HK40124184A - Extracellular vesicle comprising a biologically active molecule and a cleavable linker - Google Patents

Description

Extracellular vesicles containing bioactive molecules and cleavable linkers

Reference to electronically submitted sequence lists via EFS-WEB

The contents of the sequence list submitted electronically 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

This disclosure provides extracellular vesicles (EVs), such as exosomes, which can be used as agents for the prevention or treatment of cancer and other diseases, the extracellular vesicles containing at least one bioactive molecule linked to the extracellular vesicle, such as an exosome, via a cleavable connector and an anchoring portion.

Background Technology

Many bioactive compounds possess potent biological activity that is therapeutically significant. However, these compounds often exhibit toxicity in non-target organs. One way to limit non-target tissue exposure is to chemically conjugate small molecules with affinity-based agents such as antibodies, which can direct therapeutic compounds to specific cell types (Dosio, F. et al., Toxins (Basel) 3(7):848-883 (2011)). However, this approach is limited by the number of target compound molecules that can be attached to the antibody (typically 2 to 6 molecules per antibody) and the availability/presence of antibodies that specifically bind to the targeted, relevant disease/effect cell without binding to non-target cells. These two issues limit the use of antibody-drug conjugates (ADCs) by reducing potency and increasing systemic toxicity, respectively. Therefore, there is a need for delivery systems with higher payloads than ADCs that can selectively target specific tissues or organs while limiting overall systemic exposure to the therapeutic compound.

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 mediators, EVs, such as exosomes, offer many advantages over traditional drug delivery methods (such as peptide immunization and DNA vaccines) as novel therapeutic approaches 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 investigated as maintenance immunotherapy after first-line chemotherapy in patients with unresectable 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 met. (Besse, B., et al., Oncoimmunology 5(4):e1071008 (2015)).

Therefore, there is a need for novel and more effective engineered EVs, such as exosomes, to better realize the therapeutic uses and other applications of EV-based technologies.

Summary of the Invention

This disclosure provides an extracellular vesicle (EV) comprising a bioactive molecule (BAM) attached to the EV via an anchoring portion (AM) according to Formula I:

AM-SP ₁ -L ₁ -SP ₂ -BAM (Formula I)

Wherein _L1 is a cleavable bond containing the following: 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-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline and alanine-phenylalanine-lysine; a pyrophosphate oxy group; or a silyl ether; and _SP1 and _SP2 are optional first and second spacers, respectively.

In some respects, AM is covalently bonded to BAM at the 5′ position. In other respects, AM is covalently bonded to BAM at the 3′ position.

In some respects, _L1 comprises a polynucleotide group that is a trinucleotide or a higher nucleotide group. In some respects, the polynucleotide group is a tetranucleotide group comprising dTdTdTdT, where dT is deoxythymidine.

In some respects, _L1 contains 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-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine.

In some respects, _L1 contains pyrophosphate oxy groups.

In some aspects, _L1 comprises a silyl ether. In some aspects, the ^silyl ether comprises ^–OSiR1R2O- , wherein ^R1 and ^R2 are the same or different and are each _C1-8 alkyl or aryl. In some aspects, the ^silyl ether comprises ^–OSiR1R2O- and both ^R1 and ^R2 are isopropyl.

In some respects, AM includes sterols, lipids, vitamins, peptides, or combinations thereof.

In some respects, AM includes sterols, including cholesterol, mercaptocholesterol, ergosterol, 7-dehydrocholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fucosterol, sargastricosterol, campesterol, β-sitosterol, sitosterol, coprosterol, alfalfa, stigmasterol, or combinations thereof. In some respects, the sterol is cholesterol.

In some respects, AM includes lipids, which include fatty acids or phospholipids. In some respects, the fatty acids are straight-chain fatty acids, branched-chain fatty acids, saturated fatty acids, unsaturated fatty acids, hydroxy fatty acids, polycarboxylic acids, or any combination thereof. In some respects, the straight-chain fatty acids are butyric acid, hexanoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, or stearic acid. In some respects, the straight-chain fatty acid is palmitic acid. In some respects, phospholipids include 16:01,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[3-(2-pyridyldithio)propionate] (16:0PDP PE), 16:01,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimidemethyl)cyclohexane-formamide] (16:0PE MCC) or 16:01,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-(cyanuric acid) (16:0 cyanuric acid PE).

In some respects, AM includes vitamins, such as tocopherols, tocotrienols, vitamin D, vitamin K, riboflavin, niacin, or pyridoxine. In other respects, the vitamin is tocopherol.

In some respects, AM is attached to the outer surface of the EV.

In some respects, BAM includes peptides, polypeptides, polynucleotides, proteins, antibodies or their antigen-binding fragments, chemical compounds or any combination thereof. In some respects, BAM includes antisense oligonucleotides (ASO), siRNA, miRNA, shRNA, nucleic acids or any combination thereof. In some respects, BAM includes ASO. In some respects, ASO targets transcripts. In some respects, the transcripts are STAT6 transcripts, EGFP transcripts, CEBP/β transcripts, STAT3 transcripts, KRAS transcripts, NRAS transcripts, NLPR3 transcripts, FFLUC transcripts, RLUC transcripts, MYC transcripts or any combination thereof.

In some respects, SP ₁ and SP ₂ are the same or different, and each comprises an alkylene group, a polyoxyalkylene group, a succinimide group, a maleimide group, an aryl group, an ether, a carbonyl group, a carboxylate group, a carbamoyl group, a thioether, a sulfonyl group, a thiocarbonyl group, a thiocarbamoyl group, a thiosuccinimide group, an amino group, an amide group, a hydrazido group, a thiophosphate group, a 1,2,3-triazolyl group, a benzoylcyclooctenyl group, a bicyclononenyl group, a p-aminobenzoyl group, a p-aminobenzylcarbamate group, or a combination thereof, and at least one of SP ₁ and SP ₂ is present in formula (I). In some aspects, at least one of SP ₁ and SP ₂ comprises a _C2-8 alkylene group, a polyoxyalkylene group, a maleimide group, a carbamoyl group, a thioyl group, an amide group, a 1,2,3-triazolyl group, a dibenzoylcyclooctenyl group, a bicyclononenyl group, a 1,2,3-triazolyldibenzoylcyclooctenyl group, a 1,2,3-triazolylbicyclononenyl group, a p-aminobenzoyl group, a p-aminobenzylcarbamate group, or a combination thereof. In some aspects, at least one of SP ₁ and SP ₂ comprises a _C3 alkylene group, a _C6 alkylene group, or a _C8 alkylene group. In some aspects, at least one of SP ₁ and SP ₂ comprises a polyoxyalkylene group containing 2 to 15 –OCH ₂ CH ₂ – repeating units. In some respects, at least one of SP ₁ and SP ₂ further comprises a carbamoyl group, an amide group, a thiosuccinimide group, a 1,2,3-triazolyldibenzoylcyclooctenyl group, a 1,2,3-triazolylbicyclononenyl group, or a combination thereof.

In some respects, SP ₁ exists in equation (I). In some respects, SP ₂ exists in equation (I). In some respects, both SP ₁ and SP ₂ exist in equation (I) and are identical. In some respects, both SP ₁ and SP ₂ exist in equation (I) and are different.

In some respects, equation (I) is a construct selected from the following

as well as

This disclosure provides a pharmaceutical composition comprising the EV described herein and a pharmaceutically acceptable carrier.

This disclosure also provides a kit comprising the EV or a pharmaceutical composition thereof described herein, along with instructions for use.

This disclosure provides a method for treating or preventing a disease or condition in a subject with this need, the method comprising administering to the subject an effective amount of the EV or a pharmaceutical composition thereof described herein. In some aspects, the disease or condition is cancer, graft-versus-host disease (GvHD), autoimmune disease, infectious disease, fibrotic disease, inflammatory disease, neurodegenerative disease, central nervous system disease, muscular dystrophy, or metabolic disease.

Attached Figure Description

Figure 1 is a schematic diagram illustrating the following: the general structure of an exosome (left); an exemplary bioactive molecule (e.g., an oligonucleotide) attached to a ligand that allows attachment to the outer surface of the exosome via a linker (middle); and how a bioactive molecule (e.g., an oligonucleotide) attached to an anchoring portion (e.g., a lipid, such as cholesterol) via a linker can attach to the membrane of the exosome (right).

Figure 2 shows the sequences of exemplary ASOs. FFLUC and RLUC are named after the targeted luminescent reporter genes. MYC and STAT6ASO are named after the genes targeted by the ASOs. Nb: LNA residues (including LNA-5MeC and LNAT/LNA-5MeU). Nm: 2′-O′MOE residues (including MOE-5MeC and MOE-T/MOED-5MeU). dN: DNA residues. (5MdC): 5-methyl-dC. s: Phosphothioester backbone modification.

Figure 3 shows the chemical scheme for the multi-step synthesis of the construct of formula (I).

Figure 4 is a table showing some properties of the lipid-connector-ASO(-CPP) stock solution.

Figure 5 is a bar chart of the average diameter (nm) of the reconstructed lipid-connector-ASO(-CPP) after filtration through a 0.2 μm filter.

Figure 6 is a table showing some characteristics of ASO concentration under load.

Figure 7 is a bar chart of the load density of exemplary exoASOs, representing the number of ASOs per quantity of exosome load.

Figure 8 is a graph of the IC ₅₀ value (nm) for an exemplary exoASO.

Figure 9 is a graph of the IC ₅₀ values (nm) of an exemplary exoASO normalized to load density. "*" indicates that the ASO was loaded under unoptimized load conditions, thus there is potential for further improvement in load density.

Figure 10 is a table showing some characteristics of exoASO, including ASO concentration and _IC50 under load.

Figure 11 is a graph of gene expression percentage (hSTAT6) normalized to ASO concentration (nM) for various exoASOs numbered 1 to 11.

Figure 12 is a graph of gene expression percentage (hSTAT6) normalized to exosome concentration for each of the various exoASOs numbered 1 to 11.

Figure 13 is a graph comparing the _IC50 values of ASO numbers 1 to 11, normalized to ASO concentration (nM).

Figure 14 is a graph comparing _IC50 values for ASO numbers 1 to 11, normalized to exosome concentrations (p/mL).

Figure 15 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 Implementation

This disclosure relates to extracellular vesicles (EVs), such as exosomes, comprising at least one bioactive molecule covalently linked to the EV (e.g., exosome) via a cleavable linker and an anchoring moiety, and the uses thereof. Non-limiting examples of various aspects are shown in this disclosure.

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

The headings provided herein are not intended to limit the various aspects of this disclosure, which can be defined by reference to this specification in its entirety. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be restrictive, as the scope of this disclosure will be limited only by the appended claims.

Therefore, the terms defined below are defined more fully by referring to the entire specification.

definition

To make this description easier to understand, some terms are defined first. Other definitions are elaborated throughout the detailed description.

It is important to note that the term "a (or an)" 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 negative limitations in the statement incorporating the elements of the claims.

Furthermore, the term “and/or” as used herein is considered to be a specific disclosure of each of the two specified features or components, having or not having the other. Therefore, 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). Similarly, the term “and/or” as used in phrases such as “A, B, and/or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It should be understood that whenever an aspect is described in this document using the language “including”, other similar aspects described as “consisting of” and/or “substantially composed of” are also provided.

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 pertains. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd edition, 2002, CRC Press; *The Dictionary of Celland 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 those skilled in the art of the use of many of the terms used in this disclosure.

Units, prefixes, and symbols are represented in a form accepted by the International System of Units (SI). A range of values includes the numbers defining the range. In the case of enumerated ranges of values, it should be understood that each intermediate integer value between the enumerated upper and lower limits of the range 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 or excluded from the range, and each range that includes, excludes, or includes both limits is also covered within this disclosure. Therefore, the ranges enumerated herein should be understood as shorthand notations of all values within the range, including the enumerated endpoints. For example, the range 1 to 10 is understood to include any number, combination of numbers, or subrange of numbers derived from groups consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

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

Nucleotides are represented by their commonly accepted single-letter codes. Unless otherwise specified, nucleotide sequences are written from left to right in a 5' to 3' orientation. Nucleotides are represented in this document by the single-letter symbols they are commonly known to be recommended by the IUPAC-IUB Committee on Biochemical Nomenclature. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

Amino acid sequences are written from left to right with the amino to carboxyl orientation. Amino acids are represented in this paper using their commonly known three-letter symbols or single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature.

The term "about" is used in this document to mean approximately, roughly, around a certain area, or within a certain area. 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 value. Generally, the term "about" can modify values above or below the specified value by, for example, 10% variance upwards or downwards (higher or lower).

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

As used herein, the term "agonist" refers to a molecule that binds to and activates a 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.

The term "amino acid substitution" refers to replacing an amino acid residue present in a parental or reference sequence (e.g., a wild-type sequence) with another amino acid residue. Amino acids can be substituted in a parental or reference sequence (e.g., a wild-type polypeptide sequence), for example, via chemical peptide synthesis or by recombination methods known in the art. Therefore, reference to "substitution at position X" means replacing the amino acid present at position X with an alternative amino acid residue. In some aspects, the substitution pattern can be described according to scheme AnY, where A is a single-letter code corresponding to the amino acid naturally present or originally present at position n, and Y is the substituted amino acid residue. In other aspects, the substitution pattern can be described according to scheme An(YZ), where A is a single-letter code corresponding to the amino acid residue that replaces the amino acid naturally present or originally present at position n, and Y and Z are alternating substitutions of amino acid residues that can replace A.

As used herein, the term "antagonist" refers to a molecule that, upon binding to a receptor, blocks or inhibits an agonist-mediated response rather than eliciting the agonist's own biological response. Many antagonists exert their potency by competing with endogenous ligands or substrates at structurally defined binding sites on the receptor. Non-limiting examples of antagonists include alpha-blockers, beta-blockers, and calcium channel blockers. Antagonists can be competitive, non-competitive, or non-competitive.

As used herein, the term "antibody" encompasses naturally occurring or partially or fully 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 a framework region derived from an immunoglobulin gene or a fragment thereof, which specifically bind to and recognize an antigen. The use of the term antibody is intended to include intact antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, mouse-human antibodies, mouse-primate antibodies, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments such as, for example, scFv, (scFv) ₂ , Fab, Fab' and F(ab') ₂ , F(ab1) ₂ , Fv, dAb and Fd fragments, biantibodies, and antibody-associated polypeptides. Antibodies include bispecific and multispecific antibodies, provided they exhibit the desired biological activity or function. In some aspects of this disclosure, a biologically active molecule is an antibody or a molecule containing its antigen-binding fragment.

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 drug, pharmaceutical, or active pharmaceutical ingredient) or a drug agent. In some aspects of this disclosure, the bioactive molecule is an antibody-drug conjugate.

As used herein, the term "approximately" when applied to one or more target values refers to a value similar to a specified reference value. In some respects, the term "approximately" refers to a range of values within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction (greater than or less than) the specified reference value, unless otherwise stated or otherwise apparent from the context (except where such figures exceed 100% of possible values).

As used herein, the term "bioactive molecule" refers to any molecule that can attach to an EV (e.g., exosome) via an anchoring motif, wherein the molecule may have a therapeutic or prophylactic effect in a subject in need, or for diagnostic purposes. Thus, by way of example, the term bioactive molecule includes proteins (e.g., antibodies, proteins, peptides and their derivatives, fragments and variants), lipids and their derivatives, carbohydrates (e.g., the glycan motif in glycoproteins), or small molecules (e.g., molecules having a molecular weight of 1000 g/mol or less). In some aspects, bioactive molecules include radioisotopes. In some aspects, bioactive molecules are detectable motifs, such as radionuclides, fluorescent molecules, or contrast agents. In some aspects, bioactive molecules may be or may include targeting or tropism motifs. In some aspects, bioactive molecules may be or may include, for example, affinity ligands (e.g., biotin, digoxin, or dinitrophenol). In some aspects, bioactive molecules may be or may include motifs capable of improving pharmacokinetic or pharmacodynamic properties, such as motifs capable of increasing plasma half-life, such as the PEG motif.

"Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered conserved. Alternatively, an amino acid string can be conservatively substituted with a structurally similar string that differs in the order and/or composition of the side chain family members.

As used herein, the term "conservative" refers to nucleotide or amino acid residues in polynucleotide or polypeptide sequences that have not changed at the same position in two or more sequences being compared. Relatively conserved nucleotides or amino acids are those that are more conserved in a more relevant sequence than they are elsewhere in the sequence.

In some respects, if two or more sequences are 100% identical to each other, they are said to be "completely conserved" or "identical." In some respects, if two or more sequences are about 70% or more identical to each other, such as about 80%, about 90%, about 95%, about 98%, or about 99%, they are said to be "highly conserved." In some respects, if two or more sequences are at most about 70% identical to each other, including about 30%, about 40%, about 50%, about 60%, or about 65%, they are said to be "conserved." Sequence conservation can apply to the full length of a polynucleotide or polypeptide or to its parts, regions, or features.

As used in this article, the term "conventional EV protein" refers to proteins that are previously known to be enriched in EVs.

As used herein, the term “common exosomal protein” means proteins previously known to be enriched in exosomes, including but not limited to CD9, CD63, CD81, PDGFR, GPI-anchored proteins, lactoglucosins LAMP2 and LAMP2B, fragments thereof, or peptides bound to them.

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

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

As used herein, the terms “extracellular vesicle,” “EV,” and their grammatical variations 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, extranuclear granules, cancer bodies, or apoptotic bodies) having a diameter smaller than that derived from their cells. In some aspects, extracellular vesicles range in diameter 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 contain various macromolecular payloads located within the internal space (i.e., the lumen), displayed on the external surface of the extracellular vesicle, and/or spanning the membrane. In some aspects, the payload may comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and/or combinations thereof. In some aspects, the extracellular carrier comprises a scaffold portion. Extracellular vesicles include, but are not limited to, apoptotic bodies, cell debris, vesicles derived from cells through direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicularized organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosome fusion 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 respects, extracellular vesicles are produced by cells expressing one or more transgenic products.

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

In some aspects, the EVs disclosed herein, such as exosomes or nanovesicles, are engineered by covalently linking at least one bioactive molecule (such as a protein like an antibody or antibody-drug conjugate (ADC), ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) like an antisense oligonucleotide, a small molecule drug or a small molecule toxin) to the EV, such as an exosome or nanovesicle via an anchoring moiety.

In some aspects, the EVs of this disclosure, such as exosomes or nanovesicles, may comprise various macromolecular payloads located within an internal space (i.e., lumen), displayed on the outer (outer) surface or inner (inner) surface of the EV, and/or spanning a membrane. In some aspects, the payloads may comprise, for example, nucleic acids, proteins, carbohydrates, lipids, small molecules, and combinations thereof. In some aspects, the EVs, such as exosomes, include 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, the EVs, such as exosomes, are produced by cells expressing one or more transgenic products. In other aspects, the EVs of this disclosure are not limited to nanovesicles, microsomes, microvesicles, extracellular bodies, or apoptotic bodies.

The following schematic diagram is shown in Figure 1: the general structure of exosomes; exemplary bioactive molecules (e.g., oligonucleotides) attached to ligands (e.g., anchoring portions) that allow attachment to the outer surface of exosomes via linkers; and how bioactive molecules (e.g., oligonucleotides) attached to anchoring portions (e.g., lipids, such as cholesterol) via linkers can attach to the membrane of exosomes. In some aspects, AM attaches to the outer surface of EVs.

As used herein, the term “fragment” of a protein (e.g., a bioactive molecule such as a therapeutic protein, or a scaffold protein such as scaffold X) refers to the amino acid sequence of a protein that is shorter than the naturally occurring N- and/or C-terminal deletion sequence or any part of a naturally occurring protein compared to a naturally occurring protein.

As used herein, the term "functional fragment" refers to a protein fragment that retains the function of the protein. Thus, in some respects, functional fragments of scaffold proteins (such as scaffold X protein) retain the ability to anchor bioactive molecules to the luminal or external surfaces of EVs (such as exosomes).

Whether a fragment is a functional fragment can be assessed by any method known in the art to determine the protein content of an EV (e.g., exosome), including Western blotting, fluorescence activated cell sorting (FACS) analysis, and fusion of the fragment with an autofluorescent protein (e.g., green fluorescent protein (GFP)). In some respects, 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 bioactive molecules to the lumen or external surface of the EV (e.g., exosome).

As used herein, “anchoring” a bioactive molecule to the lumen or external surface of an EV (e.g., exosome) via a scaffold protein means attaching the bioactive molecule, either covalently or non-covalently, to portions of a scaffold molecule located on the lumen or external surface of an EV (e.g., exosome).

As used herein, the term "homology" refers to the overall correlation between polymer molecules, such as nucleic acid molecules (e.g., DNA and/or RNA molecules) and/or polypeptide molecules. In general, the term "homology" implies an evolutionary relationship between two molecules. Therefore, two homologous molecules will share a common evolutionary ancestor. In the context of this disclosure, the term homology encompasses both identity and similarity.

In some respects, 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 the same monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

In the context of this disclosure, substitution (even when referred to as amino acid substitution) occurs at the nucleic acid level, i.e., replacing an amino acid residue with a substitute amino acid residue is done by replacing the codon encoding the first amino acid with the codon encoding the second amino acid.

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

For example, the percentage of identity between two polypeptide sequences can be calculated by aligning the two sequences for optimal comparison purposes (e.g., a spacer can be introduced in one or both of the first and second polypeptide sequences for optimal alignment, and dissimilar sequences can be ignored for comparison purposes). In some respects, the length of the sequence 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.

The molecules are considered identical at that position when a position in the first sequence is occupied by the same amino acid as its corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of shared positions, taking into account the number of spacers and the length of each spacer. This function needs to be introduced for optimal alignment of the two sequences. Sequence comparison and determination of the percentage of identity between two sequences can be achieved using mathematical algorithms.

Suitable software programs are available from various sources and can be used for aligning both protein and nucleotide sequences. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST (Basic Local Alignment Search Tool) program suite and is available from the National Center for Biotechnology Information (NCBI) BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm to compare two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs include Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS bioinformatics program suite and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk/Tools/psa.

Sequence alignment can be performed using methods known in the art, such as MAFFT (multiple alignment using fast Fourier transform), Clustal (ClustalW, ClustalX, or Clustal Omega), MUSCLE (multiple sequence alignment by logarithmic expectation), etc.

Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percentage of sequence identity. Note that the percentage of sequence identity values are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. Also note that length values will always be integers.

In some respects, the percentage of identity (%ID) between the first amino acid sequence (or nucleic acid sequence) and the second amino acid sequence (or nucleic acid sequence) is calculated as %ID = 100 x (Y/Z), where Y is the number of amino acid residues (or nucleobases) that are scored as identical matches in the alignment of the first and second sequences (e.g., by visual inspection or a specific sequence alignment procedure), and Z is the total number of residues in the second sequence. If the first sequence is longer than the second sequence, the percentage of identity between the first and second sequences will be higher than the percentage of identity between the second and first sequences.

Those skilled in the art will understand that the generation of sequence alignments used to calculate the percentage of sequence identity is not limited to binary sequence-sequence comparisons driven solely by primary sequence data. It will also be understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystal protein structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable procedure for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, available at www.tcoffee.org and alternatively, for example, from EBI. It will also be understood that the final alignments used to calculate the percentage of sequence identity can be curated automatically or manually.

As used herein, the terms “isolated,” “purified,” “extracted,” and their grammatical variations are used interchangeably and refer to the state of preparation of a desired EV (e.g., multiple EVs of known or unknown amounts and/or concentrations) that has undergone one or more purification processes, such as the selection or enrichment, preparation, of a desired EV (e.g., exosomes). In some respects, isolation or purification, as used herein, is the process of removing, partially removing (e.g., a portion) EVs (e.g., exosomes) from a sample containing production cells. In some respects, the isolated EV (e.g., exosome) composition does not have detectable undesirable activity, or alternatively, the level or amount of undesirable activity is at or below an acceptable level or amount. In other respects, the isolated EV (e.g., exosome) composition has an amount and/or concentration of the desired EV (e.g., exosomes) at or above an acceptable amount and/or concentration. In other respects, the isolated EV (e.g., exosome) composition is enriched compared to the starting material (e.g., the production cell preparation) from which the composition was obtained. Compared to the starting material, this 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) formulation is substantially free of residual biological products. In some aspects, the isolated EV (e.g., exosome) formulation is 100% free of, at least 99% free of, at least 98% free of, at least 97% free of, at least 96% free of, at least 95% free of, at least 94% free of, at least 93% free of, at least 92% free of, at least 91% free of, or at least 90% free of any contaminating biological material. Residual biological products may include non-biological materials (including chemicals) or unwanted nucleic acids, proteins, lipids, and/or metabolites. "Substantially free of residual biological products" may also mean that the EV (e.g., exosome) composition does not contain detectable producing cells and only EVs (e.g., exosomes) are detectable.

The terms “link,” “fusion,” and their grammatical variations are used interchangeably and refer to a first part, such as a first amino acid sequence or nucleotide sequence, covalently or non-covalently linked to a second part, such as a second amino acid sequence, nucleotide sequence, and/or lipid (e.g., cholesterol), respectively. The first part may be directly linked to or juxtaposed to the second part, or alternatively, an intervening part may covalently link the first part to the second part. The term “link” means not only the fusion of the first and second parts at the C-terminus or N-terminus, but also includes the insertion of the entire first part (or the second part) into any two points in the second part (or, respectively, the first part), such as amino acids. In one aspect, the first part is linked to the second part via a peptide bond or a linker. The first part may be linked to the second part via a phosphodiester bond or a linker. The linker may be a peptide or polypeptide (for polypeptide chains), a nucleotide or nucleotide chain (for nucleotide chains), or any chemical part (for polypeptide or polynucleotide chains or any chemical molecule). The term “link” is also indicated by a hyphen (-). In some aspects, scaffold X proteins on EVs (e.g., exosomes) may be linked to or fused to a bioactive molecule via a linker, a spacer, or both a linker and a spacer.

The term "modified" as used herein, when used in the context of EVs (e.g., exosomes), refers to an alteration or engineering of the EV (e.g., exosome) and/or its producing cells such that the modified EV (e.g., exosome) differs from naturally occurring EVs (e.g., exosomes). In some aspects, the modified EVs (e.g., exosomes) described herein comprise membranes with compositions of proteins, lipids, small molecules, carbohydrates, etc., different from those of naturally occurring EVs (e.g., exosomes). In one aspect, the membrane contains a higher density or number of natural EVs (e.g., exosomes), and the proteins and/or the membrane contains proteins not naturally found in the EV (e.g., exosomes). In some aspects, such modifications to the membrane alter the outer surface of the EV, such as exosomes (e.g., surface-engineered EVs and exosomes described herein).

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

As used herein, the terms “modifier,” “modifier,” and their grammatical variations generally refer to the ability to alter a particular concentration, level, expression, function, or behavior by increasing or decreasing, for example, directly or indirectly promoting/stimulating/upregulating or interfering with/inhibiting/downregulating, a particular concentration, level, expression, function, or behavior, such as acting as an antagonist or agonist. In some cases, modifiers may increase and/or decrease a certain concentration, level, activity, or function relative to a control, or relative to the generally expected mean activity level or relative to the control activity level.

As used herein, the term "nanovesicle" refers to an extracellular vesicle having a diameter between 20 and 250 nm (e.g., between 30 and 150 nm) and being generated by a cell (e.g., a production cell) through direct or indirect manipulation such that the nanovesicle would not be generated by the cell without manipulation. Suitable manipulations for generating nanovesicles from cells include, but are not limited to, continuous extrusion, treatment with an alkaline solution, sonication, or combinations thereof. In some aspects, the generation of nanovesicles may lead to the destruction of the production cell. In some aspects, the population of nanovesicles described herein is substantially free of vesicles derived from the cell through 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 the production cell, the nanovesicle can be isolated from the production cell based on its size, density, biochemical parameters, or combinations thereof.

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 an EV (e.g., an exosome) of this disclosure. Non-limiting examples of payloads that may be incorporated into an EV (e.g., an exosome) include therapeutic agents such as nucleotides (e.g., nucleotides containing a detectable portion 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 portion 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 some aspects, the payload contains an antigen. As used herein, the term "antigen" refers to any agent that, when introduced into a subject, elicits an immune response (cellular or humoral) against itself. In some aspects, the payload molecule is covalently linked to an EV, such as an exosome, as disclosed herein, via a linker, a spacer, or both a linker and a spacer. In other aspects, the payload contains an adjuvant.

