLRKGPLG (SEQ ID NO: 1119)). NO: 1119)), CyLoP-1 (peptide group of the sequence CRWRWKCCKK (SEQ ID NO: 1120)), GALA (peptide group of the sequence WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 1121)), or R6 (peptide group of the sequence RRRRRR (SEQ ID NO: 1122)). Other suitable examples of cell penetrating peptides are disclosed in Kalafatovic et al., Molecules, 22(11), 1929 (2017), which is incorporated herein by reference in its entirety. ;

[0273] In some aspects, the cell penetrating peptide comprises a cyclic peptide, TAT (the peptidyl group of the sequence YGRKKRRQRRR (SEQ ID NO: 1096)), or Antp (Antennapedia; the peptidyl group of the sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 1097)).

[0274] In some aspects, the cell penetrating peptide is a cyclic peptide. Examples of cyclic peptides include, for example, cyclic Tat (cTAT, a peptide of the sequence KRRRGRKKRRE (wherein K and E are linked to form a cyclic peptide) (SEQ ID NO: 1123)), cyclic [WR]4, cyclic [WR]5, cyclic [WH]5, cyclic [KW]4, cyclic [KW]5, cyclic [CR]4, cyclic [CR]5, cyclic [HR]5, cyclic [W(RW)4K], cyclic [C(RW)4K], C16-[r 12] (r = arginine) or cyclic sC18. Other suitable examples of cyclic cell penetrating peptides are disclosed in Park et al., Molecular Pharmaceutics, 16, 3727-3743 (2019), which is incorporated herein by reference in its entirety.

[0275] In some aspects, cell penetrating peptides are classified as primary amphipathic (eg, Pep-1, TP10), secondary amphipathic (eg, penetratin, R6 / W3, CADY, sC18), or non-amphipathic.

[0276] spacer

[0277] Typically, the cleavable bond L1 is part of a spacer (e.g., SP1 and / or SP2) between the anchoring portion AM and the biologically active molecule BAM to provide optimal spacing between the anchoring portion AM and the biologically active molecule BAM. For example, in the case of antisense oligonucleotides (ASOs), one goal of the combination of a linker and a spacer is to reduce steric hindrance and position the ASO so that it can interact with the target nucleic acid (e.g., mRNA or miRNA). Other goals can include, for example, improving the loading efficiency of EVs (e.g., exosomes), increasing the number of BAMs per EV, increasing the surface density of BAMs on EVs, improving the effectiveness of BAMs after incorporation into EVs, or any combination thereof. In the context of the present disclosure, the term "linker combination" refers to a combination of a cleavable bond L1 and one or more spacers that form the construct of Formula I disclosed herein.

[0278] In some aspects, SP1 is present in formula (I). In some aspects, SP2 is present in formula (I). In some aspects, both SP1 and SP2 are present in formula (I) and are the same. In some aspects, both SP1 and SP2 are present in formula (I) and are different.

[0279] In some aspects, prior to conjugation to AM and / or BAM, the cleavable bond L1 is part of a spacer moiety, i.e., SP1 and / or SP2. In some aspects, prior to conjugation to BAM, L1 is part of SP2 and AM is part of SP1. In some aspects, prior to conjugation to each other, L1 is part of both SP1 and AM, and BAM is part of SP2. In some aspects, prior to conjugation to each other, L1 is part of both SP2 and BAM, and AM is part of SP1. In some aspects, prior to conjugation to AM and BAM, L1 is part of both SP1 and SP2. In some aspects, L1 is part of SP1 and AM prior to conjugation to BAM, and SP2 is absent. In some aspects, L1 is part of SP2 and BAM prior to conjugation to AM, and SP1 is absent. In some aspects, L1 is part of SP1 prior to conjugation to AM and BAM, and SP2 is absent. In some aspects, L1 is part of SP2 prior to conjugation to AM and BAM, and SP2 is absent. In some aspects, L1 is part of SP2 prior to conjugation to AM and BAM, and SP1 is absent.

[0280] In some aspects, SP1 and SP2 of formula (I) are the same or different and each comprises an alkylene group, a polyoxyalkylene group, a succinimidyl group, a maleimido group, an aryl group (e.g., 1,2-phenyl, 1,3-phenyl, or 1,4-phenyl), an ether (—O—), a carbonyl group (—C(O)—), a carboxyl group (—C(O)O— or —OC(O)—), a carbamoyl group (—OC(O)NR— or —NRC(O)O—), a thioether (—S—), a sulfo group (—SO2—), a thiocarbonyl group (—C(S)—), a thiocarbamoyl group (—OC(O)NR— or —NRC(O)O—), a thioether (—S—), a sulfo group (—SO2—), a thiocarbonyl group (—C(S)—), a thiocarbamoyl group (—OC(O)NR— or —NRC(O)O—), a sulfoether (—S—), a sulfocarbonyl group (—SO2—), a sulfocarbonyl group (—C(S) ... Acyl (-OC(S)NR- or -NRC(S)O-), sulfosuccinimidyl, amino (-NR-), amide (-C(O)NR- or -NRC(O)-), hydrazido (-NH-NH-), thiophosphate (-OP(=S)(OH)O-), 1,2,3-triazolyl, dibenzoylcyclooctenyl, bicyclononenyl, p-aminobenzoyl, p-aminobenzylcarbamate, or a combination thereof, and at least one of SP1 and SP2 is present in formula (I). The following parts are associated with the provided structures

[0281] Succinimidyl

[0282] Sulfosuccinimide

[0283] Maleimide

[0284] 1,2,3-Triazolyl

[0285] Dibenzoylcyclooctenyl

[0286] Bicyclononenyl

[0287] p-Aminobenzoyl and

[0288] p-Aminobenzyl carbamate in Indicates linkage to AM, BAM, L1 or the rest of the spacer.

[0289] In some aspects, at least one of SP1 and SP2 comprises C 2-8 In some aspects, at least one of SP1 and SP2 comprises a C3 alkylene, a C6 alkylene, or a C8 alkylene, as described herein. In some aspects, at least one of SP1 and SP2 comprises a polyoxyalkylene (e.g., a diol-based spacer) containing 2 to 15 -OCH2CH2- repeating units, as described herein. In some aspects, the polyoxyalkylene (e.g., a diol-based spacer) comprises 4 (TEG) or 6 (HEG) -OCH2CH2- repeating units, as described herein. In some aspects, at least one of SP1 and SP2 comprises a C3 alkylene, a C6 alkylene, or a C8 alkylene or a polyoxyalkylene containing 2 to 15 repeating units of -OCH2CH2- (e.g., a diol-based spacer), as described herein, and further comprises a carbamoyl group, an amide group, a sulfosuccinimide group, a 1,2,3-triazolyldibenzoylcyclooctenyl group, a 1,2,3-triazolylbicyclononenyl group, or a combination thereof, wherein the 1,2,3-triazolyldibenzoylcyclooctenyl group has the structure and

[0290] 1,2,3-Triazolylbicyclononenyl has the following structure:

[0291] in Indicates linkage to AM, BAM, L1 or the rest of the spacer.

[0292] Alkylene spacer

[0293] In some aspects, the spacer may include an alkylene spacer, such as a linear alkylene spacer. In some aspects, the alkylene spacer attached to the anchoring portion AM and / or BAM is selected from the group consisting of C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14 or C15, wherein C represents a divalent methylene unit (-CH2-) and the number represents the number of methylene units in the alkylene spacer. In some aspects, the alkylene spacer includes a single methylene (C1; -CH2-). In some aspects, the alkylene spacer is C2 (-CH2CH2-). In some aspects, the alkylene spacer is C3. In some aspects, the alkylene spacer is C4. In some aspects, the alkylene spacer is C5. In some aspects, the alkylene spacer is C6. In some aspects, the alkylene spacer is C7. In some aspects, the alkylene spacer is C8. In some aspects, the alkylene spacer is C9. In some aspects, the alkylene spacer is C10. In some aspects, the alkylene spacer is C11. In some aspects, the alkylene spacer is C12. In some aspects, the alkylene spacer is C13. In some aspects, the alkylene spacer is C14. In some aspects, the alkylene spacer is C15.

[0294] In some aspects, the optional first spacer of SP1 and / or the optional second spacer of SP2 independently comprise a spacer (e.g., an alkylene spacer or a diol-based spacer) or a combination thereof. In some aspects, the optional first spacer of SP1 comprises or consists of an alkylene spacer that is C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, or C15 (e.g., propyl, butyl, hexyl, C2-C15 alkyl, C2-C10 alkyl, or C2-C6 alkyl). In some aspects, SP1 is a C3 or C6 alkylene spacer. In some aspects, the optional second spacer of SP2 comprises or consists of an alkylene spacer that is C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, or C15 (e.g., propyl, butyl, hexyl, C2-C15 alkyl, C2-C10 alkyl, or C2-C6 alkyl). In some aspects, SP2 is a C3 or C6 alkylene spacer.

[0295] In some aspects, the spacer comprises a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, an arylalkyl, an arylalkenyl, an arylalkynyl, a heteroarylalkyl, a heteroarylalkenyl, a heteroarylalkynyl, a heterocyclylalkyl, a heterocyclylalkenyl, a heterocyclylalkynyl, an aryl, a heteroaryl, a heterocyclyl, a cycloalkyl, a cycloalkenyl, an alkylarylalkyl, an alkylarylalkenyl, an alkylarylalkynyl, an alkenylarylalkyl, an alkenylarylalkenyl, an alkenylarylalkynyl, an alkynylaryl wherein the alkyl radicals are alkyl, alkylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl or alkenylheterocyclylalkynyl.

[0296] Optionally, these spacers are substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) substituents. Substituents include, for example, hydroxy, alkoxy (e.g., -O-(C 1-8 Alkyl)), straight or branched alkyl (e.g. C 1-15 Alkyl or C 1-8 alkyl), amino, aminoalkyl (e.g., C1-C15 alkylamino), phosphoramidite, phosphate, phosphoramidite, phosphorodithioate, phosphorothioate, hydrazido, hydrazine, halo (e.g., F, Cl, Br, or I), aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-, NHC(O)R', -S(O)2R', -S(O)R' , -NH(R'), -N(R')2, carboxylic acid halide, ether, sulfonyl halide, imidoester, isocyanate, isothiocyanate, haloformate, carbodiimide adduct, aldehyde, ketone, sulfhydryl, haloacetyl, alkyl halide, alkyl sulfonate, C(=O)CH=CHC(=O) (maleimide), thioether, cyano, nitro, sugar (such as mannosyl, galactosyl and glucosyl), α,β-unsaturated carbonyl, alkylmercury or α,β-unsaturated sulfonyl, wherein each R' is independently H, C 1-8 Alkyl or aryl.

[0297] Examples of aliphatic linkers include the following structures: —O—CO—O—; —NH—CO—O—; —NH—CO—NH—; —NH—(CH2) n1 —;—S—(CH2) n1 —;—CO—(CH2) n1 —CO—; —CO—(CH2) n1 —NH—; —NH—(CH2) n1—NH—;—CO—NH—(CH2) n1 —NH—CO—;—C(═S)—NH—(CH2) n1 —NH—CO—;—C(═S)—NH—(CH2) n1 —NH—C—(═S)—;—CO—O—(CH2) n1 —O—CO—;—C(═S)—O—(CH2) n1 —O—CO—;—C(═S)—O—(CH2) n1 —O—C—(═S)—;—CO—NH—(CH2) n1 —O—CO—;—C(═S)—NH—(CH2) n1 —O—CO—;—C(═S)—NH—(CH2) n1 —O—C—(═S)—;—CO—NH—(CH2) n1 —O—CO—;—C(═S)—NH—(CH2) n1 —CO—;—C(═S)—O—(CH2) n1 —NH—CO—;—C(═S)—NH—(CH2) n1 —O—C—(═S)—;—NH—(CH2CH2O) n2 —CH(CH2OH)—;—NH—(CH2CH2O) n2 —CH2—;—NH—(CH2CH2O) n2 —CH2—CO—;—O—(CH2) n3 —S—S—(CH2) n4 —O—P(═O)2—;—CO—(CH2) n3 —O—CO—NH—(CH2) n4 —;—CO—(CH2) n3 —CO—NH—(CH2) n4 —;—(CH2) n1 NH—;—C(O)(CH2) n1 NH—;—C(O)—(CH2) n1 -C(O)—;—C(O)—(CH2) n1 -C(O)O—;—C(O)—O—;—C(O)—(CH2) n1 -NH—C(O)—;—C(O)—(CH2) n1 —;—C(O)—NH—;—C(O)—;—(CH2) n1 -C(O)—;—(CH2) n1 -C(O)O—;—(CH2) n1 —;—(CH2)n1 -NH—C(O)—; wherein n1 is an integer between 1 and 15 (e.g., 2 to 15, or 2 to 12); n2 is an integer between 1 and 15 (e.g., 1 to 10, or 1 to 6); n3 and n4 may be the same or different and are each an integer between 1 and 15 (e.g., 1 to 10, or 1 to 6), and wherein the total number of carbon units in the linker is C15 or less.

[0298] Diol-based spacers

[0299] In some aspects, the spacer may include a diol-based spacer. In some aspects, the diol-based spacer includes two or more –OCH2CH2- repeating units and can be formed by, for example, diethylene glycol, triethylene glycol, tetraethylene glycol (TEG), pentaethylene glycol, hexaethylene glycol (HEG), heptaethylene glycol, octaethylene glycol, nonaethylene glycol, or decaethylene glycol to provide a corresponding number of –OCH2CH2- repeating units. In some aspects, the diol-based spacer may include 11, 12, 13, 14, or 15 diol-based repeating units (–OCH2CH2-). In some aspects, the diol-based spacer has between 2 and 10, between 2 and 5, between 5 and 10, or between 10 and 15 diol-based repeating units. In some aspects, the diol-based spacer is formed by HEG (i.e., 6 diol repeating units). In some aspects, the diol-based spacer is formed by TEG (i.e., 4 diol repeating units).

[0300] In some aspects, the diol-based spacer may comprise a compound of formula (R 3 —O—(CH2—CHOR 5 —CH2—O) n —) branched polyglycerol (wherein R 5 is hydrogen) or formula (R 3 —O—(CH2—CHOH—CH2—O) n —) linear glycerol chain, and R 3 is hydrogen, methyl, or ethyl, and n is an integer from 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In some aspects, the diol-based spacer may comprise the formula (R 3 —O—(CH2—CHOR 5 —CH2—O) n —) of hyperbranched polyglycerol (wherein R 5 is hydrogen) or by formula (R 3 —O—(CH2—CHOR 6 —CH2—O) n —) the glycerol chain (wherein R 6 is hydrogen) or formula (R 3—O—(CH2—CHOR 7 —CH2—O) n —) glycerol chain (where R 7 is hydrogen) or formula (R 3 —O—(CH2—CHOH—CH2—O) n —) linear glycerol chain and R 3 is hydrogen, methyl or ethyl. In each case, n is an integer from 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15). Hyperbranched glycerol and synthesis methods are described, for example, in Oudshorn et al. (Biomaterials, 2006, 27: 5471-5479) and Wilms et al. (Acc. Chem. Res., 2010, 43, 129-41).

[0301] In some aspects, the optional first spacer of SP1 comprises or consists of a diol-based spacer having 2 (diethylene glycol), 3 (triethylene glycol), 4 (tetraethylene glycol; TEG), 5 (pentaethylene glycol), 6 (hexaethylene glycol; HEG), 7, 8, 9, 10, 11, 12, 13, 14, or 15 diol units. In some aspects, SP1 is a tetraethylene glycol (TEG) or hexaethylene glycol (HEG) diol-based spacer. In some aspects, the optional second spacer of SP2 comprises or consists of a diol-based spacer having 2 (diethylene glycol), 3 (triethylene glycol), 4 (tetraethylene glycol; TEG), 5 (pentaethylene glycol), 6 (hexaethylene glycol; HEG), 7, 8, 9, 10, 11, 12, 13, 14, or 15 diol-based units. In some aspects, SP2 is a tetraethylene glycol (TEG) or hexaethylene glycol (HEG) diol-based spacer.

[0302] In some aspects, the SP1 spacer comprises an alkylene spacer and the SP2 spacer further comprises an alkylene spacer. In some aspects, the SP1 spacer comprises a diol-based spacer and the SP2 spacer also comprises a diol-based spacer. In some aspects, the SP1 spacer comprises an alkylene spacer and the SP2 spacer comprises a diol-based spacer. In some aspects, the SP1 spacer comprises a diol-based spacer and the SP2 spacer comprises an alkylene spacer. In some aspects, the SP1 spacer consists of an alkylene spacer and the SP2 spacer also consists of an alkylene spacer. In some aspects, the SP1 spacer consists of a diol-based spacer and the SP2 spacer also consists of a diol-based spacer. In some aspects, the SP1 spacer consists of an alkylene spacer and the SP2 spacer consists of a diol-based spacer. In some aspects, the SP1 spacer consists of a diol-based spacer and the SP2 spacer consists of an alkylene spacer.

[0303] In some aspects, each non-cleavable spacer is independently selected from the group consisting of an alkyl group (e.g., C2, C3, C4, C5, C6, C7, or C8), a glycol (e.g., diethylene glycol, triethylene glycol, tetraethylene glycol (TEG), hexaethylene glycol (HEG), pentaethylene glycol, polyethylene glycol (PEG)), a glycerol (e.g., diglycerol, triglycerol, tetraglycerol (TG), pentaglycerol, hexaglycerol (HG), polyglycerol (PG)), a succinimide, a maleimide, or any combination thereof.

