Patent Application
20190151456
The instant application includes a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 15, 2018, is named 41406US_CRF_sequencelisting.txt and is 57,837 bytes in size.
Exosomes are important mediators of intercellular communication. They are also important biomarkers in the diagnosis and prognosis of many diseases, such as cancer. As drug delivery vehicles, exosomes offer many advantages over traditional drug delivery methods as a new treatment modality in many therapeutic areas.
A central feature of exosomes is their ability to contain biologically active payload within their interior space, or lumen. It is well known that exosomes contain endogenous payload including mRNA, miRNA, DNA, proteins, carbohydrates, and lipids, but the ability to direct specific loading of desired therapeutic payload is currently limited. Exosomes may be loaded by overexpressing desired therapeutic payloads in a producer cell, but this loading is often of limited efficiency due to stochastic localization of the payload to cellular exosome processing centers. Alternatively, purified exosomes may be loaded ex vivo by, for example, electroporation. These methods may suffer from low efficiency or be limited to small payloads, such as siRNAs. Therefore, suitable methods for generating highly efficient and well-defined loaded exosomes are needed to better enable therapeutic use and other applications of exosome-based technologies.
An aspect of the present invention relates to novel methods of loading exosomes for therapeutic use. Specifically, the methods use protein markers that are newly identified from the lumen of exosomes. In particular, a group of proteins (e.g., myristoylated alanine rich Protein Kinase C substrate (MARCKS); myristoylated alanine rich Protein Kinase C substrate like 1 (MARCKSL1); and brain acid soluble protein 1 (BASP1)) were identified to be highly enriched in the lumen of exosomes. Furthermore, a short sequence of the amino terminus of BASP1 was shown to be sufficient to direct high efficiency loading of fluorescent protein cargo molecules to the same extent as the full length BASP1 protein. This fragment, which is less than ten amino acids, presents a significant advance in the field of engineered exosome loading, and allows for the efficient, reproducible loading of any therapeutic protein cargo into the lumen of exosomes with no additional steps of ex vivo manipulation. The loading of exosomes using the fusion proteins described herein produces engineered exosomes with significantly higher levels of cargo compared to any other genetic engineering method described thus far.
The proteins and peptide sequences newly identified from exosomes are used in various embodiments of the present invention. For example, some embodiments relate to generating a fusion protein by conjugating the exosome protein or protein fragment and a therapeutically relevant protein and producing an exosome containing the fusion protein in the lumen of the exosome. The native full-length protein or a biologically active fragment of the therapeutically relevant protein can be transported to the lumen of exosomes by being conjugated to the exosome-enriched proteins or protein fragments.
The present invention further relates to generation or use of a lumen-engineered exosome designed for more efficient loading, or for loading of a therapeutically relevant protein in the lumen of an exosome. For example, the exosome lumen can be modified to contain a higher concentration of the native full-length exosome protein and/or a fragment or a modified protein of the native exosome protein in the lumen.
Some embodiments of the present invention relate to a producer cell or a method of generating the producer cell for producing such a lumen-engineered exosome. An exogenous polynucleotide can be introduced transiently or stably into a producer cell to generate a lumen-engineered exosome from the producer cell.
In one aspect, provided herein are exosomes comprising a target protein, wherein at least a part of the target protein is expressed from an exogenous sequence, and the target protein comprises BASP1 or a fragment thereof.
In some embodiments, the target protein is present in the lumen of the exosome at a higher density than a different target protein of a different exosome, wherein the different target protein comprises a conventional exosome protein or a variant thereof. In some embodiments, the conventional exosome protein is selected from the group consisting of CD9, CD63, CD81, PDGFR, GPI anchor proteins, lactadherin, LAMP2, LAMP2B, and a fragment thereof.
In some embodiments, the target protein comprises a polypeptide of any of SEQ ID NOs: 3-15. In some embodiments, wherein the target protein comprises a polypeptide of SEQ ID NO: 14.
In some embodiments, the exosome is produced from a cell genetically modified to comprise the exogenous sequence, optionally wherein the cell is an HEK293 cell. In some embodiments, the cell comprises a plasmid comprising the exogenous sequence. In some embodiments, the cell comprises the exogenous sequence inserted into a genome of the cell. In some embodiments, the exogenous sequence is inserted into a genomic site located 3′ or 5′ end of a genomic sequence encoding BASP1 or a fragment thereof. In some embodiments, the exogenous sequence is inserted into a genomic sequence encoding BASP1.
In some embodiments, the target protein is a fusion protein comprising BASP1 or a fragment thereof, and a therapeutic peptide. In some embodiments, the therapeutic peptide is selected from the group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, or a linker to a therapeutic compound. In some embodiments, wherein the therapeutic compound is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, and small molecules. In some embodiments, the therapeutic peptide is an antibody or a fragment thereof. In some embodiments, the therapeutic peptide is an antigen. In some embodiments, the therapeutic peptide is a component of a genome editing complex. In some embodiments, the genome editing complex is a CRISPR/Cas9 genome editing complex.
In some embodiments, the target protein is a fusion protein comprising BASP1 or a fragment thereof, and a viral capsid protein. In some embodiments, the viral capsid protein is from adeno-associated virus
In some embodiments, further comprising a second target protein, wherein the second target protein comprises PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2, ATP transporter, or a fragment thereof.
Accordingly, in an aspect, the present invention provides an exosome comprising a target protein, wherein at least a part of the target protein is expressed from an exogenous sequence, and the target protein comprises MARCKS, MARCKSL1, BASP1 or a fragment or a modification thereof.
In some embodiments, the target protein is present in the exosome at a higher density than a different target protein in a different exosome, wherein the different target protein comprises a conventional exosome protein or a variant thereof. In some embodiments, the conventional exosome protein is selected from the group consisting of CD9, CD63, CD81, PDGFR, GPI anchor proteins, lactadherin, LAMP2, LAMP2B, and a fragment thereof.
In some embodiments, the exosome is produced from a cell genetically modified to comprise the exogenous sequence, optionally wherein the cell is an HEK293 cell.
In some embodiments, the cell comprises a plasmid comprising the exogenous sequence.
In some embodiments, the exogenous sequence is inserted into a genomic site located 3′ or 5′ relative to a genomic sequence encoding MARCKS, MARCKSL1, or BASP1. In some embodiments, the exogenous sequence is inserted into a genomic sequence encoding MARCKS, MARCKSL1, or BASP1.
In some embodiments, the target protein is a fusion protein comprising MARCKS, MARCKSL1, BASP1, or a fragment thereof, and a therapeutic peptide.
In some embodiments, the therapeutic peptide is selected from the group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, or a linker to a therapeutic compound. In some embodiments, the therapeutic compound is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, and small molecules. In some embodiments, the therapeutic peptide is an antibody or a fragment or a modification thereof. In some embodiments, the therapeutic peptide is an enzyme, a ligand, a receptor, a transcription factor, or a fragment or a modification thereof. In some embodiments, the therapeutic peptide is an antimicrobial peptide or a fragment or a modification thereof.
In some embodiments, the exosome further comprises a second target protein, wherein the second target protein comprises MARCKS, MARCKSL1, BASP1, or a fragment thereof. In some embodiments, the exosome further comprises a second target protein, wherein the second target protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment thereof.
In some embodiments, the target protein comprises a peptide of (M)(G)(G/A/S)(K/Q)(L/F/S/Q)(S/A)(K)(K) (SEQ ID NO: 118). In some embodiments, the target protein comprises a peptide of (M)(G)(π)(X)(Φ/π)(π)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu). In some embodiments, the target protein comprises a peptide of (M)(G)(π)(ξ)(Φ/π)(S/A/G/N)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu).
In some embodiments, the target protein comprises a peptide of any one of SEQ ID NO: 4-110. In some embodiments, the target protein comprises a peptide of MGXKLSKKK, wherein X is any amino acid (SEQ ID NO: 116). In some embodiments, the target protein comprises a peptide of SEQ ID NO: 110. In some embodiments, the target protein comprises the peptide of SEQ ID NO: 13.
In some embodiments, the target protein further comprises a cargo peptide.
In another aspect, the present invention provides a pharmaceutical composition comprising the exosome and an excipient.
In some embodiments, the pharmaceutical composition is substantially free of macromolecules, wherein the macromolecules are selected from nucleic acids, exogenous proteins, lipids, carbohydrates, metabolites, and a combination thereof.
In yet another aspect, the present invention provides a population of cells for producing the exosome provided herein.
In some embodiments, the population of cells comprises an exogenous sequence encoding the target protein comprising MARCKS, MARCKSL1, BASP1 or a fragment or a modification thereof. In some embodiments, the population of cells further comprise a second exogenous sequence encoding a second target protein, wherein the second target protein comprises MARCKS, MARCKSL1, BASP1 or a fragment or a modification thereof. In some embodiments, the population of cells further comprises a second exogenous sequence encoding a second target protein, wherein the second target protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment thereof.
In some embodiments, the exogenous sequence is inserted into a genomic sequence encoding MARCKS, MARCKSL1, or BASP1, wherein the exogenous sequence and the genomic sequence encodes the target protein. In some embodiments, the exogenous sequence is in a plasmid.
In some embodiments, the exogenous sequence encodes a therapeutic peptide. In some embodiments, the therapeutic peptide is selected from a group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, or a linker to a therapeutic compound. In some embodiments, the therapeutic compound is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, and small molecules. In some embodiments, the therapeutic peptide is an antibody or a fragment or a modification thereof. In some embodiments, the therapeutic peptide is an enzyme, a ligand, a receptor, a transcription factor, or a fragment or a modification thereof. In some embodiments, the therapeutic peptide is an antimicrobial peptide or a fragment or a modification thereof.