The terms "pharmaceutically acceptable carrier," "pharmaceuticalally acceptable excipient," and their grammatical variations cover any pharmaceutical preparation approved by a U.S. federal regulatory agency or listed in the United States Pharmacopeia for use in animals (including humans), and any carrier or diluent that does not cause undesirable physiological effects to the extent that would preclude administration of the composition to a subject, and does not eliminate the biological activity and properties of the administered compound. This includes excipients and carriers that can be used to prepare pharmaceutical compositions and are generally safe, non-toxic, and desirable.

As used herein, the term "pharmaceutical composition" refers to one or more compounds described herein, such as, for example, EVs, such as exosomes of this disclosure, which are 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 an EV (e.g., exosome) to a subject.

As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogues, 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 polynucleotides, such as those obtained through alkylation and/or end-capping, as well as unmodified forms. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, any other type of polynucleotide, whether spliced or unspliced, of N- or C-glycosides of purine or pyrimidine bases, and other polymers containing a positive nucleotide backbone, such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholino polymers, as well as other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases that allow for base pairing and base stacking configurations, such as those found in DNA and RNA. In some aspects of this disclosure, as disclosed herein, the bioactive molecule attached to an EV (e.g., exosome) via a linker, a spacer, or both is a polynucleotide, such as an antisense oligonucleotide. In certain aspects, the polynucleotide comprises mRNA. In other aspects, the mRNA is synthetic mRNA. In some aspects, the synthetic mRNA contains 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 may be replaced with non-natural nucleobases, such as 5-methoxyuridine). In some aspects of this disclosure, the bioactive molecule is a polynucleotide (e.g., an antisense oligonucleotide, ASO).

The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to an amino acid polymer of any length. This polymer may contain modified amino acids. The term also covers amino acid polymers that are naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. Also included within this 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), and other modifications known in the art. In some aspects of this disclosure, as disclosed herein, bioactive molecules attached to EVs (e.g., exosomes) via linkers, spacers, or both linkers and spacers are polypeptides, such as antibodies or derivatives thereof, such as ADCs, proteolytic targeting chimeras (PROTACs), toxins, fusion proteins, or enzymes.

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

As used herein, the term "prevent/preventing" and its variations refer to the partial or complete delay of the onset of a disease, symptom, and/or condition; the partial or complete delay of the onset of one or more symptoms, features, or clinical manifestations of a particular disease, symptom, and/or condition; the partial or complete delay of the progression of a particular disease, symptom, and/or condition; and/or the reduction of the risk of pathological development associated with a disease, symptom, and/or condition. In some respects, preventive treatment achieves the prevention of a certain outcome.

As used herein, the term "productive cell" refers to a cell used to produce EVs (e.g., exosomes). Productive cells can be cells cultured in vitro or in vivo. Productive cells include, but are not limited to, cells known to be efficient at producing EVs (e.g., exosomes), such as HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, AGE.HN ^™ neuronal precursor cells, CAP ^™ amniotic fluid cells, adipose-derived mesenchymal stem cells, and RPTEC/TERT1 cells. In some respects, productive cells are not antigen-presenting cells. In some respects, productive cells are not dendritic cells, B cells, mast cells, macrophages, neutrophils, Kupffer-Browicz cells, cells derived from any of these cells, or any combination thereof.

As used in this article, “prevention” refers to a treatment or course of action intended to prevent the onset of a disease or condition, or to prevent or delay symptoms associated with a disease or condition.

As used in this article, “prevention” refers to measures taken to maintain health and prevent or delay the onset of bleeding, or to prevent or delay symptoms associated with a disease or condition.

“Recombinant” polypeptides or proteins refer to polypeptides or proteins produced via recombinant DNA technology. For the purposes of this disclosure, recombinant polypeptides and proteins expressed in engineered host cells are considered isolated, as are natural or recombinant polypeptides that have been isolated, 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 this disclosure, the scaffold X protein present in EVs (e.g., exosomes) is recombinantly produced by overexpressing the scaffold protein in production cells, thus resulting in an increased level of the scaffold protein in the resulting EVs (e.g., exosomes) relative to the level of the scaffold protein present in production cells that do not overexpress such scaffold proteins.

As used herein, the term "scaffold portion" refers to a molecule, such as a protein (e.g., scaffold X), that can be used to anchor a payload, such as a bioactive molecule, to an EV (e.g., exosome), for example, on the external surface of the EV. In some aspects, the scaffold portion includes synthetic molecules. In some aspects, the scaffold portion includes non-peptide portions. In other aspects, the scaffold portion includes, for example, lipids, carbohydrates, proteins, or combinations thereof (e.g., glycoproteins or proteolipids) naturally present in the EV (e.g., exosome). In some aspects, the scaffold portion includes lipids, carbohydrates, or proteins not naturally present in the EV (e.g., exosome). In some aspects, the scaffold portion includes lipids or carbohydrates naturally present in the EV (e.g., exosome) but enriched in the EV (e.g., exosome) relative to basal/natural/wild-type levels. In some aspects, the scaffold portion includes proteins naturally present in the EV (e.g., exosome) but enriched in the EV (e.g., exosome) relative to basal/natural/wild-type levels, for example, through recombinant overexpression in producing cells. In some aspects, the scaffold portion is scaffold X.

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 β-1 (“ITGB1”); integrin α-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 the entire protein or a fragment thereof (e.g., a functional fragment, such as the smallest fragment capable of anchoring another portion to the external or luminal surface of an EV (e.g., an exosome). In some aspects, scaffold X can anchor bioactive molecules to the external surface or lumen of an EV (e.g., an exosome). In some aspects of this disclosure, bioactive molecules can be covalently attached to scaffold X, for example, via a linker, a spacer, or both, as disclosed herein. Non-limiting examples of other scaffold portions that may be used with this disclosure include: aminopeptidase N (CD13); enkephalin, also known as membrane metalloendopeptidase (MME); exonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1); neuropilin-1 (NRP1); CD9, CD63, CD81, PDGFR, GPI-anchored proteins, lactoglucosins, LAMP2, and LAMP2B.

As used herein, the term "similarity" refers to the overall correlation between polymer molecules, such as between polynucleotide molecules (e.g., DNA and/or RNA molecules) and/or between polypeptide molecules. The calculation of the percentage of similarity between polymer molecules can be performed in the same manner as the calculation of the percentage of identity, except that the calculation of the percentage of similarity 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 amino acids are compared, for example, based on evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or combinations thereof.

As used herein, the term "small molecule" refers to an organic compound having a molecular weight of about 1000 g/mol or less (e.g., about 900 g/mol or less) that is capable of crossing cell membranes. Organic compounds can be, for example, biomolecules, drugs, or toxins. 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, antibacterial agents, antiviral agents, anticonvulsants, antipsychotics, antitumor agents, anti-inflammatory agents, antiobesity agents, antiparasitic drugs, contraceptives, otomedicines, ophthalmic agents, skeletal muscle relaxants, sleep disorder agents, central nervous system agents, cardiovascular agents, glycemic regulators, immunomodulators, 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, doxorubicin, taxol, and paclitaxel. Suitable toxins include, for example, bacterial toxins, including hemotoxins, phototoxins, hepatotoxins, and neurotoxins, mycotoxins, aflatoxins, ochratoxins, citrinins, and ergot alkaloids. Specific examples include monomethylreoxetine E (MMAE), botulinum toxin A, tetanus toxin A, edema toxin, exotoxin A, cholera toxin, pertussis toxin, diphtheria toxin, dioxins, muscarinic acid, and bufotoxin, or synthetic toxins such as bisphenol A, perchlorate, tetrachloroethylene, 2-butoxyethanol, and formaldehyde.

Unless otherwise stated, references to compounds having one or more stereocenters mean each stereoisomer and all combinations thereof.

The terms “subject,” “patient,” “individual,” and “host,” and their variants, are used interchangeably herein and refer to any mammalian subject, including but not limited to humans, livestock (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.), particularly humans, for the purpose of diagnosis, treatment, or therapy. The methods described herein are applicable to both human therapy and veterinary applications.

As used herein, the term "substantially free" means that a sample containing EVs (e.g., exosomes) contains less than 10% of macromolecules, such as contaminants, on a mass/volume (m/v) percentage basis. 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.

As used herein, the term “surface-engineered EV” (e.g., scaffold-engineered exosomes) refers to an EV whose membrane or surface is modified in its composition such that the surface of the engineered EV differs from that of the unmodified EV or the surface of a naturally occurring EV.

As used herein, the term “surface-engineered exosome” (e.g., scaffold-engineered exosome) refers to an exosome whose membrane or surface (external or internal surface) is modified in its composition such that the surface of the engineered exosome differs from that of the unmodified exosome or the surface of naturally occurring exosomes.

Engineering can be performed on the surface of an EV (e.g., exosome) or on the membrane of an EV (e.g., exosome) to alter the surface of the EV (e.g., exosome). For example, the composition of the membrane can be modified, such as proteins, lipids, small molecules, carbohydrates, or combinations thereof. The composition can be altered by chemical, physical, or biological methods, or by production from cells previously or simultaneously modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering, or by production from cells pre-modified by genetic engineering. In some aspects, surface-engineered EVs (e.g., exosomes) contain exogenous proteins (i.e., proteins not naturally expressed by the EV, e.g., exosome) or fragments or variants thereof that can be exposed on the surface of the EV (e.g., exosome), or can be anchoring sites (attachments) of portions exposed on the surface of the EV (e.g., exosome). In other respects, surface-engineered EVs (e.g., exosomes) comprise higher expression (e.g., higher quantity) of native EVs, such as exosomal proteins (e.g., scaffold X), or fragments or variants thereof that can be exposed on the surface of the EV (e.g., exosome), or can be anchoring sites (attachments) of portions exposed on the surface of the EV (e.g., exosome). In a specific respect, surface-engineered EVs, such as exosomes, comprise modifications to one or more membrane components, such as proteins (e.g., 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.

As used herein, the term "therapeutic effective amount" is an amount of reagent or pharmaceutical compound comprising the EV or exosomes of this disclosure that is sufficient to produce the desired therapeutic, pharmacological, and/or physiological effects in a subject in need. A therapeutic effective amount can also be a "prophylactic effective amount," as prevention can be considered a therapy.

As used herein, the term "treatment" refers to, for example, a reduction in the severity of a disease or condition; a shortening of the duration of the illness; improvement or elimination of one or more symptoms associated with the disease or condition; or providing any appropriate degree of beneficial effect to a subject suffering from a disease or condition, but not necessarily a cure for the disease or condition. The term also includes prevention or avoidance of a disease or condition or its symptoms. In one aspect, the term "treatment" means inducing an immune response against an antigen in a subject.

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 identity with another molecule when compared by methods known in the art. For example, a variant of a protein may include substitutions, insertions, deletions, frameshifts, or rearrangements in another protein.

In some respects, variants of scaffold X or its derivatives contain scaffold X variants or fragments (e.g., functional fragments) of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporters that have at least 70% identity with the full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporters.

In some respects, 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 respects, scaffold X or scaffold X derivatives include one or more mutations, such as conserved amino acid substitutions.

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

Using known protein engineering and recombinant DNA techniques, variants can be generated to improve or alter the characteristics of peptides. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secretory protein without 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 variants of the KGF protein that exhibited heparin-binding activity even after the deletion of 3, 8, or 27 amino acid residues from the N-terminus. Similarly, interferon-γ exhibited up to ten times greater activity after the deletion of 8 to 10 amino acid residues from the C-terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988), which is incorporated herein by reference in its entirety.)

Furthermore, there is ample evidence that variants often retain biological activity 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 an extensive mutational analysis of the human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants, each variant having an average of 2.5 amino acid changes across the entire length of the molecule. Multiple mutations were examined at each possible amino acid position. The researchers found that “most of the molecules could be altered with minimal effect on [binding or biological activity]” (see abstract). In fact, of the over 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins with significantly different activities from the wild type.

As described above, variants or derivatives include, for example, modified peptides. In some respects, variants or derivatives of peptides, polynucleotides, lipids, and glycoproteins are the result of chemical modification and/or endogenous modification. In some respects, variants or derivatives are the result of in vivo modification. In some respects, variants or derivatives are the result of in vitro modification. In still other respects, variants or derivatives are the result of intracellular modification in the producing cells.

Modifications present in variants and derivatives include, for example, acetylation, acylation, adenosine diphosphate ribosylation (ADP), amidation, covalent linkage of flavin, covalent linkage of heme moieties, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, covalent linkage of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cysteine, formation of pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, polyethylene glycolation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, isopreneation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginination and ubiquitination.

In some aspects, scaffold X can be modified at any convenient location. In some aspects, bioactive molecules can be modified at any convenient location. In certain aspects of this disclosure, EV (e.g., exosome) components (e.g., proteins such as scaffold X, lipids, or glycans) and/or bioactive molecules (e.g., antibodies or ADCs, PROTACs, small molecules (e.g., cyclic dinucleotides), toxins (e.g., MMAEs), STING (interferon gene stimulator) agonists (e.g., CAS Nos. 702662-50-8 and 849214-04-6), tolerators (e.g., those disclosed in U.S. Patent 7,910,113 and U.S. Patent Application Publication 2009/0169578) or antisense oligonucleotides) can be modified to produce derivatives comprising at least one linker, spacer, or both, as disclosed herein.

The term "alkyl," either itself or as part of another substituent, means, unless otherwise specified, a straight-chain or branched hydrocarbon group having a specified number of carbon atoms (e.g., _C1 - _C10 means 1 to 10 carbon atoms). Typically, an alkyl group will have 1 to 15 carbon atoms, such as 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms. A "lower alkyl" group is an alkyl group having 1 to 4 carbon atoms (e.g., 1 to 3 carbon atoms or 1 to 2 carbon atoms). The term "alkyl" includes monovalent, divalent, and polyvalent groups. For example, where appropriate, such as when the formula indicates that the alkyl group is divalent or when a substituent is attached to form a ring, the term "alkyl" includes "alkylene". Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, and homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, and n-octyl.

The term "alkylene" itself, or as part of another substituent _, refers to a divalent ( _{dimethyl) alkyl group, where alkyl is defined herein. "alkylene" is exemplified by –CH₂CH₂CH₂CH₂- ,} but is not limited to _this . Typically, an "alkylene" group will have 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 1 to 4 carbon atoms (e.g., 1 to 3 carbon atoms or 1 to 2 carbon atoms).

The term "alkenyl" itself, or as part of another substituent, refers to a straight-chain or branched hydrocarbon group having 2 to 15 carbon atoms and at least one double bond. A typical alkenyl group has 2 to 10 carbon atoms and at least one double bond. In one aspect, an 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, crotonyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), 2-isopentenyl, 1-pent-3-enyl, 1-hex-5-enyl, etc.

The term "alkynyl" itself, or as part of another substituent, refers to a straight-chain or branched, unsaturated or polyunsaturated hydrocarbon group having 2 to 15 carbon atoms and at least one triple bond. A typical "alkynyl" group has 2 to 10 carbon atoms and at least one triple bond. In one aspect of this disclosure, an alkynyl group has 2 to 6 carbon atoms and at least one triple bond. Exemplary alkynyl groups include prop-1-alkynyl, prop-2-alkynyl (i.e., propynyl), ethynyl, and 3-butynyl.

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

The term "heteroalkyl" on its own or in combination with another term means a stable straight-chain or branched hydrocarbon group consisting of a specified number of carbon atoms (e.g., _C2 - _C10 or _C2 - _C8 ) and at least one heteroatom selected from, for example, N, O, S, Si, B, and P (in one respect, N, O, S), wherein nitrogen, sulfur, and phosphorus atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The heteroatom is located at any internal position within the heteroalkyl group. Examples of heteroalkyl groups include, but are not limited to, _-CH₂ - _CH₂ -O- _CH₃ , _-CH₂ - _CH₂ -NH- _CH₃ , _-CH₂ - _CH₂ -N( _CH₃ ) _-CH₃ , _-CH₂ -S- _CH₂ - _CH₃ , _-CH₂ - _CH₂ -S(O) _-CH₃ , _-CH₂ - _CH₂ -S(O) _₂ - _CH₃ , -CH=CH-O- _CH₃ , _-CH₂ -Si( _CH₃ ) _₃ , _-CH₂ -CH=N- _OCH₃ , and -CH=CH-N( _CH₃ ) _-CH₃ . At most two heteroatoms can be consecutive, such as, for example, _-CH₂ -NH- _OCH₃ and _–CH₂ -O-Si( _CH₃ ) _₃ .

Similarly, the term "heteroalkylene" itself, or as part of another substituent, refers to a divalent group derived from a heteroalkyl group, such as, but not limited to, _-CH₂ - _CH₂ -S- _CH₂ - _CH₂- and _–CH₂ -S- _CH₂ - _CH₂ -NH- _CH₂- . Typically, the heteroalkyl group will have 3 to 24 atoms (carbons and heteroatoms, excluding hydrogen) (3 to 24-membered heteroalkyl). In another instance, the heteroalkyl group has a total of 3 to 10 atoms (3 to 10-membered heteroalkyl) or 3 to 8 atoms (3 to 8-membered heteroalkyl). Where appropriate, such as when the formula indicates that the heteroalkyl group is divalent or when the substituent is linked to form a ring, the term "heteroalkyl" includes "heteroalkylene".

As used herein, the term " _C1-8 alkyl" refers to a straight-chain or branched saturated hydrocarbon having 1 to 8 carbon atoms (e.g., 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2). Representative " _C1-8 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.

The term " _C1-10 alkylene" refers to a saturated straight-chain hydrocarbon group of the formula –( _CH2 ) _1-10- . Examples of _C1-10 alkylenes include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene.

The term "cycloalkyl" on its own or in combination with other terms refers to a saturated or unsaturated non-aromatic carbocyclic group having 3 to 24 carbon atoms, such as a _C3 - _C8 or _C3 - _C6 cycloalkyl group having 3 to 12 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3,5-cycloheptadienyl, cyclooctyl, and cyclooctadienyl. The term "cycloalkyl" also includes bridging polycyclic (e.g., bicyclic) structures such as norbornyl, adamantyl, and bicyclic [2.2.1]heptyl. The "cycloalkyl" group may be fused to at least one (e.g., 1 to 3) other rings and non-aromatic (e.g., carbocyclic or heterocyclic) rings selected from aryl (e.g., phenyl), heteroaryl (e.g., pyridyl). When the "cycloalkyl" group includes a fused aryl, heteroaryl, or heterocyclic group, the "cycloalkyl" group is attached to the rest of the molecule via a carbocyclic ring.

The terms “heterocyclic alkyl,” “heterocyclic,” “heterocyclic,” or “heterocyclic group,” alone or in combination with other terms, refer to a carbocyclic, non-aromatic ring (e.g., 3- to 8-membered rings and, in one aspect, 4, 5, 6, or 7-membered rings) containing at least one and at most five heteroatoms selected from, for example, N, O, S, Si, B, and P (in one aspect, N, O, and S), wherein 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 containing at least one and at most ten heteroatoms (e.g., 1 to 5 heteroatoms selected from N, O, and S) in stable combinations known to those skilled in the art. Exemplary heterocyclic alkyl groups include fused benzene rings. When a “heterocyclic” group includes a fused aryl, heteroaryl, or cycloalkyl ring, the “heterocyclic” group is attached to the remainder of the molecule via a heterocycle. Heteroatoms may occupy the positions where the heterocycle is attached to the remainder of the molecule.

Exemplary heterocyclic alkyl or heterocyclic groups disclosed herein include morpholino, thiomorpholino, thiomorpholino S-oxide, thiomorpholino S,S-dioxide, piperazino, homopiperazino, pyrrolyl, pyrrololino, imidazoalkyl, tetrahydropyrano, piperidino, tetrahydrofurano, tetrahydrothiopheno, piperidino, homopiperridino, homomorpholino, homothiomorpholino, homothiomorpholino S,S-dioxide, oxazolidinone, dihydropyrazolo, dihydropyrrolo, and dihydropyrazolo. Dihydropyridyl, dihydropyrimidinyl, dihydrofuranyl, dihydropyranyl, tetrahydrothiopheneyl S-oxide, tetrahydrothiopheneyl S,S-dioxide, high-thiomorpholinyl S-oxide, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, 1-piperazinyl, 2-piperazinyl, etc.

“Aryl” means a 5, 6, or 7-membered aromatic carbocyclic group having a monocyclic ring (e.g., phenyl) or fused to other aromatic or non-aromatic rings (e.g., 1 to 3 other rings). When the “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 remainder of the molecule via an aryl ring (e.g., a benzene ring). The aryl group is optionally substituted (e.g., having 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, anthracene, quinoline, indanyl, indenyl, dihydronaphthyl, fluorenyl, tetrahydronaphthyl, benzo[d][1,3]dioxacyclopentenyl, or 6,7,8,9-tetrahydro-5H-benzo[a]cycloheptenyl. In one respect, the aryl group is selected from phenyl, benzo[d][1,3]dioxanepentenyl, and naphthyl. In another respect, the aryl group is phenyl.

The term "arylene" refers to an aryl group having two open valence bonds, and it can be ortho, meta, or para configurations, as shown in the following structures:

The arylene group may be unsubstituted or substituted by up to four (e.g., 1, 2, 3, or 4) groups, including but not limited to _C1-8 alkyl, -O-( _C1-8 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, -halogen, _-N3 , _-NH2 , -NH(R'), -N(R') ₂ - _NO2 , and -CN, wherein each R' is independently H, _-C1-8 alkyl, or aryl.

The term “arylalkyl” or “arylalkyl” is intended to include those groups in which an aryl group or a heteroaryl group is attached to an alkyl group to form the groups -alkyl-aryl and -alkyl-heteroaryl, wherein alkyl, aryl, and heteroaryl are defined herein. Exemplary “arylalkyl” or “arylalkyl” groups include benzyl, phenethyl, pyridylmethyl, etc.

"Aryloxy group" refers to the group -O-aryl, where the aryl group is as defined herein. In one instance, the aryl moiety of the aryloxy group is phenyl or naphthyl. In another aspect, the aryl moiety of the aryloxy group is phenyl.

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. The "heteroaryl" group can be monocyclic or fused with other aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings (e.g., 1 to 3 other rings). When the "heteroaryl" group includes a fused aryl, cycloalkyl, or heterocycloalkyl ring, the "heteroaryl" group is attached to the remainder of the molecule via the heteroaryl ring. The heteroaryl group can be attached to the remainder of the molecule via carbon or heteroatom.

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 pyridinyl, pyrimidinyl, quinolinyl, benzothiopheneyl, indolyl, indololinyl, pyridazinyl, pyrazinyl, isoindolyl, isoquinolinyl, quinazolinyl, quinoxalolinyl, phthalazinyl, imidazolyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, indazinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, furanyl, thiopheneyl, pyrroleyl, oxadiazolyl, thiazolyl, triazolyl, tetrazolyl, isothiazolyl, naphridinyl, and isobenzodihydropyridine. Benzyl, benzodihydropyranyl, tetrahydroisoquinolinyl, isoindolinel, isobenzotetrahydrofuranyl, isobenzotetrahydrothiophenyl, isobenzothiophenyl, benzoxazolyl, pyridopyridyl, benzotetrahydrofuranyl, benzotetrahydrothiophenyl, purine, benzodioxacyclopentenyl, triazine, pteridinyl, benzothiazolyl, imidazopyridyl, imidazothiazolyl, dihydrobenzoisooxazine, benzoisooxazine, benzooxazine, dihydrobenzoisothiazolyl, benzopyranyl, benzothiaranyl, chromonel Benzodihydropyranyl, pyridyl-N-oxide, tetrahydroquinolinyl, dihydroquinolinyl, dihydroquinolinyl, dihydroisoquinolinyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindololinyl, benzodioxane, benzoxazolinyl, pyrroleyl N-oxide, pyrimidinyl N-oxide, pyrazinyl N-oxide, pyrazinyl N-oxide, quinolinyl N-oxide, indolyl N-oxide, indololinyl N-oxide, isoquinolinyl N-oxide, quinazolinyl N-oxide, quinoxalyl Phthaloazinyl N-oxide, phthalazinyl N-oxide, imidazoleyl N-oxide, isoxazolyl N-oxide, oxazolyl N-oxide, thiazolyl N-oxide, indazinyl N-oxide, indazoleyl N-oxide, benzothiazolyl N-oxide, benzimidazolyl N-oxide, pyrroleyl N-oxide, oxadiazolyl N-oxide, thiazolyl N-oxide, triazolyl N-oxide, tetrazolyl N-oxide, benzothiaranyl S-oxide, benzothiaranyl S,S-dioxide. Exemplary heteroaryl groups include imidazoleyl, pyrazolyl, thiazolyl, triazolyl, isoxazolyl, isothiazolyl, imidazoleyl, thiazolyl, oxadiazolyl, and pyridinyl. Other exemplary heteroaryl groups include 1-pyrrole, 2-pyrrole, 3-pyrrole, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isooxazolyl, 4-isooxazolyl, 5-isooxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, pyridin-4-yl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinel, 2-benzimidazolyl, 5-indolyl, 1-isoquinolinyl, 5-isoquinolinyl, 2-quinoxolinyl, 5-quinoxolinyl, 3-quinolinyl, and 6-quinolinyl.

Any organic residues 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 the case may be). Typical substituents include, but are not limited to, _C1-8 alkyl, -O-( _C1-8 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, -halogen, _-N3 , _-NH2 , -NH(R'), -N(R') ₂ , and –CN, wherein each R' is independently H, _-C1-8 alkyl, or aryl.

Extracellular vesicles of inclusion (I) construct

This disclosure provides an extracellular vesicle (EV) comprising a bioactive molecule (BAM) attached to the EV via an anchoring portion (AM) according to Formula I:

AM-SP ₁ -L ₁ -SP ₂ -BAM (Formula I)

in

_L1 is a cleavable bond containing the following: 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-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate oxy group; or a silyl ether; and

_SP1 and _SP2 are optional first and second spacers, respectively.

As used herein, the term "connector" refers to a combination of structural elements comprising, for example, "bonds" (both cleavable and non-cleavable) and "spacers" that connect the anchoring portion AM and the bioactive molecule BAM. In the absence of the cleavable bonds of this disclosure, these connectors allow for more efficient and greater loading of bioactive molecules (e.g., ASO) onto the surface of EVs (e.g., exosomes) compared to corresponding constructs comprising the same anchoring portion (AM) and bioactive molecule ( _BAM ). In other words, the constructs disclosed herein, such as those of the formula IAM- _SP1 - _L1 - _SP2- BAM, result in: (i) higher EV loading efficiency, (ii) a higher number of BAMs per EV, (iii) a higher BAM density per EV, (iv) higher BAM potency, or (v) any combination thereof, relative to constructs having the structure AM-BAM or AM-SP1-BAM.

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

As used herein, the term "spacer" refers to a chemical moiety capable of covalently linking two spacer portions (e.g., a bioactive molecule and an anchoring portion) together to form a generally stable bipartite molecule. Generally, spacers are non-cleavable. For example, spacers can be alkyl chains or polyalkoxy chains, as described herein.

In some aspects, the length of the connector connecting the anchoring portion AM and the bioactive molecule BAM is between approximately 2 nm and approximately 30 nm. In other aspects, the length of the connector connecting the anchoring portion AM and the bioactive molecule BAM is approximately 2 nm, approximately 3 nm, approximately 4 nm, approximately 5 nm, approximately 6 nm, approximately 7 nm, approximately 8 nm, approximately 9 nm, approximately 10 nm, approximately 11 nm, approximately 12 nm, approximately 13 nm, approximately 14 nm, approximately 15 nm, approximately 16 nm, approximately 17 nm, approximately 18 nm, approximately 19 nm, approximately 20 nm, approximately 21 nm, approximately 22 nm, approximately 23 nm, approximately 24 nm, approximately 25 nm, approximately 26 nm, approximately 27 nm, approximately 28 nm, approximately 29 nm, or approximately 30 nm. In some aspects, the optimized connector length is at least 2nm, at least 3nm, at least 4nm, at least 5nm, at least 6nm, at least 7nm, at least 8nm, at least 9nm, at least 10nm, at least 11nm, at least 12nm, at least 13nm, at least 14nm, at least 15nm, at least 16nm, at least 17nm, at least 18nm, at least 19nm, at least 20nm, at least 21nm, at least 22nm, at least 23nm, at least 24nm, at least 25nm, at least 26nm, at least 27nm, at least 28nm, at least 29nm, or at least 30nm. In some aspects, the optimized connector lengths are 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.