[0304] polyethylene glycol spacer

[0305] In some aspects, the spacer in the anchoring moiety AM, the first spacer SP1, the second spacer SP2, or any combination thereof, can comprise or consist of a diol-based spacer, which can be considered to be of the formula -(O-CH2-CH2) n -or-(O-CH2-CH2) n -O-, wherein n is an integer between 1 and 200 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200). These PEGs can be described as PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG11, PEG12, PEG2 10 PEG 11PEG 12 PEG 13 PEG 14 PEG 15 PEG 25 PEG 50 PEG 75 PEG 100 PEG 125 PEG 150 PEG 175 PEG 200 Or PEG1-O-, PEG2-O-, PEG3-O-, PEG4-O-, PEG5-O-, PEG6-O-, PEG7-O-, PEG8-O-, PEG9-O-, PEG 10 -O-、PEG 11 -O-、PEG 12 -O-、PEG 13 -O-、PEG 14 -O-、PEG 15 -O-、PEG 25 -O-、PEG 50 -O-、PEG 75 -O-、PEG 100 -O-、PEG 125 -O-、PEG 150 -O-、PEG 175 -O- or PEG 200 -O-. Prior to conjugation to another moiety (e.g., AM or BAM) forming a construct of Formula I, the PEG residue may be formed by R 1 -(O-CH2-CH2) n -R 1 or R 1 -(O-CH2-CH2) n -OR 1 Formed, where R 1 is hydrogen, methyl, or ethyl, and n is an integer between 1 and 200 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200).

[0306] In some aspects, PEG is a branched PEG. A branched PEG has 3 to 10 PEG chains emanating from a central core group. In some aspects, the PEG moiety is a monodisperse polyethylene glycol. In the context of the present disclosure, a monodisperse polyethylene glycol (mdPEG) is a PEG with a single, defined chain length and molecular weight.

[0307] In some aspects, the PEG is a star PEG. A star PEG has about 10 to 15 PEG chains emanating from a central core group. In some aspects, the PEG is a comb PEG. A comb PEG has multiple PEG chains, typically grafted onto a polymer backbone.

[0308] In some aspects, the spacer in the anchoring portion AM, the first spacer SP1, the second spacer SP2, or any combination thereof, can comprise or consist of a polyglycerol (PG) residue having the formula -O—(CH2—CHOH—CH2O) n - characterized, wherein n is an integer between 1 and 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). These polyglycerol residues can be described as PG1, PG2, PG3, PG4, PG5, PG6, PG7, PG8, PG9, PG 10 PG 11 PG 12 PG 13 PG 14 or PG 15 .

[0309] Vitamin spacers

[0310] In some aspects, the anchoring moiety AM comprises or consists of a vitamin as described herein and an alkylene spacer. In some aspects, the anchoring moiety AM comprises or consists of a tocopherol and an alkylene spacer. In some aspects, the anchoring moiety AM comprises or consists of a vitamin and an octyl (C8) alkylene spacer, such as tocopherol and an octyl (C8) alkylene spacer.

[0311] In some aspects, the anchoring moiety AM comprises or consists of a fatty acid and an alkylene spacer. In some aspects, the anchoring moiety AM comprises or consists of a palmitate and an alkylene spacer. In some aspects, the anchoring moiety AM comprises or consists of a fatty acid and a hexyl(C6)alkylene spacer (such as palmitate and a hexyl(C6)alkylene spacer).

[0312] In some aspects, the anchoring portion AM comprises a sterol and a diol-based spacer or consists of it. In some aspects, the anchoring portion AM comprises a cholesterol and a diol-based spacer or consists of it. In some aspects, the anchoring portion AM comprises a sterol and a TEG diol-based spacer (such as cholesterol and a TEG diol-based spacer) or consists of it. In some aspects, the anchoring portion AM comprises a sterol and an alkylene spacer or consists of it. In some aspects, the anchoring portion AM comprises a sterol and a hexyl (C6) alkylene spacer or consists of it. In some aspects, the anchoring portion AM comprises a cholesterol and an alkylene spacer (such as cholesterol and a hexyl (C6) alkylene spacer) or consists of it.

[0313] Non-cleavable linker

[0314] In some aspects, the joint combination comprises a "non-cleavable bond", which is a joint and / or an intron comprising a chemical bond that is substantially resisted from cracking. Non-cleavable joints substantially resist, for example, acid-induced cracking, light-induced cracking, peptidase-induced cracking, esterase-induced cracking and disulfide bond cleavage, particularly under conditions where cyclic dinucleotides and / or antibodies do not lose their activity. Non-cleavable joints are any chemical moieties that can connect constructs disclosed herein, for example, two or more components of a Formula I construct. In particular, non-cleavable joints can connect any two parts of a construct, for example, anchoring moiety AM and / or intron SP and / or bioactive molecule BAM. Typically, non-cleavable joints are a part for introns (for example SP1, SP2), as described herein, but in some aspects, AM or BAM are modified to comprise non-cleavable bonds.

[0315] In some aspects, the linker combination comprises a non-cleavable linker comprising, for example, tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), glycerol, a C2 to C12 alkyl, a succinimidyl group, or any combination thereof. In some aspects, the non-cleavable bond comprises a spacer as described herein to connect AM and / or BAM to the non-cleavable bond.

[0316] Bioactive molecules

[0317] Bioactive molecule BAM is an agent that acts on a target (e.g., a target cell). Contact can occur in vitro or in a subject. Non-limiting examples of bioactive molecules BAM that can be attached to EVs (e.g., exosomes) as described in the present disclosure include the following agents: such as polynucleotides (e.g., nucleotides containing a detectable portion or a toxin that destroys 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, mRNA, siRNA, shRNA, or antisense oligonucleotides (ASO)), amino acids (e.g., amino acids containing a detectable portion or a toxin that destroys translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins), antivirals, RNA decoys, or any combination thereof. In some aspects, BAM includes peptides, polypeptide groups, polynucleotide groups, proteins, antibodies, or antigen-binding fragments thereof, chemical compounds, or any combination thereof. In some aspects, BAM includes antisense oligonucleotide groups (ASOs), siRNAs, miRNAs, shRNAs, nucleic acids, or any combination thereof.

[0318] In some aspects, the EVs (e.g., exosomes) disclosed herein can comprise more than one attached bioactive molecule BAM, for example, using the constructs disclosed herein. For example, in some aspects, EVs (e.g., exosomes) can comprise multiple populations of constructs disclosed herein (e.g., constructs of Formula I), wherein each population of constructs carries a different bioactive molecule BAM.

[0319] Thus, in some aspects, a population of EVs (e.g., exosomes) disclosed herein may comprise multiple constructs, as exemplified below.

[0320] AM1-SP1-L1-SP2-BAM1

[0321] AM2-SP1-L1-SP2-BAM2

[0322] …

[0323] AM n -SP1-L1-SP2-BAM n ,

[0324] Among them [AM1]..[AM n ] can be the same or different anchoring moieties, each of SP1, L1 and SP2 can be the same or different, and [BAM1]..[BAM n ] can be the same or different biologically active molecules.

[0325] In some aspects, the bioactive molecule BAM comprises a peptide, a protein, an antibody or an antigen-binding portion thereof, or any combination thereof, or consists thereof. In some aspects, the antigen-binding portion thereof comprises scFv, (scFv)2, Fab, Fab', F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, diabodies, antibody-related polypeptides, or any fragments thereof. In some aspects, the antibody or antigen-binding portion thereof can bind to Protein X protein present in the membrane of EVs (e.g., exosomes).

[0326] In some aspects, the bioactive molecule BAM targets a tumor antigen. Non-limiting examples of tumor antigens include: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, α-folate receptor, CE7R, IL-3, cancer testis antigen (CTA), MART-1gp100, TNF-related apoptosis-inducing ligand, or a combination thereof.

[0327] In some aspects, the bioactive molecule (BAM) is a targeting moiety, such as an antibody or binding portion thereof, or a ligand that specifically binds to a marker on a muscle cell. In some aspects, the muscle cell is a smooth muscle cell. In some aspects, the muscle cell is a skeletal muscle cell. In some aspects, the muscle cell is a cardiac muscle cell. In some aspects, the muscle cell marker is selected from α-smooth muscle actin, VE-cadherin, calmodulin binding protein / CALD1, calmodulin 1, hexim 1, histamine H2 receptor; motilin receptor / GPR38, transglutamin / TAGLN, and any combination thereof. In some aspects, the marker on the muscle cell is selected from α-sarcoglycan, β-sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, ε-sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin α7, integrin α7β1, integrin β1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, troponin I, and any combination thereof. In some aspects, the marker on the muscle cell is myosin heavy chain, myosin light chain, or a combination thereof.

[0328] In some aspects, the bioactive molecule BAM is a small molecule. In some aspects, the small molecule is a proteolysis targeting chimera (PROTAC). In some aspects, the bioactive molecule BAM is a small molecule comprising a synthetic anti-tumor agent (e.g., monomethyl Rui Oxetine E (MMAE) (vedotin)), a cytokine release inhibitor (e.g., MCC950), an mTOR inhibitor (e.g., rapamycin and its analogs (rapalog)), an autocrine motility factor inhibitor (e.g., PAT409 or PAT505), a lysophosphatidic acid receptor agonist (e.g., BMS-986020), a STING antagonist (e.g., CL656), or any combination thereof. In some aspects, the bioactive molecule BAM comprises a morpholino backbone structure, as disclosed in U.S. Patent No. 5,034,506, which is incorporated herein by reference in its entirety.

[0329] In some aspects, the bioactive molecule BAM comprises a nucleotide, wherein the nucleotide is a stimulator of interferon genes (STING) agonist. STING is a cytoplasmic sensor of cyclic dinucleotides typically produced by bacteria. Upon activation, it produces type I interferon and initiates an immune response.

[0330] In some aspects, the STING agonist comprises a cyclic nucleotide STING agonist or a non-cyclic dinucleotide STING agonist. Cyclic purine dinucleotides (CDNs), such as but not limited to cGMP, cyclic di-GMP (c-di-GMP), cAMP, cyclic di-AMP (c-di-AMP), cyclic-GMP-AMP (cGAMP), cyclic di-IMP (c-di-IMP), cyclic AMP-IMP (cAIMP), and any analogs thereof, are known to stimulate or enhance a patient's immune or inflammatory response. The CDNs may have 2'2', 2'3', 2'5', 3'3', or 3'5' bonds connecting the cyclic dinucleotides, or any combination thereof. The cyclic purine dinucleotides may be modified via standard organic chemistry techniques to produce analogs of the purine dinucleotides. Suitable purine dinucleotides include, but are not limited to, adenine, guanine, inosine, hypoxanthine, xanthine, isoguanine, or any other suitable purine dinucleotide known in the art. The cyclic dinucleotides may be modified analogs. Any suitable modification known in the art can be used, including but not limited to phosphorothioate, thiodiphosphate, fluoride, and difluoride modifications. Acyclic dinucleotide agonists such as 5,6-dimethylxanthone-4-acetic acid (DMXAA), or any other acyclic dinucleotide agonist known in the art can also be used.

[0331] It is expected that any STING agonist can be used as a bioactive molecule BAM. STING agonists include DMXAA, STING agonist-1, MLRR-S2 CDA, MLRR-S2c-di-GMP, ML-RR-S2 cGAMP, 2′3′-c-di-AM(PS)2, 2′3′-cGAMP, 2′3′-cGAMPdFHS, 3′3′-cGAMP, 3′3′-cGAMPdFSH, cAIMP, cAIM(PS)2, 3′3′-cAIMP, 3′3′-cAIMPdFSH, 2′2′-cGAMP, 2′3′-cGAM(PS)2, 3′3′-cGAMP, c-di-AMP, 2′3′-c-di-AMP, 2′3′-c-di-AM(PS)2, c-di-GMP, 2′3′-c-di-GMP, c-di-IMP, c-di-UMP, or any combination thereof. In a specific aspect, the STING agonist is 3'3'-cAIMPdFSH, alternatively referred to as 3-3cAIMPdFSH. Additional STING agonists known in the art may also be used.

[0332] In some aspects, the bioactive molecule BAM is an antibody or an antigen-binding fragment thereof. In some aspects, the bioactive molecule BAM is an antibody-drug conjugate (ADC). In some aspects, the bioactive molecule BAM is a fusion peptide.

[0333] In some aspects, the bioactive molecule BAM targets macrophages. In other aspects, the bioactive molecule induces macrophage polarization. Macrophage polarization is a process by which macrophages adopt distinct functional programs in response to signals from their microenvironment. This ability is related to their diverse roles in the body: they are powerful effector cells of the innate immune system and also play important roles in clearing cellular debris, embryonic development, and tissue repair.

[0334] Using simplified classification, macrophage phenotypes have been divided into two groups: M1 (classically activated macrophages) and M2 (alternatively activated macrophages). This broad classification is based on in vitro studies in which cultured macrophages are treated with molecules that stimulate phenotypic shifts to specific states. In addition to chemical stimulation, it has been shown that the stiffness of the matrix in which macrophages grow can guide polarization states, functional roles, and migration patterns. M1 macrophages have been described as proinflammatory, playing a key role in direct host defense against pathogens, such as phagocytosis and the secretion of proinflammatory cytokines and bactericidal molecules. M2 macrophages have been described as having the opposite function: regulating the resolution phase of inflammation and repairing damaged tissues. Subsequently, more extensive in vitro and ex vivo studies have revealed that macrophage phenotypes are even more diverse, overlapping with each other in terms of gene expression and function, revealing that these many hybrid states form a continuum of activation states that depends on the microenvironment. Furthermore, in vivo, gene expression profiles vary significantly between macrophage populations in different tissues. Therefore, the spectrum of macrophage activation is thought to be broader, involving complex regulatory pathways in response to a plethora of different signals from the environment. The diversity of macrophage phenotypes remains to be fully characterized in vivo.

[0335] An imbalance in macrophage types is associated with a variety of immune-related diseases. For example, an increased M1 / M2 ratio may be associated with the development of inflammatory bowel disease as well as obesity in mice. On the other hand, in vitro experiments have shown that M2 macrophages are the main mediators of tissue fibrosis. Several studies have linked the fibrotic characteristics of M2 macrophages to the pathogenesis of systemic sclerosis. Non-limiting examples of macrophage targeting bioactive molecules are: PI3Kγ (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit γ), RIP1 (receptor interacting protein (RIP) kinase 1, RIPK1), HIF-1α (hypoxia-inducible factor 1-α), AHR1 (adhesion and hyphal regulator 1), miR146a, miR155, IRF4 (interferon regulatory factor 4), PPARγ (peroxisome proliferator-activated receptor γ), IL-4RA (interleukin 4 receptor subunit α), TLR8 (Toll-like receptor 8) and TGF-β1 (transforming growth factor β-1 proprotein)

[0336] In some aspects, bioactive molecule BAM includes antisense oligonucleotide (ASO) or is made up of it. In some aspects, ASO is spacer polymer, mixed polymer or full polymer. In some aspects, ASO can target precursor mRNA or mature mRNA, including protein coding region (exon), non-coding region (such as 5 'or 3 'untranslated region or intron), intron-exon junction or regulatory region (such as promoter). In some aspects, ASO target protein transcript, such as STAT6 transcript, EGFP transcript, CEBP / β transcript, STAT3 transcript, KRAS transcript, NRAS transcript, NLPR3 transcript, FFLUC transcript, RLUC transcript, MYC transcript or its any combination.

[0337] In some respects, ASO can include one or more nucleosides with the sugar moiety of modification, i.e. when compared with the ribose moiety found in DNA and RNA, sugar moiety is modified. Many nucleosides with the modification of ribose moiety have been prepared, and main purpose is to improve some characteristics of oligonucleotide, such as affinity and / or nuclease resistance. Such modification includes those modifications in which ribose ring structure has been modified, such as by replacing with hexose ring (HNA), dicyclo (it usually has diradical bridges between C2 ' and C4 ' carbon on ribose ring (LNA)) or unconnected ribose ring (it usually lacks key between C2 ' and C3 ' carbon) (such as UNA). Other sugar-modified nucleosides include such as dicyclohexose nucleic acids (WO2011 / 017521) or tricyclic nucleic acids (WO2013 / 154798). The nucleosides of modification also include the nucleosides in which sugar moiety is replaced by non-sugar moiety, such as in the case of peptide nucleic acid (PNA) or morpholino nucleic acids.

[0338] Sugar modification also includes the modification via the substituent group on the ribose ring is changed to the group other than hydrogen or the naturally occurring 2'-OH group in RNA nucleoside.Substituent group can be for example introduced at 2', 3', 4' and / or 5' position.Nucleosides with the sugar moiety of modification also include 2' modified nucleosides, such as 2' substituted nucleosides.2' sugar-modified nucleosides are nucleosides (2' substituted nucleosides) or the diradicals that comprise 2' connections with the substituent group other than H or –OH at the 2' position, and include 2' substituted nucleosides and LNA (2' to 4' diradical bridging) nucleosides.For example, the sugar of 2' modification can provide enhanced binding affinity (for example, enhancing the affinity of 2' sugar-modified nucleosides) and / or increase the nuclease resistance to oligonucleotide. 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, arabinoic acid (ANA), and 2'-fluoro-ANA nucleosides. Other examples are provided in Freier & 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 is a diagram of some 2'-substituted modified nucleosides.