In some embodiments, the exogenous sequence encodes a targeting moiety. In some embodiments, the targeting moiety is specific to an organ, a tissue, or a cell.
In some embodiments, the second target protein further comprises a targeting moiety. In some embodiments, the targeting moiety is specific to an organ, a tissue, or a cell.
In one aspect, the present invention provides a polypeptide for modifying an exosome, comprising a sequence of (i) (M)(G)(G/A/S)(K/Q)(L/F/S/Q)(S/A)(K)(K) (SEQ ID NO: 118); (ii) (M)(G)(π)(X)(Φ/π)(π)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu); or (iii) (M)(G)(π)(Φ/π)(S/A/G/N)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu).
In some embodiments, the polypeptide comprises a sequence of any of SEQ ID NO: 4-110. In some embodiments, the polypeptide comprises a sequence of SEQ ID NO: 13. In some embodiments, the polypeptide comprises a sequence of SEQ ID NO: 110. In some embodiments, the polypeptide comprises a sequence of MGXKLSKKK, wherein X is any amino acid (SEQ ID NO: 116).
In some embodiments, the polypeptide is fused to a cargo peptide. In some embodiments, the polypeptide is fused to the N-terminus of the cargo peptide.
In one aspect, the present invention provides a polynucleotide construct comprising a coding sequence encoding the polypeptide provided herein. In some embodiments, the coding sequence is codon optimized.
In another aspect, the present invention provides a method of making an engineered exosome, comprising the steps of: a. introducing into a cell a nucleic acid construct encoding a fusion polypeptide comprising (i) a first sequence encoding MARCKS, MARCKSL1, BASP1 or a fragment or a modification thereof, and (ii) a second sequence encoding a cargo peptide; b. maintaining the cell under conditions allowing the cell to express the fusion polypeptide; and c. obtaining the engineered exosome comprising the fusion polypeptide from said cell.
In some embodiments, the first sequence comprises a sequence of (i) (M)(G)(G/A/S)(K/Q)(L/F/S/Q)(S/A)(K)(K) (SEQ ID NO: 118); (ii) M)(G)(π)(X)(Φ/π)(π)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu); or (iii) (M)(G)(π)(Φ/π)(S/A/G/N)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu).
In some embodiments, the polynucleotide comprises a sequence of any of SEQ ID NO: 4-110. In some embodiments, the polynucleotide comprises a sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide comprises a sequence of SEQ ID NO: 110. In some embodiments, the polynucleotide comprises a sequence of MGXKLSKKK, wherein X is any amino acid (SEQ ID NO: 116).
In some embodiments, the fusion polypeptide is present in the lumen of the engineered exosome at a higher density than a different target protein in a different exosome, wherein the different target protein comprises a conventional exosome protein or a variant thereof. In some embodiments, the fusion polypeptide is present at more than 2 fold higher density than the different target protein in the different exosome. In some embodiments, the fusion polypeptide is present at more than 4 fold, 16 fold, 100 fold, or 10,000 fold higher density than the different target protein in the different exosome.
The figures depict various embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.
As used herein, the term “extracellular vesicle” or “EV” refers to a cell-derived vesicle comprising a membrane that encloses an internal space. Extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally extracellular vesicles range in diameter from 20 nm to 1000 nm, and can comprise various macromolecular payload either within the internal space, displayed on the external surface of the extracellular vesicle, and/or spanning the membrane. Said payload can comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and/or combinations thereof. By way of example and without limitation, extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane). Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, and/or cultured cells.
As used herein the term “exosome” refers to a cell-derived small (between 20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from said cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. The exosome is a species of extracellular vesicle. The exosome comprises lipid or fatty acid and polypeptide and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The exosome can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.
As used herein, the term “nanovesicle” refers to a cell-derived small (between 20-250 nm in diameter, more preferably 30-150 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from said cell by direct or indirect manipulation such that said nanovesicle would not be produced by said producer cell without said manipulation. Appropriate manipulations of said producer cell include but are not limited to serial extrusion, treatment with alkaline solutions, sonication, or combinations thereof. The production of nanovesicles may, in some instances, result in the destruction of said producer cell. Preferably, populations of nanovesicles are substantially free of vesicles that are derived from producer cells by way of direct budding from the plasma membrane or fusion of the late endosome with the plasma membrane. The nanovesicle comprises lipid or fatty acid and polypeptide, and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The nanovesicle, once it is derived from a producer cell according to said manipulation, may be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.
As used herein the term “lumen-engineered exosome” refers to an exosome with an internal luminal space modified in its composition. For example, the lumen is modified in its composition of a protein, a lipid, a small molecule, a carbohydrate, etc. The composition can be changed by a chemical, a physical, or a biological method or by being produced from a cell previously modified by a chemical, a physical, or a biological method. Specifically, the composition can be changed by a genetic engineering or by being produced from a cell previously modified by genetic engineering.
As used herein the term “a modification” of a protein refers to a protein having at least 15% identity to the non-mutant amino acid sequence of the protein. A modification of a protein includes a fragment or a variant of the protein. A modification of a protein can further include chemical, or physical modification to a fragment or a variant of the protein.
As used herein the term “a fragment” of a protein refers to a protein that is N- and/or C-terminally deleted in comparison to the naturally occurring protein. Preferably, a fragment of MARCKS, MARCKSL1, or BASP1 retains the ability to be specifically targeted to the lumen of exosomes. Such a fragment is also referred to as a “functional fragment”. Whether a fragment is a functional fragment in that sense can be assessed by any art known methods to determine the protein content of exosomes including Western Blots, FACS analysis and fusions of the fragments with autofluorescent proteins like, e.g., GFP. In a particular embodiment the fragment of MARCKS, MARCKSL1, or BASP1 retains at least 50%, 60%, 70%, 80%, 90% or 100% of the ability of the naturally occurring MARCKS, MARCKSL1, or BASP1 to be specifically targeted to exosomes. In a particular embodiment the ability of the variant of MARCKS, MARCKSL1, BASP1 or of a fragment of MARCKS, MARCKSL1, or BASP1 is at least 70%, 80%, 85%, 90%, 95% or 99% of the ability of MARCKS, MARCKSL1, and BASP1, respectively, to be specifically targeted to the lumen of exosomes. This ability can be assessed, e.g. by fluorescently labeled variants, in the assays described in the experimental section.
As used herein the term “variant” of a protein refers to a protein that shares a certain amino acid sequence identity with another protein upon alignment by a method known in the art. A variant of a protein can include a substitution, insertion, deletion, frameshift or rearrangement in another protein. In a particular embodiment, the variant is a variant having at least 70% identity to MARCKS, MARCKSL1, BASP1 or a fragment of MARCKS, MARCKSL1, or BASP1. In some embodiments variants or variants of fragments of MARCKS share at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with MARCKS according to SEQ ID NO: 1 or with a functional fragment thereof. In some embodiments variants or variants of fragments of MARCKSL1 share at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with MARCKSL1 according to SEQ ID NO: 2 or with a functional fragment thereof. In some embodiments variants or variants of fragments of BASP1 share at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with BASP1 according to SEQ ID NO: 3 or with a functional fragment thereof. In each of above cases, it is preferred that the variant or variant of a fragment retains the ability to be specifically targeted to the lumen of exosomes.
Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 15 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989) Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992); and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) [Altschul 20 et al., J. Mol. Biol. 215: 403-10 (1990) J is available from several sources, including the National Center for Biological Information (NBCl, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blasm, blastx, tblastn and tblastx. BLAST and a description of how to determine sequence identify using the program can be accessed at the official website of NCBI (National Center for Biotechnology Information) under NIH (National Institute of Health).
Recitation of any protein provided herein encompasses a functional variant of the protein. The term “functional variant” of a protein refers to a variant of the protein that retains the ability to be specifically targeted to the lumen of exosomes. In a particular embodiment the ability of the functional variant of MARCKS, MARCKSL1, BASP1 or of a fragment of MARCKS, MARCKSL1, or BASP1 is at least 70%, 80%, 85%, 90%, 95% or 99% of the ability of MARCKS, MARCKSL1, and BASP1, respectively, to be specifically targeted to the lumen of exosomes.
As used herein the term “producer cell” refers to a cell used for generating an exosome. A producer cell can be a cell cultured in vitro, or a cell in vivo. A producer cell includes, but not limited to, a cell known to be effective in generating exosomes, e.g., HEK293 cells, Chinese hamster ovary (CHO) cells, and mesenchymal stem cells (MSCs).
As used herein the term “target protein” or “target peptide” refers to a protein or peptide that can be targeted to the lumen of an exosome. The target protein or peptide can be a non-mutant protein that is naturally targeted to an exosome lumen, or a fragment or a modification of the non-mutant protein. The target protein can be a fusion protein containing a flag tag, a therapeutic peptide, a targeting moiety, or other peptide attached to the non-mutant protein or a modification or a fragment of the non-mutant protein. The target protein can comprise a modification such as myristoylation, prenylation, or palmitoylation, or a soluble protein attached to the internal leaflet of the membrane by a linker.
As used herein the term “cargo protein” or cargo peptide” refers to any protein or peptide, or fragment or modification thereof, which can be loaded into an exosome or engineered exosome. Cargo proteins or peptide may include therapeutic peptides or proteins that act on a target (e.g. a target cell) that is contacted with the exosome. Cargo proteins may be a fusion protein comprising a targeting protein or peptide or fragment or modification thereof, as described above, such that the cargo fusion protein can be targeted to an exosome lumen.