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

extracellular vesicles

Extracellular vesicles (EVs) typically have a diameter of 20 nm to 1000 nm. Exosomes are small extracellular vesicles, typically with a diameter of about 50 to 200 nm (e.g., 100 to 200 nm). EVs, such as exosomes, consist of a restrictive lipid bilayer and a variety of proteins and nucleic acids (Maas, S.L.N., 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)).

The EVs (e.g., exosomes) of this disclosure may have a diameter between about 20 and about 300 nm. In some aspects, the EVs (e.g., exosomes) of this disclosure have diameters between about 20 and about 290 nm, about 20 and about 280 nm, about 20 and about 270 nm, about 20 and about 260 nm, about 20 and about 250 nm, about 20 and about 240 nm, about 20 and about 230 nm, about 20 and about 220 nm, about 20 and about 210 nm, about 20 and about 200 nm, about 20 and about 190 nm, about 20 and about 180 nm, and about 20 and about 300 nm. Diameters ranging from 170 nm, about 20 to about 160 nm, about 20 to about 150 nm, about 20 to about 140 nm, about 20 to about 130 nm, about 20 to about 120 nm, about 20 to about 110 nm, about 20 to about 100 nm, about 20 to about 90 nm, about 20 to about 80 nm, about 20 to about 70 nm, about 20 to about 60 nm, about 20 to about 50 nm, about 20 to about 40 nm, or about 20 to about 30 nm. The size of the EVs (e.g., exosomes) described herein can be measured according to methods known in the art.

Unlike antibodies, EVs (e.g., exosomes) can accommodate a large number of molecules attached to their surface, with each EV (e.g., exosome) containing approximately thousands to tens of thousands of molecules. 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 large number of molecules accommodated on the surface of an EV (e.g., exosomes) can be influenced by factors such as the type of bioactive molecule used (e.g., antibodies are larger than antisense oligonucleotides), the type of membrane anchor used (e.g., protein anchors are larger than lipid or lipid-spacer anchors), and the combination of linkers and spacers connecting the bioactive molecule and the membrane anchor. In this regard, this 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).

In some aspects, this disclosure provides “bioactive molecules” (BAMs), such as ASOs, that are directly or indirectly attached (e.g., covalently bonded) to one or more anchoring moieties (AMs), for example via one or more adapter combinations. The anchoring moieties can be inserted into the lipid bilayer of an EV (e.g., exosome), allowing exosomes to be loaded with BAMs (e.g., ASOs). Currently, a major obstacle to the commercialization of exosomes as delivery mediators for polar BAMs (e.g., ASOs) is the highly inefficient loading process. As illustrated herein, this obstacle can be overcome by using specific adapter/spacer combinations (i.e., “adaptors”) to attach the BAM to the AM before loading it onto the EV (e.g., exosome). Therefore, as described herein, the optimized use of adapters facilitates the loading of BAMs (e.g., ASOs) onto EVs (e.g., exosomes).

Compared to previously reported loading efficiencies and BAM densities in EVs (e.g., exosomes) introduced via methods such as electroporation or cationic lipid transfection, compositions and methods using BAM-containing constructs (e.g., ASO) linked to AMs (e.g., lipids, such as sterols) via optimized linkers described herein improve loading efficiency and BAM density. The compositions and methods disclosed herein also significantly improve EV (e.g., exosome) potency via methods such as electroporation or cationic lipid transfection, compared to previously reported potency when introducing unmodified BAM into EVs (e.g., exosomes).

The EVs (e.g., exosomes) disclosed herein comprise a lipid bilayer membrane (“exosome membrane” or “EV membrane”) comprising an inner surface (luminal surface) and an outer surface. The inner surface faces the 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, glycerol phosphates, sterols, cholesterol, and phosphatidylserine. The EV or exosome membrane comprises an inner lobule and an outer lobule. The composition of the inner and outer lobules can be determined by transbilayer distribution assays known in the art, see, for example, Kuypers et al., Biohim Biophys Acta 1985 819:170.

In some aspects, the ectolobule is composed of approximately 70% to approximately 90% choline phospholipids, approximately 0% to approximately 15% acidic phospholipids, and approximately 5% to approximately 30% phosphatidylethanolamine. In some aspects, the endolobule is composed of approximately 15% to approximately 40% choline phospholipids, approximately 10% to approximately 50% acidic phospholipids, and approximately 30% to approximately 60% phosphatidylethanolamine. In some aspects, the EV or exosome membrane contains one or more polysaccharides, such as glycans. The glycans on the surface of the EV or exosome can serve as attachments to maleimide moieties or as junctions connecting the glycans and maleimide moieties. The glycans can be present on one or more proteins on the surface of the EV (e.g., exosome), such as scaffold X, such as PTGFRN peptides, or on the lipid membrane of the EV (e.g., exosome). The glycans can be modified to have thiofucose, which can serve as a functional group for attaching maleimide moieties to the glycans. In some respects, scaffold X can be modified to express a large amount of glycans to allow additional attachment to EVs (e.g., exosomes).

Anchoring part

In some aspects, the anchoring portion AM of Formula I comprises sterols, lipids (e.g., phospholipids), vitamins, peptides, or combinations thereof, and optionally spacers. Generally, AM may comprise any hydrophobic portion or combination thereof capable of intercalating into a lipid bilayer of an EV (e.g., exosome), electrostatically interacting with the surface of an EV (e.g., exosome), or a combination thereof. Suitable anchoring portions AM capable of anchoring the bioactive molecule BAM to the surface of an EV (e.g., exosome) comprise, for example, sterols (e.g., cholesterol), lipids, phospholipids, lysophospholipids, fatty acids, vitamins (e.g., fat-soluble vitamins), scaffold portions (e.g., protein X), or combinations thereof, as described herein.

In some aspects, the anchoring portion AM comprises a sterol, steroid, hopane steroid, hydroxy steroid, open-ring steroid, or analogue thereof having lipophilic properties. In some aspects, the anchoring portion comprises a sterol, such as phytosterol, fungal sterol, or animal sterol. Exemplary animal sterols include cholesterol and 24S-hydroxycholesterol; exemplary phytosterols include ergosterol (fungal sterol), campesterol, sitosterol, and stigmasterol. In some aspects, the sterol is selected from ergosterol, 7-dehydrocholesterol, cholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fucosterol, sargastric sterol, campesterol, β-sitosterol, sitosterol, coprostaphylol, alfalfa, stigmasterol, or combinations thereof. Sterols can exist as free sterols, acylated (sterol esters), alkylated (alkyl sterol ethers), sulfated (sterol sulfate esters), or linked to a glycoside moiety (sterol glycoside), which itself can be acylated (acylated sterol glycoside).

Sterols can be attached to spacers SP, for example, via available -OH groups of the sterol (via solid-phase synthesis or conjugation). Exemplary sterols have the general skeleton shown below:

For example, ergosterol has the following structure:

In another example, cholesterol has the following structure:

In some aspects, the anchoring portion AM comprises, consists of, or is substantially composed of the following: sterols, cholesterol, mercaptocholesterol, ergosterol, 7-dehydrocholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fucosterol, sargastricosterol, campesterol, β-sitosterol, sitosterol, coprosterol, alfalfa, stigmasterol, or combinations thereof. In some specific aspects, the anchoring portion AM contains cholesterol.

In some respects, the anchoring portion AM contains steroids. In some respects, the steroids are selected from dihydrotestosterone, phytosterol, sesquiterpenes, diosgenin, progesterone, or cortisol.

In some aspects, the anchored portion AM comprises or is composed of lipids. The lipid-anchored portion AM may include any lipid known in the art, such as palmitic acid or glycosylphosphatidylinositol. In some aspects, AM includes lipids, which include fatty acids or phospholipids. In some aspects, the lipid is a fatty acid, phosphatide, phospholipid (e.g., phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine) or analogues thereof (e.g., phosphatidylcholine, lecithin, phosphatidylethanolamine, cephalin, or phosphatidylserine or analogues thereof, or a portion thereof, such as a partially hydrolyzed portion thereof).

In some aspects, the anchored portion AM comprises, consists of, or is substantially composed of: fatty acids, such as straight-chain fatty acids. In some aspects, the fatty acid is a straight-chain fatty acid, branched-chain fatty acid, saturated fatty acid, unsaturated fatty acid, hydroxy fatty acid, 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 omega-3 or omega-6 fatty acids. In some aspects, the anchored portion AM comprises, consists of, or is substantially composed of: straight-chain fatty acids, branched-chain fatty acids, unsaturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, hydroxy fatty acids, polycarboxylic acids, or any combination thereof.

In some respects, lipids, such as fatty acids, have _C2 - _C18 chains. In some respects, lipids, such as fatty acids, have _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , C8, _C9 , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _C16 , _C17 , or _C18 chains. In some respects, fatty acids have a _C2 chain. In some respects, fatty acids have a _C3 chain. In some respects, fatty acids have a _C4 chain. In some respects, fatty acids have a _C5 chain. In some respects, fatty acids have a _{C6 chain} . In some respects, fatty acids have a _C7 chain. In some respects, fatty acids have a _C8 chain. In some respects, fatty acids have a _C9 chain. In some respects, fatty acids have a _C10 chain. In some respects, fatty acids have a _C11 chain. In some respects, fatty acids have a _C12 chain. In some respects, fatty acids have a _C13 chain. In some respects, fatty acids have a _C14 chain. In some respects, fatty acids have a _C15 chain. In some respects, fatty acids have a _C16 chain. In some respects, fatty acids have a _C17 chain. In some respects, fatty acids have a _C18 chain.

In some respects, fatty acids have _C4 - _C18 chains. In some respects, fatty acids possess _C2 - _C3 , _C2 - _C4 , _C2 - _C5 , _C2 - _C6 , _C2 - _C7 , _C2 - _C8 , _C2 -C9, C2- _C10 , _C2 - _C11 , _C2 - _C12 , _C2 - _C13 , _C2 - _C14 , _C2 - _C15 , _C2 - _C16 , _C2 - _C17 , _C2 - _C18 , _C3 - _C4 , _C3 - _C5 , C3- _C6 , _C3 - _C7 , _C3 - _C8 , _C3 - _C9 , _C3 - _C10 , _C3 - _C11 , _C3 - _C12 , _{and C3} - _{C4 -C5.} -C ₁₃ , C ₃ -C ₁₄ , C ₃ -C ₁₅ , C ₃ -C ₁₆ , C ₃ -C ₁₇ , C ₃ -C ₁₈ , C ₄ -C ₅ , C ₄ -C ₆ , C ₄ -C ₇ , C ₄ -C ₈ , C ₄ -C ₉ , C ₄ -C ₁₀ , C ₄ -C ₁₁ , _C4 - _C12 , _C4 - _C13 , _C4 -C14, C4- _C15 , _C4 - _C16 , _C4 - _C17 , _C4 - _C18 , _C5 - _C6 , _C5 - _C7 , _{C5 -C8 , C5 - C9} , _C5 -C ₁₀ ,C ₅ -C ₁₁ , C ₅ -C ₁₂ , C ₅ -C ₁₃ , C ₅ -C ₁₄ , C ₅ -C ₁₅ , C ₅ -C ₁₆ , C ₅ -C ₁₇ , C ₅ -C ₁₈ , C ₆ -C ₇ , C ₆ -C ₈ , C ₆ -C ₉ , C ₆ -C ₁₀ , C ₆ -C ₁₁ , C ₆ -C ₁₂ , C ₆ -C ₁₃ , C ₆ -C ₁₄ , C ₆ -C ₁₅ , C ₆ -C ₁₆ , C ₆ -C ₁₇ , C ₆ -C ₁₈ , C ₇ -C ₈ , C ₇ -C ₉ , C ₇ -C ₁₀ , C ₇ -C ₁₁ , C ₇ -C ₁₂ , _C7 - _C13 , C ₇ -C ₁₄ , C ₇ -C ₁₅ , C ₇ -C ₁₆ , C ₇ -C ₁₇ , C ₇ -C ₁₈ , C ₈ -C ₉ , C ₈ -C ₁₀ , C ₈ -C ₁₁ , C ₈ -C ₁₂ , C ₈ -C ₁₃ , C ₈ -C ₁₄ , C ₈ -C ₁₅ , _C8 - _C16 , _C8 - _C17 , _C8 -C18, C9- _C10 , _C9 - _C11 , _C9 - _C12 , _C9 - _C13 , _C9 - _C14 , _C9 - _C15 , _C9 - _C16 , _{C9 - C17 , 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 ,C ₁₃ -C ₁₅ , _C13 - _C16 , _C13 - _C17 , C13-C18, _C14 - _C15 , _C14 - _C16 , _C14 - _C17 , _C14 - _C18 , _C15 - _C16 , _C15 - _C17 , _C15 - _C18 , _{C16 - C17} , _C16 - _C18 or _C17 - _{C18 chain} .

In some respects, the anchored portion AM contains two fatty acids, each of which is independently selected from fatty acids having a chain having any of the aforementioned ranges or numbers of carbon atoms. In some respects, one of the fatty acids is independently _C2 - _C3 , _C2 - _C4 , _C2 - _C5 , _C2 - _C6 , C2-C7, _C2 - _C8 , _C2 - _C9 , _C2 - _C10 , C2- _C11 , _C2 - _C12 , _C2 - _C13 , _{C2-C14, C2 - C15 , C2 - C16} , C2- _C17 , _C2 - _C18 , _{C3-C4, C3 - C5 , C3} - _C6 , _C3 - _C7 , _{C3 - C8} , _C3 - _C9 , _{C3 -C10 , C3 - C11} , _C3 -C ₁₂ , _C3 - _C13 , _C3 - _C14 , _C3 -C15, C3- _C16 , _C3 - _C17 , _C3 - _C18 , _C4 - _C5 , _C4 - _C6 , _C4 - _C7 , _{C4 -C8 , C4} - _C9 , _{C4 -} C ₁₀ , C ₄ -C ₁₁ , C ₄ -C ₁₂ , C ₄ -C ₁₃ , C ₄ -C ₁₄ , C ₄ -C ₁₅ , C ₄ -C ₁₆ , C ₄ -C ₁₇ , C ₄ -C ₁₈ , C ₅ -C ₆ , C ₅ -C ₇ , C ₅ -C ₈ , C ₅ -C ₉ ,C ₅ -C ₁₀ , C ₅ -C ₁₁ , C ₅ -C ₁₂ , C ₅ -C ₁₃ , C ₅ -C ₁₄ , C ₅ -C ₁₅ , C ₅ -C ₁₆ , C ₅ -C ₁₇ , C ₅ -C ₁₈ , C ₆ -C ₇ , C ₆ -C ₈ , C ₆ -C ₉ , C ₆ -C ₁₀ , C ₆ -C ₁₁ , C ₆ -C ₁₂ , C ₆ -C ₁₃ , C ₆ -C ₁₄ , C ₆ -C ₁₅ , C ₆ -C ₁₆ , C ₆ -C ₁₇ , C ₆ -C ₁₈ , C ₇ -C ₈ , C ₇ -C ₉ , C ₇ -C ₁₀ , C ₇ -C ₁₁ , _C7 - _C12 , C ₇ -C ₁₃ , C ₇ -C ₁₄ , C ₇ -C ₁₅ , C ₇ -C ₁₆ , C ₇ -C ₁₇ , C ₇ -C ₁₈ , C ₈ -C ₉ , C ₈ -C ₁₀ , C ₈ -C ₁₁ , C ₈ -C ₁₂ , C ₈ -C ₁₃ , C ₈ -C ₁₄ , _C8 - _C15 , _C8 - _C16 , _C8 -C17, C8- _C18 , _C9 - _C10 , _C9 - _{C11 , C9} - _C12 , _C9 - _C13 , _C9 - _C14 , _C9 - _C15 , _{C9 -C16 , C9} -C ₁₇ , C ₉ -C ₁₈ , _C10 - _C11 , _C10 - _C12 , _C10 - _C13 , _C10 - _C14 , _C10 - _C15 , _C10 - _C16 , _C10 - _C17 , _C10 - _C18 , _C11 - _C12 , _C11 - _C13 , 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 Fatty acids with C13} - _C15 , _C13 - _C16 , _C13 - _C17 , _C13 - _C18 , C14- _C15 , _C14 - _C16 , _C14 - _C17 , _C14 - _C18 , _C15 - _C16 , _C15 -C17, _C15 - _C18 , _{C16 - C17} , _C16 - _C18 , or _{C17 -C18 chains} , and another independently having _C2 - _C3 , _C2 - _C4 , _C2 - _C5 , _C2 - _C6 , _C2 - _C7 , _C2 - _C8 , _C2 - _C9 , _C2 - _C10 , or _C2 - _C11 chains. , _C2 - _C12 , _C2 - _C13 , _C2 - _C14 ,C2-C15, _C2 - _C16 , _C2 - _C17 , _C2 - _C18 , _C3 - _C4 , _C3 - _C5 , _C3 - _C6 , _{C3 -C7 , C3 - C8} , _C3 - _C9 , _C3 - _C10 , _C3 - _C11 ,C3-C12, _C3 - _C13 , _C3 - _C14 , _C3 - _C15 , _C3 - _C16 , _C3 - _C17 , _{C3 - C18} , _C4 - _C5 , _{C4 - C6} , C ₄ -C ₇ , C ₄ -C ₈ , C ₄ -C ₉ , C ₄ -C ₁₀ , C ₄ -C ₁₁ , C ₄ -C ₁₂ , C ₄ -C ₁₃ , C ₄ -C ₁₄ , C ₄ -C ₁₅ , C ₄ -C ₁₆ , C ₄ -C ₁₇ , C ₄ -C ₁₈ , C ₅ -C ₆ , C ₅ -C ₇ , C ₅ -C ₈ , C ₅ -C ₉ , C ₅ -C ₁₀ , C ₅ -C ₁₁ , C ₅ -C ₁₂ , C ₅ -C ₁₃ , C ₅ -C ₁₄ , C ₅ -C ₁₅ , C ₅ -C ₁₆ , C ₅ -C ₁₇ , C ₅ -C ₁₈ , C ₆ -C ₇ , C ₆ -C _8. C ₆ -C ₉ , C ₆ -C ₁₀ , C ₆ -C ₁₁ , C ₆ -C ₁₂ , C ₆ -C ₁₃ , C ₆ -C ₁₄ , C ₆ -C ₁₅ , C ₆ -C ₁₆ , C ₆ -C ₁₇ , C ₆ -C ₁₈ , C ₇ -C ₈ , C ₇ -C _9. C ₇ -C ₁₀ , C ₇ -C ₁₁ , C ₇ -C ₁₂ , C ₇ -C ₁₃ , C ₇ -C ₁₄ , C ₇ -C ₁₅ , C ₇ -C ₁₆ , C ₇ -C ₁₇ , C ₇ -C ₁₈ , C ₈ -C ₉ , C ₈ -C ₁₀ , C ₈ -C ₁₁ , _C8 -C ₁₂ , C ₈ -C ₁₃ , C ₈ -C ₁₄ , C ₈ -C ₁₅ , C ₈ -C ₁₆ , C ₈ -C ₁₇ , C ₈ -C ₁₈ , C ₉ -C ₁₀ , C ₉ -C ₁₁ , C ₉ -C ₁₂ , C ₉ -C ₁₃ , C ₉ -C ₁₄ , C ₉ -C ₁₅ , C ₉ -C ₁₆ , C ₉ -C ₁₇ , C ₉ -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 , 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 Fatty acids with _C16 , _C15 - _C17 , _C15 - _C18 , _C16 - _C17 , _C16 - _C18 , or _C17 - _C18 chains. In some respects, each fatty acid independently has a chain with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms.

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), octanoic acid (C8), decanoic 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), valeric acid (C5), heptanoic acid (C7), nonanoic acid (C9), undecanoic acid (C11), tridecanoic acid (C13), pentadecanoic acid (C15), or heptadecanoic acid (C17).

Examples of suitable saturated branched-chain fatty acids include isobutyric acid, isohexanoic acid, isooctanoic acid, isodecanic acid, isolaric acid, 11-methyldodecanoic acid, isomearic acid, 13-methyltetradecanoic acid, isopalmitic acid, 15-methylhexadecanoic acid, or isostearic acid. Suitable saturated odd-chain branched-chain fatty acids include trans-isofatty acids terminated with isobutyl groups, such as 6-methyloctanoic acid, 8-methyldecanoic acid, 10-methyldodecanoic acid, 12-methyltetradecanoic acid, or 14-methylhexadecanoic acid.

Examples of suitable unsaturated fatty acids include 4-decenoic acid, 9-decenoic acid, 4-dodecenoic acid, 5-dodecenoic acid, myrcenoic acid, 4-tetradecenoic acid, 5-tetradecenoic acid, 9-tetradecenoic acid, palmitoleic acid, 6-octadecenoic acid, oleic acid, etc.

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, etc.

Suitable examples of polycarboxylic acids include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, octanoic acid, azelaic acid, sebacic acid, D,L-malic acid, etc.

In some respects, each fatty acid is independently selected from propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, or stearic acid.

In some respects, each fatty acid is independently selected from alpha-linolenic acid, octadecanoic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, gamma-linoleic acid, dihomo-gamma-linoleic acid, arachidonic acid, docosahexaenoic acid, palmitoleic acid, isoleic acid, eicosapentaenoic acid, oleic acid, transoleic acid, bosseopentaenoic acid, sardine acid, or another monounsaturated or polyunsaturated fatty acid.

In some respects, one or two of the fatty acids are essential fatty acids. Given the health benefits of certain essential fatty acids, the therapeutic benefits of exosomes carrying therapeutic agents can be increased by including such fatty acids in the therapeutic agents. In some respects, essential fatty acids are n-6 or n-3 essential fatty acids selected from the group consisting of linolenic acid, gamma-linolenic acid, dihoxy-gamma-linolenic acid, arachidonic acid, adrenaline, eicosapentaenoic acid (n-6), alpha-linolenic acid, or stearatetraenoic acid.

Fatty acid chains vary greatly in length and can be classified according to chain length, for example, from short to very long. Short-chain fatty acids (SCFAs) are fatty acids with chains of about five or fewer carbons (e.g., butyric acid). In some respects, fatty acids are SCFAs. Medium-chain fatty acids (MCFAs) include fatty acids with chains of about 6 to 12 carbons, which can form medium-chain triglycerides. In some respects, fatty acids are MCFAs. Long-chain fatty acids (LCFAs) include fatty acids with chains of 13 to 21 carbons. In some respects, fatty acids are LCFAs.

In some respects, the anchoring portion AM is formed of a linear fatty acid comprising, consisting of, or substantially consisting of the following: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, or combinations thereof. In some specific respects, the anchoring portion AM is formed of palmitic acid.

In some respects, the anchoring portion (AM) comprises, is composed of, or is essentially composed of phospholipids. The structure of a phospholipid molecule generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" composed of phosphate groups. For example, phospholipids can be lipids according to the following formula:

Rp is the phospholipid moiety, and ^R1 and ^R2 may be the same or different, and each may or may not have an unsaturated fatty acid moiety. The fatty acid moiety may be selected from the non-restrictive group consisting of, for example, lauric acid, myristic acid, myristone acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, or linoleic acid.

The phospholipid portion may be, for example, lecithin, phosphatidylcholine (e.g., 2-lysophosphatidylcholine), inositol phosphate, sphingolipid phosphate, ethanolamine phosphate, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and sphingomyelin, or any combination thereof.

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

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 for the anchoring portions disclosed herein include phosphatidylethanolamines (e.g., dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, 1-oleoyl-2-palmitoyl phosphatidylethanolamine and disqualyl phosphatidylethanolamine), phosphatidylglycerols (e.g., dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, 1-palmitoyl-2-oleoyl-phosphatidylglycerol, 1-oleoyl-2-palmitoyl-phosphatidylglycerol and disqualyl phosphatidylglycerol); phosphatidylserines (e.g., such as dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, ... Dipalmitoylphosphatidylserine, distearylphosphatidylserine, dioleoylphosphatidylserine, 1-palmitoyl-2-oleoylphosphatidylserine, 1-oleoyl-2-palmitoylphosphatidylserine, and disqualoylphosphatidylserine; phosphatidic acids (e.g., dilauroylphosphatidyl, dimyristoylphosphatidyl, dipalmitoylphosphatidyl, distearylphosphatidyl, dioleoylphosphatidyl, 1-palmitoyl-2-oleoylphosphatidylserine); phosphatidic acids (e.g., dilauroylphosphatidyl, dimyristoylphosphatidyl, dipalmitoylphosphatidyl, distearylphosphatidyl, dioleoylphosphatidyl, 1-palmitoyl-2-oleoylphosphatidylserine, disqual ... -Oleoylphosphatidic acid, 1-oleoyl-2-palmitoyl-phosphatidic acid and disqualoylphosphatidic acid); and phosphatidylinositol (e.g., dilauroyl phosphatidylinositol, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, distearoyl phosphatidylinositol, dioleoyl phosphatidylinositol, 1-palmitoyl-2-oleoyl-phosphatidylinositol, 1-oleoyl-2-palmitoyl-phosphatidylinositol and disqualoyl phosphatidylinositol).

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

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 fourteen carbons (C14). In some aspects, the anchoring moiety AM comprises a symmetrical phospholipid having sixteen carbons (C16). In some aspects, the anchoring moiety AM comprises a symmetrical phospholipid having eighteen carbons (C18). In some aspects, the phospholipid is phosphatidylethanolamine (PE). Therefore, 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. Therefore, in some aspects, the anchoring moiety AM comprises C14:0PE, C16:0PE, or C18:0PE. In some respects, the anchored portion AM contains 16:01,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (16:0PDPPE).

In some aspects, the anchoring moiety AM contains a phospholipid containing a cyanuric acid (cyanur) group. In some aspects, the phospholipid containing the cyanuric acid group is PE. In some aspects, the phospholipid containing the cyanuric acid group is C14:0PE, C16:0PE, or C18:0PE. In some aspects, the anchoring moiety AM contains 16:01,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide methyl)cyclohexane-formamide] (16:0PE MCC). In some aspects, the anchoring moiety AM contains 16:01,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-(cyanuric acid) (16:0 cyanuric acid PE).