[0339]

[0340] LNA nucleosides are modified nucleosides that contain a linker group (called a diradical or bridge) between the C2' and C4' of the nucleoside's ribose ring (i.e., the 2' to 4' bridge) that constrains or locks the conformation of the ribose ring. These nucleosides are also known as bridged nucleic acids or bicyclic nucleic acids (BNA). When LNA is incorporated into an oligonucleotide complementary to an RNA or DNA molecule, the locking of the ribose conformation is associated with enhanced hybridization affinity (duplex stability). This can be routinely determined by measuring the melting temperature of the oligonucleotide / complement duplex.

[0341] Non-limiting exemplary LNA nucleosides are disclosed in WO99 / 014226; WO00 / 66604; WO98 / 039352; WO2004 / 046160; WO00 / 047599; WO2007 / 134181; WO2010 / 077578; WO2010 / 036698; WO2007 / 090071; WO2009 / 006478; WO2011 / 156202; WO2008 / 154401; WO2009 / 0 67647; WO2008 / 150729; Morita et al., Bioorganic & Med. Chem. Lett., 12, 73-76; Seth et al., J. Org. Chem., 2010, Vol. 75(5), pp. 1569-1581; or Mitsuoka et al., Nucleic Acids Research, 2009, 37(4), 1225-1238, all of which are incorporated herein by reference in their entirety. In some aspects, the modified nucleoside or LNA nucleoside of the ASO of the present disclosure has the general structure of Formula X or Formula XI:

[0342]

[0343] in

[0344] B is a nucleobase or a modified nucleobase moiety;

[0345] W is selected from -O-, -S-, -N(R a )-、-C(R a R b )-, especially –O-;

[0346] X is O;

[0347] Y is CH2;

[0348] Z is an internucleoside bond to an adjacent nucleoside or a 5'-terminal group;

[0349] Z* is an internucleoside bond to an adjacent nucleoside or a 3′-terminal group;

[0350] R 1 、R 2 、R 3 、R 5 and R 5* are independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, hydroxy, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, azido, heterocyclyl, and aryl; and

[0351] R a and R b are independently selected from hydrogen and alkyl.

[0352] ASO targeting NLRP3

[0353] In some aspects, the bioactive molecule BAM is an anti-NLRP3 ASO. NLRP3 (NLRP3) is also known as containing the NLR family Pyrin domain 3. Unless otherwise indicated, the term "NLRP3" as used herein may refer to NLRP3 from one or more species (e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears). The sequence of the human NLRP3 gene can be found under the publicly available GenBank accession number NC_000001.11:247416156-247449108. The human NLRP3 gene is located at chromosomal position 1q44 at 247, 416, 156 to 247, 449, 108. The sequence of the human NLRP3 pre-mRNA transcript (SEQ ID NO: 1) corresponds to the reverse complement of residues 247, 416, 156 to 247, 449, 108 of chromosome 1q44. The NLRP3 mRNA sequence (GenBank Accession No. NM_001079821.2) is provided in SEQ ID NO: 3, except that the nucleotide "t" in SEQ ID NO: 3 is shown as "u" in the mRNA. The sequence of the human NLRP3 protein can be found under the following publicly available accession numbers: Q96P20 (canonical sequence, SEQ ID NO: 2), Q96P20-2 (SEQ ID NO: 4), Q96P20-3 (SEQ ID NO: 5), Q96P20-4 (SEQ ID NO: 6), Q96P20-5 (SEQ ID NO: 7), and Q96P20-6 (SEQ ID NO: 8), each of which is incorporated herein by reference in its entirety. The anti-NLRP3 ASOs disclosed herein can be designed to reduce or inhibit the expression of natural variants of the NLRP3 protein.

[0354] An example of a target nucleic acid sequence for an anti-NLRP3 ASO is NLRP3 pre-mRNA. SEQ ID NO: 1 represents the human NLRP3 genomic sequence (i.e., the reverse complement of nucleotides 247, 416, 156 to 247, 449, 108 of chromosome 1q44). SEQ ID NO: 1 is identical to the NLRP3 pre-mRNA sequence, except that the nucleotide "t" in SEQ ID NO: 1 is displayed as "u" in the pre-mRNA. In certain aspects, the "target nucleic acid" comprises an intron of an NLRP3 protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a pre-mRNA. In other aspects, the target nucleic acid comprises an exon region of an NLRP3 protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a pre-mRNA. In yet other aspects, the target nucleic acid comprises an exon-intron junction of an NLRP3 protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a pre-mRNA. The human NLRP3 protein sequence encoded by the NLRP3 pre-mRNA is shown in SEQ ID NO: 3. In other aspects, the target nucleic acid comprises an untranslated region of the NLRP3 protein-encoding nucleic acid or a naturally occurring variant thereof, such as the 5'UTR, the 3'UTR, or both.

[0355] In some aspects, the anti-NLRP3 ASOs of the present disclosure hybridize to regions within introns of the NLRP3 transcript (e.g., SEQ ID NO: 1). In certain aspects, the anti-NLRP3 ASOs of the present disclosure hybridize to regions within exons of the NLRP3 transcript (e.g., SEQ ID NO: 1). In other aspects, the anti-NLRP3 ASOs of the present disclosure hybridize to regions within exon-intron junctions of the NLRP3 transcript (e.g., SEQ ID NO: 1). In some aspects, the anti-NLRP3 ASOs of the present disclosure hybridize to regions within the NLRP3 transcript (e.g., SEQ ID NO: 1) (e.g., introns, exons, or exon-intron junctions), wherein the anti-NLRP3 ASOs have a gapmer design.

[0356] In some aspects, the anti-NLRP3 ASO targets the mRNA encoding a specific isoform of the NLRP3 protein (e.g., isoform 1). In some aspects, the anti-NLRP3 ASO targets all isoforms of the NLRP3 protein. In other aspects, the anti-NLRP3 ASO targets two isoforms of the NLRP3 protein (e.g., isoform 1 and isoform 2, isoform 3 and isoform 4, and isoform 5 and isoform 6).

[0357] In some aspects, the nucleotide sequence or contiguous nucleotide sequence of the anti-NLRP3 ASO of the present disclosure has at least 80% sequence identity with a sequence selected from SEQ ID NOs: 101 to 200, such as 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% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, such as about 100% sequence identity (homologous).

[0358] In some aspects, the anti-NLRP3 ASO (or a contiguous nucleotide portion thereof) is selected from or comprises: one of the sequences selected from the group consisting of SEQ ID NOs: 101 to 200, or a region of at least 10 contiguous nucleotides thereof, wherein the anti-NLRP3 ASO (or a contiguous nucleotide portion thereof) may optionally comprise one, two, three, or four mismatches compared to the corresponding NLRP3 transcript.

[0359] In some aspects, the anti-NLRP3 ASO comprises a sequence selected from the group consisting of SEQ ID NOs: 101-200.

[0360] In some aspects, the anti-NLRP3 ASO comprises a sequence as set forth in any one of SEQ ID NOs: 101 to 200. In some aspects, the anti-NLRP3 ASO comprises or consists of a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, 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 the sequence set forth in SEQ ID NOs: 101 to 200. In some aspects, the anti-NLRP3 ASO (or a contiguous nucleotide portion thereof) is selected from or comprises: one of the sequences selected from the group consisting of SEQ ID NOs: 101 to 200, or a region of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides thereof. In some aspects, the anti-NLRP3 ASO (or a contiguous nucleotide portion thereof) is selected from or comprises: one of the sequences selected from the group consisting of SEQ ID NOs: 101 to 200, or a region of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides thereof, wherein the anti-NLRP3 ASO (or a contiguous nucleotide portion thereof) may optionally comprise one, two, three, or four mismatches compared to the corresponding NLRP3 transcript. In some aspects, the anti-NLRP3 ASO (or a contiguous nucleotide portion thereof) is selected from or comprises: one of the sequences selected from the group consisting of SEQ ID NOs: 101 to 200, except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, wherein the substituted ASO can bind to the NLRP3 transcript. In some aspects, the anti-NLRP3 ASO (or a contiguous nucleotide portion thereof) is selected from or comprises: one of the sequences selected from the group consisting of SEQ ID NOs: 101 to 200, or a region of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides thereof, wherein the anti-NLRP3 ASO (or a contiguous nucleotide portion thereof) may optionally comprise one, two, three, or four additional 5' and / or 3' nucleotides that are complementary to the corresponding NLRP3 transcript.

[0361] In some aspects, binding of an anti-NLRP3 ASO targeting an NLRP3 transcript disclosed herein to an mRNA transcript encoding NLRP3 can reduce the expression level and / or activity level of NLRP3.

[0362] In some aspects, any anti-NLRP3 ASO described herein can be part of an EV (e.g., exosome) of the present disclosure, i.e., an EV (e.g., exosome) comprising a construct comprising a cleavable linker disclosed herein (e.g., a construct of Formula I), wherein the biologically active moiety BAM is an anti-NLRP3 ASO described herein, or a combination thereof.

[0363] ASO targeting STAT6

[0364] In some aspects, the bioactive molecule BAM is an anti-STAT6 ASO. STAT6 (STAT6) is also known as signal transducer and activator of transcription 6. Unless otherwise indicated, the term "STAT6" as used herein may refer to STAT6 from one or more species (e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).

[0365] The sequence of the human STAT6 pre-mRNA transcript (SEQ ID NO: 11) corresponds to the reverse complement of residues 57111413 to 57095404, the complement of chromosome 12q13.3. The STAT6 mRNA sequence (GenBank accession number NM_001178078.1) is provided in SEQ ID NO: 13, except that the nucleotide "t" in SEQ ID NO: 13 is shown as "u" in the mRNA. The sequence of the human STAT6 protein can be found under the following publicly available accession numbers: P42226-1 (canonical sequence, SEQ ID NO: 12), P42226-2 (SEQ ID NO: 14), and P42226-3 (SEQ ID NO: 15), each of which is incorporated herein by reference in its entirety.

[0366] Natural variants of human STAT6 gene products are known. For example, natural variants of human STAT6 protein may contain one or more amino acid substitutions selected from the group consisting of M118R, D419N, and any combination thereof. Additional variants of human STAT6 protein produced by alternative splicing are also known in the art. STAT6 isoform 2 (identifier on UniProt: P42226-2) differs from the canonical sequence (SEQ ID NO: 13) in that: relative to SEQ ID NO: 13, residues 1 to 174 and 175 PSE 177 Replaced by 175 MEQ 177 The sequence of STAT6 isoform 3 (identifier: P42226-3) differs from the canonical sequence (SEQ ID NO: 13) as follows: residues 1 to 110 are deleted relative to SEQ ID NO: 13. Therefore, the anti-STAT6 ASOs of the present disclosure can be designed to reduce or inhibit the expression of natural variants of STAT6 protein.

[0367] The example of the target nucleic acid sequence of anti-STAT6ASO is STAT6 precursor mRNA.SEQ ID NO:11 represents human STAT6 genomic sequence (i.e., the reverse complementary sequence of nucleotides 57111413 to 57095404, the complementary sequence of chromosome 12q13.3).SEQ ID NO:11 is identical to STAT6 precursor mRNA sequence, except that the nucleotide "t" in SEQ ID NO:11 is shown as "u" in precursor mRNA. In some aspects, "target nucleic acid" includes the introns of STAT6 protein-coding nucleic acids or its naturally occurring variants, and RNA nucleic acids derived therefrom, such as precursor mRNA. In other aspects, target nucleic acid includes the exon regions of STAT6 protein-coding nucleic acids or its naturally occurring variants, and RNA nucleic acids derived therefrom, such as precursor mRNA. In other aspects, target nucleic acid includes the exon-intron junctions of STAT6 protein-coding nucleic acids or its naturally occurring variants, and RNA nucleic acids derived therefrom, such as precursor mRNA. The human STAT6 protein sequence encoded by STAT6 pre-mRNA is shown in SEQ ID NO: 13. In other aspects, the target nucleic acid comprises an untranslated region of a STAT6 protein-encoding nucleic acid or a naturally occurring variant thereof, such as a 5'UTR, a 3'UTR, or both.

[0368] In some aspects, the anti-STAT6 ASO of the present disclosure is hybridized with the region in the intron of STAT6 transcript (such as SEQ ID NO: 11). In certain aspects, the anti-STAT6 ASO of the present disclosure is hybridized with the region in the exon of STAT6 transcript (such as SEQ ID NO: 11). In other aspects, the anti-STAT6 ASO of the present disclosure is hybridized with the region in the exon-intron junction of STAT6 transcript (such as SEQ ID NO: 11). In some aspects, the anti-STAT6 ASO of the present disclosure is hybridized with the region (such as intron, exon or exon-intron junction) in STAT6 transcript (such as SEQ ID NO: 11), wherein the anti-STAT6 ASO has a spaced polymer design.

[0369] In some aspects, anti-STAT6 ASO targets the mRNA of a specific isoform (e.g., isoform 1) of encoding STAT6 protein. In some aspects, ASO targets all isoforms of STAT6 protein. In other aspects, anti-STAT6 ASO targets two isoforms (e.g., isoform 1 and isoform 2, isoform 1 and isoform 3, or isoform 2 and isoform 3) of STAT6 protein.

[0370] In some aspects, the region of the payload (such as ASO) disclosed herein is hybridized with the intron of the STAT6 transcript. In some aspects, the region of the payload and the exon of the STAT6 transcript is hybridized. In some aspects, the region of the payload and the exon-intron junction of the STAT6 transcript is hybridized. In some aspects, the region (such as intron, exon or exon-intron junction) of the payload and the STAT6 transcript is hybridized. The non-limiting examples of the payload (such as ASO) in the region of the STAT6 transcript can be specifically targeted.

[0371] In some aspects, binding of an anti-STAT6 ASO targeting a STAT6 transcript disclosed herein to an mRNA transcript encoding STAT6 can reduce the expression level and / or activity level of STAT6.

[0372] In some aspects, any anti-STAT6 ASO described herein can be part of an EV (e.g., exosome) of the present disclosure, i.e., an EV (e.g., exosome) comprising a construct comprising a cleavable linker disclosed herein (e.g., a construct of Formula I), wherein the biologically active moiety BAM is an anti-STAT6 ASO described herein, or a combination thereof.

[0373] In some aspects, the anti-STAT6 ASO of the present disclosure comprises the base sequence of SEQ ID NO: 1091. In some aspects, the anti-STAT6 ASO of the present disclosure comprises Figure 2 The STAT 6 ASO sequence is shown in .

[0374] ASO targeting MYC

[0375] In some aspects, the biologically active molecule BAM is anti-MYC ASO. Unless otherwise indicated, the term "MYC" as used herein may refer to MYC from one or more species (e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).

[0376] In some aspects, the anti-MYCASO of the present disclosure comprises the base sequence of SEQ ID NO: 1092. In some aspects, the anti-MYCASO of the present disclosure comprises Figure 2 The MYCASO sequence shown in .

[0377] ASO targeting CEBP / β

[0378] In some aspects, the bioactive molecule BAM is an anti-CEBP / β ASO. Unless otherwise indicated, the term "CEBP / β" as used herein can refer to CEBP / β from one or more species (e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).

[0379] The sequence of the human CEBP / β gene can be found under the publicly available GenBank accession number NC_000020.11 (50190583..50192690).The human CEBP / β gene is located at chromosome position 20q13.13 from 50190583 to 50192690.

[0380] The sequence of the human CEBP / β precursor mRNA transcript (SEQ ID NO: 21) corresponds to the reverse complement of residues 50190583 to 50192690 of chromosome 20q13.13. The CEBP / β mRNA sequence (GenBank Accession No. NM_001285878.1) is provided in SEQ ID NO: 23, except that the nucleotide "t" in SEQ ID NO: 23 is shown as "u" in the mRNA. The sequence of the human CEBP / β protein can be found under the following publicly available accession numbers: P17676 (canonical sequence, SEQ ID NO: 22), P17676-2 (SEQ ID NO: 24), and P17676-3 (SEQ ID NO: 25), each of which is incorporated herein by reference in its entirety.

[0381] Natural variants of the human CEBP / β gene product are known. For example, a natural variant of the human CEBP / β protein may contain one or more amino acid substitutions selected from the group consisting of A241P, A253G, G195S, and any combination thereof. Additional variants of the human CEBP / β protein resulting from alternative splicing are also known in the art. CEBP / β isoform 2 (identifier on UniProt: P17676-2) differs from the canonical sequence (SEQ ID NO: 23) by the deletion of residues 1 to 23 relative to SEQ ID NO: 23. The sequence of CEBP / β isoform 3 (identifier: P17676-3) differs from the canonical sequence (SEQ ID NO: 23) by the deletion of residues 1 to 198 relative to SEQ ID NO: 23. Thus, the anti-CEBPb ASOs disclosed herein can be designed to reduce or inhibit the expression of natural variants of the protein.