As used herein the term “contaminant protein” refers to a protein that is not associated with an exosome. For example, a contaminant protein includes a protein, not enclosed in the exosome and not attached to or incorporated into the membrane of the exosome.
As used herein, the terms “isolate,” “isolated,” and “isolating” or “purify,” “purified,” and “purifying” as well as “extracted” and “extracting” are used interchangeably and refer to the state of a preparation (e.g., a plurality of known or unknown amount and/or concentration) of desired EVs, that have undergone one or more processes of purification, e.g., a selection or an enrichment of the desired exosome preparation. In some embodiments, isolating or purifying as used herein is the process of removing, partially removing (e.g., a fraction) of the exosomes from a sample containing producer cells. In some embodiments, an isolated exosome composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In other embodiments, an isolated exosome composition has an amount and/or concentration of desired exosomes at or above an acceptable amount and/or concentration. In other embodiments, the isolated exosome composition is enriched as compared to the starting material (e.g., producer cell preparations) from which the composition is obtained. This enrichment can be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to the starting material. In some embodiments, isolated exosome preparations are substantially free of residual biological products. In some embodiments, the isolated exosome preparations are 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free, or 90% free of any contaminating biological matter. Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological products can also mean that the exosome composition contains no detectable producer cells and that only exosomes are detectable.
The term “excipient” or “carrier” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. The term “pharmaceutically-acceptable carrier” or “pharmaceutically-acceptable excipient” encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound. Included are excipients and carriers that are useful in preparing a pharmaceutical composition and are generally safe, non-toxic, and desirable.
As used herein, the term “payload” refers to a therapeutic agent that acts on a target (e.g., a target cell) that is contacted with the EV. Payloads that can be introduced into an exosome and/or a producer cell include therapeutic agents such as, nucleotides (e.g., nucleotides comprising a detectable moiety or a toxin or that disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules that encode a polypeptide such as an enzyme, or RNA molecules that have regulatory function such as miRNA, dsDNA, lncRNA, and siRNA), amino acids (e.g., amino acids comprising a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, viruses and viral particles (e.g., adeno-associated viruses and viral particles, retroviruses, adenoviruses, etc.) and small molecules (e.g., small molecule drugs and toxins, including small molecule STING agonists including cyclic dinucleotides such as ML-RR S2 and 3′-3′ cAIMPdFSH).
As used herein, “a mammalian subject” includes all mammals, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like).
The terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject is a mammal, and in other embodiments the subject is a human.
As used herein, the term “substantially free” means that the sample comprising exosomes comprise less than 10% of macromolecules by mass/volume (m/v) percentage concentration. Some fractions may contain less than 0.001%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% (m/v) of macromolecules.
As used herein, the term “macromolecule” means nucleic acids, exogenous proteins, lipids, carbohydrates, metabolites, or a combination thereof.
As used herein, the term “conventional exosome protein” means a protein previously known to be enriched in exosomes, including but not limited to CD9, CD63, CD81, PDGFR, GPI anchor proteins, lactadherin LAMP2, and LAMP2B, a fragment thereof, or a peptide that binds thereto. For the avoidance of doubt, PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment or a variant thereof are not conventional exosome proteins.
Other Interpretational Conventions
Ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.
Unless otherwise indicated, reference to a compound that has one or more stereocenters intends each stereoisomer, and all combinations of stereoisomers, thereof.
Exosome Proteins
Some embodiments of the present invention relate to identification, use and modification of exosome proteins, which are highly enriched in exosome lumens. Such exosome proteins can be identified by analyzing highly purified exosomes with mass spectrometry or other methods known in the art.
The proteins include various luminal proteins or membrane proteins, such as transmembrane proteins, integral proteins and peripheral proteins, enriched on the exosome membranes. Specifically, the proteins include, but not limited to, (1) myristoylated alanine rich Protein Kinase C substrate (MARCKS); (2) myristoylated alanine rich Protein Kinase C substrate like 1 (MARCKSL1); and (3) brain acid soluble protein 1 (BASP1).
One or more exosome proteins identified herein can be selectively used depending on a producer cell, production condition, purification methods, or intended application of the exosomes. Exosome proteins enriched in the lumen of certain exosomes with a specific size range, a targeting moiety, a charge density, a payload, etc. can be identified and used in some embodiments of the present invention. In some embodiments, more than one exosome proteins can be used concurrently or subsequently for generation and isolation of therapeutic exosomes.
Lumen-Engineered Exosomes
Another aspect of the present invention relates to generation and use of lumen-engineered exosomes. Lumen-engineered exosomes have an internal space modified in its compositions. For example, the composition of the lumen can be modified by changing the protein, lipid or glycan content of the lumen.
In some embodiments, the lumen-engineered exosomes are generated by chemical and/or physical methods, such as PEG-induced fusion and/or ultrasonic fusion.
In other embodiments, the lumen-engineered exosomes are generated by genetic engineering. Exosomes produced from a genetically-modified producer cell or a progeny of the genetically-modified cell can contain modified lumen compositions. In some embodiments, lumen-engineered exosomes have the exosome protein at a higher or lower density or include a modification or a fragment of the exosome protein.
For example, lumen-engineered exosomes can be produced from a cell transformed with an exogenous sequence encoding the exosome protein or a modification or a fragment of the exosome protein. Exosomes including proteins expressed from the exogenous sequence can include modified lumen protein compositions.
Various modifications or fragments of the exosome protein can be used for the embodiments of the present invention. For example, proteins modified to be more effectively targeted to exosome lumens can be used. Proteins modified to comprise a minimal fragment required for specific and effective targeting to exosome lumens can be also used.
Fusion proteins having a therapeutic activity can be also used. For example, the fusion protein can comprise MARCKS, MARCKSL1, BASP1, or a modification thereof, in particular a fragment or variant thereof, and a therapeutic peptide or a cargo protein or peptide. In some embodiments, the fusion protein comprises a fragment of the amino terminus of BASP1.
The therapeutic peptide is selected from a group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, or a linker to a therapeutic compound. The therapeutic compound can be nucleotides, amino acids, lipids, carbohydrates, or small molecules. The therapeutic peptide can be an antibody, an enzyme, a ligand, an antigen (e.g., a tumor antigen or an antigen from an infectious agent such as a bacteria, virus, fungus, or protozoa), a receptor, an antimicrobial peptide, a transcription factor, or a fragment or a modification thereof. The fusion proteins can be presented in the lumen of exosomes and provide a therapeutic activity to the exosome.
In some embodiments, the therapeutic peptide is a component of a genome editing complex. In some embodiments, said genome editing complex is a transcription activator-like effector nuclease (TAL-effector nuclease or TALEN); a zinc finger nuclease (ZFN); a recombinase; a CRISPR/Cas9 complex, a CRISPR/Cpfl complex, a CRISPR/C2c1, C2c2, or C2c3 complex, a CRISPR/CasY or CasX complex, or any other appropriate CRISPR complex known in the art; or any other appropriate genome editing complex known in the art or any combination thereof.
In some embodiments, the therapeutic peptide is a transmembrane peptide. The transmembrane peptides described herein may be expressed as fusion proteins to any of the sequences described herein or any fragments or variants thereof. In some embodiments, the transmembrane protein has a first end fused to the luminal sequence in the lumen of the exosome, and a second terminus expressed on the surface of the exosome. In some embodiments, the transmembrane protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment or a variant thereof.
In some embodiments, the therapeutic peptide is a nucleic acid binding protein. In some embodiments, the nucleic acid binding protein is Dicer, an Argonaute protein, TRBP, MS2 bacteriophage coat protein. In some embodiments, the nucleic acid binding protein additionally comprises one or more RNA or DNA molecules. In some embodiments, the one or more RNA is a miRNA, siRNA, guide RNA, lincRNA, mRNA, antisense RNA, dsRNA, or combinations thereof.
In some embodiments, the therapeutic peptide is a part of a protein-protein interaction system. In some embodiments, the protein-protein interaction system comprises an FRB-FKBP interaction system, e.g., the FRB-FKBP interaction system as described in Banaszynski et al., J Am Chem Soc. 2005 Apr. 6; 127(13):4715-21.
The fusion proteins can be targeted to the lumen of exosomes and provide a therapeutic activity to the exosome.
In some embodiments, fusion proteins having a targeting moiety are used. For example, fusion proteins can comprise MARCKS, MARCKSL1, BASP1, or a fragment or a modification thereof, and a targeting moiety. The targeting moiety can be used for targeting the exosome to a specific organ, tissue, or cell for a treatment using the exosome. In some embodiments, the targeting moiety is an antibody or antigen-binding fragment thereof. Antibodies and antigen-binding fragments thereof include whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments, such as, e.g., scFv, (scFv)2, Fab, Fab′, and F(ab′)2, F(ab1)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides. Antibodies and antigen-binding fragments thereof also includes bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function.
In some embodiments, fusion proteins comprising viral proteins are used. In some embodiments, the fusion protein comprises viral capsid or envelope proteins. In some embodiments, the fusion proteins allow for the assembly of intact viruses that are retained in the lumen of an exosome.