In some respects, the anchoring portion AM comprises at least one symmetrical phospholipid. The symmetrical phospholipid may be selected from the non-restricted group consisting of: 1,2-dipropionyl sn-glycerol 3-phosphate choline (O3:OPC), 1,2-dibutyryl sn-glycerol 3-phosphate choline (O4:OPC), 1,2-divalloyl sn-glycerol 3-phosphate choline (O5:OPC), 1,2-dihexanoyl sn-glycerol 3-phosphate choline (O6:OPC), 1,2-diheptanoyl sn-glycerol 3-phosphate choline (O7:OPC), 1,2-dioctanoyl sn-glycerol Choline-3-phosphate (08:0PC), 1,2-nonanoyl sn-glycerol choline-3-phosphate (09:0PC), 1,2-decanoyl sn-glycerol choline-3-phosphate (10:0PC), 1,2-undecanoyl sn-glycerol choline-3-phosphate (11:0PC, DUPC), 1,2-lauroyl sn-glycerol choline-3-phosphate (12:0PC), 1,2-tridecanoyl sn-glycerol choline-3-phosphate (13:0PC), 1,2-myristoyl 1,2-Dipentadecanoyl 1,2-sn-glycerol 3-phosphate choline (14:0PC, DMPC), 1,2-Dipalmitoyl 1,2-sn-glycerol 3-phosphate choline (15:0PC), 1,2-Dipalmitoyl 1,2-sn-glycerol 3-phosphate choline (16:0PC, DPPC), 1,2-Diphytyl 1,2-sn-glycerol 3-phosphate choline (4ME 16:0PC), 1,2-Diheptadecanoyl 1,2-sn-glycerol 3-phosphate choline (17:0PC), 1,2-Distearyl 1,2-sn-glycerol 3-phosphate choline (18:0PC) C, DSPC), 1,2-Dimyristoyl sn-glycerol 3-phosphate choline (14:1 (Δ9-trans)PC), 1,2-Dipalmitoyl sn-glycerol 3-phosphate choline (16:1 (Δ9-cis)PC), 1,2-Dipalmitoyl sn-glycerol 3-phosphate choline (16:1 (Δ9-trans)PC), 1,2-Dioctadecylselenoyl sn-glycerol 3-phosphate choline (18:1 (Δ6-cis)PC), 1,2-Dioleoyl sn-glycerol 3-phosphate Choline (18:1(Δ9-cis)PC, DOPC), 1,2-dioleoylsn-glycerol 3-phosphate choline (18:1(Δ9-trans)PC), 1,2-dilinoleoylsn-glycerol 3-phosphate choline (18:2(cis)PC, DLPC), 1,2-dilinoleoylsn-glycerol 3-phosphate choline (18:3(cis)PC, DLnPC), 1,2-dihexanoylsn-glycerol 3-phosphate ethanolamine (06:0PE), 1,2-dioct ... 1,2-Didecanoyl sn-glycerol 3-phosphate ethanolamine (08:0PE), 1,2-Dilauroyl sn-glycerol 3-phosphate ethanolamine (10:0PE), 1,2-Dilauroyl sn-glycerol 3-phosphate ethanolamine (12:0PE), 1,2-Dimyristoyl sn-glycerol 3-phosphate ethanolamine (14:0PE), 1,2-Dipentadecanoyl sn-glycerol 3-phosphate ethanolamine (15:0PE), 1,2-Dipalmitoyl sn-glycerol 3-phosphate ethanolamine (16:0PE), 1,2 Diphyllyl sn-glycerol ethanolamine 3-phosphate (4ME 16:0PE), 1,2-Diheptadecanoyl sn-glycerol ethanolamine 3-phosphate (17:0PE), 1,2-Distearatel sn-glycerol ethanolamine 3-phosphate (18:0PE, DSPE), 1,2-Dipalmitoyl sn-glycerol ethanolamine 3-phosphate (16:1PE), 1,2-Dioleoyl sn-glycerol ethanolamine 3-phosphate (18:1(Δ9-cis)PE, DO ..., 1,2-Dioleoyl sn-glycerol ethanolamine 3-phosphate (18:1(Δ9-cis)PE, DOPE), 1,2-Dioleoyl sn-glycerol ethanolamine 3-phosphate (18:1(Δ9-cis)PE, DOPE, 1,2-Dioleoyl sn-glycerol ethanolamine 3-phosphate (18:1(Δ9-cis)PE, DOPE, 1,2-Dioleoyl sn-glycerol ethanolamine Acyl sn-glycerol 3-phosphate ethanolamine (18:1(Δ9-trans)PE), 1,2-dilinoleoyl sn-glycerol 3-phosphate ethanolamine (18:2PE, DLPE), 1,2-dilinoleoyl sn-glycerol 3-phosphate ethanolamine (18:3PE, DLnPE), 1,2-bisoctadecenyl sn-glycerol 3-phosphate choline (18:0 diether PC), 1,2-dioleoyl sn-glycerol 3-phosphate rac(1-glycerol) sodium salt (DOPG), and any combination thereof.

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

In some aspects, the anchoring portion AM comprises at least one asymmetric phospholipid. The asymmetric phospholipid may be selected from the non-restricted group consisting of: 1 myristoyl 2 palmitoyl sn glycerol 3 phosphoric acid choline (14:0-16:0 PC, MPPC), 1 myristoyl 2 stearyl sn glycerol 3 phosphoric acid choline (14:0-18:0 PC, MSPC), 1 palmitoyl 2 acetyl sn glycerol 3 phosphoric acid choline (16:0-02:0 PC), 1 palmitoyl 2 myristoyl sn glycerol 3 phosphoric acid choline (16:0-14:0 PC, PMPC), 1 palmitoyl 2 stearyl sn glycerol 3 phosphoric acid choline (16:0-18:0 PC, PSPC), 1 palmitoyl 2 oleoyl sn glycerol 3 phosphoric acid choline (16:0-18:1 PC, POPC), 1 palmitoyl 2 linoleic acid Acyl sn glycerol 3-phosphate choline (16:0-18:2PC, PLPC), 1 palmitoyl 2 arachidonicoyl sn glycerol 3-phosphate choline (16:0-20:4PC), 1 palmitoyl 2 docosahexaenooyl sn glycerol 3-phosphate choline (14:0-22:6PC), 1 stearoyl 2 myristoyl sn glycerol 3-phosphate choline (18:0-14:0PC, SMPC), 1 stearoyl 2 palmitoyl sn glycerol 3-phosphate choline (18:0-16:0PC, SPPC), 1 stearoyl 2 oleoyl sn glycerol 3-phosphate choline (18:0-18:1PC, SOPC), 1 stearoyl 2 linoleoyl sn glycerol 3-phosphate choline (18:0-18:2PC), 1 stearoyl 2-Arachidonicoyl Sn-glycerol 3-phosphate choline (18:0-20:4PC), 1-Stearyl 2-docosahexaenoicoyl Sn-glycerol 3-phosphate choline (18:0-22:6PC), 1-Oleoyl 2-Myristoyl Sn-glycerol 3-phosphate choline (18:1-14:0PC, OMPC), 1-Oleoyl 2-Palmyloyl Sn-glycerol 3-phosphate choline (18:1-16:0PC, OPPC), 1-Oleoyl 2-Stearyl Sn-glycerol 3-phosphate choline (18:1-18:0PC, OSPC), 1-Palmyl 2-Oleoyl Sn-glycerol 3-phosphate ethanolamine (16:0-18:1PE, POPE), 1-Palmyl 2-Linoleoyl Sn-glycerol 3-phosphate ethanolamine (16:0-18:2PE), 1 Palmitoyl-2-arachidonico-sn-glycerol-3-phosphate ethanolamine (16:0-20:4PE), 1 palmitoyl-2-docosahexaenoo-sn-glycerol-3-phosphate ethanolamine (16:0-22:6PE), 1 stearoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (18:0-18:1PE), 1 stearoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (18:0-18:2PE), 1 stearoyl-2-arachidonico-sn-glycerol-3-phosphate ethanolamine (18:0-20:4PE), 1 stearoyl-2-docosahexaenoo-sn-glycerol-3-phosphate ethanolamine (18:0-22:6PE), 1 oleoyl-2-cholesterol-6-hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC) and any combination thereof.

To provide improved nuclease resistance, cellular uptake efficiency, and more significant RNA interference effects, phosphatidylethanolamines (such as dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, and dioleoyl phosphatidylethanolamine) can be used as the anchoring moiety AM.

In some respects, the anchored portion AM comprises or consists of the following: lysolipin, such as lysophospholipid. Lysolipin is a lipid derivative in which one or both fatty acyl chains have typically been removed by hydrolysis. Lysophospholipid is a phospholipid derivative in which one or both fatty acyl chains have been removed by hydrolysis.

In some respects, the anchored portion contains any phospholipid disclosed herein, one or both of which have been removed by hydrolysis, and the resulting lysophospholipid contains one or no fatty acid acyl chain.

In some respects, the anchoring moiety includes lysophosphatidylglycerol, lysosphingolipid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, or lysophosphatidylserine.

In some respects, the anchored portion AM comprises lysins selected from the non-restrictive group of the following: 1 hexanoyl 2-hydroxysn-glycerol 3-phosphate choline (0.6:0 Lyso PC), 1 heptayl 2-hydroxysn-glycerol 3-phosphate choline (0.7:0 Lyso PC), 1 capryloyl 2-hydroxysn-glycerol 3-phosphate choline (0.8:0 Lyso PC), 1 nonanoyl 2-hydroxysn-glycerol 3-phosphate choline (0.9:0 Lyso PC), 1 decanyl 2-hydroxysn-glycerol 3-phosphate choline (10:0 Lyso PC), 1 undecanoyl 2-hydroxysn-glycerol 3-phosphate choline (11:0 Lyso PC), 1 lauroyl 2-hydroxysn-glycerol 3-phosphate choline (12:0 Lyso PC), 1 tridecanoyl 2-hydroxysn-glycerol 3-phosphate choline (13:0 Lyso PC), 1 myristoyl 2-hydroxysn-glycerol 3-phosphate choline (14:0 Lyso PC), 1 pentadecanoyl 1-Palmitoyl-2-hydroxy-sn-glycerol-3-phosphate choline (15:0 Lyso PC), 1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphate choline (16:0 Lyso PC), 1-heptadecyl-2-hydroxy-sn-glycerol-3-phosphate choline (17:0 Lyso PC), 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate choline (18:0 Lyso PC) or 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate choline (18:1 Lyso PC), 1-myristoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine (14:0 Lyso PE), 1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine (16:0 Lyso PE), 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine (18:0 Lyso PE), 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine (18:1 Lyso PE), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC) and any combination thereof.

In some respects, the anchored portion AM comprises, consists of, or is substantially composed of: vitamins, such as lipophilic vitamins. 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 combinations thereof), and vitamin K, or combinations thereof. In some respects, the vitamin is tocopherol, tocotrienol, vitamin D, vitamin K, riboflavin, niacin, or pyridoxine. In some respects, the vitamin is tocopherol.

In some respects, the anchored portion AM contains or is composed of vitamin D. Vitamin D is a group of fat-soluble open-ring steroids responsible for increasing the 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).

In some respects, the anchoring portion AM contains vitamin B9 (folic acid) or is composed of it.

In some respects, the anchoring portion AM contains vitamin B2 (riboflavin) or is composed of it.

In some respects, the anchoring portion AM contains vitamin B3 (niacin) or is composed of it.

In some respects, the anchoring portion AM contains vitamin B6 (pyridoxine) or is composed of it.

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

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

Tocotrienols also possess vitamin E activity. The structural difference between tocotrienols and tocopherols lies in the fact that tocotrienols have unsaturated isoprene-like 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 combinations thereof. Tocotrienols can be represented by the following formula.

α(α)-Tocotrienols: ^R1 = Me, ^R2 = Me, ^R3 = Me;

β(β)-Tocotrienols: ^R1 = Me, ^R2 = H, ^R3 = Me;

γ(γ)-Tocotrienol: ^R1 = H, ^R2 = Me, ^R3 = Me;

δ(δ)-Tocotrienols: ^R1 = H, ^R2 = H, ^R3 = Me.

In some respects, the anchored portion AM contains or is composed of vitamin K. Chemically, the vitamin K family includes 2-methyl-1,4-naphthoquinone (3-) derivatives. Vitamin K comprises two naturally occurring isotopes: vitamin _K1 and vitamin _K2 . The structure of vitamin _K1 (also known as phytonaphthoquinone, phylloquinone, or (E)-phytonaphthoquinone) is characterized by the presence of a phytoyl group. The structure of vitamin _K2 (methylnaphthoquinones) is characterized by the presence of a polyisoprene side chain in the molecule, which may contain six to 13 isoprene units. Thus, vitamin _K2 consists of many related chemical isotopes with carbon side chains of varying lengths composed of isoprene-like atomic groups. MK-4 is the most common form of vitamin _K2 . Longer-chain forms, such as MK-7, MK-8, and MK-9, dominate in fermented foods. Longer-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 the natural form of vitamin K, there are many synthetic forms of vitamin K, such as vitamin _K3 (menaquinone; 2-methylnaphthyl-1,4-dione), vitamin _K4 , and vitamin _K5 .

Therefore, in some respects, the anchoring portion contains vitamins _K1 , _K2 (e.g., MK-4, MK-5, MK-6, MK-7, MK-8, MK-9, MK-10, MK-11, MK-12 or MK-13), _K3 , _K4 , _K5 or any combination thereof.

Chemically, the vitamin K family includes 2-methyl-1,4-naphthoquinone (3-) derivatives. Vitamin K comprises two naturally occurring isoforms: vitamin K1 (phylloquinone) and vitamin K2 (menadione). In turn, vitamin K2 is composed of many related chemical isoforms with carbon side chains of varying lengths consisting of isoprene-like atomic groups. The two most studied are menadione-4 (MK-4) and menadione-7 (MK-7). Thus, in some respects, vitamin K is MK-4, MK-5, or a combination thereof.

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

In some aspects, one or more scaffold portions may be CD47, CD55, CD49, CD40, CD133, CD59, phosphatidylinositol proteoglycan-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 portions that may be used with this disclosure include: aminopeptidase N (CD13); enkephalin, also known as membrane metalloendopeptidase (MME); exonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1); neurocilitin-1 (NRP1); or any combination thereof.

In other respects, one or more scaffold motifs are expressed in the membrane of EVs (e.g., exosomes) by recombinant expression of the scaffold motif in production cells. EVs (e.g., exosomes) obtained from production cells can be further modified to conjugate maleimide motifs or linkers. In other respects, the scaffold motif, such as scaffold X, is deglycosylated. In some respects, the scaffold motif, such as scaffold X, is highly glycosylated, for example, more so than naturally occurring scaffold X under the same conditions.

In some respects, 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 β-1 (ITGB1 protein); integrin α-4 (ITGA4 protein); 4F2 cell surface antigen heavy chain (SLC3A2 protein); a class of ATP transport proteins (ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4 proteins); their functional fragments; and any combination thereof.

In some respects, scaffold X contains a prostaglandin F2 receptor negative regulator (PTGFRN peptide). PTGFRN peptide may also be referred to as CD9 chaperone 1 (CD9P-1), protein F containing the Glu-Trp-Ile EWI motif (EWI-F), prostaglandin F2-α receptor regulator, prostaglandin F2-α receptor-associated protein, or CD315.

In some aspects, scaffold X is a PTGFRN protein or a functional fragment thereof. In some aspects, scaffold X comprises the amino acid sequence as set forth in SEQ ID NO:302. In some aspects, scaffold X comprises 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 amino acid sequence to that of SEQ ID NO:302.

Other non-limiting examples of scaffold X proteins can be found in U.S. Patent No. 10195290B1, granted February 5, 2019, which is incorporated herein by reference in its entirety.

In the context of this disclosure, combinations of lipid portions in the anchored AM, such as different fatty acids, different sterols, different vitamins, or combinations thereof, refer to some of the constructs disclosed herein within a group of constructs, such as the construct of Formula I, which may have different anchored AM portions and/or connectors. For example, some constructs of Formula I will contain fatty acids, while other constructs may contain vitamins or sterols. In another instance, the anchored AM portion may contain two lipids, such as phospholipids and fatty acids, or two phospholipids, or two fatty acids, or lipids and vitamins, or cholesterol and vitamins, etc., which together have 6 to 30 carbon atoms (i.e., an equivalent carbon number (ECN) of 6 to 30). Choosing construct combinations with different anchored AM portions and/or connectors generally results in better lipid packaging in the membrane on EVs (e.g., exosomes), which can lead to increased loading efficiency and BAM density.

Generally, anchoring moieties are chemically attached, for example, via solid-phase synthesis. However, anchoring moieties AM can be enzymatically attached to the bioactive molecule BAM. Anchoring moieties AM can be conjugated directly or indirectly to the bioactive molecule BAM via linkers or spacer combinations, as described herein, at any chemically feasible location, such as at the 5′ and/or 3′ ends of a nucleotide sequence (e.g., ASO). In some aspects, anchoring moieties AM are conjugated only to the 3′ end of the bioactive molecule BAM, directly or indirectly, via the cleavable linkers disclosed herein. In some aspects, anchoring moieties AM are conjugated only to the 5′ end of a nucleotide sequence (e.g., ASO). In one aspect, anchoring moieties AM are conjugated at locations other than the 3′ or 5′ ends of a nucleotide sequence (e.g., ASO).

In some aspects, the anchoring portion AM of this disclosure may include any hydrophobic group modification disclosed below:

In some aspects, the anchored portion AM may include spacers SP (e.g., SP ₁ or SP ₂ ) that enable connection to the BAM and include cleavable bonds, as described herein (e.g., L ₁ ). Suitable spacers are described herein.

Decomposable bonds

This disclosure provides a construct of formula I containing a cleavable bond: L _1. The term "cleavable linker" refers to a linker or spacer containing 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 nucleases, peptidases, proteases, phosphatases, oxidases, or reductases, or by specific physicochemical conditions such as redox environments, pH, the presence of reactive oxygen species, or light of a specific wavelength.

In some respects, _L1 is a cleavable bond containing the following: 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-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate oxy group; or a silyl ether.

In some respects, _L1 contains a polynucleotide group. In some respects, the polynucleotide group is a trinucleotide group or a higher nucleotide group, such as a tetranucleotide group or a higher nucleotide group, a pentanucleotide group or a higher nucleotide group, a hexanucleotide group or a higher nucleotide group, a heptanucleotide group or a higher nucleotide group, an octanucleotide group or a higher nucleotide group, a nonanucleotide group or a higher nucleotide group, or a decanucleotide group or a higher nucleotide group. Typically, the length of the polynucleotide group is no longer than 50 nucleotides (e.g., 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 contains a tetranucleotide.

Generally, the polynucleotide base will contain adenosine monophosphate (AMP, dAMP), guanosine monophosphate (GMP, dGMP), cytidine monophosphate (CMP, dCMP), thymidine monophosphate (dTMP), uridine monophosphate (UMP), or any combination thereof. In one example, the polynucleotide base is a tetranucleotide containing dT, where dT is thymidine monophosphate.

In some aspects, _L1 is a peptidyl group containing the following cleavable bonds: 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-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine. In some aspects, the peptidyl group contains alanine-alanine-asparagine. In some aspects, the peptidyl group contains valine-glycine. In some aspects, the peptidyl group contains glycine-glycine. In some aspects, the peptidyl group contains glutamic acid-valine-citrulline. In some aspects, the peptidyl group contains aspartic acid-valine-citrulline. In some aspects, the peptidyl group contains serine-valine-citrulline. In some aspects, the peptidyl group contains lysine-valine-citrulline. In some respects, the peptide group contains glycine-glycine-glycine-valine-citrulline. In some respects, the peptide group contains cyclobutane-1,1-dicarboxamide-citrulline (cBu-Cit). In some respects, the peptide group contains alanine-phenylalanine-lysine.

In some respects, _L1 is a cleavable bond containing a pyrophosphate oxy group. In some respects, the pyrophosphate oxy group has the following formula:

Where X+ is a monovalent cation, such as a proton (H ⁺ ) or a group I cation (e.g., Li ⁺ , Na ⁺ , K ⁺ , or Rb ⁺ ) and indicates a connection with AM, BAM, or a spacer.

In some aspects, _L1 is a cleavable bond containing a silyl ether. In some aspects, the ^silyl ether comprises ^–OSiR1R2O- , wherein ^R1 and ^R2 are the same or different, and each is a _C1-8 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl) or aryl (e.g., phenyl). In one example, both ^R1 and ^R2 are isopropyl.

In some respects, _L1 is a cleavable bond containing 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 facilitate the transfer of BAM across the plasma membrane. On the one hand, cell-penetrating peptides may contain 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).

In some respects, cell-penetrating peptides can be linear or cyclic. In some respects, cell-penetrating peptides are linear.

In some respects, cell-penetrating peptides can be protein-derived, synthetic, or chimeric. In some respects, cell-penetrating peptides can be classified as cationic, amphiphilic (e.g., primary or secondary), non-amphiphilic, hydrophilic, and/or hydrophobic. In some respects, cell-penetrating peptides can be classified as both cationic and hydrophobic. In some respects, cell-penetrating peptides can be classified as both cationic and hydrophilic.

On the one hand, cell-penetrating peptides are cationic, including polycationic. The charge on a cationic cell-penetrating peptide can be +1 or more (e.g., +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). Generally, the charge will be +30 or less (e.g., +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 respects, 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 respect, the cationic cell-penetrating peptide may contain 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, glutamic acid, or combinations thereof. In another respect, the cationic cell-penetrating peptide contains Tat or Arg9.

Cell-penetrating peptides may contain naturally occurring and/or non-natural amino acid residues. The term "naturally occurring amino acid" 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). “Non-natural amino acids” (i.e., amino acids that do not exist naturally) include, by way of non-limiting examples, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, leucine (“Nle”), valine (“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 plasminases.

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

In some respects, cell-penetrating peptides or fragments thereof may contain low sequence diversity, for example, containing 50% or more of a single amino acid, such as arginyl, cysteyl, prolyl, or lysyl. In other respects, cell-penetrating peptides or fragments thereof may be rich in arginyl, cysteyl, prolyl, or lysyl, wherein the sequence or fragment contains 50% or more of that particular amino acid.

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

In some aspects, the cell-penetrating peptide may comprise three or more argininoyl moieties (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, ₁₅ , or 16 argininoyl moieties). In other aspects, the cell-penetrating peptide may comprise three (Arg ₃ ), six (Arg ₆ ), eight (Arg 8), or nine (Arg ₉ ) argininoyl moieties. In some aspects, the cell-penetrating peptide further comprises at least one amino acid other than argininoyl, such as cysteyl, glycyl, or combinations thereof. In some aspects, the at least one amino acid other than argininoyl is cysteyl, glycyl, lysyl, glutamine acyl, isoleucyl, tryptophanyl, phenylalanyl, valine, threonyl, serine, or combinations thereof.

In some respects, cell-penetrating peptides include cyclic peptides, TAT (a peptide of sequence YGRKKRRQRRR (SEQ ID NO:1096)), Antp (an antennal peptide of sequence RQIKIWFQNRRMKWKK (SEQ ID NO:1097)), DPV3 (a peptide of sequence RKKRRRESRKKRRRES (SEQ ID NO:1098)), DPV6 (a peptide of sequence GRPRESGKKRKRKRLKP (SEQ ID NO:1099)), penetrin (a peptide of sequence RQIKIWFQNRRMKWKK (SEQ ID NO:1100)), R9-TAT (a peptide of sequence GRRRRRRRRRPPQ (SEQ ID NO:1101)), pVEC (a peptide of sequence LLIILRRRIRKQAHAHSK (SEQ ID NO:1102)), and ARF(19-31) (a peptide of sequence RVRVFVVHIPRLT (SEQ ID NO:1098)). Peptides of sequence NO:1103), MPG (sequence GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO:1104)), MAP (sequence KLALKLALKALKAALKLA (SEQ ID NO:1105)), transportan (sequence GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:1106)), Bip4 (sequence VSAILK (SEQ ID NO:1107)), C105Y (sequence CSIPPEVKFNPFVYLI (SEQ ID NO:1108)), bee venom peptide (sequence GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO:1109)), gH625 (sequence HGLASTLTRWAHYNALIRAF (SEQ ID NO:1103)). Peptides of (SEQ ID NO: 1110), Pep-1 (sequence KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 1111)), SAP (sweet arrow peptide; sequence (VRLPPP) ₃ (SEQ ID NO: 1112)), gH 625 (sequence HGLASTLTRWAHYNALIRAF (SEQ ID NO: 1113)), crot (27-29) (sequence KMDCRWRWKCCKK (SEQ ID NO: 1114)), R6/W3 (sequence RRWWRWRR (SEQ ID NO: 1115)), TP10 (sequence AGYLLGKINLKALAALAKKIL (SEQ ID NO: 1116)), CATY (sequence Ac-GLWRALWRLLRSLWRLLWRA-cysteine (SEQ ID NO: 1116)). The peptides of sC18 (sequence GLRKRLRKFRNKIKEK (SEQ ID NO:1118)), YTA4 (sequence IAWVKAFIRKLRKGPLG (SEQ ID NO:1119)), CyLoP-1 (sequence CRWRWKCCKK (SEQ ID NO:1120)), GALA (sequence WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO:1121)), or R6 (sequence RRRRRR (SEQ ID NO:1122)) are disclosed. 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.

In some respects, cell-penetrating peptides comprise cyclic peptides, TAT (a peptide of the sequence YGRKKRRQRRR (SEQ ID NO: 1096)), or Antp (an antennal leg; a peptide of the sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 1097)).

In some respects, cell-penetrating peptides are cyclic peptides. Examples of cyclic peptides include, for example, cyclic Tat (cTAT, peptide of the sequence KRRRGRKKRRE (where 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-[ _r12 ] (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.

In some respects, cell-penetrating peptides are classified as primary amphiphilic (e.g., Pep-1, TP10), secondary amphiphilic (e.g., penetratin, R6/W3, CATY, sC18), or non-amphiphilic.

spacer

Typically, the cleavable bond _L1 is part of a spacer (e.g., _SP1 and/or _SP2 ) between the anchoring portion AM and the bioactive molecule BAM to provide an optimal spacing between the anchoring portion AM and the bioactive molecule BAM. For example, in the case of antisense oligonucleotides (ASOs), one goal of the combination of linker and 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 may 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 potency of BAMs incorporated into EVs, or any combination thereof. In the context of this disclosure, the term "linker combination" refers to the combination of the cleavable bond _L1 and one or more spacers that form the construct of Formula I disclosed herein.

In some respects, SP ₁ exists in equation (I). In some respects, SP ₂ exists in equation (I). In some respects, both SP ₁ and SP ₂ exist in equation (I) and are identical. In some respects, both SP ₁ and SP ₂ exist in equation (I) and are different.

In some aspects, prior to conjugation with AM and/or BAM, the cleavable bond _L1 is a spacer portion, i.e., part of _SP1 and/or _SP2 . In some aspects, prior to conjugation with BAM, _L1 is part of _SP2 and AM is part of _SP1 . In some aspects, prior to conjugation with each other, _L1 is part of both _SP1 and AM and BAM is part of _SP2 . In some aspects, prior to conjugation with each other, _L1 is part of both _SP2 and BAM and AM is part of _SP1 . In some aspects, prior to conjugation with AM and BAM, _L1 is part of both _SP1 and _SP2 . In some aspects, _L1 is part of _SP1 and AM prior to conjugation with BAM, and _SP2 is not present. In some aspects, _L1 is part of _SP2 and BAM prior to conjugation with AM, and _SP1 is not present. In some aspects, _L1 is part of _SP1 prior to conjugation with AM and BAM, and _SP2 is not present. In some respects, _L1 is part of _SP2 before being conjugated with AM and BAM, and _SP1 does not exist.

In some respects, SP ₁ and SP ₂ of formula (I) may be the same or different, and each contains an alkylene group, a polyoxyalkylene group, a succinimide group, a maleimide 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-), or a sulfonyl group (-SO _2). -), thiocarbonyl (-C(S)-), thiocarbamoyl (-OC(S)NR- or -NRC(S)O-), thiosuccinimide, 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 combinations thereof, and at least one of SP ₁ and SP ₂ is present in formula (I). The following parts are associated with the provided structure.

Succinimide group

Thiosuccinimide

maleimide group

1,2,3-Triazolyl

Dibenzoylcyclooctenyl

Bicyclononenyl

p-Aminobenzoyl and

p-Aminobenzylcarbamate, where the connection is indicated to the remainder of AM, BAM, _L1 , or spacer.

In some aspects, at least one of SP ₁ and SP ₂ comprises a _C2-8 alkylene group, a polyoxyalkylene group, a maleimide group, a carbamoyl group, a thio group, an amide 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. In some aspects, at least one of SP ₁ and SP ₂ comprises a _C3 alkylene group, a _C6 alkylene group, or a _C8 alkylene group, as described herein. In some aspects, at least one of SP ₁ and SP ₂ comprises a polyoxyalkylene group (e.g., a diol-based spacer) containing 2 to 15 –OCH ₂ CH ₂ – repeating units, as described herein. In some aspects, the polyoxyalkylene group (e.g., a diol-based spacer) comprises a 4 (TEG) or 6 (HEG) –OCH ₂ CH ₂ – repeating unit, as described herein. In some aspects, at least one of SP ₁ and SP ₂ comprises a _C3 alkylene, _C6 alkylene, or _C8 alkylene or a polyoxyalkylene (e.g., a diol-based spacer) containing 2 to 15 –OCH ₂ CH ₂ – repeating units, as described herein, and further comprises a carbamoyl group, an amide group, a thiosuccinimide 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 following structure and

1,2,3-Triazolylbicyclononenyl has the following structure:

This indicates the connection to AM, BAM, _L1 , or the rest of the spacer.

alkylene spacers

In some aspects, the spacer may comprise an alkylene spacer, such as a linear alkylene spacer. In some aspects, the alkylene spacer attached to the anchor 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, where C represents a divalent methylene unit ( _-CH2- ) and the number indicates the number of methylene units in the alkylene spacer. In some aspects, the alkylene spacer comprises a single methylene group (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.

In some aspects, the optional first spacer of SP ₁ and/or the optional second spacer of SP ₂ independently comprise spacers (e.g., alkylene spacers or diol-based spacers) or combinations thereof. In some aspects, the optional first spacer of SP ₁ comprises or is composed of alkylene spacers that are 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, SP ₁ is a C3 or C6 alkylene spacer. In some aspects, the optional second spacer of SP ₂ comprises or is composed of alkylene spacers that are 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, SP ₂ is a C3 or C6 alkylene spacer.