[0382] An example of a target nucleic acid sequence for an anti-CEBPb ASO is CEBP / β precursor mRNA. SEQ ID NO: 21 represents the human CEBP / β genomic sequence (i.e., the reverse complement of nucleotides 50190583-50192690 of chromosome 20q13.13). SEQ ID NO: 21 is identical to the CEBP / β precursor mRNA sequence, except that the nucleotide "t" in SEQ ID NO: 21 is displayed as "u" in the precursor mRNA. In certain aspects, the "target nucleic acid" comprises an intron of a CEBP / β protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a precursor mRNA. In other aspects, the target nucleic acid comprises an exon region of a CEBP / β protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a precursor mRNA. In yet other aspects, the target nucleic acid comprises an exon-intron junction of a CEBP / β protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a precursor mRNA. The human CEBP / β protein sequence encoded by CEBP / β pre-mRNA is shown in SEQ ID NO: 23. In other aspects, the target nucleic acid comprises an untranslated region of a CEBP / β protein encoding nucleic acid or a naturally occurring variant thereof, such as a 5'UTR, a 3'UTR, or both.

[0383] In some aspects, the anti-CEBPb ASOs of the present disclosure hybridize to regions within introns of the CEBP / β transcript (e.g., SEQ ID NO: 21). In certain aspects, the anti-CEBPb ASOs of the present disclosure hybridize to regions within exons of the CEBP / β transcript (e.g., SEQ ID NO: 21). In other aspects, the anti-CEBPb ASOs of the present disclosure hybridize to regions within exon-intron junctions of the CEBP / β transcript (e.g., SEQ ID NO: 21). In some aspects, the anti-CEBPb ASOs of the present disclosure hybridize to regions within CEBP / β transcripts (e.g., SEQ ID NO: 21) (e.g., introns, exons, or exon-intron junctions), wherein the anti-CEBPb ASOs have a gapmer design.

[0384] In some aspects, anti-CEBPb ASOs target mRNA encoding a specific isoform of CEBP / β protein (e.g., isoform 1). In some aspects, anti-CEBPb ASOs target all isoforms of CEBP / β protein. In other aspects, anti-CEBPb ASOs target two isoforms of CEBP / β protein (e.g., isoform 1 and isoform 2, isoform 1 and isoform 3, or isoform 2 and isoform 3).

[0385] In some aspects, binding of an anti-CEBPb ASO targeting a CEBPb transcript disclosed herein to an mRNA transcript encoding CEBPb can reduce the expression level and / or activity level of CEBPb.

[0386] In some aspects, any anti-CEBPbASO described herein can be part of an EV (e.g., exosome) of the present disclosure, i.e., an EV (e.g., exosome) comprising a construct comprising a cleavable linker disclosed herein (e.g., a construct of Formula I), wherein the biologically active moiety BAM is an anti-CEBPbASO described herein, or a combination thereof.

[0387] ASO targeting STAT3

[0388] In some aspects, the bioactive molecule BAM is an anti-STAT3 ASO. Signal transducer and activator of transcription 3 (STAT3) is a signal transducer and activator of transcription that transmits signals from cell surface receptors to the cell nucleus. STAT3 is often overactivated in many human cancers.

[0389] Signal transducer and activator of transcription 3 (STAT3) is known in the art by various names. Such names include: DNA binding protein APRF and acute phase response factor. The mRNA encoding human STAT3 can be found in Genbank accession number NM_003150.3 and is represented by the sequence (SEQ ID NO:43).

[0390] Natural variants of the human STAT3 gene product are known. Therefore, the ASOs of the present disclosure can be designed to reduce or inhibit the expression of natural variants of the STAT3 protein.

[0391] SEQ ID NO: 41 is identical to the STAT3 pre-mRNA sequence, except that the nucleotide "t" in SEQ ID NO: 41 is shown as "u" in the pre-mRNA. In certain aspects, the "target nucleic acid" comprises an intron of a STAT3 protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a pre-mRNA. In other aspects, the target nucleic acid comprises an exon region of a STAT3 protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a pre-mRNA. In yet other aspects, the target nucleic acid comprises an exon-intron junction of a STAT3 protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a pre-mRNA. The human STAT3 protein sequence encoded by the STAT3 pre-mRNA is shown in SEQ ID NO: 42. In other aspects, the target nucleic acid comprises an untranslated region of a STAT3 protein-encoding nucleic acid or a naturally occurring variant thereof, such as a 5'UTR, a 3'UTR, or both.

[0392] In yet other aspects, the target nucleic acid comprises an exon-intron junction of a STAT3 protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a pre-mRNA. The human STAT3 protein sequence encoded by the STAT3 pre-mRNA is shown in SEQ ID NO: 43. In other aspects, the target nucleic acid comprises an untranslated region of a STAT3 protein-encoding nucleic acid or a naturally occurring variant thereof, such as a 5'UTR, a 3'UTR, or both.

[0393] In some aspects, the anti-STAT3 ASOs of the present disclosure hybridize to regions within introns of STAT3 transcripts (e.g., SEQ ID NO: 41 or SEQ ID NO: 43). In certain aspects, the anti-STAT3 ASOs of the present disclosure hybridize to regions within exons of STAT3 transcripts (e.g., SEQ ID NO: 41 or SEQ ID NO: 43). In other aspects, the anti-STAT3 ASOs of the present disclosure hybridize to regions within exon-intron junctions of STAT3 transcripts (e.g., SEQ ID NO: 41 or SEQ ID NO: 43). In some aspects, the anti-STAT3 ASOs of the present disclosure hybridize to regions within STAT3 transcripts (e.g., SEQ ID NO: 41 or SEQ ID NO: 43), such as introns, exons, or exon-intron junctions, wherein the anti-STAT3 ASOs have a spaced polymer design.

[0394] In some aspects, anti-STAT3 ASOs target mRNA encoding a specific isoform of STAT3 protein (e.g., isoform 1). In some aspects, ASOs target all isoforms of STAT3 protein. In other aspects, ASOs target two isoforms of STAT3 protein (e.g., isoform 1 (UniProt ID: P40763-1) and isoform 2 (UniProt ID: P40763-2), isoform 2, and isoform 3 (UniProt ID: P40763-3).

[0395] In some aspects, the anti-STAT3 ASOs of the present disclosure hybridize to regions within introns of STAT3 transcripts (e.g., SEQ ID NO: 41 or SEQ ID NO: 43). In certain aspects, the anti-STAT3 ASOs of the present disclosure hybridize to regions within exons of STAT3 transcripts (e.g., SEQ ID NO: 41 or SEQ ID NO: 43). In other aspects, the anti-STAT3 ASOs of the present disclosure hybridize to regions within exon-intron junctions of STAT3 transcripts (e.g., SEQ ID NO: 41 or SEQ ID NO: 43). In some aspects, the anti-STAT3 ASOs of the present disclosure hybridize to regions within STAT3 transcripts (e.g., SEQ ID NO: 41 or SEQ ID NO: 43), e.g., introns, exons, or exon-intron junctions, wherein the ASO has a spacer design.

[0396] In some aspects, the anti-STAT3 ASO of the present disclosure hybridizes with multiple target regions in a STAT3 transcript (e.g., genomic sequence, SEQ ID NO: 41). In some aspects, ASO hybridizes with two different target regions in a STAT3 transcript. In some aspects, anti-STAT3 ASO hybridizes with three different target regions in a STAT3 transcript. In some aspects, compared to the anti-STAT3 ASO hybridized with a single region in a STAT3 transcript (e.g., genomic sequence, SEQ ID NO: 41), the anti-STAT3 ASO hybridized with multiple regions in a STAT3 transcript (e.g., genomic sequence, SEQ ID NO: 41) is more effective (e.g., with a lower EC50) in reducing STAT3 expression.

[0397] The anti-STAT3 ASOs of the present disclosure comprise a contiguous nucleotide sequence corresponding to the complementary sequence of a region of the STAT3 transcript, for example, a nucleotide sequence corresponding to SEQ ID NO:41.

[0398] In some aspects, binding of an anti-STAT3 ASO targeting a STAT3 transcript disclosed herein to an mRNA transcript encoding STAT3 can reduce the expression level and / or activity level of STAT3.

[0399] In some aspects, any anti-STAT3 ASO described herein can be part of an EV (e.g., exosome) of the present disclosure, i.e., an EV (e.g., exosome) comprising a construct comprising a cleavable linker disclosed herein (e.g., a construct of Formula I), wherein the biologically active moiety BAM is an anti-STAT3 ASO described herein, or a combination thereof.

[0400] ASO targeting NRAS

[0401] In some aspects, the bioactive molecule BAM is an anti-NRasASO. NRas is an oncogene encoding a membrane protein that shuttles between the Golgi apparatus and the plasma membrane. NRas-encoding genomic DNA can be found at chromosome position 1p13.2 (i.e., nucleotides 5001 to 17438 of GenBank accession number NG_007572). Specifically, the combination of time-lapse microscopy and photobleaching technology has shown that, in the absence of palmitoylation, GFP-tagged N-Ras is rapidly exchanged between the cytosol and the ER / Golgi membrane, and wild-type GFP-N-Ras is recovered to the Golgi complex by a non-vesicular mechanism. N-ras mutations have been described in melanoma, thyroid cancer, teratocarcinoma, fibrosarcoma, neuroblastoma, rhabdomyosarcoma, Burkitt's lymphoma, acute promyelocytic leukemia, T-cell leukemia, and chronic myeloid leukemia. Oncogenic N-Ras can induce acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CMML)-like diseases in mice.

[0402] Neuroblastoma RAS viral oncogene (NRas) is known in the art by various names. Such names include: GTPase NRas, N-ras protein portion 4, neuroblastoma RAS viral (v-ras) oncogene homolog, neuroblastoma RAS viral oncogene homolog, transforming protein N-Ras, and v-ras neuroblastoma RAS viral oncogene homolog.

[0403] The NRAS gene provides instructions for making a protein called N-Ras, which is primarily involved in regulating cell division. The mRNA sequence encoding human NRAS can be found in NCBI reference sequence NM_002524.5 and is represented by the coding sequence (SEQ ID NO: 53).

[0404] The natural variants of people's NRas gene products are known. For example, the natural variants of people's NRas protein can contain one or more amino acid substitutions selected from: G12D, G13D, T50I, G60E and any combination thereof. The other variants of people's NRas protein produced by alternative splicing are also known in the art, such as: G13R, Q61K, Q61R and P34L. Therefore, the anti-NRas ASO of the present disclosure can be designed to reduce or inhibit the expression of the natural variants of STAT3 protein.

[0405] SEQ ID NO:51 is identical to the NRas precursor mRNA sequence, except that the nucleotide " t " in SEQ ID NO:51 is shown as " u " in the precursor mRNA. In some aspects, " target nucleic acid " comprises the intron of NRas protein-coding nucleic acid or its naturally occurring variant, and the RNA nucleic acid derived therefrom, such as precursor mRNA. In other aspects, the target nucleic acid comprises the exon region of NRas protein-coding nucleic acid or its naturally occurring variant, and the RNA nucleic acid derived therefrom, such as precursor mRNA. In other aspects, the target nucleic acid comprises the exon-intron junction of NRas protein-coding nucleic acid or its naturally occurring variant, and the RNA nucleic acid derived therefrom, such as precursor mRNA. The human NRas protein sequence encoded by NRas precursor mRNA is as shown in SEQ ID NO:52. In other aspects, the target nucleic acid comprises the untranslated region of NRas protein-coding nucleic acid or its naturally occurring variant, such as 5'UTR, 3'UTR or both.

[0406] In some aspects, the anti-NRasASO of the present disclosure can also lower (for example, reduce or remove) NRas mRNA or protein expression.In this respect, the anti-NRasASO of the present disclosure can affect the indirect inhibition of NRas protein by reducing NRasmRNA level, usually in mammalian cells (such as human cells, such as tumor cells).Especially, the present disclosure is related to the anti-NRasASO of one or more regions (such as intron region, exon region and / or exon-intron junction region) of targeting NRas precursor mRNA.Unless otherwise indicated, otherwise as used herein, term " NRas " can refer to NRas from one or more species (such as mankind, non-human primate, dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle and bear).

[0407] In some aspects, the anti-NRasASO of the present disclosure hybridizes with the region in the intron of NRAS transcript (such as SEQ ID NO:51 or SEQ ID NO:53). In some aspects, the ASO of the present disclosure hybridizes with the region in the exon of NRAS transcript (such as SEQ ID NO:51 or SEQ ID NO:53). In other aspects, the ASO of the present disclosure hybridizes with the region in the exon-intron junction of NRAS transcript (such as SEQ ID NO:51 or SEQ ID NO:53). In some aspects, the anti-NRasASO of the present disclosure hybridizes with the region (such as intron, exon or exon-intron junction) in NRAS transcript (such as SEQ ID NO:51 or SEQ ID NO:53), wherein the ASO has a spaced polymer design.

[0408] In some respects, the anti-NRasASO of the present disclosure hybridizes with multiple target regions in NRas transcript (such as genomic sequence, SEQ ID NO:51).In some respects, the anti-NRasASO hybridizes with two different target regions in NRas transcript.In some respects, the anti-NRasASO hybridizes with three different target regions in NRas transcript.In some respects, compared to the anti-NRas ASO hybridized with the single region in NRas transcript (such as genomic sequence, SEQ ID NO:51), the anti-NRas ASO hybridized with multiple regions in NRas transcript (such as genomic sequence, SEQ ID NO:51) is more effective (such as having lower EC50) in reducing NRas expression.

[0409] In some aspects, ASO targets the mRNA of the specific isoform (such as isoform 1, NCBI ID: NP_001229821.1) of coding NRAS protein. In some aspects, ASO targets all isoforms of NRAS protein. In other aspects, ASO targets two isoforms of NRAS protein (such as isoform 1 and isoform 2 (NCBI ID: NP_009089.4), isoform 2 and isoform 3 (NCBI ID: NP_001123995) and isoform 3 and isoform 4 (NCBI ID: NP_001229820.1)).

[0410] The anti-NRas ASOs of the present disclosure comprise a contiguous nucleotide sequence corresponding to the complementary sequence of a region of the NRas transcript, for example, a nucleotide sequence corresponding to SEQ ID NO: 51.

[0411] In some aspects, binding of an anti-NRas ASO targeting a NRas transcript disclosed herein to an mRNA transcript encoding NRas can reduce the expression level and / or activity level of NRas.

[0412] In some aspects, any of the anti-NRasASOs described herein can be part of an EV (e.g., exosome) of the present disclosure, i.e., an EV (e.g., exosome) comprising a construct comprising a cleavable linker disclosed herein (e.g., a construct of Formula I), wherein the biologically active moiety BAM is an anti-NRasASO described herein, or a combination thereof.

[0413] ASO targeting KRAS

[0414] In some aspects, the biologically active molecule BAM is an anti-KRASASO. The sequence of the human KRAS gene can be found at chromosome position 12p12.1 and under the publicly available GenBank accession number NC_000012 (25,204,789 to 25,250,936). The genomic sequence of the human wild-type KRAS transcript corresponds to the reverse complement of residues 25,204,789 to 25,250,936 of NC_000012 (SEQ ID NO: 35). The KRAS G12D genomic sequence provided in SEQ ID NO: 31 differs from SEQ ID NO: 35 in that it has a guanine to adenine substitution at nucleotide position 5,587. An exemplary KRAS G12D mRNA sequence is provided in SEQ ID NO: 33, except that the nucleotide "t" in SEQ ID NO: 33 is shown as "u" in the mRNA. The KRAS G12D mRNA provided in SEQ ID NO:33 differs from the wild-type mRNA sequence (e.g., GenBank Accession No. NM_004985.5; SEQ ID NO:37) in that it has a guanine to adenine substitution at nucleotide position 225. The sequence of the human KRAS protein can be found under the following publicly available accession numbers: P01116 (canonical sequence), A8K8Z5, B0LPF9, P01118, and Q96D10, each of which is herein incorporated by reference in its entirety.

[0415] Human KRAS protein (P01116) has two isoforms, resulting from alternative splicing. Isoform 2A (Accession No.: P01116-1; SEQ ID NO: 38) is the canonical sequence. It is also known as K-Ras4A. Isoform 2B (Accession No.: P01116-2; also known as K-Ras4B; SEQ ID NO: 36) differs from the canonical sequence as follows: (i) 151 to 153: RVE→GVD; and (ii) 165-189: QYRLKKISKEEKTPGCVKIKKCIIM (SEQ ID NO: 599)→KHKEKMSKDGKKKKKKSKTKCVIM (SEQ ID NO: 600). In some aspects, the anti-KRAS ASOs disclosed herein can reduce or inhibit the expression of KRAS protein isoform 2A, isoform 2B, or both.