In some embodiments, fusion proteins that comprise MARCKS, MARCKSL1, BASP1, any of SEQ ID NO: 4-109 or a modification thereof, in particular a fragment or variant thereof, resulting in enrichment of the fusion protein in exosomes compared to expression of the fusion protein lacking MARCKS, MARCKSL1, BASP1, any of SEQ ID NO: 4-109 or a modification thereof, in particular a fragment or variant thereof, or compared to fusion proteins that comprise conventional exosome proteins. In some embodiments, the fusion proteins that comprise MARCKS, MARCKSL1, BASP1, any of SEQ ID NO: 4-109 or a fragment or a modification thereof comprise a peptide with the sequence MGXKLSKKK, where X is alanine or any other amino acid (SEQ ID NO: 117); or a peptide with sequence of (M)(G)(π)(Φ/π)(S/A/G/N)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu). In some embodiments, the fusion protein comprises a peptide with sequence of (M)(G)(π)(X)(Φ/π)(π)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, 1 is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu). In some embodiments, the conventional exosome protein is selected from the list consisting of CD9, CD63, CD81, PDGFR, GPI anchor proteins, LAMP2, LAMP2B, and a fragment thereof. In some embodiments, the enrichment of the fusion protein comprising MARCKS, MARCKSL1, BASP1, any of SEQ ID NO: 4-109 or a fragment or a modification thereof in exosomes is >2-fold, >4-fold, >8-fold, >16-fold, >25-fold, >50-fold, >100-fold, >200-fold, >500-fold, >750-fold, >1,000-fold, >2,000-fold, >5,000-fold, >7,500-fold, >10,000-fold higher than the fusion protein lacking MARCKS, MARCKSL1, BASP1, any of SEQ ID NO: 4-109 or a fragment or a modification thereof, or compared to fusion proteins that comprise conventional exosome proteins. In some embodiments, the protein sequence of any of SEQ ID NO: 1-109 is sufficient to load the exosomes with the fusion protein.
In some embodiments, the lumen-engineered exosome comprising a fusion protein containing an exogenous sequence and an exosome lumen protein newly-identified herein has a higher density of the fusion protein than similarly engineered exosomes comprising an exogenous sequence conjugated to a conventional exosome protein known in the art (e.g., CD9, CD63, CD81, PDGFR, GPI anchor proteins, lactadherin, LAMP2, and LAMP2B, a fragment thereof, or a peptide that binds thereto). In some embodiments, said fusion protein containing an exosome protein newly-identified herein is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density in the exosome lumen than fusion proteins in other exosome lumens similarly modified using a conventional exosome protein. In some embodiments, said fusion protein containing an exosome protein newly-identified herein is present at 2 to 4-fold, 4 to 8-fold, 8 to 16-fold, 16 to 32-fold, 32 to 64-fold, 64 to 100-fold, 100 to 200-fold, 200 to 400-fold, 400 to 800-fold, 800 to 1,000-fold or to a higher density in the exosome lumen than fusion proteins in other exosome lumens similarly modified using a conventional exosome protein.
In some embodiments, a fusion protein comprising MARCKS, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD9. In some embodiments, a fusion protein comprising MARCKS, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD63. In some embodiments, a fusion protein comprising MARCKS, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD81. In some embodiments, a fusion protein comprising MARCKS, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using PDGFR. In some embodiments, a fusion protein comprising MARCKS, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using GPI anchor proteins. In some embodiments, a fusion protein comprising MARCKS, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using lactadherin. In some embodiments, a fusion protein comprising MARCKS, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using LAMP2. In some embodiments, a fusion protein comprising MARCKS, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using LAMP2B. In some embodiments, a fusion protein comprising MARCKS, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using an conventional protein. In some embodiments, a fusion protein comprising MARCKS, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using a variant of a conventional exosome protein.
In some embodiments, a fusion protein comprising MARCKSL1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD9. In some embodiments, a fusion protein comprising MARCKSL1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD63. In some embodiments, a fusion protein comprising MARCKSL1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD81. In some embodiments, a fusion protein comprising MARCKSL1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using PDGFR. In some embodiments, a fusion protein comprising MARCKSL1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using GPI anchor proteins. In some embodiments, a fusion protein comprising MARCKSL1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using lactadherin. In some embodiments, a fusion protein comprising MARCKSL1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using LAMP2. In some embodiments, a fusion protein comprising MARCKSL1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using LAMP2B. In some embodiments, a fusion protein comprising MARCKSL1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using an conventional protein. In some embodiments, a fusion protein comprising MARCKSL1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using a variant of a conventional exosome protein.
In some embodiments, a fusion protein comprising BASP1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD9. In some embodiments, a fusion protein comprising BASP1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD63. In some embodiments, a fusion protein comprising BASP1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD81. In some embodiments, a fusion protein comprising BASP1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using PDGFR. In some embodiments, a fusion protein comprising BASP1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using GPI anchor proteins. In some embodiments, a fusion protein comprising BASP1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using lactadherin. In some embodiments, a fusion protein comprising BASP1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using LAMP2. In some embodiments, a fusion protein comprising BASP1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using LAMP2B. In some embodiments, a fusion protein comprising BASP1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using an conventional protein. In some embodiments, a fusion protein comprising BASP1, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using a variant of a conventional exosome protein.
In some embodiments, a fusion protein comprising any of SEQ ID NO: 1-109, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD9. In some embodiments, a fusion protein comprising any of SEQ ID NO: 1-109, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD63. In some embodiments, a fusion protein comprising any of SEQ ID NO: 1-109, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using CD81. In some embodiments, a fusion protein comprising any of SEQ ID NO: 1-109, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using PDGFR. In some embodiments, a fusion protein comprising any of SEQ ID NO: 1-109, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using GPI anchor proteins. In some embodiments, a fusion protein comprising any of SEQ ID NO: 1-109, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using lactadherin. In some embodiments, a fusion protein comprising any of SEQ ID NO: 1-109, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using LAMP2. In some embodiments, a fusion protein comprising any of SEQ ID NO: 1-109, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using LAMP2B. In some embodiments, a fusion protein comprising any of SEQ ID NO: 1-109, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using an conventional protein. In some embodiments, a fusion protein comprising any of SEQ ID NO: 1-109, a variant, a fragment, a variant of a fragment, or a modification thereof is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or a higher density than the exosomes similarly modified using a variant of a conventional exosome protein.
In some embodiments, the lumen-engineered exosomes described herein demonstrate superior characteristics compared to lumen-engineered exosomes known in the art. For example, lumen-engineered exosomes produced by using the newly-identified exosome proteins provided herein contain modified proteins more highly enriched in their lumen than exosomes in prior art, e.g., those produced using conventional exosome proteins. Moreover, the lumen-engineered exosomes of the present invention can have greater, more specific, or more controlled biological activity compared to lumen-engineered exosomes known in the art. For example, a lumen-engineered exosome comprising a therapeutic or biologically relevant exogenous sequence fused to an exosome protein or a fragment thereof described herein (e.g., BASP1 or a fragment thereof) can have more of the desired engineered characteristics than fusion to scaffolds known in the art. Scaffold proteins known in the art include tetraspanin molecules (e.g., CD63, CD81, CD9 and others), lysosome-associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI anchor proteins, lactadherin and fragments thereof, and peptides that have affinity to any of these proteins or fragments thereof. For the avoidance of doubt, PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment or a variant thereof are not conventional exosome proteins. Previously, overexpression of exogenous proteins relied on stochastic or random disposition of the exogenous proteins into the exosome for producing lumen-engineered exosomes. This resulted in low-level, unpredictable density of the exogenous proteins in exosomes. Thus, the exosome proteins and fragments thereof described herein provide important advancements in novel exosome compositions and methods of making the same.
Fusion proteins provided herein can comprise MARCKS, MARCKSL1, BASP1, or a fragment or a variant thereof, and an additional peptide. The additional peptide can be attached to either the N terminus or the C terminus of the exosome protein or a fragment or a variant thereof.
In some embodiments, fusion proteins provided herein comprise MARCKS, MARCKSL1, BASP1, or a fragment or a variant thereof, and two additional peptides. Both of the two additional peptides can be attached to either the N terminus or the C terminus of the exosome protein or a fragment or a variant thereof. In some embodiments, one of the two additional peptides is attached to the N terminus and the other of the two additional peptides is attached to the C terminus of the exosome protein or a fragment or a variant thereof.
In some embodiments, the compositions and methods of generating lumen-engineered extracellular vesicles described herein comprise nanovesicles.
Producer Cell for Production of Lumen-Engineered Exosomes
Exosomes of the present invention can be produced from a cell grown in vitro or a body fluid of a subject. When exosomes are produced from in vitro cell culture, various producer cells, e.g., HEK293 cells, can be used for the present invention. Additional cell types that can be used for the production of the lumen-engineered exosomes described herein include, without limitation, mesenchymal stem cells, T-cells, B-cells, dendritic cells, macrophages, and cancer cell lines.
The producer cell can be genetically modified to comprise one or more exogenous sequences to produce lumen-engineered exosomes. The genetically-modified producer cell can contain the exogenous sequence by transient or stable transformation. The exogenous sequence can be transformed as a plasmid. The exogenous sequences can be stably integrated into a genomic sequence of the producer cell, at a targeted site or in a random site. In some embodiments, a stable cell line is generated for production of lumen-engineered exosomes.
The exogenous sequences can be inserted into a genomic sequence of the producer cell, located within, upstream (5′-end) or downstream (3′-end) of an endogenous sequence encoding the exosome protein. Various methods known in the art can be used for the introduction of the exogenous sequences into the producer cell. For example, cells modified using various gene editing methods (e.g., methods using a homologous recombination, transposon-mediated system, loxP-Cre system, CRISPR/Cas9 or TALEN) are within the scope of the present invention.