In some respects, the spacer includes substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalenyl, arylalkynyl, heteroarylalkyl, heteroarylalenyl, heteroarylalkynyl, heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alkynyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalenyl, alkenylarylalkynyl, alkynylarylalkyl Alkyl, alkynylaryl-alkenyl, alkynylaryl-alkynyl, alkyl heteroarylalkyl, alkyl heteroarylalkyl, alkyl heteroaryl-alkenyl, alkyl heteroaryl-alkynyl, alkenyl heteroarylalkyl, alkenyl heteroaryl-alkenyl, alkenyl heteroaryl-alkynyl, alkynyl heteroarylalkyl, alkynyl heteroaryl-alkenyl, alkynyl heteroaryl-alkynyl, alkyl heterocyclic alkyl, alkyl heterocyclic alkenyl, alkyl heterocyclic alkynyl, alkenyl heterocyclic alkyl, alkenyl heterocyclic alkenyl or alkenyl heterocyclic alkynyl.

Optionally, these spacers are substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) substituents. Substituents include, for example, hydroxyl, alkoxy (e.g., -O-( _C1-8 alkyl)), straight-chain or branched alkyl (e.g., _C1-15 alkyl or _C1-8 alkyl), amino, aminoalkyl (e.g., C1-C15 alkylamino), phosphoramidyl, phosphate, phosphoramidyl, dithiophosphate, thiophosphate, hydrazido, hydrazine, halogen (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 halides, ethers, sulfonyl halides, imine esters, isocyanates, isothiocyanates, halocarbamates, carbodiimide adducts, aldehydes, ketones, thiohydrogen groups, haloacetyl groups, alkyl halides, alkyl sulfonates, C(=O)CH=CHC(=O) (maleimide), thioethers, cyano groups, nitro groups, sugars (e.g., mannose, galactose, and glucose), α,β-unsaturated carbonyl groups, alkylmercuric groups, or α,β-unsaturated sulfonyl groups, wherein each R' is independently H, _C1-8 alkyl, or aryl.

Examples of aliphatic linkers include the following structures: —O—CO—O—; —NH—CO—O—; —NH—CO—NH—; —NH—(CH ₂ ) _n1 —; —S—(CH ₂ ) _n1 —; —CO—(CH ₂ ) _n1 —CO—; —CO—(CH ₂ ) _n1 —NH—; —NH—(CH ₂ ) _n1 —NH—; —CO—NH—(CH ₂ ) _n1 —NH—CO—; —C(═S)—NH—(CH ₂ ) _n1 —NH—CO—; —C(═S)—NH—(CH ₂ ) _n1 —NH—C—(═S)—; —CO—O—(CH ₂ ) _n1 —O—CO—; —C(═S)—O—(CH ₂ ) _n1 —O—CO—; —C(═S)—O—(CH ₂ ) _n1 —O—C—(═S)—;—CO—NH—(CH ₂ ) _n1 —O— _CO ——C(═S)—NH—(CH _{2 ) n1} —O—CO——C(═S)—NH—(CH ₂ ) _{n1 2} ) _n1 —CO—;—C(═S)—O—(CH ₂ ) _n1 —NH—CO—;—C(═S)—NH—(CH ₂ ) _n1 —O—C—(═S)—;—NH—(CH ₂ CH ₂ O) _n2 —CH(CH ₂ OH)—;—NH—(CH ₂ CH ₂ O) _n2 —CH ₂ —;—NH—(CH ₂ CH ₂ O) _n2 —CH ₂ —CO—; —O—(CH ₂ ) _n3 —S—S—(CH ₂ ) _n4 —O—P(═O) ₂ —;—CO—(CH ₂ ) _n3 —O—CO—NH—(CH ₂ ) _n4 —;—CO—(CH ₂ ) _n3 —CO—NH—(CH ₂ ) _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)O—;—(CH2) _n1 —；—(CH2) _n1 -NH—C(O)—；where 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 each is an integer between 1 and 15 (e.g., 1 to 10, or 1 to 6), and the total number of carbon units in the joint is C15 or less.

Diol-based spacers

In some aspects, the spacers may comprise diol-based spacers. In some aspects, the _diol -based spacers comprise two or more _{–OCH₂CH₂-} repeating units and may be formed from, 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 the corresponding number of _{–OCH₂CH₂-} repeating units. In some aspects, the diol-based spacers may comprise 11, 12, 13, 14, or 15 diol-based repeating units ( _{–OCH₂CH₂-} ). In some aspects, the _diol -based spacers have between 2 and 10, 2 and 5, 5 and 10, or 10 and 15 diol-based repeating units. In some aspects, the diol-based spacers are formed from HEG (i.e., 6 diol repeating units). In some respects, diol-based spacers are formed by TEG (i.e., four diol repeating units).

In some respects, the diol-based spacer may comprise a branched polyglycerol of the formula ( ^R3 —O—( _CH2 —CHOR ⁵ —CH2 _—O ) _n —) (where ^R5 is hydrogen) or a linear glycerol chain of the formula ( ^R3 —O—( _CH2 —CHOH— _CH2 —O) _n —), where ^R3 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 a hyperbranched polyglycerol of formula ( ^R3 -O-( _CH2 - ^CHOR5 - _CH2 -O) _n- ) (where ^R5 is hydrogen) or a glycerol chain of formula ( ^R3 -O-(CH2 _- ^CHOR6 - _CH2 -O) _n- ) (where ^R6 is hydrogen) or a glycerol chain of formula (R3-O-( _CH2- ^CHOR7 - _CH2 -O) _n- ) (where ^R7 is hydrogen) or a linear glycerol chain of formula ( ^R3 -O-( _CH2 -CHOH- _CH2 -O) _n- ) where R3 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 its synthetic methods are described, for example, by Oudshorn et al. (Biomaterials, 2006, 27: 5471-5479) and Wilms et al. (Acc.Chem.Res., 2010, 43, 129-41).

In some aspects, the first spacer of SP ₁ optionally comprises or consists of diol-based spacers 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, SP ₁ is a spacer based on tetraethylene glycol (TEG) or hexaethylene glycol (HEG) diol. In some aspects, the optional second spacer of SP ₂ comprises or consists of diol-based spacers 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, SP ₂ is based on tetraethylene glycol (TEG) or hexaethylene glycol (HEG) diol spacers.

In some aspects, the SP ₁ spacer comprises an alkylene spacer and the SP ₂ spacer also comprises an alkylene spacer. In some aspects, the SP ₁ spacer comprises a glycol-based spacer and the SP ₂ spacer also comprises a glycol-based spacer. In some aspects, the SP ₁ spacer comprises an alkylene spacer and the SP ₂ spacer comprises a glycol-based spacer. In some aspects, the SP ₁ spacer comprises a glycol-based spacer and the SP ₂ spacer comprises an alkylene spacer. In some aspects, the SP ₁ spacer is composed of alkylene spacers and the SP ₂ spacer is also composed of alkylene spacers. In some aspects, the SP ₁ spacer is composed of glycol-based spacers and the SP ₂ spacer is also composed of glycol-based spacers. In some aspects, the SP ₁ spacer is composed of alkylene spacers and the SP ₂ spacer is composed of glycol-based spacers. In some aspects, the SP ₁ spacer is composed of glycol-based spacers and the SP ₂ spacer is composed of alkylene spacers.

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

polyethylene glycol spacers

In some respects, the spacers, first spacer _SP1 , second spacer _SP2 , or any combination thereof in the anchoring portion AM may comprise or consist of diol-based spacers, which may be considered to be of the formula -(O- _CH2 - _CH2 ) _n- or -(O- _CH2 - _CH2 ) _n. -O- polyethylene glycol (PEG) residues, where 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 PEG ₁ , PEG ₂ , PEG ₃ , PEG ₄ , PEG ₅ , PEG ₆ , PEG ₇ , PEG ₈ , PEG ₉ , PEG ₁₀ , PEG ₁₁ , PEG ₁₂ , PEG ₁₃ , PEG ₁₄ , PEG ₁₅ , PEG ₂₅ , PEG ₅₀ , PEG ₇₅ , PEG ₁₀₀ , PEG ₁₂₅ , PEG ₁₅₀ , PEG ₁₇₅ , PEG ₂₀₀ or PEG ₁ -O-, PEG ₂ -O-, PEG ₃ -O-, PEG ₄ -O-, PEG ₅ -O-, PEG ₆ -O-, PEG ₇ -O-, PEG ₈ -O-, PEG ₉ -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-. Before conjugation with another part of the formula I construct (e.g., AM or BAM), the PEG residues can be formed from ^R1- (O- _CH2 - _CH2 ) _n - ^R1 or ^R1- (O- _CH2 - _CH2 ) _n - ^OR1 , 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).

In some respects, PEG is branched PEG. Branched PEG has 3 to 10 PEG chains emanating from a central core group. In some respects, the PEG moiety is monodisperse polyethylene glycol. In the context of this disclosure, monodisperse polyethylene glycol (mdPEG) is PEG having a single, defined chain length and molecular weight.

In some respects, PEG is star-shaped PEG. Star-shaped PEG has about 10 to 15 PEG chains emanating from a central core group. In other respects, PEG is comb-shaped PEG. Comb-shaped PEG has multiple PEG chains that are typically grafted onto the polymer backbone.

In some aspects, the spacers, first spacer _SP1 , second spacer _SP2 , or any combination thereof in the anchoring portion AM may contain or be composed of polyglycerol (PG) residues characterized by the formula -O—( _CH2 —CHOH— _CH2O ) _n- , where 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 may be described as _PG1 , _PG2 , _PG3 , _PG4 , _PG5 , _PG6 , _PG7 , _PG8 , _PG9 , _PG10 , _PG11 , _PG12 , _PG13 , _PG14 , or _PG15 .

Vitamin spacers

In some aspects, the anchoring portion AM comprises or consists of vitamins and alkylene spacers as described herein. In some aspects, the anchoring portion AM comprises tocopherols and alkylene spacers or consists of them. In some aspects, the anchoring portion AM comprises vitamins and octyl (C8) alkylene spacers (such as tocopherols and octyl (C8) alkylene spacers) or consists of them.

In some aspects, the anchoring portion AM comprises or consists of fatty acids and alkylene spacers. In some aspects, the anchoring portion AM comprises palmitate and alkylene spacers. In some aspects, the anchoring portion AM comprises fatty acids and hexyl (C6) alkylene spacers (such as palmitate and hexyl (C6) alkylene spacers) or consists of them.

In some aspects, the anchoring portion AM comprises or consists of sterols and diol-based spacers. In some aspects, the anchoring portion AM comprises cholesterol and diol-based spacers. In some aspects, the anchoring portion AM comprises sterols and TEG-based diol-based spacers (such as cholesterol and TEG-based diol-based spacers) or consists of them. In some aspects, the anchoring portion AM comprises sterols and alkylene spacers or consists of them. In some aspects, the anchoring portion AM comprises sterols and hexyl (C6) alkylene spacers or consists of them. In some aspects, the anchoring portion AM comprises cholesterol and alkylene spacers (such as cholesterol and hexyl (C6) alkylene spacers) or consists of them.

Non-disintegrable joint

In some respects, the linker assembly comprises "unbreakable bonds," which are linkers and/or spacers that include chemical bonds that are substantially resistant to cleavage. Unbreakable linkers are substantially resistant to, for example, acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, particularly under conditions where cyclic dinucleotides and/or antibodies do not lose their activity. An unbreakable linker is any chemical part capable of linking two or more components of a construct disclosed herein, such as a construct of formula I. In particular, an unbreakable linker can link any two parts of a construct, such as the anchoring part AM and/or the spacer SP and/or the bioactive molecule BAM. Typically, an unbreakable linker is part of a spacer (e.g., _SP1 , _SP2 ), as described herein, but in some respects, AM or BAM is modified to include unbreakable bonds.

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

Bioactive molecules

A bioactive molecule (BAM) is a pharmaceutical agent that acts on a target (e.g., target cells). Exposure can occur in vitro or in a subject. Non-limiting examples of bioactive molecules (BAMs) that can attach to EVs (e.g., exosomes) as described in this disclosure include agents such as polynucleotides (e.g., nucleotides containing toxins that can detect partial or disrupt 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 (ASOs)), amino acids (e.g., amino acids containing toxins that can detect partial or disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins), antiviral agents, RNA decoys, or any combination thereof. In some aspects, BAMs include peptides, polypeptide groups, polynucleotide groups, proteins, antibodies, or antigen-binding fragments thereof, chemical compounds, or any combination thereof. In some aspects, BAMs comprise antisense oligonucleotide groups (ASOs), siRNA, miRNA, shRNA, nucleic acids, or any combination thereof.

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

Therefore, in some respects, the population of EVs (e.g., exosomes) disclosed herein may comprise multiple constructs, as exemplified below.

AM ₁ -SP ₁ -L ₁ -SP ₂ -BAM ₁

AM ₂ -SP ₁ -L ₁ -SP ₂ -BAM ₂

…

AM _n -SP ₁ -L ₁ -SP ₂ -BAM _n ,

[ _AM1 ]..[ _AMn ] can be the same or different anchoring parts, each _SP1 , _L1 and _SP2 can be the same or different, and [ _BAM1 ]..[ _BAMn ] can be the same or different bioactive molecules.

In some respects, the bioactive molecule BAM comprises peptides, proteins, antibodies or their antigen-binding moieties, or any combination thereof, or is composed of them. In some respects, its antigen-binding moieties comprise scFv, (scFv)2, Fab, Fab', F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments, biantibodies, antibody-associated peptides, or any fragment thereof. In some respects, the antibody or its antigen-binding moieties may bind to protein X, which is present in the membrane of an EV (e.g., an exosome).

In some respects, the bioactive molecule BAM targets tumor antigens. 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-associated apoptosis-inducing ligands, or combinations thereof.

In some respects, the bioactive molecule BAM is a targeting moiety, such as an antibody or its binding moiety, or a ligand that specifically binds to a marker on a muscle cell. In some respects, the muscle cell is a smooth muscle cell. In some respects, the muscle cell is a skeletal muscle cell. In some respects, the muscle cell is a cardiomyocyte. In some respects, the marker on the muscle cell is selected from α-smooth muscle actin, VE-cadherin, calmodulin-binding protein/CALD1, calcium-regulatory protein 1, hexim 1, histamine H2 R; motilin R/GPR38, taglN, and any combination thereof. In some respects, markers on muscle cells are selected from α-carnitine, β-carnitine, calpain inhibitors, creatine kinase MM/CKMM, eIF5A, enolase 2/neuron-specific enolase, ε-carnitine, FABP3/H-FABP, GDF-8/myosin, GDF-11/GDF-8, integrin α7, integrin α7β1, integrin β1/CD29, MCAM/CD146, MyoD, myopoietin, myosin light chain kinase inhibitors, NCAM-1/CD56, troponin I, and any combination thereof. In some respects, markers on muscle cells are myosin heavy chain, myosin light chain, or combinations thereof.

In some respects, the bioactive molecule BAM is a small molecule. In some respects, the small molecule is a proteolytic targeting chimera (PROTAC). In some respects, the bioactive molecule BAM is a small molecule that comprises synthetic antitumor agents (e.g., monomethylreoxetine E (MMAE) (vedotin)), cytokine release inhibitors (e.g., MCC950), mTOR inhibitors (e.g., rapalog and its analogues), autocrine motor factor inhibitors (e.g., PAT409 or PAT505), lysophosphatidylcholine receptor agonists (e.g., BMS-986020), STING antagonists (e.g., CL656), or any combination thereof. In some respects, 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.

In some respects, the bioactive molecule BAM contains a nucleotide, in which the nucleotide is an interferon gene protein stimulator (STING) agonist. STING is a cyclic dinucleotide cytoplasmic sensor, typically produced by bacteria. Upon activation, it produces type I interferon and initiates an immune response.

In some respects, STING agonists include cyclic nucleotide STING agonists or noncyclic dinucleotide STING agonists. 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 analogues thereof, are known to stimulate or enhance a patient's immune or inflammatory response. CDNs may have 2′2′, 2′3′, 2′5′, 3′3′, or 3′5′ bonds linking the cyclic dinucleotide, or any combination thereof. Cyclic purine dinucleotides may be modified via standard organic chemical techniques to produce purine dinucleotide analogs. 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. Cyclic dinucleotides may be modified analogues. Any suitable modification known in the art may be used, including but not limited to thiophosphate, thiodiphosphate, fluoride, and difluoride modifications. Noncyclic dinucleotide agonists, such as 5,6-dimethylxanthonone-4-acetic acid (DMXAA), or any other noncyclic dinucleotide agonist known in the art may also be used.

It is anticipated that any STING agonist can be used as the 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 instance, the STING agonist is 3′3′-cAIMPdFSH, alternatively referred to as 3-3cAIMPdFSH. Other STING agonists known in the art may also be used.

In some respects, the bioactive molecule BAM is an antibody or its antigen-binding fragment. In some respects, the bioactive molecule BAM is an antibody-drug conjugate (ADC). In some respects, the bioactive molecule BAM is a fusion peptide.

In some respects, the bioactive molecule BAM targets macrophages. In other respects, the bioactive molecule induces macrophage polarization. Macrophage polarization is a process by which macrophages respond to signals from their microenvironment by employing different functional programs. This ability is related to their multiple roles in the organism: they are powerful effector cells of the innate immune system and also play important roles in clearing cellular debris, embryonic development, and tissue repair.

By simplifying the classification, macrophage phenotypes were divided into two groups: M1 (classically activated macrophages) and M2 (alternatively activated macrophages). This broad classification was based on in vitro studies in which cultured macrophages were treated with molecules that stimulated their phenotype to shift to a specific state. In addition to chemical stimulation, it was shown that the stiffness of the underlying matrix in which macrophages grow can guide polarization states, functional roles, and migration patterns. M1 macrophages were described as pro-inflammatory macrophages, playing a crucial role in the host's direct defense against pathogens, such as phagocytosis, and the secretion of pro-inflammatory cytokines and bactericidal molecules. M2 macrophages were described as having completely opposite functions: regulating the resolution phase of inflammation and repairing damaged tissue. Later, more extensive in vitro and in vitro studies revealed even greater macrophage phenotype diversity, with overlapping gene expression and function, revealing that these many mixed states form a continuum of microenvironment-dependent activation states. Furthermore, in vivo, there is a high degree of diversity in gene expression profiles among macrophage populations from different tissues. Therefore, the macrophage activation profile is considered to be broader, involving complex regulatory pathways to respond to a wide variety of signals from the environment. The diversity of macrophage phenotypes still needs to be fully characterized in vivo.

Imbalances in macrophage types are 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 and obesity in mice. On the other hand, in vitro experiments have shown that M2 macrophages are the main mediators of tissue fibrosis. Multiple studies have linked the fibrotic characteristics of M2 macrophages to the pathogenesis of systemic sclerosis. Non-limiting examples of macrophages targeting bioactive molecules include: 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 hyphae regulator 1), miR146a, miR155, IRF4 (interferon regulator 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 preprotein).

In some respects, the bioactive molecule BAM contains or is composed of antisense oligonucleotides (ASOs). In some respects, ASOs are spacer polymers, mixed polymers, or holopolymers. In some respects, ASOs can target precursor or mature mRNAs, including protein-coding regions (exons), non-coding regions (e.g., 5′ or 3′ untranslated regions, or introns), intron-exon junctions, or regulatory regions (e.g., promoters). In some respects, ASOs target protein transcripts, such as STAT6 transcripts, EGFP transcripts, CEBP/β transcripts, STAT3 transcripts, KRAS transcripts, NRAS transcripts, NLPR3 transcripts, FFLUC transcripts, RLUC transcripts, MYC transcripts, or any combination thereof.

In some respects, an oligonucleotide (ASO) can contain one or more nucleosides with modified sugar moieties, i.e., the sugar moieties are modified when compared to those found in DNA and RNA. Many modified nucleosides with ribose moieties have been prepared, primarily to improve certain properties of the oligonucleotide, such as affinity and/or nuclease resistance. Such modifications include those in which the ribocycle structure has been modified, for example by replacing it with a hexose ring (HNA), a bicyclic ring (which typically has a double-base bridge between the C2' and C4' carbons on the ribocycle (LNA), or an unconnected ribocycle (which typically lacks a bond between the C2' and C3' carbons) (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclic hexosennucleotides (WO2011/017521) or tricyclic nucleotides (WO2013/154798). Modified nucleosides also include nucleosides in which the sugar moieties are replaced by non-sugar moieties, such as in the case of peptide nucleotides (PNA) or morpholinonucleotides.

Sugar modification also includes modifications made by changing a substituent group on the ribose ring to a group other than hydrogen or a naturally occurring 2'-OH group in the RNA nucleoside. Substituents can be introduced, for example, at the 2', 3', 4', and/or 5' positions. Nucleosides with modified sugar moieties also include 2'-modified nucleosides, such as 2'-substituted nucleosides. 2'-sugar-modified nucleosides are nucleosides having a substituent other than H or –OH at the 2' position (2'-substituted nucleosides) or containing a 2'-linked bimolecular junction, and include 2'-substituted nucleosides and LNA (2' to 4' bimolecular bridging) nucleosides. For example, 2'-modified sugars can provide enhanced binding affinity (e.g., enhanced affinity of 2'-sugar-modified nucleosides) and/or increased nuclease resistance to oligonucleotides. Examples of 2'-substituted nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, 2'-fluoro-DNA, arabinonucleotide (ANA), and 2'-fluoro-ANA nucleosides. 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 are illustrations of some 2'-substituted nucleosides.

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

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 respects, the ASO-modified nucleosides or LNA nucleosides of this disclosure have the general structure of formula X or formula XI:

in

B is a nucleobase or a modified nucleobase portion;

W is selected from -O-, -S-, -N( ^Ra )-, -C( ^{RaRb )} -, especially –O-;

X is O;

Y is _CH2 ;

Z is an internucleotide bond with an adjacent nucleoside or a 5'-terminal group;

Z* is a nucleoside bond with an adjacent nucleoside or a 3'-terminal group;

^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, azide, heterocyclic, and aryl; and

^Ra and ^Rb are independently selected from hydrogen and alkyl groups.

ASO targeting NLRP3

In some respects, the bioactive molecule BAM is an anti-NLRP3 ASO. NLRP3 (NLRP3) is also known as containing the NLR family Pyrin domain 3. Unless otherwise stated, 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 in 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 precursor mRNA transcript (SEQ ID NO:1) corresponds to the reverse complementary sequence of residues 247, 416, 156 to 247, 449, 108 on chromosome 1q44. The NLRP3 mRNA sequence (GenBank accession number 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. Sequences 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-NLRP3ASO of this disclosure can be engineered to reduce or inhibit the expression of native variants of the NLRP3 protein.

An example of a target nucleic acid sequence for anti-NLRP3 ASO is NLRP3 precursor mRNA. SEQ ID NO:1 represents the human NLRP3 genome sequence (i.e., the reverse complementary sequence of nucleotides 247, 416, 156 to 247, 449, 108 on chromosome 1q44). SEQ ID NO:1 is identical to the NLRP3 precursor mRNA sequence, except that the nucleotide “t” in SEQ ID NO:1 is shown as “u” in the precursor mRNA. In some respects, the “target nucleic acid” comprises an intron of an NLRP3 protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In other respects, the target nucleic acid comprises an exon region of an NLRP3 protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In still other respects, the target nucleic acid comprises an exon-intron junction of an NLRP3 protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. The sequence of the human NLRP3 protein encoded by the NLRP3 precursor mRNA is shown in SEQ ID NO:3. In other respects, the target nucleic acid contains the untranslated region of the NLRP3 protein-encoding nucleic acid or a naturally occurring variant thereof, such as the 5'UTR, 3'UTR, or both.

In some aspects, the anti-NLRP3 ASO of this disclosure hybridizes with regions within introns of NLRP3 transcripts (e.g., SEQ ID NO:1). In some aspects, the anti-NLRP3 ASO of this disclosure hybridizes with regions within exons of NLRP3 transcripts (e.g., SEQ ID NO:1). In other aspects, the anti-NLRP3 ASO of this disclosure hybridizes with regions within exon-intron junctions of NLRP3 transcripts (e.g., SEQ ID NO:1). In some aspects, the anti-NLRP3 ASO of this disclosure hybridizes with regions (e.g., introns, exons, or exon-intron junctions) within NLRP3 transcripts (e.g., SEQ ID NO:1), wherein the anti-NLRP3 ASO has a spacer-merged design.

In some respects, anti-NLRP3 ASO targets the mRNA of a specific isoform (e.g., isoform 1) of the NLRP3 protein. In other respects, anti-NLRP3 ASO targets all isoforms of the NLRP3 protein. In still other respects, 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).

In some respects, the nucleotide sequence or continuous nucleotide sequence of the anti-NLRP3 ASO of this disclosure has at least 80% sequence identity with the sequence selected from SEQ ID NO: 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%, at least 97%, at least 98%, at least 99%, such as about 100% sequence identity (homologous).

In some respects, anti-NLRP3 ASO (or a continuous nucleotide portion thereof) is selected from or comprises: a region of at least 10 continuous nucleotides of a sequence selected from the group consisting of SEQ ID NO: 101 to 200, wherein anti-NLRP3 ASO (or a continuous nucleotide portion thereof) may optionally contain one, two, three or four mismatches compared to the corresponding NLRP3 transcript.

In some respects, anti-NLRP3 ASO comprises sequences selected from the group consisting of SEQ ID NO: 101 to 200.

In some aspects, anti-NLRP3ASO comprises a sequence as set forth in any one of SEQ ID NO: 101 to 200. In some aspects, anti-NLRP3ASO comprises or consists of 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 or composed of the sequence set forth in SEQ ID NO: 101 to 200. In some aspects, anti-NLRP3ASO (or a continuous nucleotide portion thereof) is selected from or comprises: a region of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 continuous nucleotides thereof selected from the group consisting of SEQ ID NO: 101 to 200. In some aspects, the anti-NLRP3 ASO (or its continuous nucleotide portion) is selected from or comprises: a region of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 continuous nucleotides thereof, wherein the anti-NLRP3 ASO (or its continuous nucleotide portion) may optionally contain one, two, three, or four mismatches compared to the corresponding NLRP3 transcript. In some aspects, the anti-NLRP3 ASO (or its continuous nucleotide portion) is selected from or comprises: a sequence selected from the group consisting of SEQ ID NO: 101 to 200, except for substitutions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the substituted ASO can bind to the NLRP3 transcript. In some respects, anti-NLRP3ASO (or a continuous nucleotide portion thereof) is selected from or comprises: one of the sequences selected from the group consisting of SEQ ID NO: 101 to 200 or a region of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 continuous nucleotides thereof, wherein anti-NLRP3 ASO (or a continuous nucleotide portion thereof) may optionally comprise one, two, three or four additional 5' and/or 3' nucleotides complementary to the corresponding NLRP3 transcript.

In some respects, the binding of anti-NLRP3 ASO targeting the NLRP3 transcript disclosed in this paper to the mRNA transcript encoding NLRP3 can reduce the expression level and/or activity level of NLRP3.

In some respects, any anti-NLRP3 ASO described herein may be an EV (e.g., an exosome) of this disclosure, i.e., an EV (e.g., an exosome) comprising a construct containing a cleavable linker of the disclosed herein (e.g., the Formula I construct), wherein the bioactive portion BAM is an anti-NLRP3 ASO or a combination thereof described herein.

ASO targeting STAT6

In some respects, the bioactive molecule BAM is anti-STAT6ASO. STAT6 (STAT6) is also known as signal transducer and activator of transcription 6. Unless otherwise stated, the term “STAT6” as used herein may refer to STAT6 from one or more species (e.g., human, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).

The sequence of the human STAT6 precursor mRNA transcript (SEQ ID NO:11) corresponds to the inverse complementary sequence of residues 57111413 to 57095404, the complementary sequence 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.

Natural variants of the human STAT6 gene product are known. For example, natural variants of the human STAT6 protein may contain one or more amino acid substitutions selected from M118R, D419N, and any combination thereof. Other variants of the human STAT6 protein generated by alternative splicing are also known in the art. STAT6 isotype 2 (identifier on UniProt: P42226-2) differs from the canonical sequence (SEQ ID NO: 13) as follows: residues 1 to 174 are deleted relative to SEQ ID NO: 13, and ₁₇₅ PSE ₁₇₇ is substituted with ₁₇₅ MEQ _177. The sequence of STAT6 isotype 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-STAT6ASO of this disclosure can be designed to reduce or inhibit the expression of natural variants of the STAT6 protein.