[0416] Natural variants of human KRAS gene products are known. For example, a natural variant of the human KRAS protein may contain one or more amino acid substitutions selected from the group consisting of K5E, K5N, G10GG, G10V, G12A, G12C, G12F, G12I, G12L, G12R, G12S, G12V, G13C, G13D, G13E, G13R, G13V, V14I, L19F, T20M, Q22E, Q22H, Q22K, Q22R, Q25H, N26Y, F28L, E31K, D33E, P34L, P34Q, P34R, I36M, R41K, D57 N, T58I, A59T, G60D, G60R, G60S, G60V, Q61A, Q61H, Q61K, Q61L, Q61P, Q61R, E63K, S65N, R68S, Y71H, T74A, L79I, R97I, Q99E, M111L, K117N, K117R, D119G, S122F, T144P, A146P, A146T, A146V, K147E, K147T, R149K, L159S, I163S, R164Q, I183N, I84M, or a combination thereof. Natural variants specific to KRAS protein isoform 2B contain one or more amino acid substitutions selected from the following: V152G, D153V, F156I, F156L or a combination thereof. The anti-KRASASO disclosed herein can be designed to reduce or inhibit the expression of one or more variants of KRAS protein (e.g., any variant known in the art). In some aspects, the KRAS mutant has an amino acid substitution of G12D. In some aspects, the anti-KRAS ASO disclosed herein targets one or more KRAS mutants. In other aspects, the KRAS mutant targeted by the anti-KRAS ASO is KRAS G12D (SEQ ID NO: 32). Exemplary sequences of KRAS G12D mRNA and KRAS G12D protein are provided in SEQ ID NO: 33 and SEQ ID NO: 32.

[0417] In some aspects, the target nucleic acid sequence of the anti-KRAS ASO disclosed herein comprises one or more regions of KRAS precursor mRNA. For example, SEQ ID NO: 31 (as described above) is identical to the KRAS precursor mRNA sequence, except that the nucleotide "t" in SEQ ID NO: 31 is displayed as "u" in the precursor mRNA. As used herein, the term "target nucleic acid sequence" refers to a nucleic acid sequence complementary to the anti-KRAS ASO disclosed herein. In certain aspects, the target nucleic acid sequence comprises an exon region of a KRAS protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a precursor mRNA. In some aspects, the target nucleic acid sequence comprises an intron of a KRAS protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a precursor mRNA. In a further aspect, the target nucleic acid sequence comprises an exon-intron junction of a KRAS protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a precursor mRNA. In some aspects, for example, when used for research or diagnosis, the target nucleic acid can be a cDNA or synthetic oligonucleotide derived from a DNA or RNA nucleic acid target described herein. In some aspects, the target nucleic acid comprises an untranslated region of a KRAS protein-encoding nucleic acid or a naturally occurring variant thereof, such as the 5'UTR, 3'UTR, or both.

[0418] Thus, in some aspects, the anti-KRAS ASOs disclosed herein hybridize to exonic regions of a KRAS transcript (e.g., SEQ ID NO: 31 or SEQ ID NO: 33). In some aspects, the anti-KRAS ASOs disclosed herein hybridize to intronic regions of a KRAS transcript (e.g., SEQ ID NO: 31). In some aspects, the anti-KRAS ASOs hybridize to exon-intron junctions of a KRAS transcript (e.g., SEQ ID NO: 31). In some aspects, the anti-KRAS ASOs disclosed herein hybridize to regions within a KRAS transcript (e.g., SEQ ID NO: 31) (e.g., introns, exons, or exon-intron junctions).

[0419] In some aspects, the target nucleic acid sequence of the ASO disclosed herein is KRAS mRNA, such as SEQ ID NO: 33. Therefore, in some aspects, the anti-KRAS ASO disclosed herein can hybridize with one or more regions of KRAS mRNA. In some aspects, the anti-KRAS ASO disclosed herein targets mRNA encoding a specific isoform of KRAS protein. In some aspects, the anti-KRAS ASO disclosed herein can target all isoforms of KRAS protein, including any variants thereof (such as those described herein). In some aspects, the KRAS protein that can be targeted by the anti-KRAS ASO disclosed herein comprises a G12D amino acid substitution.

[0420] In some aspects, binding of an anti-KRAS ASO targeting a KRAS transcript disclosed herein to an mRNA transcript encoding KRAS can reduce the expression level and / or activity level of KRAS.

[0421] In some aspects, any of the anti-KRAS ASOs described herein can be part of an EV (e.g., exosome) of the present disclosure, i.e., an EV (e.g., exosome) comprising a construct comprising a cleavable linker disclosed herein (e.g., a construct of Formula I), wherein the biologically active moiety BAM is an anti-KRAS ASO described herein, or a combination thereof.

[0422] ASO targeting Pmp22

[0423] In some aspects, the bioactive molecule BAM is an anti-Pmp22 ASO. Peripheral myelin protein 22 (PMP22), also known as growth arrest-specific protein 3 (GAS-3), is encoded by the PMP22 gene. PMP22 is a 22kDa transmembrane glycoprotein consisting of 160 amino acids and is mainly expressed in Schwann cells of the peripheral nervous system. Schwann cells show high expression of PMP22, of which PMP22 can account for 2% to 5% of the total protein content of dense myelin. Dense myelin is the main part of the myelin sheath of peripheral neurons, and myelin is a protective fat layer that provides electrical insulation for neuronal axons. The level of PMP22 expression in the central nervous system of adults is relatively low.

[0424] PMP22 plays an important role in the formation and maintenance of dense myelin. When Schwann cells come into contact with neuronal axons, PMP22 expression is significantly upregulated, while PMP22 is downregulated during axonal degeneration or transection. PMP22 has been shown to associate with zonula-occludens 1 and occludin, proteins involved in adhesion to other cells and the extracellular matrix, and also supporting the function of myelin. In addition to its cell adhesion function, PMP22 is also upregulated during Schwann cell proliferation, suggesting that PMP22 plays a role in cell cycle regulation. PMP22 is detectable in non-neural tissues, where its expression has been shown to function as a growth arrest-specific (gas-3).

[0425] Improper gene dosage of the PMP22 gene can lead to abnormal myelin protein synthesis and function. Because myelin components are stoichiometrically determined, irregular expression of any component can lead to myelin instability and neurological disorders. Altered PMP22 gene expression has been associated with various neuropathies, such as Charcot–Marie–Tooth type 1A (CMT1A), Dejerine-Sottas disease, and hereditary peripheral neuropathy with pressure-proneness (HNPP). Excessive PMP22 (e.g., caused by gene duplication) causes CMT1A. PMP22 gene duplication is the most common genetic cause of CMT, where overproduction of PMP22 leads to defects in multiple signaling pathways and dysfunction of transcription factors such as KNOX20, SOX10, and EGR2.

[0426] The sequence of the human PMP22 gene can be found under the publicly available NCBI RefSeq accession number NM_000304. Alternative RefSeq mRNA transcripts have accession numbers NM_001281455, NM-001281456, NM-153321, and NM_153322, respectively. The human PMP22 gene is located at chromosome position 17p12 at 15,229,777 to 15,265,326.

[0427] The sequence of the human PMP22 pre-mRNA transcript (SEQ ID NO: 264) corresponds to the reverse complement of residues 15,229,777 to 15,265,326 of chromosomal position 17p12. The PMP22 mRNA sequence (GenBank accession number NM_000304.4) is provided in SEQ ID NO: 58. The sequence of the human PMP22 protein can be found under the publicly available Uniprot accession number Q01453 (canonical sequence, SEQ ID NO: 60). Potential PMP22 isoforms have Uniprot accession numbers A8MU75, J3KQW0, A0A2R8Y5L5, J3KT36, and J3QS08, respectively. The publicly available content of the database entries corresponding to the accession numbers disclosed herein are incorporated by reference in their entirety.

[0428] The anti-PMP22 ASOs of the present disclosure can be designed to reduce or inhibit the expression of natural variants of the PMP22 protein.

[0429] An example of a target nucleic acid sequence for an anti-PMP22 ASO is PMP22 pre-mRNA. SEQ ID NO: 58 represents the human PMP22 genomic sequence (i.e., the reverse complement of nucleotides 15229777 to 15265326, the complement of chromosome 17p12). SEQ ID NO: 58 is identical to the PMP22 pre-mRNA sequence, except that the nucleotide "t" in SEQ ID NO: 58 is represented as "u" in the pre-mRNA.

[0430] In some aspects, the anti-PMP22 ASO comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within nucleotides 1 to 1828 of the PMP22 transcript corresponding to the nucleotide sequence set forth in SEQ ID NO: 264 (PMP22 complete mRNA transcript) or nucleotides 208 to 690 of the PMP22 transcript corresponding to the nucleotide sequence set forth in SEQ ID NO: 59 (PMP22 coding sequence).

[0431] In some aspects, the contiguous nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or about 100% complementary to a nucleic acid sequence within a PMP22 transcript. In some aspects, the anti-PMP22 ASO is capable of reducing PMP22 protein expression in human cells (e.g., Schwann cells), wherein the human cells express PMP22 protein.

[0432] In some aspects, PMP22 protein expression is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% compared to PMP22 protein expression in human cells not exposed to the anti-PMP22 ASO.

[0433] In some aspects, anti-PMP22 ASO can reduce the level of PMP22 mRNA in human cells (e.g., immune cells), wherein human cells express PMP22 mRNA. In some aspects, compared to the level of PMP22 mRNA in human cells not exposed to anti-PMP22 ASO, the level of PMP22 mRNA is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or about 100%.

[0434] In certain aspects, the target nucleic acid comprises an intron of a PMP22 protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a pre-mRNA. In other aspects, the target nucleic acid comprises an exon region of a PMP22 protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a pre-mRNA. In yet other aspects, the target nucleic acid comprises an exon-intron junction of a PMP22 protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as a pre-mRNA. In some aspects, such as when used for research or diagnosis, the target nucleic acid can be a cDNA or synthetic oligonucleotide derived from the above-mentioned DNA or RNA nucleic acid target. The human PMP22 coding sequence (CDS) is shown in SEQ ID NO: 59, and the protein sequence encoded by the coding sequence in the PMP22 pre-mRNA is shown in SEQ ID NO: 60. In other aspects, the target nucleic acid comprises an untranslated region of a PMP22 protein-encoding nucleic acid or a naturally occurring variant thereof, such as a 5'UTR, a 3'UTR, or both.

[0435] In some aspects, the anti-PMP22 ASOs of the present disclosure hybridize to regions within introns of the PMP22 transcript (e.g., SEQ ID NO: 58). In certain aspects, the ASOs of the present disclosure hybridize to regions within exons of the PMP22 transcript (e.g., SEQ ID NO: 58). In other aspects, the anti-PMP22 ASOs of the present disclosure hybridize to regions within exon-intron junctions of the PMP22 transcript (e.g., SEQ ID NO: 58).

[0436] In some aspects, any anti-PMP22 ASO described herein can be part of an EV (e.g., exosome) of the present disclosure, i.e., an EV (e.g., exosome) comprising a construct comprising a cleavable linker disclosed herein (e.g., a construct of Formula I), wherein the biologically active moiety BAM is an anti-PMP22 ASO described herein, or a combination thereof.

[0437] In some aspects, the construct of formula (I) comprises an ASO targeting STAT6 as a BAM conjugated at the 5' or 3' end, and L1 comprises a tetranucleotide group, such as dTdTdTdT. In some aspects, the construct of formula (I) comprises an ASO targeting STAT6 as a BAM conjugated at the 5' or 3' end, L1 comprises an Ala-Ala-Asn peptidyl group, and SP1 or SP2 comprises a pyrophosphate acyloxy bond. In some aspects, the construct of formula (I) comprises an ASO targeting STAT6 as a BAM conjugated at the 5' or 3' end, L1 comprises a Glu-Val-Cit peptidyl group, and SP1 or SP2 comprises a silyl ether bond. In some aspects, the construct of formula (I) comprises an ASO targeting CEBP / β as a BAM conjugated at the 5' or 3' end, L1 comprises a Val-Cit peptidyl group, and SP1 or SP2 comprises a disulfide bond. In any of the foregoing examples, the anchoring moiety AM can be a sterol, such as cholesterol, including cholesterol-TEG. In any of the foregoing embodiments, SP1 and / or SP2 comprise C 2-8 an alkylene group (e.g., a C3 alkylene group, a C6 alkylene group, or a C8 alkylene group), a polyoxyalkenyl group (e.g., a polyoxyalkylene group comprising 2 to 15 repeating –OCH2CH2- units), a carbamoyl group, an amide group, a sulfosuccinimide group, a 1,2,3-triazolylbicyclononenyl group, or a combination thereof.

[0438] In some aspects, Formula (I) is a construct selected from

[0439]

[0440] as well as

[0441]

[0442] In some aspects, the EVs are exosomes, such as natural exosomes or recombinant exosomes. In some aspects where the exosomes are natural exosomes, the loading density of the ASOs attached to the exosomes is increased by at least 1.5-fold relative to the control (see above). In some aspects where the exosomes are exosomes overexpressing PTGFRN, the loading density of the ASOs attached to the exosomes is increased by at least 2-fold relative to the control, the control being, for example, a construct without a cleavable linker (such as AM-BAM or AM-SP1-BAM).

[0443] In some aspects where the exosomes are natural exosomes and the anchor moiety AM (e.g., cholesterol, tocopherol, or palmitate), the average number of ASO molecules per natural exosome is between about 500 and about 10,000. In some aspects, the average number of ASO molecules per natural exosome is between about 1,000 and about 7,000. In some aspects, the average number of ASO molecules per natural exosome is between about 700 and about 9,500, between about 800 and about 9,000, between about 850 and about 8,500, between about 900 and about 8,000, between about 950 and about 7,500, or between about 1,000 and about 7,000. In some aspects, the average number of ASO molecules per native exosome is at least 500, at least 600, at least 700, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1050, at least 1100, at least 1150, at least 1200, at least 1250, or at least 1300 and / or 10,000 or less, 9,000 or less, 8,000 or less, 7,500 or less, 7,000 or less, 6,500 or less, 6,000 or less, 5,500 or less, 5,000 or less, 4,500 or less, 4,000 or less, 3,500 or less, 3,000 or less, 2,500 or less, 2,000 or less, 1,500 or less, or 1,000 or less. In some aspects, the loading efficiency of natural exosomes is between about 70% and about 95%. In some aspects, the loading efficiency of natural exosomes is between about 70% and about 75%, between about 75% and about 80%, between about 80% and about 85%, between about 85% and about 90%, or between about 90% and about 95%. In some aspects, the loading efficiency of natural exosomes is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0444] In some aspects where the exosomes are Scaffold-X exosomes and the anchoring moiety AM is cholesterol, the average number of ASO molecules per exosome is 2442 + / - 339. In some aspects, the average number of ASO molecules per Scaffold-X exosome is between about 2000 and about 3000. In some aspects, the average number of ASO molecules per Scaffold-X exosome is between about 2000 and about 2100, between about 2100 and about 2200, between about 2200 and about 2300, between about 2300 and about 2400, between about 2400 and about 2500, between about 2500 and about 2600, between about 2600 and about 2700, between about 2700 and about 2800, between about 2800 and about 2900, or between about 2900 and about 3000. In some aspects, the average number of ASO molecules per Scaffold-X exosome is at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, at least 2500, at least 2600, at least 2700, at least 2800, at least 2900, or at least 3000. In some aspects, the loading efficiency of the Scaffold-X exosomes is between 27% and 46%. In some aspects, the loading efficiency of the Scaffold-X exosomes is between about 25% and about 50%. In some aspects, the loading efficiency of the Scaffold-X exosomes is between about 25% and about 30%, between about 30% and about 35%, between about 35% and about 40%, between about 40% and about 45%, or between about 45% and about 50%. In some aspects, the loading efficiency of the Scaffold-X exosomes is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0445] In some aspects, the average number of ASO molecules per Scaffold-X exosome is greater than 5000, greater than 6000, greater than 7000, greater than 8000, greater than 9000, greater than 10,000, greater than 11,000, greater than 12,000, greater than 13,000, greater than 14,000, greater than 15,000, greater than 16,000, greater than 17,000, greater than 18,000, greater than 19,000, or greater than 20,000. In some aspects, the average number of ASO molecules per Scaffold-X exosome is between about 5000 and about 6,000, about 6,000 and about 7,000, about 7,000 and about 8,000, about 8,000 and about 9,000, about 9,000 and about 10,000, about 10,000 and about 11,000, about 11,000 and about 12,000, about Between about 12,000 and about 13,000, about 13,000 and about 14,000, about 14,000 and about 15,000, about 15,000 and about 16,000, about 16,000 and about 17,000, about 17,000 and about 18,000, about 18,000 and about 19,000, or about 19,0000 and about 20,000.

[0446] Preparation method

[0447] The present disclosure provides a method of attaching a bioactive molecule BAM to an EV (e.g., an exosome), the method comprising linking an anchoring moiety AM to the EV, wherein the anchoring moiety AM is attached to the bioactive moiety BAM according to Formula I:

[0448] AM-SP1-L1-SP2-BAM (Formula 1),

[0449] Wherein BAM is a biologically active molecule; AM is an anchoring moiety; L1 is a cleavable bond comprising: a polynucleotide group; a peptide group selected from alanine-alanine-asparagine, valine-glycine, glycine-glycine, glutamic acid-valine-citrulline, aspartic acid-valine-citrulline, serine-valine-citrulline, lysine-valine-citrulline, glycine-glycine-glycine-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate acyloxy group; or a silyl ether; and SP1 is an optional first spacer; and SP2 is an optional second spacer. In some aspects, SP1 is present and comprises C3, C6, TEG, or HEG. In some aspects, SP2 is present and comprises C3, C6, TEG, or HEG. In some aspects, BAM is an antisense oligonucleotide (ASO), such as a gapmer.