The exogenous sequences can comprise a sequence encoding the exosome protein or a modification or a fragment of the exosome protein. An extra copy of the sequence encoding the exosome protein can be introduced to produce a lumen-engineered exosome having a higher density of the exosome protein. An exogenous sequence encoding a modification or a fragment of the exosome protein can be introduced to produce a lumen-engineered exosome containing the modification or the fragment of the exosome protein. An exogenous sequence encoding an affinity tag can be introduced to produce a lumen-engineered exosome containing a fusion protein comprising the affinity tag attached to the exosome protein.
In some embodiments, a lumen-engineered exosome has a higher density of the exosome protein than native exosomes isolated from the same or similar producer cell types. In some embodiments, said exosome protein is present at 2-, 4-, 8-, 16-, 32-, 64-, 100-, 200-, 400-, 800-, 1,000-fold or to a higher density on said lumen-engineered exosome than said native exosome. In some embodiments, said exosome protein is present at 2 to 4-fold, 4 to 8-fold, 8 to 16-fold, 16 to 32-fold, 32 to 64-fold, 64 to 100-fold, 100 to 200-fold, 200 to 400-fold, 400 to 800-fold, 800 to 1,000-fold or to a higher density on said lumen-engineered exosome than said native exosome. In some embodiments, a fusion protein comprising the exosome protein is present at 2 to 4-fold, 4 to 8-fold, 8 to 16-fold, 16 to 32-fold, 32 to 64-fold, 64 to 100-fold, 100 to 200-fold, 200 to 400-fold, 400 to 800-fold, 800 to 1,000-fold or to a higher density on said lumen-engineered exosome than the unmodified exosome protein on said native exosome. In some embodiments, a fragment or a variant of the exosome protein is present at 2 to 4-fold, 4 to 8-fold, 8 to 16-fold, 16 to 32-fold, 32 to 64-fold, 64 to 100-fold, 100 to 200-fold, 200 to 400-fold, 400 to 800-fold, 800 to 1,000-fold or to a higher density on said lumen-engineered exosome than the unmodified exosome protein on said native exosome.
In particular embodiments, MARCKS, a fragment or a variant of MARCKS, or a modification thereof is present at 2 to 4-fold, 4 to 8-fold, 8 to 16-fold, 16 to 32-fold, 32 to 64-fold, 64 to 100-fold, 100 to 200-fold, 200 to 400-fold, 400 to 800-fold, 800 to 1,000-fold or to a higher density on said lumen-engineered exosome than the unmodified MARCKS on said native exosome. In some embodiments, MARCKSL1, a fragment or a variant of MARCKSL1, or a modification thereof is present at 2 to 4-fold, 4 to 8-fold, 8 to 16-fold, 16 to 32-fold, 32 to 64-fold, 64 to 100-fold, 100 to 200-fold, 200 to 400-fold, 400 to 800-fold, 800 to 1,000-fold or to a higher density on said lumen-engineered exosome than the unmodified MARCKSL1 on said native exosome. In some embodiments, BASP1, a fragment or a variant of BASP1, or a modification thereof is present at 2 to 4-fold, 4 to 8-fold, 8 to 16-fold, 16 to 32-fold, 32 to 64-fold, 64 to 100-fold, 100 to 200-fold, 200 to 400-fold, 400 to 800-fold, 800 to 1,000-fold or to a higher density on said lumen-engineered exosome than the unmodified BASP1 on said native exosome.
In some embodiments, the producer cell is further modified to comprise an additional exogenous sequence. For example, an additional exogenous sequence can be included to modulate endogenous gene expression, or produce an exosome including a certain polypeptide as a payload. In some embodiments, the producer cell is modified to comprise two exogenous sequences, one encoding the exosome protein or a modification or a fragment of the exosome protein, and the other encoding a payload.
More specifically, lumen-engineered exosomes can be produced from a cell transformed with a sequence encoding one or more exosome lumen proteins including, but not limited to, (1) myristoylated alanine rich Protein Kinase C substrate (MARCKS); (2) myristoylated alanine rich Protein Kinase C substrate like 1 (MARCKSL1); and (3) brain acid soluble protein 1 (BASP1). Any of the one or more exosome lumen proteins described herein can be expressed from a plasmid, an exogenous sequence inserted into the genome or other exogenous nucleic acid such as a synthetic messenger RNA (mRNA).
In some embodiments, the one or more exosome lumen protein is expressed in a cell transformed with an exogenous sequence encoding its full length, endogenous form. In some embodiments, such an exogenous sequence encodes MARCKS protein of SEQ ID NO: 1. In some embodiments, such an exogenous sequence encodes MARCKSL1 protein of SEQ ID NO: 2. In some embodiments, such an exogenous sequence encodes BASP1 protein of SEQ ID NO:
3.
Lumen-engineered exosomes can be produced from a cell transformed with a sequence encoding a fragment of one or more exosome lumen proteins including, but not limited to, (1) myristoylated alanine rich Protein Kinase C substrate (MARCKS); (2) myristoylated alanine rich Protein Kinase C substrate like 1 (MARCKSL1); and (3) brain acid soluble protein 1 (BASP1). In some embodiments, the sequence encodes a fragment of the exosome lumen protein lacking at least 5, 10, 50, 100, 200, or 300 amino acids from the N-terminus of the native protein. In some embodiments, the sequence encodes a fragment of the exosome lumen protein lacking at least 5, 10, 50, 100, 200, or 300 amino acids from the C-terminus of the native protein. In some embodiments, the sequence encodes a fragment of the exosome lumen protein lacking at least 5, 10, 50, 100, 200, or 300 amino acids from both the N-terminus and C-terminus of the native protein. In some embodiments, the sequence encodes a fragment of the exosome lumen protein lacking one or more functional or structural domains of the native protein. In some embodiments, the fusion protein comprises a peptide of SEQ ID NO: 4-109. In some embodiments, the fusion protein comprises the peptide of SEQ ID NO: 13. In some embodiments, the fusion protein comprises a peptide with the sequence MGXKLSKKK, where X is alanine or any other amino acid (SEQ ID NO: 117). In some embodiments, the fusion protein comprises a peptide with sequence of (M)(G)(π)(ξ)(Φ/π)(S/A/G/N)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu). In some embodiments, the fusion protein comprises a peptide with sequence of (M)(G)(π)(X)(Φ/π)(π)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu).
In some embodiments, lumen-engineered exosomes can be produced from a cell transformed with a sequence encoding an exosome protein or a fragment or a modification thereof fused to one or more heterologous proteins. In some embodiments, the one or more heterologous proteins are fused to the N-terminus of the exosome protein or a modification thereof, in particular a fragment or variant thereof. In some embodiments, the one or more heterologous proteins are fused to the C-terminus of the exosome protein or a modification thereof, in particular a fragment or variant thereof. In some embodiments, the one or more heterologous proteins are fused to the N-terminus and the C-terminus of the exosome protein or a modification thereof, in particular a fragment or variant thereof. In some embodiments, the one or more heterologous proteins are mammalian proteins. In some embodiments, the one or more heterologous proteins are human proteins.
In some embodiments lumen-engineered exosomes are produced from a cell transformed with a sequence encoding a polypeptide of a sequence identical or similar to a full-length or a fragment of a native exosome lumen protein including, but not limited to, (1) myristoylated alanine rich Protein Kinase C substrate (MARCKS); (2) myristoylated alanine rich Protein Kinase C substrate like 1 (MARCKSL1); and (3) brain acid soluble protein 1 (BASP1). In some embodiments, said polypeptide is 50% identical to a full-length or a fragment of a native exosome lumen protein, e.g., 50% identical to SEQ ID NO: 1-3. In some embodiments, said polypeptide is 60% identical to a full-length or a fragment of a native exosome lumen protein, e.g., 60% identical to SEQ ID NO: 1-3. In some embodiments, said polypeptide is 70% identical to a full-length or a fragment of a native exosome lumen protein, e.g., 70% identical to SEQ ID NO: 1-3. In some embodiments, said polypeptide is 80% identical to a full-length or a fragment of a native exosome lumen protein, e.g., 80% identical to SEQ ID NO: 1-3. In some embodiments, said polypeptide is 90% identical to a full-length or a fragment of a native exosome lumen protein, e.g., 90% identical to SEQ ID NO: 1-3. In some embodiments, said polypeptide is 95% identical to a full-length or a fragment of a native exosome lumen protein, e.g., 95% identical to SEQ ID NO: 1-3. In some embodiments, said polypeptide is 99% identical to a full-length or a fragment of a native exosome lumen protein, e.g., 99% identical to SEQ ID NO: 1-3. In some embodiments, said polypeptide is 99.9% identical to a full-length or a fragment of a native exosome lumen protein, e.g., 99.9% identical to SEQ ID NO: 1-3.
In some embodiments, lumen-engineered exosomes produced from the cell comprise a polypeptide of a sequence identical or similar to a fragment of brain acid soluble protein 1 (BASP1). In some embodiments, said polypeptide is 50% identical to a full-length or a fragment of BASP1, e.g., 50% identical to SEQ ID NO: 4-109. In some embodiments, said polypeptide is 60% identical to a full-length or a fragment of BASP1, e.g., 60% identical to SEQ ID NO: 4-109. In some embodiments, said polypeptide is 70% identical to a full-length or a fragment of BASP1, e.g., 70% identical to SEQ ID NO: 4-109. In some embodiments, said polypeptide is 80% identical to a full-length or a fragment of BASP1, e.g., 80% identical to SEQ ID NO: 4-109. In some embodiments, said polypeptide is 90% identical to a full-length or a fragment of BASP1, e.g., 90% identical to SEQ ID NO: 4-109. In some embodiments, said polypeptide is 95% identical to a full-length or a fragment of BASP1, e.g., 95% identical to SEQ ID NO: 4-109. In some embodiments, said polypeptide is 99% identical to a full-length or a fragment of BASP1, e.g., 99% identical to SEQ ID NO: 4-109. In some embodiments, said polypeptide is 99.9% identical to a full-length or a fragment of BASP1, e.g., 99.9% identical to SEQ ID NO: 4-109. In some embodiments, said polypeptide is 100% identical to a fragment of BASP1, e.g., 100% identical to SEQ ID NO: 4-109.