An example of a target nucleic acid sequence against STAT6ASO is STAT6 precursor mRNA. SEQ ID NO:11 represents the 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 the STAT6 precursor mRNA sequence, except that the nucleotide "t" in SEQ ID NO:11 is shown as "u" in the precursor mRNA. In some respects, the "target nucleic acid" comprises an intron of a STAT6 protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In other respects, the target nucleic acid comprises an exon region of a STAT6 protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In still other respects, the target nucleic acid comprises an exon-intron junction of a STAT6 protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. The sequence of the human STAT6 protein encoded by the STAT6 precursor mRNA is shown in SEQ ID NO:13. In other respects, the target nucleic acid contains the untranslated region of the STAT6 protein-encoding nucleic acid or a naturally occurring variant thereof, such as the 5'UTR, 3'UTR, or both.

In some aspects, the anti-STAT6ASO of this disclosure hybridizes with regions within introns of STAT6 transcripts (e.g., SEQ ID NO: 11). In some aspects, the anti-STAT6ASO of this disclosure hybridizes with regions within exons of STAT6 transcripts (e.g., SEQ ID NO: 11). In other aspects, the anti-STAT6ASO of this disclosure hybridizes with regions within exon-intron junctions of STAT6 transcripts (e.g., SEQ ID NO: 11). In some aspects, the anti-STAT6ASO of this disclosure hybridizes with regions (e.g., introns, exons, or exon-intron junctions) within STAT6 transcripts (e.g., SEQ ID NO: 11), wherein the anti-STAT6ASO has a spacer-merged design.

In some respects, anti-STAT6ASO targets the mRNA of a specific isoform (e.g., isoform 1) of the STAT6 protein. In other respects, ASO targets all isoforms of the STAT6 protein. In still other respects, anti-STAT6ASO targets two isoforms of the STAT6 protein (e.g., isoform 1 and isoform 2, isoform 1 and isoform 3, or isoform 2 and isoform 3).

In some aspects, the payload of this disclosure (e.g., ASO) hybridizes with regions within introns of the STAT6 transcript. In some aspects, the payload hybridizes with regions within exons of the STAT6 transcript. In some aspects, the payload hybridizes with regions within exon-intron junctions of the STAT6 transcript. In some aspects, the payload hybridizes with regions within the STAT6 transcript (e.g., introns, exons, or exon-intron junctions). Non-limiting examples of payloads (e.g., ASO) that can specifically target regions of the STAT6 transcript.

In some respects, the binding of anti-STAT6ASO targeting the STAT6 transcript disclosed in this paper to the mRNA transcript encoding STAT6 can reduce the expression level and/or activity level of STAT6.

In some respects, any anti-STAT6ASO described herein may be an EV (e.g., an exosome) of this disclosure, i.e., a part of an EV (e.g., an exosome) containing a cleavable linker of the disclosed herein (e.g., Formula I construct), wherein the bioactive portion BAM is an anti-STAT6ASO or a combination thereof described herein.

In some aspects, the anti-STAT6ASO of this disclosure comprises the base sequence of SEQ ID NO:1091. In some aspects, the anti-STAT6ASO of this disclosure comprises the STAT 6ASO sequence shown in FIG2.

ASO targeting MYC

In some respects, the bioactive molecule BAM is anti-MYCASO. Unless otherwise stated, the term "MYC" as used herein may refer to MYC derived from one or more species (e.g., human, non-human primate, dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, and bear).

In some aspects, the anti-MYCASO of this disclosure comprises the base sequence of SEQ ID NO:1092. In some aspects, the anti-MYCASO of this disclosure comprises the MYCASO sequence shown in Figure 2.

ASO targeting CEBP/β

In some respects, the bioactive molecule BAM is an anti-CEBP/βASO. Unless otherwise stated, the term "CEBP/β" as used herein may 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).

The sequence of the human CEBP/β gene can be found in the publicly available GenBank accession number NC_000020.11 (50190583..50192690). The human CEBP/β gene is located at chromosome 20q13.13, between 50190583 and 50192690.

The sequence of the human CEBP/β precursor mRNA transcript (SEQ ID NO:21) corresponds to the reverse complementary sequence of residues 50190583 to 50192690 on chromosome 20q13.13. The CEBP/β mRNA sequence (GenBank accession number 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.

Natural variants of the human CEBP/β gene product are known. For example, natural variants of the human CEBP/β protein may contain one or more amino acid substitutions selected from the following: A241P, A253G, G195S, and any combination thereof. Other variants of the human CEBP/β protein generated by alternative splicing are also known in the art. CEBP/β isotype 2 (identifier on UniProt: P17676-2) differs from the canonical sequence (SEQ ID NO: 23) as follows: residues 1 to 23 are deleted relative to SEQ ID NO: 23. The sequence of CEBP/β isotype 3 (identifier: P17676-3) differs from the canonical sequence (SEQ ID NO: 23) as follows: residues 1 to 198 are deleted relative to SEQ ID NO: 23. Therefore, the anti-CEBPbASO of this disclosure can be designed to reduce or inhibit the expression of natural variants of the protein.

An example of a target nucleic acid sequence against CEBPbASO is CEBP/β precursor mRNA. SEQ ID NO:21 represents the human CEBP/β genome sequence (i.e., the reverse complementary sequence of nucleotides 50190583-50192690 on 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 shown as “u” in the precursor mRNA. In some respects, the “target nucleic acid” comprises an intron of the CEBP/β protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In other respects, the target nucleic acid comprises an exon region of the CEBP/β protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In still other respects, the target nucleic acid comprises an exon-intron junction of the CEBP/β protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. The human CEBP/β protein sequence encoded by the CEBP/β precursor mRNA is shown in SEQ ID NO:23. In other respects, the target nucleic acid contains the untranslated region of the CEBP/β protein-encoding nucleic acid or a naturally occurring variant thereof, such as the 5'UTR, 3'UTR, or both.

In some aspects, the anti-CEBPbASO of this disclosure hybridizes with regions within introns of CEBP/β transcripts (e.g., SEQ ID NO: 21). In some aspects, the anti-CEBPbASO of this disclosure hybridizes with regions within exons of CEBP/β transcripts (e.g., SEQ ID NO: 21). In other aspects, the anti-CEBPbASO of this disclosure hybridizes with regions within exon-intron junctions of CEBP/β transcripts (e.g., SEQ ID NO: 21). In some aspects, the anti-CEBPbASO of this disclosure hybridizes with regions (e.g., introns, exons, or exon-intron junctions) within CEBP/β transcripts (e.g., SEQ ID NO: 21), wherein the anti-CEBPbASO has a spacer aggregate design.

In some respects, anti-CEBPbASO targets the mRNA of a specific isoform (e.g., isoform 1) of the CEBP/β protein. In other respects, anti-CEBPbASO targets all isoforms of the CEBP/β protein. In still other respects, anti-CEBPbASO targets two isoforms of the CEBP/β protein (e.g., isoform 1 and isoform 2, isoform 1 and isoform 3, or isoform 2 and isoform 3).

In some respects, the binding of anti-CEBPbASO targeting the CEBPb transcript disclosed in this paper to the mRNA transcript encoding CEBPb can reduce the expression level and/or activity level of CEBPb.

In some respects, any anti-CEBPbASO described herein may be an EV (e.g., an exosome) of this disclosure, i.e., a part of an EV (e.g., an exosome) containing a cleavable linker of the disclosed herein (e.g., Formula I construct), wherein the bioactive portion BAM is an anti-CEBPbASO or a combination thereof described herein.

ASO targeting STAT3

In some respects, the bioactive molecule BAM is an anti-STAT3ASO. 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 frequently overactivated in many human cancers.

Signal transducer and activator of transcription 3 (STAT3) is known in the art under various names. These 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).

Natural variants of the human STAT3 gene product are known. Therefore, the ASO disclosed herein can be engineered to reduce or suppress the expression of natural variants of the STAT3 protein.

SEQ ID NO:41 is identical to the STAT3 precursor mRNA sequence, except that the nucleotide "t" in SEQ ID NO:41 is shown as "u" in the precursor mRNA. In some respects, 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 the precursor mRNA. In other respects, 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 the precursor mRNA. In still other respects, 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 the precursor mRNA. The human STAT3 protein sequence encoded by the STAT3 precursor mRNA is shown in SEQ ID NO:42. In other respects, the target nucleic acid comprises an untranslated region of a STAT3 protein-encoding nucleic acid or a naturally occurring variant thereof, such as the 5'UTR, 3'UTR, or both.

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

In some aspects, the anti-STAT3 ASO of this disclosure hybridizes with regions within introns of STAT3 transcripts (e.g., SEQ ID NO:41 or SEQ ID NO:43). In some aspects, the anti-STAT3 ASO of this disclosure hybridizes with regions within exons of STAT3 transcripts (e.g., SEQ ID NO:41 or SEQ ID NO:43). In other aspects, the anti-STAT3 ASO of this disclosure hybridizes with 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 ASO of this disclosure hybridizes with regions (e.g., introns, exons, or exon-intron junctions) within STAT3 transcripts (e.g., SEQ ID NO:41 or SEQ ID NO:43), wherein the anti-STAT3 ASO has a spacer-merged design.

In some respects, anti-STAT3ASO targets the mRNA of a specific isoform (e.g., isoform 1) of the STAT3 protein. In other respects, ASO targets all isoforms of the STAT3 protein. In still other respects, ASO targets two isoforms of the STAT3 protein (e.g., isoform 1 (UniProt ID: P40763-1) and isoform 2 (UniProt ID: P40763-2), and isoforms 2 and 3 (UniProt ID: P40763-3).

In some aspects, the anti-STAT3 ASO of this disclosure hybridizes with regions within introns of STAT3 transcripts (e.g., SEQ ID NO:41 or SEQ ID NO:43). In some aspects, the anti-STAT3 ASO of this disclosure hybridizes with regions within exons of STAT3 transcripts (e.g., SEQ ID NO:41 or SEQ ID NO:43). In other aspects, the anti-STAT3 ASO of this disclosure hybridizes with 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 ASO of this disclosure hybridizes with regions (e.g., introns, exons, or exon-intron junctions) within STAT3 transcripts (e.g., SEQ ID NO:41 or SEQ ID NO:43), wherein the ASO has a spacer aggregate design.

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

The anti-STAT3 ASO disclosed herein comprises a continuous nucleotide sequence of complementary sequences to regions corresponding to STAT3 transcripts, such as the nucleotide sequence corresponding to SEQ ID NO:41.

In some respects, the binding of anti-STAT3 ASO targeting the STAT3 transcript disclosed in this paper to the mRNA transcript encoding STAT3 can reduce the expression level and/or activity level of STAT3.

In some respects, any anti-STAT3ASO described herein may be an EV (e.g., an exosome) of this disclosure, i.e., an EV (e.g., an exosome) comprising a construct containing a cleavable linker of the disclosed herein (e.g., the Formula I construct), wherein the bioactive portion BAM is an anti-STAT3 ASO or a combination thereof described herein.

ASO targeting NRAS

In some respects, the bioactive molecule BAM is an anti-NRasASO agent. NRas is an oncogene encoding a membrane protein that shuttles between the Golgi apparatus and the plasma membrane. The NRas-encoding genomic DNA can be found at chromosome position 1p13.2 (nucleotides 5001 to 17438 of GenBank accession number NG_007572). Specifically, a combination of time-lapse microscopy and photobleaching techniques has shown that, in the absence of palmitoylation, GFP-labeled N-Ras undergoes rapid exchange between the cytosol and the ER/Golgi membrane, and wild-type GFP-N-Ras is recovered to the Golgi complex via a non-vesicular mechanism. N-ras mutations have been described in melanoma, thyroid cancer, teratoma, fibrosarcoma, neuroblastoma, rhabdomyosarcoma, Burkitt lymphoma, acute promyelocytic leukemia, T-cell leukemia, and chronic myeloid leukemia. Carcinogenic N-Ras can induce acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CMML)-like disease in mice.

Neuroblastoma RAS virus oncogenes (NRas) are known in the art under various names. These names include: GTPase NRas, N-ras protein part 4, neuroblastoma RAS virus (v-ras) oncogene homolog, neuroblastoma RAS virus oncogene homolog, converting protein N-Ras and v-ras neuroblastoma RAS virus oncogene homolog.

The NRAS gene provides instructions for producing 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).

Natural variants of the human NRas gene product are known. For example, natural variants of the human NRas protein may contain one or more amino acid substitutions selected from the following: G12D, G13D, T50I, G60E, and any combination thereof. Other variants of the human NRas protein generated by alternative splicing are also known in the art, such as G13R, Q61K, Q61R, and P34L. Therefore, the anti-NRasASO of this disclosure can be engineered to reduce or inhibit the expression of natural variants of the STAT3 protein.

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 respects, the "target nucleic acid" comprises an intron of a NRas protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as the precursor mRNA. In other respects, the target nucleic acid comprises an exon region of a NRas protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as the precursor mRNA. In still other respects, the target nucleic acid comprises an exon-intron junction of a NRas protein-encoding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as the precursor mRNA. The sequence of the human NRas protein encoded by the NRas precursor mRNA is shown in SEQ ID NO:52. In other respects, the target nucleic acid comprises an untranslated region of a NRas protein-encoding nucleic acid or a naturally occurring variant thereof, such as the 5'UTR, 3'UTR, or both.

In some respects, the anti-NRasASO of this disclosure is also capable of downregulating (e.g., reducing or eliminating) the expression of NRas mRNA or protein. In this respect, the anti-NRasASO of this disclosure can affect the indirect inhibition of NRas proteins by reducing NRas mRNA levels, typically in mammalian cells (such as human cells, such as tumor cells). In particular, this disclosure relates to anti-NRasASOs targeting one or more regions of NRas precursor mRNA (e.g., intronic regions, exon regions, and/or exon-intron junction regions). Unless otherwise stated, the term "NRas" as used herein can refer to NRas from one or more species (e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).

In some aspects, the anti-NRasASO of this disclosure hybridizes with regions within introns of NRAS transcripts (e.g., SEQ ID NO:51 or SEQ ID NO:53). In some aspects, the ASO of this disclosure hybridizes with regions within exons of NRAS transcripts (e.g., SEQ ID NO:51 or SEQ ID NO:53). In other aspects, the ASO of this disclosure hybridizes with regions within exon-intron junctions of NRAS transcripts (e.g., SEQ ID NO:51 or SEQ ID NO:53). In some aspects, the anti-NRasASO of this disclosure hybridizes with regions (e.g., introns, exons, or exon-intron junctions) within NRAS transcripts (e.g., SEQ ID NO:51 or SEQ ID NO:53), wherein the ASO has a spacer aggregate design.

In some aspects, the anti-NRasASO of this disclosure hybridizes to multiple target regions within the NRas transcript (e.g., genomic sequence, SEQ ID NO:51). In some aspects, the anti-NRasASO hybridizes to two different target regions within the NRas transcript. In some aspects, the anti-NRasASO hybridizes to three different target regions within the NRas transcript. In some aspects, the anti-NRasASO that hybridizes to multiple regions within the NRas transcript (e.g., genomic sequence, SEQ ID NO:51) is more effective in reducing NRas expression (e.g., having a lower EC50) compared to the anti-NRas ASO that hybridizes to a single region within the NRas transcript (e.g., genomic sequence, SEQ ID NO:51).

In some respects, ASO targets the mRNA of a specific isoform of the NRAS protein (e.g., isoform 1, NCBI ID: NP_001229821.1). In other respects, ASO targets all isoforms of the NRAS protein. In still other respects, ASO targets two isoforms of the NRAS protein (e.g., 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)).

The anti-NRasASO disclosed herein comprises a continuous nucleotide sequence of complementary sequences to regions corresponding to NRas transcripts, such as the nucleotide sequence corresponding to SEQ ID NO:51.

In some respects, the binding of anti-NRasASO targeting the NRas transcripts disclosed in this paper to the mRNA transcripts encoding NRas can reduce the expression level and/or activity level of NRas.

In some respects, any anti-NRasASO described herein may be an EV (e.g., an exosome) of this disclosure, i.e., a part of an EV (e.g., an exosome) containing a cleavable linker of the disclosed herein (e.g., Formula I construct), wherein the bioactive portion BAM is an anti-NRasASO or a combination thereof described herein.

ASO targeting KRAS

In some respects, the bioactive molecule BAM is anti-KRASASO. The sequence of the human KRAS gene can be found at chromosome position 12p12.1 and under 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 complementary sequence 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 because 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 number NM_004985.5; SEQ ID NO:37) because 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 incorporated herein by reference in its entirety.

Human KRAS protein (P01116) has two isoforms, generated by alternative splicing. Isoform 2A (accession number: P01116-1; SEQ ID NO:38) is the canonical sequence. It is also known as K-Ras4A. Isoform 2B (accession number: 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 respects, the anti-KRAS ASO disclosed herein can reduce or inhibit the expression of KRAS protein isoform 2A, isoform 2B, or both.

Natural variants of the human KRAS gene product are known. For example, natural variants of the human KRAS protein may contain one or more amino acid substitutions selected from the following: 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 combinations thereof. Natural variants specific to KRAS protein isotype 2B contain one or more amino acid substitutions selected from the group consisting of V152G, D153V, F156I, F156L, or combinations thereof. The anti-KRASASO of this disclosure can be programmed to reduce or inhibit the expression of one or more variants of the 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 of this disclosure 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.

In some aspects, the target nucleic acid sequences for anti-KRAS ASO disclosed herein comprise 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 shown 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 some aspects, the target nucleic acid sequence comprises an exon region of a KRAS protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In some aspects, the target nucleic acid sequence comprises an intron of a KRAS protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In a further aspect, the target nucleic acid sequence comprises an exon-intron junction of a KRAS protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In some respects, for example when used for research or diagnosis, the target nucleic acid may be cDNA or a synthetic oligonucleotide derived from the DNA or RNA nucleic acid targets described herein. In some respects, the target nucleic acid contains the untranslated region of a KRAS protein-encoded nucleic acid or a naturally occurring variant thereof, such as the 5'UTR, 3'UTR, or both.

Therefore, in some aspects, the anti-KRAS ASO disclosed herein hybridizes with exon regions of KRAS transcripts (e.g., SEQ ID NO:31 or SEQ ID NO:33). In some aspects, the anti-KRAS ASO disclosed herein hybridizes with intron regions of KRAS transcripts (e.g., SEQ ID NO:31). In some aspects, the anti-KRAS ASO hybridizes with exon-intron junctions of KRAS transcripts (e.g., SEQ ID NO:31). In some aspects, the anti-KRAS ASO disclosed herein hybridizes with regions within KRAS transcripts (e.g., SEQ ID NO:31) (e.g., introns, exons, or exon-intron junctions).

In some respects, the target nucleic acid sequence of the ASO disclosed herein is KRAS mRNA, such as SEQ ID NO:33. Therefore, in some respects, the anti-KRAS ASO disclosed herein can hybridize to one or more regions of KRAS mRNA. In some respects, the anti-KRAS ASO disclosed herein targets a specific isotype of mRNA encoding the KRAS protein. In some respects, the anti-KRAS ASO disclosed herein can target all isotypes of the KRAS protein, including any variants thereof (e.g., those described herein). In some respects, the KRAS protein targeted by the anti-KRAS ASO disclosed herein may contain a G12D amino acid substitution.

In some respects, the binding of anti-KRAS ASO targeting the KRAS transcripts disclosed in this paper to the mRNA transcripts encoding KRAS can reduce the expression and/or activity levels of KRAS.

In some respects, any anti-KRAS ASO described herein may be an EV (e.g., an exosome) of this disclosure, i.e., an EV (e.g., an exosome) comprising a construct containing a cleavable linker of the disclosed herein (e.g., the Formula I construct), wherein the bioactive portion BAM is an anti-KRAS ASO or a combination thereof described herein.

ASO targeting Pmp22

In some respects, the bioactive molecule BAM is an anti-Pmp22ASO. Peripheral myelin protein 22 (PMP22), also known as growth arrest-specific protein 3 (GAS-3), is encoded by the PMP22 gene. PMP22 is a 22 kDa transmembrane glycoprotein composed of 160 amino acids and is primarily expressed in Schwann cells of the peripheral nervous system. Schwann cells exhibit high expression of PMP22, which can account for 2% to 5% of the total protein content of dense myelin. Dense myelin is the main component of the myelin sheath of peripheral neurons, a protective fatty layer that provides electrical insulation for neuronal axons. The level of PMP22 expression in the adult central nervous system is relatively low.

PMP22 plays a crucial role in the formation and maintenance of dense myelin. PMP22 expression is significantly upregulated when Schwann cells contact neuronal axons, while it is downregulated during axonal degeneration or transection. PMP22 has been shown to be associated with zonula-occludens 1 and occludin, proteins involved in adhesion to other cells and the extracellular matrix, and also support myelin function. In addition to its cell adhesion function, PMP22 is also upregulated during Schwann cell proliferation, suggesting a role in cell cycle regulation. PMP22 is detectable in non-neural tissues, where its expression has been shown to serve a growth arrest-specific (gas-3) function.

Inappropriate gene dosage of the PMP22 gene can lead to abnormalities in myelin protein synthesis and function. Since the components of myelin are stoichiometric, irregular expression of any component can result in myelin instability and neurological disorders. Altered PMP22 gene expression is associated with a variety of neuropathies, such as Charcot–Marie–Tooth type 1A (CMT1A), Dejerine-Sottas disease, and hereditary stress-predisposed peripheral neuropathy (HNPP). Excessive PMP22 (e.g., caused by gene duplication) leads to CMT1A. PMP22 gene duplication is the most common genetic cause of CMT, where excessive PMP22 production leads to defects in multiple signaling pathways and dysfunction of transcription factors such as KNOX20, SOX10, and EGR2.

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 17p12, between 15,229,777 and 15,265,326.

The sequence of the human PMP22 precursor mRNA transcript (SEQ ID NO: 264) corresponds to the reverse complementary sequence of residues 15,229,777 to 15,265,326 at chromosome 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 isotypes have Uniprot accession numbers A8MU75, J3KQW0, A0A2R8Y5L5, J3KT36, and J3QS08. Publicly available contents of database entries corresponding to the accession numbers disclosed herein are incorporated herein by reference in their entirety.

The disclosed anti-PMP22ASO can be designed to reduce or inhibit the expression of natural variants of the PMP22 protein.

An example of a target nucleic acid sequence for anti-PMP22ASO is the PMP22 precursor mRNA. SEQ ID NO:58 represents the human PMP22 genomic sequence (i.e., the reverse complementary sequence of nucleotides 15229777 to 15265326, the complementary sequence of chromosome 17p12). SEQ ID NO:58 is identical to the PMP22 precursor mRNA sequence, except that the nucleotide "t" in SEQ ID NO:58 is shown as "u" in the precursor mRNA.

In some respects, anti-PMP22ASO comprises a continuous nucleotide sequence of 10 to 30 nucleotides that is complementary to nucleotides 1 to 1828 of the PMP22 transcript corresponding to the nucleotide sequence set forth in SEQ ID NO:264 (complete mRNA transcript of PMP22) or nucleotides 208 to 690 of the PMP22 transcript corresponding to the nucleotide sequence set forth in SEQ ID NO:59 (coding sequence of PMP22).

In some respects, the continuous nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or about 100% complementary to the nucleic acid sequence within the PMP22 transcript. In some respects, anti-PMP22 ASO can reduce PMP22 protein expression in human cells (e.g., Schwan cells) that express the PMP22 protein.

In some respects, PMP22 protein expression was 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 anti-PMP22 ASO.

In some respects, anti-PMP22ASO can reduce the level of PMP22 mRNA in human cells (e.g., immune cells) that express PMP22 mRNA. In some respects, 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% compared to the level of PMP22 mRNA in human cells not exposed to anti-PMP22ASO.

In some respects, the target nucleic acid comprises an intron of a PMP22 protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In other respects, the target nucleic acid comprises an exon region of a PMP22 protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In still other respects, the target nucleic acid comprises an exon-intron junction of a PMP22 protein-coding nucleic acid or a naturally occurring variant thereof, and an RNA nucleic acid derived therefrom, such as precursor mRNA. In some respects, such as when used for research or diagnosis, the target nucleic acid may be cDNA or a synthetic oligonucleotide derived from the aforementioned DNA or RNA nucleic acid targets. 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 precursor mRNA is shown in SEQ ID NO:60. In other respects, the target nucleic acid comprises an untranslated region of a PMP22 protein-coding nucleic acid or a naturally occurring variant thereof, such as a 5'UTR, a 3'UTR, or both.

In some aspects, the anti-PMP22 ASO of this disclosure hybridizes with regions within introns of the PMP22 transcript (e.g., SEQ ID NO: 58). In some aspects, the ASO of this disclosure hybridizes with regions within exons of the PMP22 transcript (e.g., SEQ ID NO: 58). In other aspects, the anti-PMP22 ASO of this disclosure hybridizes with regions within exon-intron junctions of the PMP22 transcript (e.g., SEQ ID NO: 58).

In some respects, any anti-PMP22ASO described herein may be an EV (e.g., an exosome) of this disclosure, i.e., a part of an EV (e.g., an exosome) containing a cleavable linker of the disclosed herein (e.g., the Formula I construct), wherein the bioactive portion BAM is an anti-PMP22ASO or a combination thereof described herein.

In some aspects, the construct of formula (I) contains an ASO targeting STAT6 as a BAM conjugated at the 5′ or 3′ end, and _L1 contains a tetranucleotide group, such as dTdTdTdT. In some aspects, the construct of formula (I) contains an ASO targeting STAT6 as a BAM conjugated at the 5′ or 3′ end, _L1 contains an Ala-Ala-Asn peptide group, and _SP1 or _SP2 contains a pyrophosphate oxy group. In some aspects, the construct of formula (I) contains an ASO targeting STAT6 as a BAM conjugated at the 5′ or 3′ end, _L1 contains a Glu-Val-Cit peptide group, and _SP1 or _SP2 contains a silyl ether group. In some aspects, the construct of formula (I) contains an ASO targeting CEBP/β as a BAM conjugated at the 5′ or 3′ end, _L1 contains a Val-Cit peptide group, and _SP1 or _SP2 contains 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 examples, SP ₁ and/or SP ₂ contain a _C2-8 alkylene group (e.g., a _C3 alkylene group, a _C6 alkylene group, or a _C8 alkylene group), a polyoxyalkylene group (e.g., a polyoxyalkylene group containing ₂ to 15 _–OCH2CH2- repeating units), a carbamoyl group, an amide group, a thiosuccinimide group, a 1,2,3-triazolylbicyclononenyl group, or a combination thereof.

In some respects, equation (I) is a construct selected from the following

as well as

In some respects, EVs are exosomes, such as natural or recombinant exosomes. In some respects where the exosomes are natural exosomes, the loading density of ASOs attached to the exosomes is at least 1.5-fold higher than the control (see above). In some respects where the exosomes are exosomes overexpressing PTGFRN, the loading density of ASOs attached to the exosomes is at least 2-fold higher than the control, which is, for example, a construct without cleavable linkers (such as AM-BAM or AM- _SP1 -BAM).

In some aspects, the exosomes are natural exosomes and anchored to 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, about 800 and about 9,000, about 850 and about 8,500, about 900 and about 8,000, about 950 and about 7,500, or about 1,000 and about 7,000. In some respects, the average number of ASO molecules per natural 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 respects, the loading efficiency of natural exosomes is between approximately 70% and approximately 95%. In other respects, the loading efficiency of natural exosomes is between approximately 70% and approximately 75%, between approximately 75% and approximately 80%, between approximately 80% and approximately 85%, between approximately 85% and approximately 90%, or between approximately 90% and approximately 95%. In some respects, 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%.