[0450] The present disclosure also provides a method for increasing the loading density of a biologically active molecule BAM attached to an EV, the method comprising screening a library of anchoring moieties AM attached to the biologically active moiety BAM according to Formula I:

[0451] AM-SP1-L1-SP2-BAM (Formula 1),

[0452] Wherein BAM is a biologically active molecule; AM is an anchoring moiety; L1 is a cleavable bond comprising: a polynucleotide group; a peptide group selected from alanine-alanine-asparagine, valine-glycine, glycine-glycine, glutamic acid-valine-citrulline, aspartic acid-valine-citrulline, serine-valine-citrulline, lysine-valine-citrulline, glycine-glycine-glycine-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate acyloxy group; or a silyl ether; and SP1 is an optional first spacer; and SP2 is an optional second spacer. In some aspects, SP1 is present and comprises C3, C6, TEG, or HEG. In some aspects, SP2 is present and comprises C3, C6, TEG, or HEG. In some aspects, BAM is an antisense oligonucleotide (ASO), such as a gapmer. In some aspects, the loading density of the bioactive molecule BAM attached to EVs (e.g., exosomes) is increased by at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least about 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, or at least 10-fold relative to a control. In some aspects, the control is a corresponding construct lacking the cleavable linker L1. For example, in some aspects, a control for a construct having the structure AM-SP1-L1-SP2-BAM is a control construct having the structure AM-BAM or AM-SP1-BAM.

[0453] The EVs (e.g., exosomes) of the present disclosure can be produced by chemical synthesis, recombinant DNA technology, biochemical or enzymatic fragmentation of larger molecules, a combination of the foregoing methods, or by any other method. In one aspect, the present disclosure provides a method for attaching a biologically active molecule to an EV (e.g., exosome) via a cleavable linker disclosed herein, e.g., via solid phase synthesis or conjugation.

[0454] Exosome production: In some aspects, the EVs disclosed herein (e.g., exosomes) can be produced from cells grown in vitro or from body fluids of a subject. When producing exosomes from in vitro cell culture, various production cells can be used, such as HEK293 cells, CHO cells, and MSCs. In some aspects, the production cells are not dendritic cells, macrophages, B cells, mast cells, neutrophils, Kupffer cells, cells derived from any of these cells, or any combination thereof.

[0455] In certain aspects, the producer cells are HEK293 cells. Human embryonic kidney 293 cells, also commonly referred to as HEK 293, HEK-293, 293 cells, or less accurately as HEK cells, are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture.

[0456] A comprehensive study of the genome and transcriptome of HEK 293 and five derived cell lines compared the HEK 293 transcriptome with that of human kidney, adrenal gland, pituitary gland, and central nervous system tissues. The HEK 293 pattern most closely resembles that of adrenal gland cells, which share many neuronal properties. HEK 293 cells have a complex karyotype, with two or more copies of each chromosome and a modal chromosome number of 64. They are described as hypotriploid, containing less than three times the number of chromosomes as haploid human gametes. Chromosomal abnormalities include a total of three copies of the X chromosome and four copies of chromosomes 17 and 22. Variants of HEK293 cells that can be used for EV production include, but are not limited to, HEK 293F, HEK293FT, and HEK 293T.

[0457] Solid phase synthesis: solid phase synthesis known in the art can be used additionally or alternatively to produce constructs disclosed in the present application. In some aspects, two or more components of the cleavable linker disclosed herein can be attached to each other (e.g., in series) using solid phase synthesis. For example, ASO can be synthesized, and different spacers or combinations thereof can be added to ASO via conventional synthesis steps. Spacers such as C3-phosphoramidites, TEG-phosphoramidites or HEG-phosphoramidites can be used. In some aspects, the combination of spacers or spacers can be further extended to incorporate a membrane anchor portion via synthesis. In an example, phosphoramidites can be used to produce optimized linkers of the present disclosure via solid phase synthesis, such as octyl-tocopheryl phosphoramidite, tocopheryl phosphoramidite, palmitate-C6 phosphoramidite, cholesterol-TEG phosphoramidite or cholesterol-C6 phosphoramidite. Suitable solid-phase techniques, including automated synthesis techniques, are described, for example, in F. Eckstein (ed.), Oligonucleotides and Analogues, a Practical Approach, Oxford University Press, New York (1991) and Toy, PH; Lam, Y (eds.), Solid-Phase Organic synthesis, concepts, Strategies, and Applications, John Wiley & Sons, Inc. New Jersey (2012).

[0458] Conjugation: In some aspects, two or more components of the linkers disclosed herein can be attached to each other (e.g., in series) using conjugation. In addition to amine-reactive compounds, compounds with chemical groups that form bonds with sulfhydryl (-SH) groups can also be used as crosslinkers and modification reagents for proteins and other bioconjugation techniques. Sulfhydryl groups, also known as thiols, are present in the side chains of cysteine (Cys, C) amino acids in proteins.

[0459] Thiol groups are useful targets for protein conjugation and labeling. First, thiol groups are present in most proteins, but in smaller quantities than primary amines; therefore, cross-linking via thiol groups is more selective and precise. Second, thiol groups in proteins are often involved in disulfide bonds, so cross-linking at these sites generally does not significantly alter the underlying protein structure or block binding sites. Third, the number of available (i.e., free) thiol groups can be easily controlled or modified; they can be generated by reducing native disulfide bonds, or they can be introduced into molecules by reacting with primary amines using thiol addition reagents such as 2-iminothiolane (Traut's reagent), N-succinimidyl S-acetylthioacetate (SATA), N-succinimidyl S-acetylthiopropionate (SATP), or N-succinimidyl S-acetyl (thiotetraethylene glycol) (SAT(PEG)). Finally, combining thiol-reactive groups with amine-reactive groups to prepare heterobifunctional cross-linkers provides greater flexibility and control over the cross-linking procedure. For example, using 3-maleimido-propionic acid N-hydroxysuccinimide (NHS) ester, which contains a maleimide group and an NHS ester, NHS esters can be used to label primary amines (-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The maleimide group will react with a thiol group to form a covalent bond, thereby enabling the attachment of biomolecules to thiols.

[0460] When the pH of the reaction mixture is between 6.5 and 7.5, the maleimide group reacts specifically with the sulfhydryl group; the result is the formation of an irreversible, stable thioether bond (i.e., the bond cannot be cleaved by a reducing agent). Under more alkaline conditions (pH>8.5), the reaction favors primary amines and also increases the rate at which the maleimide group hydrolyzes to non-reactive maleamic acid. Maleimide does not react with tyrosine, histidine, or methionine.

[0461] Thiol-containing compounds, such as dithiothreitol (DTT) and β-mercaptoethanol (BME), must be excluded from reaction buffers used with maleimides because they compete for coupling sites. For example, if DTT is used to reduce disulfide bonds in proteins to make sulfhydryl groups available for conjugation, the DTT must be thoroughly removed using a desalting column before initiating the maleimide reaction. Interestingly, the disulfide reducing agent tris(2-carboxyethyl)phosphine (TCEP) does not contain thiols and does not need to be removed before reactions involving maleimide reagents.

[0462] Excess maleimide can be quenched at the end of the reaction by adding a free thiol. Ethylenediaminetetraacetic acid (EDTA) can be included in the coupling buffer to chelate stray divalent metals that would otherwise promote oxidation of the (non-reactive) sulfhydryl groups.

[0463] In one aspect, the ligation comprises treating the EV (e.g., exosome) with a reducing agent. Suitable reducing agents include, for example, TCEP (tris(2-carboxyethyl)phosphine), DTT (dithiothreitol), BME (2-mercaptoethanol), a thiolation agent, and any combination thereof. The thiolation agent may include, for example, Traut's reagent (2-iminothiolane).

[0464] After treatment with a reducing agent, the ligation reaction further comprises contacting the reduced EV (e.g., exosome) with a maleimide moiety. In one aspect, the maleimide moiety is connected to the bioactive molecule before being connected to the EV (e.g., exosome). In some aspects, the maleimide moiety is further attached to a linker to connect the maleimide moiety to the bioactive molecule. Thus, in some aspects, one or more linkers or spacers are inserted between the maleimide moiety and the bioactive molecule.

[0465] Any anchoring moiety AM, spacer SP or spacer combination disclosed herein, or biologically active molecule BAM can be conjugated to a reactive moiety, such as an amino reactive moiety (e.g., NHS-ester, p-nitrophenol, isothiocyanate, isocyanate or aldehyde), a thiol reactive moiety (e.g., acrylate, maleimide or pyridyl disulfide), a hydroxyl reactive moiety (e.g., isothiocyanate or isocyanate), a carboxylic acid reactive moiety (e.g., epoxide) or an azide reactive moiety (e.g., alkyne). Reactive groups include carboxyl, activated ester, sulfonyl halide, sulfonate, isocyanate, isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amine, acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxylamine, alkyl halide, imidazole, pyridine, phenol, alkyl sulfonate, halotriazine, imino ester, maleimide, hydrazide, hydroxyl and photoreactive azidoaryl groups. As understood in the art, activated esters generally include esters of succinimidyl, benzotriazolyl, or aryl substituted with electron withdrawing groups such as sulfo, nitro, cyano, or halide; or carboxylic acids activated with carbodiimides.

[0466] Exemplary reactive groups that can be used to covalently bind two components disclosed herein (e.g., an anchoring moiety AM and a spacer SP, two spacers SP, a spacer SP and a biologically active molecule BAM, or an anchoring moiety AM and a biologically active moiety BAM) via chemical conjugation include, for example, N-succinimidyl-3-(2-pyridyldithio) propionate, N-4-maleimidobutyric acid, S-(2-pyridyldithio) cysteamine, iodoacetoxysuccinimide, N-(4-maleimidobutyryloxy) succinimide, N-[5-(3'-maleimidopropionamide)-1-carboxypentyl] iminodiacetic acid, N-(5-aminopentyl) iminodiacetic acid, and 1'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. Bifunctional linkers (linkers containing two reactive groups) can also be used.

[0467] In some aspects, the anchoring moiety AM, the spacer SP, or the bioactive molecule BAM can comprise a terminal oxyamino group (-ONH2), a hydrazine group (-NHNH2), a sulfhydryl group (i.e., SH or thiol), or an olefin (e.g., -CH=CH2). In some aspects, the anchoring moiety AM, the spacer SP, or the bioactive molecule BAM can comprise, for example, an electrophilic moiety such as an aldehyde, an alkyl halide, a mesylate, a tosylate, a p-nitrobenzenesulfonate, or a p-bromobenzenesulfonate, or an activated carboxylate (e.g., an NHS ester, a phosphoramidite, or a pentafluorophenyl ester) at the terminal position.

[0468] As used herein, term "blocking group" refers to an unstable chemical moiety for protecting reactive groups known in the art, including but not limited to hydroxyl, amino and thiol groups, to prevent undesirable reactions from occurring during building-up. Blocking group is typically selectively and / or orthogonally used for protecting sites during reactions at other reaction sites, and can then be removed to leave unprotected groups as is or can be used for further reactions. Blocking groups as known in the art are generally described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).

[0469] In addition, the various synthetic steps can be carried out in an alternating order or sequence to obtain the desired compound. Synthetic chemical transformations 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 R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. Wuts, Protective Groups in Organic Synthesis, 2nd edition, 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.

[0470] Amine reactive portion: In some aspects, the reactive portion is an amine reactive portion. As used herein, the term "amine reactive portion" refers to a chemical group that can react with a reactive group having an amino portion, such as a primary amine. Exemplary amine reactive portions are N-hydroxysuccinimide esters (NHS-esters), p-nitrophenol, isothiocyanates, isocyanates, and aldehydes. Alternative reactive portions that react with primary amines are also well known in the art. In some aspects, the amine reactive portion can be attached to the terminal position of the anchor portion AM, spacer SP, or bioactive molecule BAM disclosed herein. In some aspects, the amine reactive portion is an NHS-ester. Typically, the NHS-ester reactive portion reacts with the primary amine of the reactive group to produce a stable amide bond and N-hydroxysuccinimide (NHS). In some aspects, the amine reactive portion is a p-nitrophenol group. Typically, the p-nitrophenol reactive portion is an activated carbamate that reacts with the primary amine of the reactive group to produce a stable carbamate portion and p-nitrophenol. In some aspects, the amine reactive moiety is an isothiocyanate. Typically, isothiocyanate reacts with the primary amine of the reactive group to produce a stable thiourea moiety. In some aspects, the amine reactive moiety is an isocyanate. Typically, isocyanate reacts with the primary amine of the reactive group to produce a stable urea moiety. In some aspects, the amine reactive moiety is an aldehyde. Typically, aldehyde reacts with the primary amine to form a Schiff base, which can be further reduced to form a covalent bond by reductive amination.

[0471] Thiol-reactive moiety: In some aspects, the reactive moiety is a thiol-reactive moiety. As used herein, the term "thiol-reactive moiety" refers to a chemical group that can react with a reactive group having a thiol moiety (or sulfhydryl group). Exemplary thiol-reactive moieties are acrylates, maleimides, and pyridyl disulfides. Alternative reactive moieties that react with thiols are also well known in the art. In some aspects, the thiol-reactive moiety can be attached to the terminal position of the anchoring moiety AM, spacer SP, or bioactive molecule BAM disclosed herein. In some aspects, the thiol-reactive moiety is an acrylate. Typically, acrylates react with thiols on the carbonyl β-carbon of acrylates to form stable sulfide bonds. In some aspects, the thiol-reactive moiety is maleimide. Typically, maleimide reacts with thiols on the β-carbon or carbonyl groups to form stable sulfide bonds. In some aspects, the thiol-reactive moiety is pyridyl disulfide. Typically, pyridyl disulfides react with thiols on the sulfur atom at the β position of the pyridyl group to form a stable disulfide bond and pyridine-2-thione.

[0472] Hydroxyl reactive moiety: In some aspects, the reactive moiety is a hydroxyl reactive moiety. As used herein, the term "hydroxyl reactive moiety" refers to a chemical group that can react with a reactive group having a hydroxyl moiety. Exemplary hydroxyl reactive moieties are isothiocyanates and isocyanates. Alternative reactive moieties that react with hydroxyl moieties are also well known in the art. In some aspects, the hydroxyl reactive moiety can be attached to the terminal position of the anchoring moiety AM, spacer SP, or bioactive molecule BAM disclosed herein. In some aspects, the hydroxyl reactive moiety is an isothiocyanate. Typically, an isothiocyanate reacts with the hydroxyl group of the reactive group to produce a stable aminothioate moiety. In some aspects, the amine reactive moiety is an isocyanate. Typically, an isocyanate reacts with the hydroxyl group of the reactive group to produce a stable carbamate moiety.

[0473] Carboxylic acid reactive portion: In some aspects, the reactive portion is a carboxylic acid reactive portion. As used herein, the term "carboxylic acid reactive portion" refers to a chemical group that can react with a reactive group having a carboxylic acid portion. An exemplary carboxylic acid reactive portion is an epoxide. Alternative reactive portions that react with carboxylic acid moieties are also well known in the art. In some aspects, the carboxylic acid reactive portion can be attached to the terminal position of the anchoring portion AM, spacer SP, or bioactive molecule BAM disclosed herein. In some aspects, the carboxylic acid reactive portion is an epoxide. Typically, the epoxide reacts with the carboxylic acid of the reactive group on any carbon atom of the epoxide to form a 2-hydroxyethyl acetate portion.

[0474] Azide reactive portion: In some aspects, the reactive portion is an azide reactive portion. As used herein, the term "azide reactive portion" refers to a chemical group that can react with a reactive group having an azide portion. An exemplary azide reactive portion is an alkyne. Alternative reactive portions that react with the azide portion are also well known in the art. In some aspects, the carboxylic acid reactive portion can be attached to the terminal position of the anchor portion AM, spacer SP, or bioactive molecule BAM of the present disclosure. In some aspects, the azide reactive portion is an alkyne. Typically, alkynes react with the azide of the reactive group by a 1,3-dipolar cycloaddition reaction (also known as "click chemistry") to form a 1,2,3-triazole portion.

[0475] In some aspects, the exosomes disclosed herein can be prepared and / or stored under conditions that maintain exosome stability and / or promote higher loading density. In some aspects, exosomes comprising an ASO attached to its surface via a membrane-anchored construct of Formula I can be maintained in a low salt buffer (e.g., comprising about 150 mM NaCl) for about 2, 4, 6, or 8 days. In some aspects, exosomes comprising an ASO attached to its surface via a membrane-anchored construct of Formula I comprising cholesterol as an anchoring moiety can be maintained in a low salt buffer, a high salt buffer, or a high salt buffer (e.g., comprising about 150 mM NaCl) further comprising sucrose at 4° C. or 25° C. for about 2, 4, 6, or 8 days.

[0476] The present disclosure also provides a method for increasing the loading density of exosomes, the method comprising loading exosomes under high salt conditions (e.g., using a high salt buffer comprising about 150 mM NaCl). In some aspects, the exosomes are loaded with an ASO attached (e.g., covalently attached) via a membrane-anchored construct of Formula I.

[0477] Therapeutic uses

[0478] The present disclosure provides a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising EVs (e.g., exosomes) of the present disclosure. The present disclosure also provides a method for preventing or ameliorating the symptoms of a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising EVs (e.g., exosomes) of the present disclosure. The present disclosure also provides a method for diagnosing a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising EVs (e.g., exosomes) of the present disclosure.