Characterization of Exosomes
In some embodiments, the methods described herein further comprise the step of characterizing exosomes contained in each collected fraction. In some embodiments, contents of the exosomes can be extracted for study and characterization. In some embodiments, exosomes are isolated and characterized by metrics including, but not limited to, size, shape, morphology, or molecular compositions such as nucleic acids, proteins, metabolites, and lipids
Exosomes can include proteins, peptides, RNA, DNA, and lipids. Total RNA can be extracted using acid-phenol:chloroform extraction. RNA can then be purified using a glass-fiber filter under conditions that recover small-RNA containing total RNA, or that separate small RNA species less than 200 nucleotides in length from longer RNA species such as mRNA. Because the RNA is eluted in a small volume, no alcohol precipitation step may be required for isolation of the RNA.
Exome compositions may be assessed by methods known in the art including, but not limited to, transcriptomics, sequencing, proteomics, mass spectrometry, or HP-LC.
The composition of nucleotides associated with isolated exosomes (including RNAs and DNAs) can be measured using a variety of techniques that are well known to those of skill in the art (e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis, solution hybridization detection). In a particular embodiment, the level of at least one RNA is measured by reverse transcribing RNA from the exosome composition to provide a set of target oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to one or more RNA-specific probe oligonucleotides (e.g., a microarray that comprises RNA-specific probe oligonucleotides) to provide a hybridization profile for the exosome composition, and comparing the exosome composition hybridization profile to a hybridization profile generated from a control sample. An alteration in the signal of at least one RNA in the test sample relative to the control sample is indicative of the RNA composition.
Also, a microarray can be prepared from gene-specific oligonucleotide probes generated from known RNA sequences. The array can contain two different oligonucleotide probes for each RNA, one containing the active, mature sequence and the other being specific for the precursor of the RNA (for example miRNA and pre-miRNAs). The array can also contain controls, such as one or more mouse sequences differing from human orthologs by only a few bases, which can serve as controls for hybridization stringency conditions. tRNAs and other RNAs (e.g., rRNAs, mRNAs) from both species can also be printed on the microchip, providing an internal, relatively stable, positive control for specific hybridization. One or more appropriate controls for non-specific hybridization can also be included on the microchip. For this purpose, sequences are selected based upon the absence of any homology with any known RNAs.
The microarray can be fabricated using techniques known in the art. For example, probe oligonucleotides of an appropriate length, e.g., 40 nucleotides, are 5′-amine modified at position C6 and printed on activated slides using commercially available microarray systems, e.g., the GeneMachine OmniGrid.™ 100 Microarrayer and Amersham CodeLink.™ Labeled cDNA oligomer corresponding to the target RNAs is prepared by reverse transcribing the target RNA with labeled primer. Following first strand synthesis, the RNA/DNA hybrids are denatured to degrade the RNA templates. The labeled target cDNAs thus prepared are then hybridized to the microarray chip under hybridizing conditions, e.g., 6.times. SSPE/30% formamide at 25° C. for 18 hours, followed by washing in 0.75.times. TNT at 37° C. for 40 minutes. At positions on the array where the immobilized probe DNA recognizes a complementary target cDNA in the sample, hybridization occurs. The labeled target cDNA marks the exact position on the array where binding occurs, allowing automatic detection and quantification. The output consists of a list of hybridization events, indicating the relative abundance of specific cDNA sequences, and therefore the relative abundance of the corresponding complementary RNAs, in the exosome preparation. According to one embodiment, the labeled cDNA oligomer is a biotin-labeled cDNA, prepared from a biotin-labeled primer. The microarray is then processed by direct detection of the biotin containing transcripts using, e.g., Streptavidin-Alexa647 conjugate, and scanned utilizing conventional scanning methods. Image intensities of each spot on the array are proportional to the abundance of the corresponding RNA in the exosome.
Data mining work is completed by bioinformatics, including scanning chips, signal acquisition, image processing, normalization, statistic treatment and data comparison as well as pathway analysis. As such, microarray can profile hundreds and thousands of polynucleotides simultaneously with high throughput performance. Microarray profiling analysis of mRNA expression has successfully provided valuable data for gene expression studies in basic research. And the technique has been further put into practice in the pharmaceutical industry and in clinical diagnosis. With increasing amounts of miRNA data becoming available, and with accumulating evidence of the importance of miRNA in gene regulation, microarray becomes a useful technique for high through-put miRNA studies. The analysis of miRNA levels utilizing polynucleotide probes can be carried out in a variety of physical formats as well. For example, the use of microtiter plates or automation can be used to facilitate the processing of large numbers of test samples.
In some embodiments, the methods described herein comprise measuring the size of exosomes and/or populations of exosomes included in the purified fractions. In some embodiments, exosome size is measured as the longest measurable dimension. Generally, the longest general dimension of an exosome is also referred to as its diameter.
Exosome size can be measured using various methods known in the art, for example, nanoparticle tracking analysis, multi-angle light scattering, single angle light scattering, size exclusion chromatography, analytical ultracentrifugation, field flow fractionation, laser diffraction, tunable resistive pulse sensing, or dynamic light scattering.
Exosome size can be measured using dynamic light scattering (DLS) and/or multiangle light scattering (MALS). Methods of using DLS and/or MALS to measure the size of exosomes are known to those of skill in the art, and include the nanoparticle tracking assay (NTA, e.g., using a Malvern Nanosight NS300 nanoparticle tracking device). In a specific embodiment, the exosome size is determined using a Malvern NanoSight NS300. In some embodiments, the exosomes described herein have a longest dimension of about 20-1000 nm as measured by NTA (e.g., using a Malvern NanosightNS300). In other embodiments, the exosomes described herein have a longest dimension of about 40-1000 nm as measured by NTA (e.g., using a Malvern NanosightNS300). In other embodiments, the exosome populations described herein comprise a population, wherein 90% of said exosomes have a longest dimension of about 20-1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome populations described herein comprise a population, wherein 95% of said exosomes have a longest dimension of about 20-1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome populations described herein comprise a population, wherein 99% of said exosomes have a longest dimension of about 20-1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome populations described herein comprise a population, wherein 90% of said exosomes have a longest dimension of about 40-1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome populations described herein comprise a population, wherein 95% of said exosomes have a longest dimension of about 40-1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome populations described herein comprise a population, wherein 99% of said exosomes have a longest dimension of about 40-1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300).
Exosome size can be measured using tunable resistive pulse sensing (TRPS). In a specific embodiment, exosome size as measured by TRPS is determined using an iZON qNANO Gold. In some embodiments, the exosomes described herein have a longest dimension of about 20-1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosomes described herein have a longest dimension of about 40-1000 nm as measured by TRPS (e.g., an iZON qNano Gold). In other embodiments, the exosome populations described herein comprise a population, wherein 90% of said exosomes have a longest dimension of about 20-1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosome populations described herein comprise a population, wherein 95% of said exosomes have a longest dimension of about 20-1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosome populations described herein comprise a population, wherein 99% of said exosomes have a longest dimension of about 20-1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosome populations described herein comprise a population, wherein 90% of said exosomes have a longest dimension of about 40-1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosome populations described herein comprise a population, wherein 95% of said exosomes have a longest dimension of about 40-1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosome populations described herein comprise a population, wherein 99% of said exosomes have a longest dimension of about 40-1000 nm as measured by TRPS (e.g., using an iZON qNano Gold).
Exosome size can be measured using electron microscopy. In some embodiments, the method of electron microscopy used to measure exosome size is transmission electron microscopy. In a specific embodiment, the transmission electron microscope used to measure exosome size is a Tecnai™ G2 Spirit BioTWIN. Methods of measuring exosome size using an electron microscope are well-known to those of skill in the art, and any such method can be appropriate for measuring exosome size. In some embodiments, the exosomes described herein have a longest dimension of about 20-1000 nm as measured by a scanning electron microscope (e.g., a Tecnai™ G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosomes described herein have a longest dimension of about 40-1000 nm as measured by a scanning electron microscope (e.g., a Tecnai™ G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome populations described herein comprise a population, wherein 90% of said exosomes have a longest dimension of about 20-1000 nm as measured by a scanning electron microscope (e.g., a Tecnai™ G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome populations described herein comprise a population, wherein 95% of said exosomes have a longest dimension of about 20-1000 nm as measured by a scanning electron microscope (e.g., a Tecnai™ G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome populations described herein comprise a population, wherein 99% of said exosomes have a longest dimension of about 20-1000 nm as measured by a scanning electron microscope (e.g., a Tecnai™ G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome populations described herein comprise a population wherein 90% of said exosomes have a longest dimension of about 40-1000 nm as measured by a scanning electron microscope (e.g., a Tecnai™ G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome populations described herein comprise a population wherein 95% of said exosomes have a longest dimension of about 40-1000 nm as measured by a scanning electron microscope (e.g., a Tecnai™ G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome populations described herein comprise a population wherein 99% of said exosomes have a longest dimension of about 40-1000 nm as measured by a scanning electron microscope (e.g., a Tecnai™ G2 Spirit BioTWIN scanning electron microscope).