In some cases, the exosome is a scaffold-X exosome and the anchoring portion AM is cholesterol. The average number of ASO molecules per exosome is 2442 +/- 339. In other cases, the average number of ASO molecules per scaffold-X exosome is between approximately 2000 and approximately 3000. In still other cases, the average number of ASO molecules per scaffold-X exosome is between approximately 2000 and approximately 2100, approximately 2100 and approximately 2200, approximately 2200 and approximately 2300, approximately 2300 and approximately 2400, approximately 2400 and approximately 2500, approximately 2500 and approximately 2600, approximately 2600 and approximately 2700, approximately 2700 and approximately 2800, approximately 2800 and approximately 2900, or approximately 2900 and approximately 3000. In some aspects, the average number of ASO molecules per stent-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 stent-X exosome is between 27% and 46%. In some aspects, the loading efficiency of the stent-X exosome is between about 25% and about 50%. In some aspects, the loading efficiency of the stent-X exosome is between about 25% and about 30%, about 30% and about 35%, about 35% and about 40%, about 40% and about 45%, or about 45% and about 50%. In some aspects, the loading efficiency of the stent-X exosome is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

In some respects, the average number of ASO molecules per scaffold-X exosome exceeds 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000. In some respects, the average number of ASO molecules per scaffold-X exosome is approximately 5,000 to approximately 6,000, approximately 6,000 to approximately 7,000, approximately 7,000 to approximately 8,000, approximately 8,000 to approximately 9,000, approximately 9,000 to approximately 10,000, approximately 10,000 to approximately 11,000, approximately 11,000 to approximately 12,000, and approximately Between 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 19,000 and about 20,000.

Preparation method

This disclosure provides a method for attaching a bioactive molecule BAM to an EV (e.g., an exosome), the method comprising attaching an anchoring portion AM to the EV, wherein the anchoring portion AM is attached to the bioactive portion BAM according to Formula I:

AM-SP ₁ -L ₁ -SP ₂ -BAM (Formula I),

Wherein BAM is a bioactive molecule; AM is the anchoring moiety; _L1 is a cleavable bond containing the following: 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-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate oxy group; or a silyl ether; and; _SP1 is optionally a first spacer; and _SP2 is optionally a second spacer. In some aspects, _SP1 is present and contains C3, C6, TEG, or HEG. In some aspects, _SP2 is present and contains C3, C6, TEG, or HEG. In some aspects, BAM is an antisense oligonucleotide (ASO), such as a spacer polymer.

This disclosure also provides a method for increasing the loading density of the bioactive molecule BAM attached to the EV, the method comprising screening a library of anchoring portions AM attached to the bioactive portion BAM according to Formula I:

AM-SP ₁ -L ₁ -SP ₂ -BAM (Formula I),

Wherein BAM is a bioactive molecule; AM is the anchoring moiety; _L1 is a cleavable bond containing the following: 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-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate oxy group; or a silyl ether; and; _SP1 is optionally a first spacer; and _SP2 is optionally a second spacer. In some aspects, _SP1 is present and contains C3, C6, TEG, or HEG. In some aspects, _SP2 is present and contains C3, C6, TEG, or HEG. In some aspects, BAM is an antisense oligonucleotide (ASO), such as a spacer polymer. In some aspects, the loading density of the bioactive molecule BAM attached to EVs (e.g., exosomes) is increased by at least 1, 1.5, 2, 2.5, 3, 3.5, about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times relative to the control. In some aspects, the control is a corresponding construct lacking the cleavable linker _L1 . For example, in some aspects, the control of a construct having the structure AM- _SP1 - _L1 - _SP2 -BAM is a control construct having the structure AM-BAM or AM- _SP1 -BAM.

The EVs (e.g., exosomes) disclosed herein can be produced by chemical synthesis, recombinant DNA technology, biochemical or enzymatic cleavage of larger molecules, a combination of the foregoing methods, or by any other method. In one aspect, this disclosure provides a method for attaching a bioactive molecule to an EV (e.g., exosome) via a cleavable adapter disclosed herein, for example via solid-phase synthesis or conjugation.

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

In some respects, the producing cells are HEK293 cells. Human embryonic kidney 293 cells, commonly also known as HEK 293, HEK-293, 293 cells, or less precisely HEK cells, are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture.

A comprehensive study of the genome and transcriptome of HEK 293 and five derived cell lines compared the HEK 293 transcriptome with those of human kidney, adrenal gland, pituitary, and central nervous system tissues. The HEK 293 pattern is most closely similar to that of adrenal cells, which possess many neuronal characteristics. 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 subtriploid, containing less than three times the number of chromosomes in 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 HEK 293 cells that can be used to produce EVs include, but are not limited to, HEK 293F, HEK 293FT, and HEK 293T.

Solid-phase synthesis: Solid-phase synthesis known in the art can be used, additionally or alternatively, to produce the constructs disclosed herein. In some aspects, two or more components of the disintegrable 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 synthetic steps. Spacers such as C3-phosphorous amide, TEG-phosphorous amide, or HEG-phosphorous amide can be used. In some aspects, spacers or combinations of spacers can be further extended via synthesis to incorporate into the membrane anchoring portion. In examples, phosphorous amides can be used via solid-phase synthesis to produce the optimized linkers of this disclosure, such as octyl-tocopherol phosphorous amide, tocopherol phosphorous amide, palmitate-C6 phosphorous amide, cholesterol-TEG phosphorous amide, or cholesterol-C6 phosphorous amide. 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, P.H.; Lam, Y (ed.), Solid-Phase Organic Synthesis, Concepts, Strategies, and Applications, John Wiley & Sons, Inc., New Jersey (2012).

Conjugation: In some respects, two or more components of the connectors disclosed herein can be attached to each other using conjugation (e.g., tandem). In addition to amine-reactive compounds, compounds having chemical groups that form bonds with thiol groups (–SH) can also be used as cross-linking agents and modifying agents for proteins and other bioconjugation techniques. Thiol groups, also known as mercaptans, are present in the side chains of cysteine (Cys,C) amino acids in proteins.

Thiol groups are useful targets for protein conjugation and labeling. First, thiol groups are present in most proteins, but in fewer numbers than in primary amines; therefore, crosslinking via thiol groups is more selective and precise. Second, thiol groups in proteins are usually involved in disulfide bonds, so crosslinking at these sites typically 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 the molecule by reacting a thiol addition reagent with a primary amine, such as 2-iminothiacyclopentane (Traut 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 crosslinking agents provides greater flexibility and control over the crosslinking process. For example, using 3-maleimide-propionic acid N-hydroxysuccinimide (NHS) ester containing a maleimide group and an NHS ester, the NHS ester can be used to label primary amines ( _-NH2 ) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The maleimide group will react with the thiol group to form a covalent bond, thereby enabling the linkage of biomolecules to thiols.

When the pH of the reaction mixture is between 6.5 and 7.5, the maleimide group reacts specifically with the thiol group; the result is the formation of an irreversible, stable thioether bond (i.e., this 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 of hydrolysis of the maleimide group to unreactive maleamic acid. Maleimides do not react with tyrosine, histidine, or methionine.

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 a protein to make the thiol group available for conjugation, 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.

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

In one aspect, the connection involves treating EVs (e.g., exosomes) with a reducing agent. Suitable reducing agents include, for example, TCEP (tris(2-carboxyethyl)phosphine), DTT (dithiothreitol), BME (2-mercaptoethanol), thiolation agents, and any combination thereof. Thiollation agents may include, for example, Traut reagent (2-iminothiacyclopentane).

Following treatment with a reducing agent, the ligation reaction further involves contacting the reduced EV (e.g., exosome) with the maleimide moiety. In one aspect, the maleimide moiety is ligated to the bioactive molecule prior to ligation to the EV (e.g., exosome). In some aspects, the maleimide moiety is further attached to a linker to ligate 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.

Any anchoring moiety AM, spacer SP, or combination of spacers disclosed herein, or bioactive molecule BAM, may be conjugated to reactive moieties such as amino reactive moieties (e.g., NHS-esters, p-nitrophenol, isothiocyanates, isocyanates, or aldehydes), thiol reactive moieties (e.g., acrylates, maleimides, or pyridyl disulfides), hydroxyl reactive moieties (e.g., isothiocyanates or isocyanates), carboxylic acid reactive moieties (e.g., epoxides), or azide reactive moieties (e.g., alkynes). Reactive groups include carboxyl groups, activated esters, sulfonyl halides, sulfonates, isocyanates, isothiocyanates, epoxides, aziridines, halides, aldehydes, ketones, amines, acrylamides, thiols, acyl azides, acyl halides, hydrazides, hydroxylamines, alkyl halides, imidazoles, pyridines, phenols, alkyl sulfonates, halotriazines, imino esters, maleimides, hydrazides, hydroxyl groups, and photoreactive azide aryl groups. As understood in the art, activated esters typically include esters of succinimide, benzotriazolyl, or aryl groups substituted with electron-withdrawing groups such as sulfonyl, nitro, cyano, or halogenated groups; or carboxylic acids activated by carbodiimide.

Exemplary reactive groups that can be used to chemically conjugate and covalently bind the two components disclosed herein (e.g., anchoring moiety AM and spacer SP, two spacers SP, spacer SP and bioactive molecule BAM, or anchoring moiety AM and bioactive moiety BAM) include, for example, N-succinimide-3-(2-pyridyldithio)propionate, N-4-maleimidebutyric acid, S-(2-pyridyldithio)cysteine, iodoacetoxysuccinimide, N-(4-maleimidebutyryloxy)succinimide, N-[5-(3'-maleimidepropionamide)-1-carboxypentyl]iminodiacetic acid, N-(5-aminopentyl)iminodiacetic acid, and 1'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide). Bifunctional linkers (linkers containing two reactive groups) can also be used.

In some aspects, the anchoring moiety AM, spacer SP, or bioactive molecule BAM may contain a terminal oxyamino group (-ONH ₂ ), a hydrazine group (-NHNH ₂ ), a thiol group (i.e., SH or thiol), or an olefin (e.g., -CH=CH ₂ ). In some aspects, the anchoring moiety AM, spacer SP, or bioactive molecule BAM may contain, at the terminal position, an electrophilic moiety such as an aldehyde, alkyl halide, methanesulfonate, toluenesulfonate, p-nitrobenzenesulfonate, or p-bromobenzenesulfonate, or an activated carboxylic acid ester (e.g., NHS ester, phosphoramide, or pentafluorophenyl ester).

As used herein, the term "protecting group" refers to the unstable chemical moiety of a reactive group, including but not limited to hydroxyl, amino, and thiol groups, known in the art to prevent unwanted reactions during synthesis. Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reaction sites and can then be removed to leave the unprotected group intact or usable for further reactions. Protecting groups known in the art are generally described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York (1999).

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

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

Thiol Reactive Mole: In some aspects, the reactive mole is a thiol reactive mole. As used herein, the term "thiol reactive mole" refers to a chemical group that can react with a reactive group having a thiol mole (or mercapto group). Exemplary thiol reactive moles are acrylates, maleimides, and pyridyl disulfides. Alternative reactive moles that react with thiols are also well known in the art. In some aspects, the thiol reactive mole may be attached to the terminal position of the anchoring mole AM, spacer SP, or bioactive molecule BAM of this disclosure. In some aspects, the thiol reactive mole is an acrylate. Typically, acrylates react with thiols on the carbonyl β-carbon of the acrylate to form a stable sulfur bond. In some aspects, the thiol reactive mole is a maleimide. Typically, maleimides react with thiols on the β-carbon or carbonyl group to form a stable sulfur bond. In some aspects, the thiol reactive mole is a pyridyl disulfide. Typically, pyridyl disulfides react with thiols on the β-thio atom of the pyridyl group to form stable disulfide bonds and pyridin-2-thiones.

Hydroxyl Reactive Molecular Part: In some aspects, the reactive mole is a hydroxyl reactive mole. As used herein, the term "hydroxyl reactive mole" refers to a chemical group that can react with a reactive group having a hydroxyl mole. Exemplary hydroxyl reactive moles are isothiocyanates and isocyanates. Alternative reactive moles that react with the hydroxyl mole are also well known in the art. In some aspects, the hydroxyl reactive mole may be attached to the terminal position of the anchoring mole AM, spacer SP, or bioactive molecule BAM of this disclosure. In some aspects, the hydroxyl reactive mole is an isothiocyanate. Typically, isothiocyanates react with the hydroxyl group of the reactive group to produce a stable aminothioester mole. In some aspects, the amine reactive mole is an isocyanate. Typically, isocyanates react with the hydroxyl group of the reactive group to produce a stable carbamate mole.

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

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

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

This 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 containing about 150 mM NaCl). In some aspects, the exosomes are loaded with ASOs attached (e.g., covalently attached) via a membrane anchoring construct of Formula I.

Therapeutic uses

This disclosure provides methods for treating a disease or condition in a subject with this need, the method comprising administering to the subject a composition comprising an EV (e.g., exosome) of this disclosure. This disclosure also provides methods for preventing or improving symptoms of a disease or condition in a subject with this need, the method comprising administering to the subject a composition comprising an EV (e.g., exosome) of this disclosure. This disclosure further provides methods for diagnosing a disease or condition in a subject with this need, the method comprising administering to the subject a composition comprising an EV (e.g., exosome) of this disclosure.

This disclosure also provides methods for preventing and/or treating diseases or conditions in subjects who require such treatment, the method comprising administering an EV (e.g., exosome) of this disclosure to the subject. In some aspects, diseases or conditions treatable using the methods of this disclosure include cancer, graft-versus-host disease (GvHD), autoimmune diseases, infectious diseases, fibrotic diseases, inflammatory diseases, neurodegenerative diseases, central nervous system diseases, muscular dystrophy, or metabolic diseases. In some aspects, the treatment is prophylactic. In other aspects, the EV (e.g., exosome) of this disclosure is used to induce an immune response. In other aspects, the EV (e.g., exosome) of this disclosure is used to vaccinate subjects.

In some respects, the disease or condition is cancer. When administered to a subject with cancer, in some respects, the EVs (e.g., exosomes) of this disclosure can upregulate the immune response and enhance tumor targeting of the subject's immune system. In some respects, the treated cancer is characterized by the infiltration of leukocytes (T cells, B cells, macrophages, dendritic cells, monocytes) into the tumor microenvironment, or so-called "hot tumors" or "inflammatory tumors." In some respects, the treated cancer is characterized by low or undetectable levels of leukocyte infiltration into the tumor microenvironment, or so-called "cold tumors" or "non-inflammatory tumors." In some respects, the EVs (e.g., exosomes) are administered in an amount and for a time sufficient to convert a "cold tumor" into a "hot tumor," i.e., administration that results in the infiltration of leukocytes (such as T cells) into the tumor microenvironment. In some respects, cancers include 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 combinations thereof. In other respects, the term "distal tumor" refers to a tumor that has spread from the original (or primary) tumor to a distant organ or tissue, such as a lymph node. In some respects, the EVs (e.g., exosomes) of this disclosure can treat tumors that have spread metastatically.

In some respects, the disease or symptom is graft-versus-host disease (GvHD).

In some respects, diseases or conditions that can be treated with this disclosure are autoimmune diseases. Non-limiting examples of autoimmune diseases include: multiple sclerosis, peripheral neuritis, Sjogren's syndrome, rheumatoid arthritis, alopecia, autoimmune pancreatitis, Behçet's disease, bullous pemphigoid, celiac disease, Dervian disease (neuromyelitis optica), glomerulonephritis, IgA nephropathy, various types of vasculitis, scleroderma, diabetes, arteritis, vitiligo, ulcerative colitis, irritable bowel syndrome, psoriasis, uveitis, systemic lupus erythematosus, and combinations thereof.

In some respects, diseases or conditions that can be treated with this disclosure are inflammatory diseases. 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.

In some respects, diseases or conditions that can be treated with this disclosure are central nervous system (CNS) diseases. 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 (ALS), and Huntington's disease.

In some respects, diseases or conditions that can be treated with this disclosure are fibrotic diseases. Non-limiting examples of fibrotic diseases include pulmonary fibrosis, liver fibrosis, cardiac fibrosis, mediastinal fibrosis, myelofibrosis, skin fibrosis, scleroderma, retroperitoneal fibrosis, and keloids.

In some respects, the disease or condition is an infectious disease. In some respects, the disease or condition is a carcinogenic virus. In some respects, infectious diseases that can be treated with this disclosure include, but are not limited to, human gamma herpesvirus 4 (Epstein-Barr virus), influenza A virus, influenza B virus, cytomegalovirus, Staphylococcus aureus, Mycobacterium tuberculosis, Chlamydia trachomatis, HIV-1, HIV-2, coronaviruses (e.g., MERS-CoV and SARS-CoV), filamentous viruses (e.g., Marburg virus and Ebola virus), Streptococcus pyogenes, Streptococcus pneumoniae, Plasmodium species (e.g., Plasmodium vivax and Plasmodium falciparum), Chikungunya virus, human papillomavirus (HPV), hepatitis B, hepatitis C, human herpesvirus 8, herpes simplex virus 2 (HSV2), Klebsiella spp., Pseudomonas aeruginosa, Enterococcus spp., Proteus spp., Enterobacter spp., Actinomyces spp., coagulase-negative staphylococci (CoNS), Mycoplasma spp., or combinations thereof.

In some respects, diseases or conditions that can be treated by the method of the present invention include Pompe disease, Gaucher disease, lysosomal storage diseases, myxoviral 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 transcarbamate (OTC) deficiency, glycogen storage disease 1A, Criggler-Najjar syndrome, primary hyperoxaluria type 1, acute intermittent porphyria, phenylketonuria, and familial hypercholesterolemia. Blood disorders, mucopolysaccharidosis type VI, α1-antitrypsin deficiency, Rett syndrome, Dravet syndrome, Angelman syndrome, DM1 disease, Fragile X disease, Huntington's disease, Friedrich's ataxia, Charcot-Marie-Tooth (CMT) disease (also known as hereditary motor sensory neuropathy (HMSN) or peroneal muscular atrophy), CMT1X (Charcot-Marie-Tooth 1X type) disease, catecholamine polymorphic ventricular tachycardia, spinocerebellar ataxia type 3 (SCA3) disease, limb-girdle muscular dystrophy, or hypercholesterolemia. In some respects, treatment for these diseases or conditions is preventative.

In some respects, a disease or symptom is a neurodegenerative disease. In some respects, a 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.

In some respects, diseases or conditions include muscular dystrophy. In some respects, muscular dystrophy is selected from 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.

In some respects, the disease or condition is selected from aromatic L-amino acid decarboxylase (AADC) deficiency (CNS), adenosine deaminase severe combined immunodeficiency (ADA-SCID), α-1 antitrypsin deficiency, β-thalassemia (severe sickle cell anemia), cancer (squamous cell carcinoma of the head and neck), Niemman-Pick type C disease, cerebral ALD, choroidal vas deferens, 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 brain disease. 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, latent dystrophic epidermolysis bullosa, RPE65 gene defect (visual 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.

Pharmaceutical Compositions and Administration

This disclosure also provides pharmaceutical compositions comprising EVs (e.g., exosomes) of this disclosure, suitable for administration to a subject. Pharmaceutical compositions typically comprise multiple EVs (e.g., exosomes) containing a bioactive molecule covalently linked to the multiple 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 various suitable formulations of pharmaceutical compositions comprising multiple 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 fully compliant with all Good Manufacturing Practices (GMP) regulations of the U.S. Food and Drug Administration. In some aspects, pharmaceutical compositions comprise one or more chemical compounds, such as small molecules covalently linked to EVs (e.g., exosomes) of this disclosure.

This disclosure provides pharmaceutical compositions comprising an EV (e.g., exosome) of the present disclosure with desired purity and a pharmaceutically acceptable carrier or excipient in a form suitable for administration to a subject. The pharmaceutically acceptable excipient or carrier may be determined in part by the specific composition being administered and by the specific method used to administer the composition. Thus, there are various 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 generally formulated aseptically and fully compliant with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

Acceptable carriers, excipients, or stabilizers are non-toxic to recipients (e.g., animals or humans) at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight peptides (e.g., about 10 or fewer amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; and 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 dextrin; 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 those sold as PLURONIC ^™ ), or polyethylene glycol (PEG).

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

Typically, pharmaceutical compositions are formulated to be compatible with their intended route of administration. The EVs (e.g., exosomes) of this disclosure can be administered via parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intratumoral, intrathecal, intraocular, intraocular, intramuscular, or as an inhaler. In some aspects, pharmaceutical compositions containing EVs (e.g., exosomes) are administered intravenously, for example by injection.

In some respects, EVs (e.g., exosomes) are administered intravenously to the circulatory system of a subject. In some respects, EVs (e.g., exosomes) are infused in a suitable fluid and administered into the veins of a subject. In some respects, EVs (e.g., exosomes) are administered intraarterially to the circulatory system of a subject. In some respects, EVs (e.g., exosomes) are infused in a suitable fluid and administered into the arteries of a subject. In some respects, EVs (e.g., exosomes) are administered to a subject via intrathecal administration. In some respects, EVs (e.g., exosomes) are administered via injection into the spinal canal or into the subarachnoid space to reach the cerebrospinal fluid (CSF). In some respects, EVs (e.g., exosomes) are administered intratumorally to one or more tumors of a subject. In some respects, EVs (e.g., exosomes) are administered to a subject via intranasal administration. In some respects, EVs (e.g., exosomes) may be administered via nasal inhalation in the form of local or systemic administration. In some respects, EVs (e.g., exosomes) are administered as a nasal spray. In some aspects, EVs (e.g., exosomes) are administered to subjects via intraperitoneal administration. In some aspects, EVs (e.g., exosomes) are infused in a suitable fluid and injected into the peritoneum of the subject. In some aspects, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) to lymphatic vessels. In some aspects, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) to the thymus, spleen, and/or bone marrow. In some aspects, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) to one or more lymph nodes. In some aspects, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) to one or more of the following: cervical lymph nodes, inguinal lymph nodes, mediastinal lymph nodes, or sternal lymph nodes. In some aspects, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) to the pancreas. In some aspects, EVs (e.g., exosomes) are administered to subjects via periocular administration. In some aspects, EVs (e.g., exosomes) are injected into the periocular tissue. Periocular drug administration includes subconjunctival, anterior subtenonic, posterior subtenonic, and retroocular routes.

The solution or suspension may contain the following components: a sterile diluent, such as water, a salt solution, a fixing oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial compound, such as benzyl alcohol or methylparaben; 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 tonic compound, such as sodium chloride or dextrose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. The formulation may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders. For intravenous administration, suitable carriers include physiological saline, antibacterial water, mixtures of polyoxyethylated triglycerides prepared by reacting ethylene oxide with castor oil (e.g., Cremophor EL ^™ (BASF, Parsippany, NJ)), or phosphate-buffered saline (PBS). The composition is generally sterile and fluid in a manner conducive to injection. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, or liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Antimicrobial action can be achieved by various antibacterial and antifungal compounds such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. If desired, isotonic compounds such as sugars, polyols such as mannitol, sorbitol, and sodium chloride can be added to the composition. Extended absorption of injectable compositions can be achieved by including compounds that delay absorption, such as aluminum monostearate and gelatin, in the composition.

As needed, sterile injectable solutions can be prepared by incorporating the EVs (e.g., exosomes) of this disclosure with one or a combination of components listed herein in an effective amount and in a suitable solvent. Generally, dispersions are prepared by incorporating EVs (e.g., exosomes) into a sterile medium containing a basic dispersion medium and any other desired components. In the case of sterile powders used to prepare sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, which produce an active ingredient powder plus any additional desired components from a previously sterile filtered solution. EVs (e.g., exosomes) can be administered in the form of reservoir injections or implantable formulations, which can be formulated in such a manner to allow for sustained or pulsatile release of the EVs (e.g., exosomes).

Systemic administration of compositions containing EVs (e.g., exosomes) of this disclosure can also be performed transmucosally. 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 used for transmucosal administration. Transmucosal administration can be accomplished by using, for example, a nasal spray.

In some respects, a pharmaceutical composition comprising an EV (e.g., exosome) of the present disclosure is administered intravenously to a subject who will benefit from the pharmaceutical composition. In some other respects, the composition is administered to the lymphatic system, for example by intralymphatic injection or intranodal injection (see, for example, 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.

In some aspects, pharmaceutical compositions comprising EVs (e.g., exosomes) of this disclosure are administered as liquid suspensions. In other aspects, the pharmaceutical compositions are administered as formulations capable of forming reservoirs after administration. In some preferred aspects, the reservoir slowly releases the EVs (e.g., exosomes) into circulation or maintains the reservoir form.

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

In some respects, pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and/or mineral oil. The pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavor enhancers, emulsifiers, suspending agents, and/or preservatives.

In some aspects, the formulations of the EVs (e.g., exosomes) of this disclosure are subjected to radiation, such as X-rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, and ultraviolet light, to destroy residual replicative nucleic acids. In some aspects, the formulations of the EVs (e.g., exosomes) of this disclosure are subjected to gamma irradiation with radiation doses exceeding 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or exceeding 100 kGy. In some respects, the formulations of the EVs (e.g., exosomes) disclosed herein are subjected to X-ray irradiation with a radiation dose (Gy) greater than 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 2000, 3000, 4000, 5000, 600, 7000, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 Gy.

In some aspects, the pharmaceutical composition comprises one or more therapeutic agents and an EV (e.g., exosome), specifically an EV containing a cleavable linker disclosed herein. In some aspects, the EV (e.g., exosome) is co-administered with one or more additional therapeutic agents in a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition comprising an EV (e.g., exosome) is administered prior to the administration of the additional 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 immunotherapy agent, an anti-inflammatory agent, or a drug that inhibits the action of cell growth factors or cell growth factor receptors. In other aspects, the pharmaceutical composition comprising an EV (e.g., exosome) is administered after the administration of the additional therapeutic agent. In a further aspect, the pharmaceutical composition comprising an EV (e.g., exosome) is administered concurrently with the additional therapeutic agent.

Reagent test kit

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

In some aspects, the kit comprises: an EV (e.g., exosome) containing one or more bioactive molecules; reagents for covalently attaching one or more bioactive molecules to the EV (e.g., exosome) via a cleavable linker, as disclosed herein, such as via conjugation or solid-phase synthesis; and instructions for performing the reaction to covalently attach one or more bioactive molecules to the EV (e.g., exosome) via the cleavable linker disclosed herein.

Unless otherwise stated, the practice of this disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, all of which are within the scope of the art. Such techniques are well explained in the literature. See, for example, Sambrook et al., eds. (1989) Molecular Cloning: A Laboratory Manual (2nd edition; Cold Springs 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, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al., U.S. Patent No. 4,683,195; H Edited by ames and Higgins, (1984) Nucleic Acid Hybridization; Edited by Hames and Higgins, (1984) Transcription AndTranslation; 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 Pr ess, Inc., N.Y.); Miller and Calos, eds., (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods in Enzymology, Vol. 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 Im munology, Volume I to IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); Crooke, Antisense Drug Technology: Principles, Strategies and Applications, 2nd edition CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Database entries and electronic publications disclosed in this disclosure are incorporated herein by reference in their entirety. The versions of database entries or electronic publications incorporated herein by reference are the most recent versions of those publicly available at the time of filing of this application. Database entries disclosed in this application corresponding to gene or protein identifiers (e.g., genes or proteins identified by accession numbers or database identifiers of public databases such as Genbank, Refseq, or Uniprot) are incorporated herein by reference in their entirety. The incorporated information related to genes or proteins is not limited to sequence data contained in the database entries. Information incorporated by reference includes the entire contents of database entries in the most recent versions of databases publicly available at the time of filing of this application. In the event of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Example

The following examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of the invention in any way. Unless otherwise stated, the practice of this invention will be carried out using conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology within the art. Such techniques are well explained in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); Green & Sambrook et al., Molecular Cloning: ALaboratory Manual, 4th ed. (Cold Spring Harbor Laboratory Press, 2012); Colowic k&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).

Unless otherwise specified, all reactions were performed using commercially available materials and reagents without further purification. Reactions were monitored by ultra-high performance liquid chromatography (UPLC) and mass spectrometry. All products were characterized by ^1H NMR, UPLC, and mass spectrometry. Chemical shifts are expressed in parts per million (ppm) and designated as s (singlet); br s (broad singlet); d (doublet); t (tripplex); q (quartet); quint (quintet); or m (multiplex). Rapid column chromatography was performed on a Biotage Isolera Four using either a Silicycle Siliasep column (pre-packed with 25 μm spherical silica) for reversed-phase chromatography purification or a Biotage SNAP C18 Ultra column (pre-packed with 25 μm spherical silica) and reagent-grade water and acetonitrile (0.1% formic acid). Further purification was performed using a semi-preparative HPLC system; column: CSH C1819 x 150 mm; eluent A: 0.1% formic acid, eluent B: acetonitrile, eluent C: 50% aqueous solution of 50% acetonitrile; gradient 0 to 15 min 5% to 40%; UV = 217 nm.