[0479] The present disclosure also provides a method for preventing and / or treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an EV (e.g., exosome) of the present disclosure. In some aspects, the diseases or conditions treatable by the disclosed methods include cancer, graft-versus-host disease (GvHD), autoimmune diseases, infectious diseases, fibrotic diseases, inflammatory diseases, neurodegenerative disorders, central nervous system diseases, muscular dystrophy, or metabolic diseases. In some aspects, the treatment is preventive. In other aspects, the EV (e.g., exosome) of the present disclosure is used to induce an immune response. In other aspects, the EV (e.g., exosome) of the present disclosure is used to vaccinate a subject.

[0480] In some aspects, the disease or illness is cancer. When applied to a subject with cancer, in some aspects, EV (e.g., exosomes) disclosed herein can raise the immune response and enhance the tumor targeting of the subject's immune system. In some aspects, the cancer treated is characterized by leukocytes (T cells, B cells, macrophages, dendritic cells, monocytes) infiltrating into the tumor microenvironment, or so-called "hot tumors" or "inflammatory tumors". In some aspects, the cancer treated is characterized by low or undetectable levels of leukocytes infiltrating into the tumor microenvironment, or so-called "cold tumors" or "non-inflammatory tumors". In some aspects, EV (e.g., exosomes) are administered in an amount and time sufficient to convert "cold tumors" into "hot tumors", i.e., administration causes leukocytes (such as T cells) to infiltrate into the tumor microenvironment. In some aspects, cancer includes bladder cancer, cervical cancer, renal cell carcinoma, breast cancer, prostate cancer, testicular cancer, colorectal cancer, lung cancer, head and neck cancer, ovarian cancer, lymphoma, pancreatic cancer, liver cancer, glioblastoma, melanoma, myeloma, leukemia or a combination thereof. In other aspects, the term "distant tumor" refers to a tumor that has spread from the original (or primary) tumor to a distal organ or distal tissue, such as a lymph node. In some aspects, EVs (e.g., exosomes) disclosed herein can treat tumors after metastatic spread.

[0481] In some aspects, the disease or disorder is graft-versus-host disease (GvHD).

[0482] In some aspects, the disease or condition treatable by the present disclosure is an autoimmune disease. Non-limiting examples of autoimmune diseases include: multiple sclerosis, peripheral neuritis, Sjogren's syndrome, rheumatoid arthritis, alopecia, autoimmune pancreatitis, Behçet's disease, bullous pemphigoid, diarrheal celiac disease, Devic's disease (neuromyelitis optica), glomerulonephritis, IgA nephropathy, various vasculitis, scleroderma, diabetes, arteritis, vitiligo, ulcerative colitis, irritable bowel syndrome, psoriasis, uveitis, systemic lupus erythematosus, and combinations thereof.

[0483] In some aspects, the disease or condition treatable by the present disclosure is an inflammatory disease. Non-limiting examples of inflammatory diseases include inflammation, fatty liver disease, endometriosis, type I diabetes, type II diabetes, inflammatory bowel disease, asthma, rheumatoid arthritis, psoriatic arthritis, gouty arthritis, obesity, chronic peptic ulcer, ulcerative colitis, sinusitis, active hepatitis, psoriasis, chronic obstructive pulmonary disease (COPD), allergies, bronchitis, and appendicitis.

[0484] In some aspects, the disease or condition treatable by the present disclosure is a central nervous system (CNS) disease. Non-limiting examples of CNS diseases include Alzheimer's disease, Bell's palsy, cerebral palsy, epilepsy, motor neuron disease, multiple sclerosis, neurofibromatosis, Parkinson's disease, sciatica, herpes zoster, stroke, transient ischemic attack, subdural hemorrhage, hematoma, meningitis, encephalitis, poliomyelitis, epidural abscess, cervical spondylosis, carpal tunnel syndrome, peripheral neuropathy, Guillain-Barré syndrome, headache, neuralgia, amyotrophic lateral sclerosis, and Huntington's disease.

[0485] In some aspects, the disease or condition treatable with the present disclosure is a fibrotic disease. Non-limiting examples of fibrotic diseases include pulmonary fibrosis, liver fibrosis, cardiac fibrosis, mediastinal fibrosis, myelofibrosis, skin fibrosis, scleroderma, retroperitoneal fibrosis, and keloids.

[0486] In some aspects, disease or illness are infectious diseases. In some aspects, disease or illness are oncogenic viruses. In some aspects, the infectious diseases for which the disclosure can be used for treatment include but are not limited to human gamma herpes virus 4 (Epstein Barr virus), influenza A virus, influenza B virus, cytomegalovirus, Staphylococcus aureus, Mycobacterium tuberculosis, Chlamydia trachomatis, HIV-1, HIV-2, coronavirus (such as MERS-CoV and SARS CoV), filovirus (such as Marburg virus and Ebola virus), Streptococcus pyogenes, Streptococcus pneumoniae, Plasmodium species (such as Plasmodium vivax and Plasmodium falciparum), Chikungunya virus, human papillomavirus (HPV), hepatitis B, hepatitis C, human herpes virus 8, herpes simplex virus 2 (HSV2), Klebsiella, Pseudomonas aeruginosa, Enterococcus, Proteus, Enterobacter, Actinomycetes (actinomycetes) belong to, coagulase negative staphylococci (CoNS), Mycoplasma or its combination.

[0487] In some aspects, diseases or conditions treatable by the methods of the invention include Pompe disease, Gaucher disease, lysosomal storage diseases, myxovirus diseases, cystic fibrosis, Duchenne muscular dystrophy, Becker muscular dystrophy, transthyretin amyloidosis, hemophilia A, hemophilia B, adenosine deaminase deficiency, Leber congenital amaurosis, X-linked adrenoleukodystrophy, metachromatic leukodystrophy, ornithine transcarbamylase (OTC) deficiency, glycogen storage disease 1A, Criggler-Najjar syndrome, primary Hyperoxaluria type 1, acute intermittent porphyria, phenylketonuria, familial hypercholesterolemia, mucopolysaccharidosis type VI, alpha 1 antitrypsin deficiency, Rett syndrome, Dravet syndrome, Angelman syndrome, DM1 disease, fragile X disease, Huntington disease, Friedreich's ataxia, Charcot-Marie-Tooth (CMT) disease (also known as hereditary motor sensory neuropathy (HMSN) or Charcot-Marie-Tooth 1X disease), CMT1X (Charcot-Marie-Tooth type 1X) disease, catecholaminergic polymorphic ventricular tachycardia, spinocerebellar ataxia type 3 (SCA3) disease, limb-girdle muscular dystrophy, or hypercholesterolemia. In some aspects, treatment of such diseases or conditions is prophylactic.

[0488] In some aspects, the disease or disorder is a neurodegenerative disease. In some aspects, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, prion disease, motor neuron disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, and any combination thereof.

[0489] In some aspects, the disease or condition comprises a muscular dystrophy. In some aspects, the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy (DMD), myotonic dystrophy, facioscapulohumeral muscular dystrophy (FSHD), congenital muscular dystrophy, limb-girdle muscular dystrophy (including but not limited to LGMD2B, LGMD2D, LGNMD2L, LGMD2C, LGMD2E, and LGMD2A), and any combination thereof.

[0490] In some aspects, the disease or condition is selected from aromatic L-amino acid decarboxylase (AADC) deficiency (CNS), adenosine deaminase severe combined immunodeficiency (ADA-SCID), alpha-1 antitrypsin deficiency, beta-thalassemia (severe sickle cell anemia), cancer (head and neck squamous cell), Niemman-Pick C disease, cerebral ALD, choroideremia, congestive heart failure, cystic fibrosis, Duchenne muscular dystrophy (DMD), Fabry disease, glaucoma, glioma (cancer), hemophilia A, hemophilia B, HoFH (hypercholesterolemia), Huntington's disease, lipoprotein lipase deficiency, Leber hereditary optic neuropathy (LHON), metachromatic leukodystrophy, mucopolysaccharidosis type I (MPS I - Hurler syndrome), mucopolysaccharidosis type II (MPS II - Hunter syndrome), mucopolysaccharidosis type III (MPS III-Sanfilippo syndrome), mucopolysaccharidosis type IIIA (MPS IIIA), Parkinson's disease, Pompe disease, recessive dystrophic epidermolysis bullosa, RPE65 gene defect (vision loss), spinal muscular atrophy (SMAI), wet age-related macular degeneration (wet AMD), Wiskott-Aldrich syndrome (WAS), X-linked myotubular myopathy, X-linked retinitis pigmentosa, and any combination thereof.

[0491] Pharmaceutical compositions and methods of administration

[0492] The present disclosure also provides a pharmaceutical composition comprising the EVs (e.g., exosomes) of the present disclosure, which is suitable for administration to a subject. The pharmaceutical composition typically comprises a plurality of EVs (e.g., exosomes), the plurality of EVs comprising a bioactive molecule covalently linked to the plurality of EVs (e.g., exosomes) via a cleavable linker disclosed herein and a pharmaceutically acceptable excipient or carrier in a form suitable for administration to a subject. The pharmaceutically acceptable excipient or carrier is determined in part by the specific composition being administered and by the specific method used to administer the composition. Thus, there are a variety of suitable formulations for pharmaceutical compositions comprising a plurality of EVs (e.g., exosomes). See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th edition (1990). Pharmaceutical compositions are typically formulated as sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. In some aspects, the pharmaceutical composition comprises one or more chemical compounds, such as, for example, small molecules covalently linked to the EVs (e.g., exosomes) of the present disclosure.

[0493] The present disclosure provides a pharmaceutical composition comprising EVs (e.g., exosomes) of the present disclosure having a desired purity and a pharmaceutically acceptable carrier or excipient in a form suitable for administration to a subject. The pharmaceutically acceptable excipient or carrier can be determined in part by the specific composition being administered and by the specific method for administering the composition. Thus, there are a variety of suitable formulations of pharmaceutical compositions comprising multiple extracellular vesicles. (See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st edition (2005)). Pharmaceutical compositions are typically formulated as sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0494] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g., about 10 or fewer amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone ketones; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polysorbates (e.g., polysorbate 20, polysorbate 80), poloxamers (e.g., block copolymers composed of hydrophilic poly(ethylene oxide) (PEO) and hydrophobic poly(propylene oxide) (PPO), arranged in an ABA triblock structure to form PEO-PPO-PEO, such as PLURONIC TM those sold as such), or polyethylene glycol (PEG).

[0495] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except in the case where any conventional media or compounds are incompatible with the extracellular vesicles described herein, the use of such media or compounds in the compositions is contemplated.

[0496] Typically, the pharmaceutical composition is formulated to be compatible with its intended route of administration. The EVs (e.g., exosomes) disclosed herein can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intraperitoneally, intranasally, intratumorally, intrathecally, intraocularly, intramuscularly, or as an inhalant. In some aspects, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered intravenously, for example, by injection.

[0497] In some aspects, EV (e.g., exosomes) are administered intravenously to the circulatory system of the subject. In some aspects, EV (e.g., exosomes) are infused in a suitable liquid and administered to the vein of the subject. In some aspects, EV (e.g., exosomes) are administered intra-arterially to the circulatory system of the subject. In some aspects, EV (e.g., exosomes) are infused in a suitable liquid and administered to the artery of the subject. In some aspects, EV (e.g., exosomes) are administered to the subject by intrathecal administration. In some aspects, EV (e.g., exosomes) are administered via injection into the spinal canal or into the subarachnoid space so that they reach the cerebrospinal fluid (CSF). In some aspects, EV (e.g., exosomes) are administered intratumorally to one or more tumors of the subject. In some aspects, EV (e.g., exosomes) are administered to the subject by intranasal administration. In some aspects, EV (e.g., exosomes) can be blown into the nose in the form of local administration or systemic administration. In some aspects, EV (e.g., exosomes) are administered as nasal spray. In some aspects, EV (e.g., exosomes) are administered to a subject via intraperitoneal administration. In some aspects, EV (e.g., exosomes) are infused in a suitable liquid and injected into the peritoneum of the subject. In some aspects, intraperitoneal administration causes EV (e.g., exosomes) to be distributed to the lymphatic vessels. In some aspects, intraperitoneal administration causes EV (e.g., exosomes) to be distributed to the thymus, spleen, and / or bone marrow. In some aspects, intraperitoneal administration causes EV (e.g., exosomes) to be distributed to one or more lymph nodes. In some aspects, intraperitoneal administration causes EV (e.g., exosomes) to be distributed to one or more of the cervical lymph nodes, inguinal lymph nodes, mediastinal lymph nodes, or sternal lymph nodes. In some aspects, intraperitoneal administration causes EV (e.g., exosomes) to be distributed to the pancreas. In some aspects, EV (e.g., exosomes) are administered to a subject via periocular administration. In some aspects, EV (e.g., exosomes) are injected into periocular tissue. Periocular drug administration includes subconjunctival, anterior subTenon's, posterior subTenon's, and retrobulbar routes.

[0498] The solution or suspension may contain the following components: a sterile diluent such as water, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; an antibacterial compound such as benzyl alcohol or methyl paraben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating compound such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate or phosphate, and a compound for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The formulation can be sealed in an ampoule, disposable syringe or multiple-dose vial made of glass or plastic.

[0499] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water soluble) or dispersions and sterile powders. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, mixtures of polyoxyethylated triglycerides prepared by reacting ethylene oxide with castor oil (e.g., Cremophor EL TM (BASF, Parsippany, NJ)) or phosphate buffered saline (PBS). The composition is typically sterile and fluid to the extent that it is easy to inject. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol or liquid polyethylene glycol, etc.) and a suitable mixture thereof. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using a surfactant. By various antibacterial and antifungal compounds, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc., it is possible to achieve the effect of preventing microorganisms. If desired, isotonic compounds such as sugars, polyols such as mannitol, sorbitol, and sodium chloride can be added to the composition. The extended absorption of the injectable composition can be achieved by including a compound that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0500] As needed, sterile injectable solutions can be prepared by incorporating the EVs (e.g., exosomes) of the present disclosure with one or a combination of ingredients listed herein in effective amounts and in an appropriate solvent. Generally, dispersions are prepared by incorporating EVs (e.g., exosomes) into a sterile vehicle containing a basic dispersion medium and any desired other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution. EVs (e.g., exosomes) can be administered in the form of a depot injection or implant formulation that can be formulated in such a way as to allow for sustained or pulsatile release of EVs (e.g., exosomes).

[0501] Systemic administration of compositions comprising EVs (e.g., exosomes) of the present disclosure can also be via transmucosal administration. For transmucosal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished using, for example, nasal sprays.

[0502] In certain aspects, a pharmaceutical composition comprising EVs (e.g., exosomes) of the present disclosure is administered intravenously to a subject who will benefit from the pharmaceutical composition. In certain other aspects, the composition is administered to the lymphatic system, for example, by intralymphatic injection or by intranodal injection (see, e.g., Senti et al., PNAS 105(46):17908(2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, by direct injection into the thymus, or by injection into the liver.

[0503] In certain aspects, the pharmaceutical composition comprising the EVs (e.g., exosomes) of the present disclosure is administered as a liquid suspension. In certain aspects, the pharmaceutical composition is administered as a formulation capable of forming a reservoir upon administration. In certain preferred aspects, the reservoir slowly releases the EVs (e.g., exosomes) into the circulation, or maintains the reservoir form.

[0504] Typically, pharmaceutically acceptable compositions are highly purified, free of contaminants, biocompatible, non-toxic, and suitable for administration to a subject. If water is a component of the carrier, the water is highly purified and treated to be free of contaminants, such as endotoxins.

[0505] In some aspects, pharmaceutically acceptable carriers may include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and / or mineral oil, but are not limited thereto. The pharmaceutical composition may further include a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifier, a suspending agent and / or a preservative.

[0506] In certain aspects, the preparations of EVs (e.g., exosomes) disclosed herein are subjected to radiation, e.g., X-rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, ultraviolet light, to destroy residual replication-competent nucleic acids. In certain aspects, the preparations of EVs (e.g., exosomes) disclosed herein are subjected to gamma irradiation using an irradiation dose exceeding 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more than 100 kGy. In certain aspects, preparations of EVs (e.g., exosomes) of the present disclosure are subjected to X-ray irradiation using an irradiation dose (Gy) of greater than 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 Gy.

[0507] In some aspects, the pharmaceutical composition comprises one or more therapeutic agents and EVs (e.g., exosomes) of the present disclosure, i.e., EVs comprising a cleavable linker disclosed herein. In certain aspects, EVs (e.g., exosomes) are co-administered with one or more additional therapeutic agents in a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered before administering another therapeutic agent. In one aspect, the additional therapeutic agent may be a nucleic acid agent or a small molecule therapeutic agent, such as a hormone therapy agent, a chemotherapeutic agent, an immunotherapeutic agent, an anti-inflammatory agent, or a drug that inhibits the action of a cell growth factor or a cell growth factor receptor. In other aspects, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered after administering another therapeutic agent. In a further aspect, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered simultaneously with another therapeutic agent.