Individual exosome size can be determined on a particle-by-particle basis by nano-flow cytometry. In some embodiments, the nano-flow cytometer is the Flow NanoAnalyzer (NanoFCM, Inc.; Xiamen, China). In some embodiments, the exosomes described herein have a longest dimension of about 20-1000 nm as measured by nano-flow cytometry (e.g., using a Flow NanoAnalyzer). In some embodiments, the exosomes described herein have a longest dimension of about 40-1000 nm as measured by nano-flow cytometry (e.g., using a Flow NanoAnalyzer). In some embodiments, the exosome populations described herein comprise a population, wherein 90% of said exosomes have a longest dimension of about 20-1000 nm as measured by nano-flow cytometry (e.g., using a Flow NanoAnalyzer). In some embodiments, the exosome populations described herein comprise a population, wherein 95% of said exosomes have a longest dimension of about 20-1000 nm as measured by nano-flow cytometry (e.g., using a Flow NanoAnalyzer). In some embodiments, the exosome populations described herein comprise a population, wherein 99% of said exosomes have a longest dimension of about 20-1000 nm as measured by nano-flow cytometry (e.g., using a Flow NanoAnalyzer). In some embodiments, the exosome populations described herein comprise a population, wherein 90% of said exosomes have a longest dimension of about 40-1000 nm as measured by nano-flow cytometry (e.g., using a Flow NanoAnalyzer). In some embodiments, the exosome populations described herein comprise a population, wherein 95% of said exosomes have a longest dimension of about 40-1000 nm as measured by nano-flow cytometry (e.g., using a Flow NanoAnalyzer). In some embodiments, the exosome populations described herein comprise a population, wherein 99% of said exosomes have a longest dimension of about 40-1000 nm as measured by nano-flow cytometry (e.g., using a Flow NanoAnalyzer).
In some embodiments, the methods described herein comprise measuring the charge density of exosomes and/or populations of exosomes included in the purified fractions. In some embodiments, the charge density is measured by potentiometric titration, anion exchange, cation exchange, isoelectric focusing, zeta potential, capillary electrophoresis, capillary zone electrophoresis, or gel electrophoresis.
In some embodiments, the methods described herein comprise measuring the density of exosome proteins on the exosome surface. The surface density can be calculated or presented as the mass per unit area, the number of proteins per area, number of molecules or intensity of molecule signal per exosome, molar amount of the protein, etc. The surface density can be experimentally measured by methods known in the art, for example, by using bio-layer interferometry (BLI), FACS, Western blotting, fluorescence (e.g., GFP-fusion protein) detection, nano-flow cytometry, ELISA, alphaLISA, and/or densitometry by measuring bands on a protein gel.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations can be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt, nucleotide(s); and the like.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 21th Edition (Easton, Pa.: Mack Publishing Company, 2005); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B(1992).
Exosomes were collected from the supernatant of high density suspension cultures of HEK293 SF cells after 9 days. The supernatant was filtered and fractionated by anion exchange chromatography and eluted in a step gradient of sodium chloride. The peak fraction with the highest protein concentration contained exosomes and contaminating cellular components. The peak fraction was isolated and further fractionated on an Optiprep™ density gradient by ultracentrifugation.
For the Optiprep™ gradient, a 4-tier sterile gradient was prepared with 4 mL 45% Optiprep™, 3 mL 30% Optiprep™, 2 mL 22.5% Optiprep™, 2 mL 17.5% Optiprep™, and 1 mL PBS in a 12 mL Ultra-Clear (344059) tube for a SW 41 Ti rotor. The exosome fraction was added to the Optiprep™ gradient and ultracentrifuged at 200,000×g for 16 hours at 4° C. to separate the exosome fraction. Ultracentrifugation resulted in a Top Fraction known to contain exosomes, a Middle Fraction containing cell debris of moderate density, and a Bottom Fraction containing high density aggregates and cellular debris (
The exosome fraction was diluted in ˜32 mL PBS in a 38.5 mL Ultra-Clear (344058) tube and ultracentrifuged at 133,900×g for 3 hours at 4° C. to pellet the purified exosomes. The pelleted exosomes were then resuspended in a minimal volume of PBS (˜200 μL) and stored at 4° C.
To determine proteins specific to exosomes, the Top Fraction and Bottom Fraction of the Optiprep™ gradient were analyzed by liquid chromatography-tandem mass spectrometry. Prior to analysis, the total protein concentration of the two samples was determined by bicinchoninic acid (BCA) assay, after which each sample was appropriately diluted to 125 μg/mL in PBS buffer. Next, 50.0 μL of each sample was added to a separate 1.5 mL microcentrifuge tube containing an equal volume of exosome lysis buffer (60 mM Tris, 400 mM GdmCl, 100 mM EDTA, 20 mM TCEP, 1.0% Triton X-100) followed by the transfer of 2.0 μL 1.0% Triton X-100 solution. All samples were then incubated at 55° C. for 60 minutes.
Protein precipitation was performed by adding 1250 μL of ethanol at −20° C. To improve efficiency, samples were vigorously vortexed and then sonicated in a water bath for 5 minutes. Precipitated material was pelleted by centrifuging for 5 minutes at 15,000 g at room temperature. The supernatant was decanted, and the pelleted material was thoroughly dried using nitrogen gas. Pellets were resuspended in 30.0 μL digestion buffer (30 mM Tris, 1.0 M GdmCl, 100 mM EDTA, 50 mM TCEP, pH 8.5) which also reduced disulfide bonds. Free cysteine residues were alkylated by adding 5.0 μL alkylation solution (375 mM iodoacetamide, 50 mM Tris, pH 8.5) and incubating the resulting solution at room temperature in the dark for at least 30 minutes.
After incubation, each sample was diluted using 30.0 μL 50 mM Tris pH 8.5, and proteolytic digestion was initiated by adding 2.0 μg trypsin. All samples were mixed and then incubated overnight at 37° C. After the incubation, trypsin activity was ceased by adding 5.0 μL 10% formic acid. Prior to analysis by LC-MS/MS, each sample was desalted using Pierce C18 spin columns. At the end of this process, each sample was dried down and reconstituted in 75.0 μL of 95:5 water:acetonitrile with 0.1% formic acid and transferred to an HPLC vial for analysis.
LC-MS/MS Analysis
Samples were injected into an UltiMate 3000 RSCLnano (Thermo Fisher Scientific) low flow chromatography system, and tryptic peptides were loaded onto an Acclaim PepMap 100 C18 trapping column (75 μm×2 cm, 3 μm particle size, 100 Å pore size, Thermo Fisher Scientific) using loading mobile phase (MPL: 95% water, 5% acetonitrile, 0.1% formic acid) at a flowrate of 2.500 μL/min. Peptides were eluted and separated with a gradient of mobile phase A (MPA: water, 0.1% formic acid) and mobile phase B (MPB: acetonitrile, 0.1% formic acid) at a flowrate of 300 nL/min across an EASY-Spray LC C18 analytical column (75 μm×25 cm, 2 μm particle size, 100 Å pore size, Thermo Fisher Scientific). The stepwise gradient used for elution began at 5% MPB, where it was held for 15 minutes during loading. The percentage MPB then increased from 5-17% over 30 minutes, again from 17-25% over 45 minutes, and finally from 25-40% over 5 minutes. The most hydrophobic species were removed by increasing to 90% MPB over 5 minutes, then holding there for 9 minutes. The total runtime for the method was 130 minutes and allowed a sufficient amount of time for column re-equilibration. Wash cycles were performed in between analytical injections to minimize carry-over.
Mass analyses were performed with a Q Exactive Basic (Thermo Fisher Scientific) mass spectrometer. Precursor ion mass spectra were measured across an m/z range of 400-1600 Da at a resolution of 70,000. The 10 most intense precursor ions were selected and fragmented in the HCD cell using a collision energy of 27, and MS/MS spectra were measured across an m/z range of 200-2000 Da at a resolution of 35,000. Ions with charge states from 2-4 were selected for fragmentation and the dynamic exclusion time was set to 30 seconds. An exclusion list containing 14 common polysiloxanes was utilized to minimize misidentification of known contaminants.
Data Processing
Proteins were first identified and quantified (label-free) using Proteome Discoverer software (version 2.1.1.21, Thermo Fisher Scientific) and the Sequest HT algorithm combined with the Target Decoy PSM Validator. Searches were performed against either the full Uniprot Homo sapiens (taxonomy 9606: 127,783 entries) or Swiss-Prot Homo sapiens (taxonomy 9606 version 2017-05-10: 42,153 entries) reference database, as well as a custom Uniprot database containing E1a proteins (7 entries). The following search parameters were used: enzyme, trypsin; maximum of 2 missed cleavages; minimum peptide length of 6 residues; 10 ppm precursor mass tolerance; and 0.02 Da fragment mass tolerance. The search also included specific dynamic modifications (oxidation of M; deamidation of N or Q; phosphorylation of S, T, or Y; pyro-glutamation of peptide-terminal E; and acetylation of protein N terminus) and static modifications (carbamidomethylation of C).
In the Target Decoy PSM Validator, the maximum delta Cn and both strict and relaxed target false discovery rates (FDRs) were set to 1 because the data were searched again using Scaffold software (version 4.8.2, Proteome Software Inc.). In Scaffold, the data were also searched using the X! Tandem open source algorithm to identify proteins using a protein threshold of 99.0%, a minimum of 2 peptides, and a peptide threshold of 95%.