Analytical methods

UPLC4-MS: (CSH C18 long acid 2 to 95) - WatersAcquity UPLC H-class system; Column: AcquityCSH C18 1.7μm 2.1x 50mm; Eluent A: Water, Eluent B: Acetonitrile, Eluent C: 2 vol% ammonia (28%) aqueous solution, Eluent D: 2 vol% formic acid aqueous solution; Gradient: 0 to 4.0 min 2% to 95% B, always with A and 5% D, 4.0 to 4.6 min 95% B; Flow rate: 0.8 mL/min; Temperature: 40 °C; PDA: 215 to 350 nm.

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; Elution A: Water, Elution B: Acetonitrile, Elution C: 2 vol% ammonia (28%) aqueous solution, Elution D: 2 vol% formic acid aqueous solution; Gradient: 0 to 4.5 min 50% to 95% B, always with A and 5% D, 4.5 to 5.0 min 95% B; Flow rate 0.8 mL/min; Temperature: 40 °C; PDA: 215 to 350 nm.

Example 1

This example relates to the synthesis of a Chinese (I) construct according to one aspect of this disclosure. The complete synthesis is shown in Figure 3.

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

Pyrrole-2,5-dione (500 mg, 5.15 mmol) was dissolved in ethyl acetate (15.0 mL) and then cooled to 0 °C in ice water. After 10 minutes, N-methylmorpholine (521 mg, 5.15 mmol, 566 μL) was slowly added dropwise. A solution of methyl chloroformate (487 mg, 5.15 mmol, 398 μL) in ethyl acetate (1.20 mL) was then slowly added dropwise, and the reaction was continued at room temperature for 1 hour. After the reaction was complete, insoluble salts were removed by filtration. The filtrate was concentrated to give crude methyl 2,5-dioxopyrrole-1-carboxylate (700 mg, crude), which was obtained as a red oily solid and could be used directly for the next step without purification. ^¹H NMR (400 MHz, _CDCl₃ ) δ [ppm] = 6.84 (s, 2H), 3.98 (s, 3H).

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

2-(2-aminoethoxy)ethanol (427 mg, 4.06 mmol) was dissolved in _NaHCO3 (15.0 mL), and the solution was cooled to 0 °C and stirred for 10 min. Methyl 2,5-dioxopyrrole-1-carboxylate (700 mg, 4.51 mmol) was then added, and the mixture was stirred at 0 °C for 20 min, followed by warming 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 under 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 give 1-[2-(2-hydroxyethoxy)ethyl]pyrrole-2,5-dione (500 mg, 2.70 mmol, 60% yield) as a white solid. ^¹H NMR (400MHz, _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 bases 2 to 20%, NBK2289-69-P1-1) _Rt = 0.55 min (97.1%), MS (ESIpos): m/z = [M+H] ⁺ 186.08.

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

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-dodecylhydro-1H-cyclopentadien[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 organic matter was then dried over magnesium sulfate and concentrated to dryness under vacuum. The substance was purified by normal-phase purification (Biotage Isolera, 25 g siliasep column; eluent: 0 to 50% ethyl acetate in a solution exceeding heptane). The desired fractions were combined and concentrated to give a white solid 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-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]thioalkyl]-1-[2-(2-hydroxyethoxy)ethyl]pyrrolidine-2,5-dione (264 mg, 449 μmol, 83% yield). ^¹H NMR (400 MHz, 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),2.49-2.31(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.

Step 4: 4-(((2-(2-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecanohydro-1H-cyclopentadien[a]phenanthrene-3-yl)thio)-2,5-dioxopyrrolidine-1-yl)ethoxy)ethoxy)diisopropylsilyl)oxy)benzaldehyde

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. Then, a solution of p-hydroxybenzaldehyde (54.2 mg, 444 μmol, 48.0 μL) in THF (1.20 mL) was slowly added dropwise, and the mixture was stirred at 0 °C for 30 minutes. Then, 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-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]thioalkyl]-1-[2-(2-hydroxyethoxy)ethyl]pyrrolidine-2,5-dione (260 mg, 442 μmol) in dichloromethane (2.50 mL) was added dropwise and the solution was stirred at 0 °C for 20 minutes. The solution was then concentrated to dryness. The substance was then purified by normal-phase purification (Biotage Isolera, 25 g siliasep column; eluent 0 to 25% ethyl acetate in a solution of more than heptane). The desired fractions were combined and concentrated to give 4-[2-[2-[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-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]thioalkyl]-2,5-dioxo-pyrrolidine-1-yl]ethoxy]ethoxy-diisopropyl-silyl]oxybenzaldehyde (140 mg, 170 μmol, 39% yield) as a colorless oil. ¹ H NMR (400MHz, CDCl ₃ )δ[ppm]=9.87(s,1H),7.79(d,2H),7.07(d,2H),5.33(dd,1H),3.91(t,2H ),3.81(dt,1H),3.72-3.68(m,2H),3.62(t,2H),3.56(t,2H),3.14-2.96( m,2H),2.50(td,1H),2.36(dd,1H),2.24(d,1H),2.04-1.76(m,5H),1.62- 1.21(m,12H),1.21-0.93(m,26H),0.90(d,3H),0.85(dd,6H),0.65(s,3H). The data is too non-polar to obtain suitable UPLC data.

Step 5: 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecano-1H-cyclopentadien[a]phenanthrene-3-yl)thio)-1-(2-(2-(((4-(hydroxymethyl)phenoxy)diisopropylsilyl)oxy)ethoxy)ethyl)pyrrolidine-2,5-dione

4-[2-[2-[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-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]thioalkyl]-2,5-dioxo-pyrrolidine-1-yl]ethoxy]ethoxy-diisopropyl-silyl]oxybenzaldehyde (136 mg, 165 μmol) was dissolved in tetrahydrofuran (3.60 mL) and the solution temperature was lowered to -10 °C. Then sodium borohydride (2.50 mg, 66.1 μmol, 2.33 μL) was added and the mixture was stirred at -7 °C for 2 hours. Then, additional sodium borohydride (2.50 mg, 66.1 μmol, 2.33 μL) was added and the mixture was stirred at -5 °C for 2 hours. Then, dried acetone (2.00 mL) was added, and the solution was concentrated to dryness. The substance was then purified by normal-phase purification (Biotage Isolera, 12 g siliasep column; eluent of more than 12 CV heptane solutions of 0 to 20% ethyl acetate). The desired fractions were combined and concentrated under reduced pressure 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-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]thioalkyl]-1-[2-[2-[[4-(hydroxymethyl)phenoxy]-diisopropyl-silyl]oxyethoxy]ethyl]pyrrolidine-2,5-dione (75.0 mg, 91.0 μmol, 55.0% yield, 95% purity). UPLC: NBK2289-16-P2-1 (C850 to 95D2Kinetex BEH C82.1x50mm 1.7um), R _{_t} = 4.05 min (95.3%), MS (ESInegr): m/z = [MH] ^- 822.70. ^1H NMR (400MHz, CDCl_₃) )δ[ppm]＝7.23(d,2H),6.93(d,2H),5.34(dd,1H),4.60(s,2H),3.90(t,2H),3.79(td,1H),3.71-3.67(m,2H),3.61(t,2H),3.55(t,2H),3.12-2.98(m,2H),2.49(td,1H),2.37(dd,1H),2.26(d,1H),2.04-1.78(m,6H),1.63-1.21(m,12H),1.18-0.94(m,25H),0.91(d,3H),0.86(dd,6H),0.67(s,3H),No 1x exchangeable protons were observed.

Step 6: 4-(((2-(2-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecano-1H-cyclopentadien[a]phenanthrene-3-yl)thio)-2,5-dioxopyrrolidine-1-yl)ethoxy)ethoxy)diisopropylsilyl)oxy)benzyl(4-nitrophenyl)carbonate (CLS Lab Book NBK2289-19)

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-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]thioalkyl]-1-[2-[2-[[4-(hydroxymethyl)phenoxy] [25.0 mg, 30.3 μmol]-diisopropyl-silyl]oxyethoxy]ethyl]pyrrolidine-2,5-dione (25.0 mg, 30.3 μmol) was dissolved in dichloromethane (1.00 mL), triethylamine (3.63 mg, 35.9 μmol, 5.00 μL), and then 4-nitrobenzene chloroformate (7.00 mg, 34.7 μmol) was added. The mixture was stirred at room temperature for 1.5 hours. Then, additional triethylamine (3.63 mg, 35.9 μmol, 5.00 μL) and 4-nitrobenzene chloroformate (7.00 mg, 34.7 μmol) were added, and the mixture was stirred at room temperature for 3 hours. The solution was then concentrated to dryness. The crude mixture was then ground with diethyl ether (10.0 mL), filtered, and the filtrate was concentrated to dryness. The substance was then purified by normal-phase purification (Biotage Isolera, 12 g siliasep column; eluent of more than 15 CV heptane solution of 0 to 20% ethyl acetate). The desired fractions were combined and concentrated under reduced pressure to give [4-[2-[2-[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-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]thioalkyl]-2,5-dioxo-pyrrolidine-1-yl]ethoxy]ethoxy-diisopropyl-silyl]oxyphenyl]methyl(4-nitrophenyl) carbonate (12.0 mg, 12.1 μmol, 40% yield). ^¹H NMR (400MHz, _CDCl₃ ) δ [ppm＝8.27(d,2H),7.37(d,2H),7.33(d,2H),6.98(d,2H),5.37-5.30(m,1H),5.21(s,2H),3.92(t,2H),3.82-3.78(m,1H),3.72-3.63(m,4H),3.57(t,2H),3.15-2.98(m,2H),2.51(td,1H),2.38(dd,1H),2.25(d,1H),2.03-1.76(m,5H),1.62-0.82(m,47H – also contains overlapping impurities),0.66(s,3H).

Step 7: Final Goal

Dissolve ASO (15.0 mg) in 50 mM sodium carbonate/sodium bicarbonate buffer (1 mL). Then treat the mixture at room temperature with a solution of [4-[2-[2-[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-dodecylhydro-1H-cyclopentadieno[a]phenanthrene-3-yl]thioalkyl]-2,5-dioxo-pyrrolidine-1-yl]ethoxy]ethoxy-diisopropyl-silyl]oxyphenyl]methyl(4-nitrophenyl) carbonate (5.00 mg, 5.05 μmol) in DMSO (1.00 mL). The mixture is then stirred overnight at room temperature. The oligonucleotides were precipitated using a mixture of 5M NaCl (0.2 mL) and ethanol (6.00 mL). The mixture was briefly vortexed and placed in a refrigerator (-80°C) for at least 2 hours to ensure complete precipitation. The sample was removed from the refrigerator and centrifuged at 4000 rpm for 15 minutes at 4°C. Subsequently, the oligonucleotide precipitate was washed with a 70% aqueous ethanol solution by briefly vortexing and then centrifuging again at 4000 rpm for 15 minutes at 4°C. The precipitate was then dissolved in water and freeze-dried overnight to give a white solid (25.0 mg).

Example 2

This example describes the loading of lipid-connector-ASO (-CPP) and the purification of exoASO.

The following materials were prepared.

Buffer CEF-01: 15mM _Na₂HPO₄ , 5mM _KH₂PO₄ , 50mM _NaCl , 5% w/v sucrose, _pH 7.2.

Buffer CEF-05: 15mM _Na₂HPO₄ , 5mM _KH₂PO₄ , 150mM NaCl, 5 _% w/v sucrose, _pH 7.2.

Buffer CEF-04: 15mM _Na₂HPO₄ , 5mM _KH₂PO₄ , 100mM NaCl, 5 _% w/v sucrose, _pH 7.2.

ULTRAFREE ^™ -MC centrifugal filter unit 0.22um GV durapore (Millipore#UFC30GV0S, Burlington, MA) CAPTO ^™ Core 700 resin (50% v/v slurry) (Cytiva#17548102, Marlborough, MA).

Filter microplate, 96 pores, polypropylene, with 0.45μm polyvinylidene fluoride membrane (Agilent #200959-100, Santa Clara, CA).

Prepare a lipid-linker-ASO stock solution. Dissolve the lipid-linker-ASO (-CPP) solid in CEF-04 buffer to achieve a final concentration of 500 μM. After vortex mixing, filter the solution using an ULTRAFREE ^™ -MC (Millipore, Burlington, MA) centrifuge filter unit at 2000 x g for 5 minutes. The actual stock solution concentration was determined by the absorbance at A260 (UA).

Loading. An equal volume of lipid-connector-ASO(-CPP) stock solution (350 μL) was thoroughly mixed with exosomes (1.26E13 p/mL, 350 μL) and incubated at 37 °C for 24 h. For each example, the ASO sequence was targeted at mouse STAT6 (mSTAT6): 5′-TbsGbsAbsdGs(5MdC)-sdGsdAsdAsdTsdGsdGsdAs(5MdC)sdAsdGsdGsdTsCbsTbsTb-3′ (SEQ ID NO: 1124).

Purification. Add 150 μL of Captocore 700 resin (50% v/v slurry) to the wells of a filter microplate, followed by 300 μL of CEF-05 buffer. Centrifuge the microplate at 2000 x g for 5 min to remove the resin stock solution and equilibrate the resin with CEF-05 buffer. Repeat this process at least 3 times to ensure complete resin equilibration.

Cool the reaction mixture to room temperature for 30 minutes. Then add it to the resin on the filter microplate and mix on a plate shaker for at least 30 minutes to provide sufficient contact time. Afterward, centrifuge the microplate at 2000 x g for 5 minutes and collect the filtrate (purified exoASO). Then filter the filtrate using an ULTRAFREE ^™ -MC (Millipore, Burlington, MA) centrifuge filter unit at 2000 x g for 5 minutes to ensure sterility.

Purified exoASO was characterized by the following parameters: (i) particle count/concentration/yield by nanoparticle tracking analysis (NTA); (ii) particle size and size distribution by dynamic light scattering (DLS); and (iii) loading density by RIBOGREEN ^™ assay (QUANT-IT ^™ RIBOGREEN ^™ RNA assay kit; Thermofisher, Waltham, MA).

The average size and polydispersity index (PDI) of lipid-connector-ASO stock solutions are good indicators of aggregation. The lipid moiety is hydrophobic, while the ASO and CPP moieties are hydrophilic. When conjugated together, lipid-connector-ASO (-CPP) may aggregate. Aggregation was evaluated by dynamic light scattering (DLS) using average diameter (nm) and PDI. Lipid-connector-ASO stock solutions with an average diameter <20 nm and a PDI >0.25 indicate sufficient solubility in the reconstitution buffer. Aggregation can prevent lipid-connector-ASO loading into exosomes and may interfere with resin purification steps. Characterization of the lipid-connector-ASO stock solutions is shown in Figure 4. The solubility of exemplary ASOs as a function of the average diameter (nm) of the ASO stock solution is shown in Figure 5.

The ASO concentration of the load was characterized using a RIBOGREEN ^™ instrument (Thermofisher, Waltham, MA). Particle concentration was measured by NTA. Loading density was calculated by dividing the ASO quantity by the number of exosomes at a given concentration/volume. Mean size and PDI were measured by a DLS instrument. A PDI < 0.25 is typical for a fairly homogeneous exosome population, meaning there is no significant aggregation caused by surface ASO loading. The results are shown in Figure 6. Exemplary ASO/exosome loading densities are shown in Figure 7.

The ASO concentration of the payload, characterized by RIBOGREEN ^™ assay (Thermofisher, Waltham, MA), can be used to determine dosing for in vitro and in vivo potency assays. Mean particle size and size distribution (PDI value) are important characteristics of exosomes. Since the surface of exosomes is modified with various ASOs containing cleavable linkers and cell-penetrating peptides, monitoring particle size and distribution is crucial to ensure that the payload does not negatively impact the physicochemical properties of the exosomes.

The loading density of each individual lipid-linker-ASO(-CPP) varied from 886 to 3777, even when the structures were loaded into exosomes under the same conditions. Different linker and CPP structures affected molecular interactions during loading. The hydrophobicity/hydrophilicity of the structures determined their solubility. Generally, if the lipid-linker-ASO(-CPP) exhibited good water solubility, it was more likely to produce higher loading densities and good filterability.

Example 3

The potency/efficacy of exoASO was assessed using an in vitro assay with H1299. H1299 is a lung cancer cell line containing the STAT6 target gene. Cells were allowed to proliferate for several days. Once the target cell density was reached, the cells were treated with exoASO. The exoASO dosage was calculated based on the ASO concentration loaded. After incubating cells with exoASO at 37°C for 48 to 72 hours, STAT6 gene knockdown was measured as a potency indicator. H1299 was used as the primary in vitro assay system for high-throughput potency evaluation. Exemplary adapters from the H1299 assay were further evaluated in macrophages.

Figure 8 illustrates the _IC50 values of exemplary exoASOs modified with various cleavable linkers and/or cell-penetrating peptides (CPPs) compared to the control 5′-Chol-TEG-HEG with phosphodiester bonds.

Cleavable linkers provide enhanced exoASO potency. Each cleavable linker mechanism has a unique effect on potency. As shown in Figure 8, the potency enhancement driven by lipid and cleavable linker modifications follows this order: phospholipid-SS > Chol-Val-citrulline > Chol-SS = Chol-tetranucleotide.

A comparison was made between exoASO 5′-Chol-valine-citrulline-PAB (p-aminobenzoyl) and 5′-Chol-valine-citrulline. As shown in Figure 8, although both exoASOs contain the cathepsin-cleavable linker Val-Cit, the linker Val-Cit-PAB (where PAB is a self-destructing linker) is more efficient than Val-Cit alone. The proposed mechanism of the dual-cleavable linker is explained below.

In comparisons of (5′-Chol-SS, 3′-Chol-Arg3 vs. 3′-Chol-Arg3) and (5′-Chol-SS, 3′-Chol-Arg6 vs. 3′-Chol-Arg6), the addition of disulfide bonds (SS) or combinations of SS with cell-penetrating peptides (CPP) significantly improved potency. The increased exoASO potency is attributed to two unique mechanisms of CPP: enhanced uptake into target cells and endosome escape. Endosomal escape facilitated by CPP is crucial for exoASO to fully realize its potency potential, as most nanoparticle drug delivery systems are affected by endosome/lysosome encapsulation.

In exoASO 5′-16:0PDP PE and 5′-16:0 cyanuric acid PE, the phospholipid (PE) containing PDP or cyanuric acid is used as the anchoring moiety, rather than cholesterol. 16:0PDP PE is a phospholipid containing both phosphodiester bonds and disulfide bonds. 16:0 cyanuric acid PE contains only phosphodiester bonds. As shown in Figure 8, exoASO with PDP PE exhibits higher potency than cyanuric acid PE, indicating that disulfide bonds in phospholipids are important in enhancing the potency of exoASO.

The _IC50 values for the normalized loading density of ASO per exosome are shown in Figure 9. The overall efficacy of exoASO is determined by two factors: individual connector efficacy and loading density. Efficacy is the product of the two. Higher exoASO efficacy means that a smaller dose is required to treat subjects with a specific disease or condition requiring treatment. Reducing the effective dose can significantly reduce the dose required during manufacturing, thereby reducing the overall cost of production and treatment.

Example 4

Following the procedure described in Example 2, the loads and characterization of exemplary exoASOs containing various detachable joints are illustrated in the table of Figure 10.

Figures 11 and 12 show the percentage of gene expression (hSTAT6) normalized to ASO concentration (nM) for various exoASOs numbered 1 to 11, as illustrated in Figure 10.

Figures 13 and 14 are graphs comparing the _IC50 values of ASO numbers 1 to 11, normalized to ASO concentration (nM), as illustrated in Figure 10. Figure 13 corresponds to the concentrations shown in Figure 11, while Figure 14 corresponds to the concentrations shown in Figure 12.

For Chol-TEG-HEG (ASO#1 to 4), there was no significant difference in efficacy by changing the position of the phosphate diester (PO) or thiophosphate (PS) bond.

For Chol-tetranucleotide bonds (ASO#5 to 6), there is no significant difference in efficacy when the cleavable linker is attached to the 5′ or 3′ end of the ASO.

For Chol-(PO)SS(PO) (ASO#9 and #11), the 3′ modification provides better efficacy than the 5′ modification.

For Chol-SS (ASO#7 to 10), exoASOs using one or two PO bonds exhibited better potency than exoASOs with non-cleavable PS bonds (ASO#7). This suggests that even though PO bonds are less efficient than other cleavable linkers, cleavable phosphodiester (PO) bonds are still slightly superior to non-cleavable linkers such as phosphate thioides (PS). Based on these potency data and without being bound by any theory, it is believed that the release of ASOs from lipid anchors or exosomes is important for ASOs to be transported to their targets and exert their potency/function. On the other hand, if ASOs are tightly bound or trapped to their exosomal carriers or endosomes, they will not be able to exert their full potency.

Example 5

Based on overall potency results obtained from both H1299 and macrophage assays, exemplary adaptor selection was investigated in vivo. ExoASOs consisting of one or two cleavable mechanisms or in combination with CPP were evaluated in vivo. The percentage of STAT6 knockdown (KD) was measured in a single-dose, 7-day treatment study. Dosage was calculated based on the weight of the loaded lipid-adaptor-ASO (-CPP). Each exoASO was administered at 5 μg of ASO, except for Chol-TEG-HEG, which was administered at both 5 μg and 10 μg. Mice were sacrificed after seven days, and liver tissue was collected to measure the percentage of STAT6 gene knockdown. The percentage of STAT6 KD was measured in five mice for each specific adaptor structure, and the resulting percentage of STAT6 KD was taken as the average of the five mice.

Figure 15 shows the STAT6 knockdown percentage, with the average of five measurements plotted on the graph. A higher KD percentage is an indicator of higher potency or efficacy of exoASOs. ExoASOs with reducible linkers, such as 5′-Chol-SS and 5′-16:0PDPPE, showed increased STAT6 KD at a 5 μg dose level compared to the Chol-TEG-HEG control. Among these exoASOs, the combination of disulfide and CPP showed the highest potency, roughly a 2-fold increase compared to Chol-TEG-HEG. These data suggest that CPP incorporation may improve cellular uptake of exoASOs and promote endosome escape, leading to enhanced potency.

***

It should be understood that the Detailed Description section, and not the Summary and Abstract section, is intended to be used to interpret the claims. The Summary and Abstract section may set forth one or more, but not all, exemplary aspects of this disclosure as contemplated by the inventors, and therefore is not intended to limit this disclosure and the appended claims in any way.

The present disclosure has been described above using functional building blocks that illustrate implementations of specified functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined herein. Alternative boundaries may be imposed, provided that the specified functions and their relationships are properly performed.

The foregoing description of the specific aspects so fully reveals the general nature of this disclosure that others, by applying knowledge within the art, can easily modify and/or adapt various applications of such specific aspects without excessive experimentation or departing from the general conception of this disclosure. Therefore, based on the teachings and guidance presented herein, such adaptations and modifications are intended to fall within the meaning and scope of equivalents of the disclosed aspects. It should be understood that phrases or terms in this document are for descriptive rather than limiting purposes, and that the terminology or phrases in this specification will be interpreted by those skilled in the art based on the teachings and guidance.

The breadth and scope of this disclosure should not be limited by any of the exemplary aspects described above, but should be defined solely by the following claims and their equivalents.

All references cited in the entirety of this application (including bibliographic references, patents, patent applications and websites) are hereby expressly incorporated in their entirety by reference for any purpose, as are the references cited therein.

Claims (40)

1. An extracellular vesicle (EV) comprising a bioactive molecule (BAM) attached to the EV via an anchoring portion (AM) according to Formula I: AM-SP ₁ -L ₁ -SP ₂ -BAM(Formula I) in _L1 is a cleavable bond containing the following: 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-valine-citrulline, cyclobutane-1,1-dicarboxamide-citrulline, and alanine-phenylalanine-lysine; a pyrophosphate oxy group; or a silyl ether; and _SP1 and _SP2 are optional first and second spacers, respectively. 2. The EV according to claim 1, wherein AM is covalently connected to BAM at the 5′ position. 3. The EV according to claim 1, wherein AM is covalently connected to BAM at position 3′. 4. The EV according to any one of claims 1 to 3, wherein _L1 comprises a polynucleotide group that is a trinucleotide group or a higher nucleotide group. 5. The EV according to claim 4, wherein the polynucleotide base is a tetranucleotide base containing 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-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 oxy group. 8. The EV according to any one of claims 1 to 3, wherein _L1 comprises a silyl ether. 9. The EV according to claim 8, wherein the ^silyl ether comprises ^-OSiR1R2O- , wherein ^R1 and ^R2 are the same or different and are each _C1-8 alkyl or aryl. 10. The EV according to claim 9, wherein both ^R1 and ^R2 are isopropyl. 11. The EV according to any one of claims 1 to 10, wherein AM comprises sterols, lipids, vitamins, peptides, or combinations thereof. 12. The EV according to claim 11, wherein the sterol is cholesterol, mercaptocholesterol, ergosterol, 7-dehydrocholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fucosterol, sargastricosterol, campesterol, β-sitosterol, sitosterol, coprosterol, alfalfa, stigmasterol, or a combination thereof. 13. The EV according to claim 11 or 12, wherein the sterol is cholesterol. 14. 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, hexanoic 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-glycerol-3-phosphate ethanolamine-N-[3-(2-pyridyl dithio)propionate] (16:0 PDP PE), 16:0 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide methyl)cyclohexane-formamide] (16:0 PE MCC) or 16:0 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-(cyanuric acid) (16:0 cyanuric acid PE). 19. The EV according to claim 11, wherein the vitamin is tocopherol, tocotrienol, vitamin D, vitamin K, riboflavin, niacin, or pyridoxine. 20. The EV according to 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 according to any one of claims 1 to 21, wherein BAM comprises peptides, polypeptides, polynucleotides, proteins, antibodies or antigen-binding fragments thereof, chemical compounds or any combination thereof. 23. The EV according to any one of claims 1 to 22, wherein BAM comprises an antisense oligonucleotide (ASO), siRNA, miRNA, shRNA, nucleic acid, or any combination thereof. 24. The EV of claim 27, wherein the BAM includes an ASO. 25. The EV of claim 24, wherein the ASO targets transcripts. 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, an NLPR3 transcript, a FFLUC transcript, an RLUC transcript, a MYC transcript, or any combination thereof. 27. The EV according to any one of claims 1 to 26, wherein SP ₁ and SP ₂ are the same or different, and each comprises an alkylene group, a polyoxyalkylene group, a succinimide group, a maleimide group, an aryl group, an ether, a carbonyl group, a carboxylate group, a carbamoyl group, a thioether, a sulfonyl group, a thiocarbonyl group, a thiocarbamoyl group, a thiosuccinimide group, an amino group, an amide group, an acylhydrazine group, a thiophosphate group, a 1,2,3-triazolyl group, a benzoylcyclooctenyl group, a bicyclononenyl group, a p-aminobenzoyl group, a p-aminobenzylcarbamate group, or a combination thereof, and at least one of SP ₁ and SP ₂ is present in formula (I). 28. The EV according to claim 27, wherein at least one of SP ₁ and SP ₂ comprises _C2-8 alkylene, polyoxyalkylene, maleimide, carbamoyl, thio, amide, 1,2,3-triazolyl, dibenzoylcyclooctenyl, bicyclononenyl, 1,2,3-triazolyldibenzoylcyclooctenyl, 1,2,3-triazolylbicyclononenyl, p-aminobenzoyl, p-aminobenzylcarbamate, or combinations thereof. 29. The EV according to claim 28, wherein at least one of SP ₁ and SP ₂ comprises a _C3 alkylene, a _C6 alkylene, or a _C8 alkylene. 30. The EV of claim 28, wherein at least one of SP ₁ and SP ₂ comprises a polyoxyalkylene group containing 2 to 15 -OCH ₂ CH ₂ - repeating units. 31. The EV according to claim 29 or 30, wherein at least one of SP ₁ and SP ₂ further comprises carbamoyl, amide, thiosuccinimide, 1,2,3-triazolylbicyclononenyl or a combination thereof. 32. The EV according to any one of claims 27 to 31, wherein SP ₁ is present. 33. The EV according to any one of claims 27 to 32, wherein SP ₂ is present. 34. The EV according to claim 32 or 33, wherein both _SP1 and _SP2 are present and identical. 35. The EV according to claim 32 or 33, wherein both _SP1 and _SP2 are present and distinct. 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 for treating or preventing a disease or condition in a subject who requires such treatment, the method 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 condition is cancer, graft-versus-host disease (GvHD), autoimmune disease, infectious disease, fibrotic disease, inflammatory disease, neurodegenerative disease, central nervous system disease, muscular dystrophy, or metabolic disease.