[0508] Reagent test kit

[0509] The present disclosure also provides a kit comprising one or more EVs (e.g., exosomes) of the present disclosure and instructions for use. In some aspects, the kit or manufactured product contains a pharmaceutical composition as described herein, which comprises at least one EV (e.g., exosome) of the present disclosure and instructions for use. In some aspects, the kit or manufactured product comprises at least one EV (e.g., exosome) of the present disclosure or a pharmaceutical composition comprising EVs (e.g., exosomes) in one or more containers. Those skilled in the art will readily recognize that the EVs (e.g., exosomes) of the present disclosure, pharmaceutical compositions comprising EVs (e.g., exosomes) of the present disclosure, or combinations thereof can be easily incorporated into one of the well-known established kit formats in the art.

[0510] In some aspects, the kit comprises: EVs (e.g., exosomes) containing one or more biologically active molecules; reagents for covalently attaching one or more biologically active molecules to EVs (e.g., exosomes) via a cleavable linker, as disclosed herein, e.g., via conjugation or solid phase synthesis; and instructions for performing a reaction to covalently attach one or more biologically active molecules to EVs (e.g., exosomes) via a cleavable linker disclosed herein.

[0511] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art and are fully explained in the literature. See, e.g., Sambrook et al., eds. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., eds. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover, ed., (1985) DNA Cloning, Vol. I and Vol. II; Gait, ed., (1984) Oligonucleotide Synthesis; Mullis et al., U.S. Pat. No. 4,683,195; Hames and Higgins, eds., (1984) Nucleic Acid Hybridization; Hames and Higgins, eds., (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., NY); Miller and Calos, eds., (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Vols. I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); Crooke, Antisense drug Technology: Principles, Strategies and Applications, 2nd ed. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.). .

[0512] All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. The database entries and electronic publications disclosed in this disclosure are incorporated by reference in their entirety. The version of the database entries or electronic publications incorporated by reference in this application is the latest version of the publicly available database entries or electronic publications when submitting this application. The database entries corresponding to the gene or protein identifier (for example, the gene or protein identified by the accession number or database identifier of the public databases such as Genbank, Refseq or Uniprot) disclosed in this application are incorporated by reference in their entirety. The information incorporated to is not limited to the sequence data included in the database entries. The information incorporated by reference is included in the full content of the database entries in the latest version of the publicly available databases when submitting this application. In the event of a conflict, this specification (comprising definitions) shall prevail. In addition, materials, methods and examples are only illustrative and are not intended to be limiting.

[0513] Examples

[0514] The following examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present invention in any way. Unless otherwise indicated, the practice of the present invention will adopt the conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the technology of this area. Such technology is fully explained in the literature. See, for example, T. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); Green & Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition (Cold Spring Harbor Laboratory Press, 2012); Colowick & Kaplan, Methods in Enzymology (Academic Press); Remington: The Science and Practice of Pharmacy, 22nd edition (Pharmaceutical Press, 2012); Sundberg & Carey, Advanced Organic Chemistry: Parts A and B, 5th edition (Springer, 2007).

[0515] Unless otherwise stated, all reactions were performed using commercially available materials and reagents without further purification. Reactions were monitored by ultra-performance liquid chromatography (UPLC) and mass spectrometry. All products were obtained by 1 Characterization was performed by HNMR, UPLC, and mass spectrometry. Chemical shifts are expressed in parts per million (ppm) and are designated as s (singlet); br s (broad singlet); d (doublet); t (triplet); q (quartet); quint (quintet), or m (multiplet). Flash column chromatography was performed on a Biotage Isolera Four using a Silicycle Siliasep column (prepacked with spherical silica gel 25 μm) for reversed-phase chromatography or a Biotage SNAP C18 Ultra column (prepacked with spherical silica gel 25 μm) with reagent-grade water and acetonitrile (0.1% formic acid). Further purification was performed using a semi-preparative HPLC system; column: CSH C18 19 x 150 mm; eluent A: 0.1% formic acid, eluent B: acetonitrile, eluent C: 50% acetonitrile in 50% water; gradient 5% to 40% over 0 to 15 minutes; UV = 217 nm.

[0516] Analytical methods

[0517] UPLC4-MS: (CSH C18 Long Acid 2 to 95) - Waters Acquity UPLC H-class system; column: Acquity CSH C18 1.7 μm 2.1 x 50 mm; eluent A: water, eluent B: acetonitrile, eluent C: 2 vol% ammonia (28%) in water, eluent D: 2 vol% formic acid in water; gradient: 0 to 4.0 minutes 2% to 95% B, with A and 5% D throughout, 4.0 to 4.6 minutes 95% B; flow rate 0.8 mL / min; temperature: 40°C; PDA: 215 to 350 nm.

[0518] UPLC3-MS: (Kinetex C8 50 to 95) - Waters Acquity UPLC - Sample Manager SM-FTN, Pump QSM, Column Manager, Detector PDA (200 to 400 nm) and SQD range 2 to 2048 Da; Eluent A: water, Eluent B: acetonitrile, Eluent C: 2 vol% ammonia (28%) in water, Eluent D: 2 vol% formic acid in water; Gradient: 50% to 95% B from 0 to 4.5 minutes, always with A and 5% D, 95% B from 4.5 to 5.0 minutes; Flow rate 0.8 mL / min; Temperature: 40°C; PDA: 215 to 350 nm.

[0519] Example 1

[0520] This example relates to the synthesis of constructs of formula (I) in one aspect of the present disclosure. The total synthesis is shown in Figure 3 middle.

[0521]

[0522] Step 1: Methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate

[0523]

[0524] Pyrrole-2,5-dione (500mg, 5.15mmol) is dissolved in ethyl acetate (15.0mL) and then cooled to 0 ° C in ice water. After 10 minutes, N-methylmorpholine (521mg, 5.15mmol, 566μL) is slowly added dropwise. A solution of methyl chloroformate (487mg, 5.15mmol, 398μL) in ethyl acetate (1.20mL) is slowly added dropwise, and the reaction is continued at room temperature for 1 hour. After the reaction is complete, insoluble salts are removed by filtration. The filtrate is concentrated to obtain a crude product, 2,5-dioxopyrrole-1-methyl formate (700mg, crude product), which is obtained as a red oily solid and can be directly used in the next step without purification. 1H NMR (400MHz, CDCl3) δ [ppm] = 6.84 (s, 2H), 3.98 (s, 3H).

[0525] Step 2: 1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrrole-2,5-dione

[0526]

[0527] 2- (2-aminoethoxy) ethanol (427 mg, 4.06 mmol) was dissolved in NaHCO (15.0 mL), and the solution temperature was lowered to 0 ° C and stirred for 10 minutes. Then 2,5-dioxopyrrole-1-carboxylic acid methyl ester (700 mg, 4.51 mmol) was added and the mixture was stirred at 0 ° C for 20 minutes, and then the mixture was warmed to room temperature over 1 hour. The mixture was then washed with DCM (3 × 20 mL). The organic matter was combined and concentrated to dryness in a vacuum. The substance was then purified by normal phase purification (Biotage Isolera, 40 g siliasep column; eluent 0 to 100% ethyl acetate in heptane). The desired fractions were combined and concentrated to obtain 1- [2- (2-hydroxyethoxy) ethyl] pyrrole -2,5- dione (500 mg, 2.70 mmol, 60% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ [ppm] = 6.72 (s, 2H), 3.75 (t, 2H), 3.70-3.64 (m, 4H), 3.57 (t, 2H), 2.08 (s, 1H). C_UPLC2-MS: (BEH-C18 short base 2 to 20%, NBK2289-69-P1-1) R t = 0.55 min (97.1%), MS (ESIpos): m / z = [M+H] + 186.08.

[0528] Step 3: 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)thio)-1-(2-(2-hydroxyethoxy)ethyl)pyrrolidine-2,5-dione

[0529]

[0530] 1-[2-(2-hydroxyethoxy)ethyl]pyrrole-2,5-dione (100 mg, 540 μmol), (3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3-thiol (255 mg, 633 μmol) and triethylamine (110 mg, 1.08 mmol, 151 μL) were dissolved in dichloromethane (12.0 mL) and stirred at room temperature for 2.5 hours. The solution was then diluted with dichloromethane (10.0 mL) and washed with water (5.00 mL). The organics were then dried over magnesium sulfate and concentrated to dryness in vacuo. The material was purified by normal phase purification (Biotage Isolera, 25 g siliasep column; eluent 0 to 50% ethyl acetate over heptane). The desired fractions were combined and concentrated to give 3-[[(3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]sulfanyl]-1-[2-(2-hydroxyethoxy)ethyl]pyrrolidine-2,5-dione (264 mg, 449 μmol, 83% yield) as a white solid. 1 H NMR (400MHz, CDCl3) δ [ppm] = 5.39-5.33 (m, 1H), 3.86 (dq, 1H), 3.79-3.72 (m, 2H), 3.70-3 .64(m,4H),3.60-3.54(m,2H),3.16(dd,1H),3.11-3.03(m,1H),2.55(td,1H),2.49-2.3 1 (m, 1H), 2.28 (d, 1H), 2.07-1.99 (m, 2H), 1.97-1.71 (m, 6H), 1.64-1.42 (m, 6H), 1.40-1.23 (m, 4H), 1.23-1.06 (m, 6H), 1.05-0.95 (m, 6H), 0.91 (d, 3H), 0.87 (q, 6H), 0.68 (s, 3H). Too nonpolar to obtain suitable UPLC data.

[0531] Step 4: 4-(((2-(2-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)thio)-2,5-dioxopyrrolidin-1-yl)ethoxy)ethoxy)diisopropylsilyl)oxy)benzaldehyde

[0532]

[0533] Dichlorodiisopropylsilane (98.5 mg, 532 μmol, 96.0 μL) was dissolved in dichloromethane (4.00 mL), and the solution temperature was lowered to 0° C. A solution of p-hydroxybenzaldehyde (54.2 mg, 444 μmol, 48.0 μL) in THF (1.20 mL) was then slowly added dropwise, and the mixture was stirred at 0° C. for 30 minutes. A solution of 3-[[(3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]sulfanyl]-1-[2-(2-hydroxyethoxy)ethyl]pyrrolidine-2,5-dione (260 mg, 442 μmol) in dichloromethane (2.50 mL) was then added dropwise and the solution was stirred at 0° C. for 20 minutes. The solution was then concentrated to dryness. The material was then purified by normal phase purification (Biotage Isolera, 25 g siliasep column; eluent 0 to 25% ethyl acetate over heptane). The desired fractions were combined...

Claims

1. An extracellular vesicle (EV) comprising a biologically active molecule (BAM) attached to the EV via an anchoring moiety (AM) according to formula I: AM-SP1-L1-SP2-BAM (Formula I) in L1 is a cleavable bond comprising: a polynucleotide group; a peptidyl group selected from alanine-alanine-asparagine, valine-glycine, glycine-glycine, glutamic acid-valine-citrulline, aspartic acid-valine-citrulline, serine-valine-citrulline, lysine-valine-citrulline, glycine-glycine-glycine-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate acyloxy group; or a silyl ether; and SP1 and SP2 are optional first and second spacers, respectively.

2. The EV of claim 1, wherein AM is covalently linked to BAM at the 5' position.

3. The EV of claim 1, wherein AM is covalently linked to BAM at the 3' position.

4. The EV according to any one of claims 1 to 3, wherein L1 comprises a polynucleotide group that is a trinucleotide group or higher nucleotide group.

5. The EV of claim 4, wherein the polynucleotide base is a tetranucleotide base comprising dTdTdTdT, wherein dT is deoxythymidine.

6. The EV according to any one of claims 1 to 3, wherein L1 comprises a peptide group selected from alanine-alanine-asparagine, valine-glycine, glycine-glycine, glutamic acid-valine-citrulline, aspartic acid-valine-citrulline, serine-valine-citrulline, lysine-valine-citrulline, glycine-glycine-glycine-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline and alanine-phenylalanine-lysine.

7. The EV according to any one of claims 1 to 3, wherein L1 comprises a pyrophosphate acyloxy group.

8. The EV according to any one of claims 1 to 3, wherein L1 comprises a silyl ether.

9. The EV of claim 8, wherein the silyl ether comprises -OSiR 1 R 2 O-, where R 1 and R 2 The same or different and each is C 1-8 Alkyl or aryl.

10. The EV of claim 9, wherein R 1 and R 2 Both are isopropyl.

11. The EV according to any one of claims 1 to 10, wherein the AM comprises a sterol, a lipid, a vitamin, a peptide or a combination thereof.

12. The EV of claim 11, wherein the sterol is cholesterol, mercaptocholesterol, ergosterol, 7-dehydrocholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fucoxosterol, sargassum sterol, campesterol, β-sitosterol, sitostanol, coprostanol, avenasterol, stigmasterol, or a combination thereof.

13. The EV according to claim 11 or 12, wherein the sterol is cholesterol. The EV of claim 11 , wherein the lipid is a fatty acid or a phospholipid. 15 . The EV according to claim 14 , wherein the fatty acid is a straight-chain fatty acid, a branched-chain fatty acid, a saturated fatty acid, an unsaturated fatty acid, a hydroxy fatty acid, a polycarboxylic acid, or any combination thereof.

16. The EV according to claim 15, wherein the straight-chain fatty acid is butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid or stearic acid.

17. The EV of claim 16, wherein the straight-chain fatty acid is palmitic acid.

18. The EV of claim 14, wherein the phospholipid comprises 16:0 1,2-(i.e., C1 alkylene, C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, or C6 alkylene) acyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (16:0 PDP PE), 16:0 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (16:0 PE MCC), or 16:0 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cyanuric acid) (16:0 cyanuric acid PE).

19. The EV of claim 11, wherein the vitamin is tocopherol, tocotrienol, vitamin D, vitamin K, riboflavin, niacin, or pyridoxine.

20. The EV of claim 19, wherein the vitamin is tocopherol.

21. The EV according to any one of claims 1 to 20, wherein the AM is attached to the outer surface of the EV.

22. The EV of any one of claims 1 to 21, wherein the BAM comprises a peptide, a polypeptide moiety, a polynucleotide moiety, a protein, an antibody or an antigen-binding fragment thereof, a chemical compound, or any combination thereof.

23. The EV of any one of claims 1 to 22, wherein the BAM comprises an antisense oligonucleotide (ASO), siRNA, miRNA, shRNA, nucleic acid, or any combination thereof.

24. The EV of claim 27, wherein the BAM comprises an ASO.

25. The EV of claim 24, wherein the ASO targets a transcript.

26. The EV of claim 25, wherein the transcript is a STAT6 transcript, an EGFP transcript, a CEBP / β transcript, a STAT3 transcript, a KRAS transcript, a NRAS transcript, a NLPR3 transcript, a FFLUC transcript, a RLUC transcript, a MYC transcript, or any combination thereof.

27. The EV according to any one of claims 1 to 26, wherein SP1 and SP2 are the same or different and each comprises an alkylene group, a polyoxyalkylene group, a succinimidyl group, a maleimido group, an aryl group, an ether, a carbonyl group, a carboxylate group, a carbamoyl group, a thioether group, a sulfo group, a thiocarbonyl group, a thiocarbamoyl group, a thiosuccinimidyl group, an amino group, an amide group, a hydrazide group, a thiophosphate group, a 1,2,3-triazolyl group, a dibenzoylcyclooctenyl group, a bicyclononenyl group, a p-aminobenzoyl group, a p-aminobenzylcarbamate group, or a combination thereof, and at least one of SP1 and SP2 is present in formula (I).

28. The EV of claim 27, wherein at least one of SP1 and SP2 comprises C 2-8 Alkylene, polyoxyalkenyl, maleimido, carbamoyl, thio, amido, 1,2,3-triazolyl, dibenzoylcyclooctenyl, bicyclononenyl, 1,2,3-triazolyldibenzoylcyclooctenyl, 1,2,3-triazolylbicyclononenyl, p-aminobenzoyl, p-aminobenzylcarbamate or a combination thereof.

29. The EV of claim 28, wherein at least one of SP1 and SP2 comprises a C3 alkylene group, a C6 alkylene group, or a C8 alkylene group.

30. The EV of claim 28, wherein at least one of SP1 and SP2 comprises a polyoxyalkylene group comprising 2 to 15 -OCH2CH2- repeating units.

31. The EV of claim 29 or 30, wherein at least one of SP1 and SP2 further comprises a carbamoyl group, an amide group, a sulfosuccinimide group, a 1,2,3-triazolylbicyclononenyl group, or a combination thereof.

32. The EV according to any one of claims 27 to 31 , wherein SP1 is present.

33. The EV of any one of claims 27 to 32, wherein SP2 is present.

34. The EV according to claim 32 or 33, wherein both SP1 and SP2 are present and are identical.

35. The EV of claim 32 or 33, wherein both SP1 and SP2 are present and different.

36. The EV according to claim 1, wherein formula (I) is 37. A pharmaceutical composition comprising the EV according to any one of claims 1 to 36 and a pharmaceutically acceptable carrier.

38. A kit comprising the EV according to any one of claims 1 to 36 or the pharmaceutical composition according to claim 37 and instructions for use.

39. A method of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of the EV according to any one of claims 1 to 36 or the pharmaceutical composition according to claim 37.

40. The method of claim 39, wherein the disease or disorder is cancer, graft-versus-host disease (GvHD), an autoimmune disease, an infectious disease, a fibrotic disease, an inflammatory disease, a neurodegenerative disorder, a central nervous system disease, a muscular dystrophy, or a metabolic disease.

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