To determine the identity of novel exosome-specific proteins, total peptide spectral matches (PSMs) were compared for proteins found in the top exosome fraction of the Optiprep™ gradient versus those in the lower fraction. As shown in
To confirm that the exosome-specific proteins identified in the mass spectrometry studies were highly expressed in the lumen of exosomes, Western blotting was carried out on total cell lysate and purified exosome populations from HEK293 cells. As shown in
Scaffolds
To confirm the utility of MARCKS, MARCKSL1, and/or BASP1 as luminal loading scaffolds, each of the proteins was fused to the N-terminus of GFP. Additionally, the first 30 amino acids of each of these proteins were also fused to GFP to determine whether a shorter protein fragment could drive loading of engineered exosomes. Exosomes engineered to contain CD81 (a well-established exosome marker) or PDGFR (a transmembrane protein with moderate exosome loading efficiency) fused to GFP were used as reference standards.
Engineered HEK293SF cells containing each of the expression constructs were stably selected and grown to high density in 200 ml cultures. The supernatants were collected and purified by Optiprep™ density gradient ultracentrifugation as described in Example 1. The resulting GFP-containing exosomes were measured in 96-well format on a Synergy H1 plate reader (BioTek®). As shown in
The results in Example 3 suggest that the N-terminal sequence of BASP1 is sufficient to load protein cargo into the lumen of exosomes. To determine the minimal peptide sequence with this activity, engineered GFP loading experiments were carried out by generating a variety of BASP1 truncations fused to the N-terminus of GFP and measuring the degree of their loading into exosomes.
BASP1 has been reported to be myristoylated, which may play a role in its localization to the exosome lumen. To test the role of myristolyation in BASP1 loading, glycine to alanine point mutations at predicted myristolyation sites were also tested in the GFP loading experiments. Single mutations at position 2 (sequence pCB 692), position 3 (pCB 693), or a double mutation (pCB 694) were included with BASP1 1-30 (pCB 540) and tested with fusion proteins containing various truncations of BASP1 (pCB 683-691).
HEK293 SF cells were transfected and selected in the presence of puromycin to stably express the plasmids encoding each of the sequences in
To confirm the results shown in
Western blotting with an anti-FLAG antibody (M2 monoclonal antibody, Millipore-Sigma) showed equal amounts of BASP1-GFP in pCB540 (amino acids 1-30) and pCB683-689 (
To identify the minimal BASP1 amino acid sequence between the twelve-amino acid truncation that facilitated loading and the six-amino acid truncation that failed to facilitate loading as shown above, individual truncation mutants of the N-terminus of BASP1 fused to a FLAG tag and GFP were generated and stably expressed in HEK293 SF cells (
The serine at position 6 of BASP1 is highly conserved across species and in MARCKS and MARCKSL1. To determine whether this amino acid was required for cargo loading into exosomes, HEK293SF cells were stably transfected with expression plasmids encoding BASP1 1-30-FLAG-GFP or BASP1 1-30-FLAG-GFP including a point mutant, replacing serine six with aspartic acid (S6D; polar charged substitution) or alanine (S6A; small nonpolar substitution). Additionally, the lysine at position five was mutated to a glutamic acid (L5Q) to test the potential role of this position in modulating myristoylation, palmitoylation, and other membrane functions of several membrane-associated proteins (Gottlieb-Abraham et al., Mol. Biol. Cell. 2016 Dec. 1; 27(24):3926-3936) (
The first thirty amino acids of BASP1 contain the N-terminal leader sequence identified above, followed by a lysine-rich stretch of amino acids. To understand whether MARCKS and MARCKSL1 N-termini can load exosomes similarly to BASP1, HEK293SF cells were stably transfected with MARCKS and MARCKSL1 full-length proteins or amino acids 1-30 fused to FLAG-GFP. Purified exosomes were analyzed by SDS PAGE and Coomassie staining to determine the extent of loading. Full-length MARCKS and MARCKSL1 were able to load exosomes with GFP, but amino acids 1-30 were inferior to the full-length proteins, suggesting that there are additional structural or sequence features in distal regions of the MARCKS and MARCKSL1 proteins required for loading (
The narrowest motif, Motif 1, allows for a protein sequence of (M)(G)(G/A/S)(K/Q)(L/F/S/Q)(S/A)(K)(K) (SEQ ID NO: 118), where each parenthetical letter or group of letters is an amino acid position, and wherein additionally position five cannot be a positively charged amino acid (K/R/H) and position six cannot be a negatively charged amino acid (D/E). Sub-motifs of Motif 1 include, without being limiting, the protein sequences: (M)(G)(G)(K/Q)(L/F/S/Q)(S/A)(K)(K), (M)(G)(A)(K/Q)(L/F/S/Q)(S/A)(K)(K), (M)(G)(S)(K/Q)(L/F/S/Q)(S/A)(K)(K), (M)(G)(G/A/S)(K)(L/F/S/Q)(S/A)(K)(K), (M)(G)(G/A/S)(Q)(L/F/S/Q)(S/A)(K)(K), (M)(G)(G/A/S)(K/Q)(L)(S/A)(K)(K), (M)(G)(G/A/S)(K/Q)(F)(S/A)(K)(K), (M)(G)(G/A/S)(K/Q)(S)(S/A)(K)(K), (M)(G)(G/A/S)(K/Q)(Q)(S/A)(K)(K), (M)(G)(G/A/S)(K/Q)(L/F/S/Q)(S)(K)(K) and (M)(G)(G/A/S)(K/Q)(L/F/S/Q)(A)(K)(K), where position five cannot be a positively charged amino acid (K/R/H) and position six cannot be a negatively charged amino acid (D/E).
Motif 2, a broader motif, can be expressed as (M)(G)(π)(ξ)(Φ/π)(S/A/G/N)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu).
Motif 3, the broadest motif, can be expressed as (M)(G)(π)(X)(Φ/π)(π)(+)(+), wherein each parenthetical position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, 41) is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu). In all cases of Motifs 1-3, the sequence may be truncated by one amino acid to be seven total amino acids in length (i.e., consisting of amino acids 1-7 in the order presented in Motifs 1-3). Any of the sequences derived from any of Motifs 1, 2, or 3 (or these motifs lacking amino acid 7), can be used to load cargo into exosomes to the same extent as, or comparable to, full length BASP1 or natural truncation sequences of BASP1. This deep analysis of amino acid sequence-structure-function provides novel insights into the requirements for directing biologically expressed cargo into exosomes by producer cells.
The results in Example 4 suggest that the N-terminus of BASP1 may be a useful engineering scaffold for generating luminally loaded exosomes directly from producer cells. To test this hypothesis, stable HEK293 SF cells were generated to express full-length Cas9 protein with codon optimization (as described in Zetsche B, Volz S E, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol. 2015 February; 33(2):139-42) fused to amino acids 1-30 or 1-10 of BASP1. Exosomes were purified from cell culture as described above and analyzed by SDS-PAGE and Western blotting using an anti-Cas9 antibody (Abcam; Catalog # ab191468, clone 7A9-3A3). As shown in
As an additional validation of the diversity of cargo proteins that can be loaded as a fusion to the N-terminus of BASP1, ovalbumin was stably expressed in HEK293SF cells as a fusion to amino acids 1-10 of BASP1 (“BASP1(1-10)-OVA”). A separate cell line was co-transfected with the same plasmid and a second plasmid encoding trimeric CD40L fused to an exosome-specific surface glycoprotein PTGFRN (“3xCD40L-PTGFRN”) using a second selectable marker. Exosomes were purified from the two transfected cell cultures and analyzed by SDS-PAGE (
Another class of proteins that may be useful in the context of therapeutic exosomes are antibodies and antibody fragments. A single chain camelid nanobody targeting GFP (as described in Caussinus E, Kanca O, Affolter M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat Struct Mol Biol. 2011 Dec. 11; 19(1):117-21) was stably expressed in HEK293SF cells as a fusion protein to amino acids 1-10 of BASP1 and a FLAG tag (“BASP1(1-10)-Nanobody”) or a FLAG tag alone (“Nanobody”) (
Acids in the Lumen of Exosomes
Nucleic acids, and in particular RNAs (e.g., mRNAs, siRNAs, miRNAs) are an attractive class of therapeutic cargo to be loaded in the lumen of therapeutic exosomes. Exosome loading of RNA may protect the RNA from degradation in the extracellular environment and the loaded exosome can be directed to certain cells and/or tissues through additional levels of exosome engineering, e.g., surface expression of a targeting construct. To understand whether the exosome proteins (or protein fragments) identified above can be used to generate mRNA-loaded exosomes, combinatorial engineered exosomes were generated. As shown in
The cells stably expressing the BASP1-MCP and Luciferase-MS2 mRNA were isolated and total Luciferase mRNA was quantified by RT-qPCR (FWD Primer: 5′-TGGAGGTGCTCAAAGAGTTG-3′ (SEQ ID NO: 119); REV Primer: 5′-TTGGGCGTGCACTTGAT-3′ (SEQ ID NO: 120); PROBE: 5′-/56-FAM/CAGCTTTCC/ZEN/GGGCATTGGCTTC/3IABkFQ/-3′ (SEQ ID NO: 121)). Untransfected cells expressed lower levels of Luciferase than all of the 811-expressing cells, which expressed comparable levels of Luciferase (
Generate Surface-Decorated Exosomes
The results in the previous experiments demonstrate that full-length and N-terminal regions of MARCKS, MARCKSL1, and BASP1 can be used to generate luminally loaded exosomes. To further explore the potential of these proteins for exosome engineering, amino acids 1-30 of MARCKS, MARCKSL1 and BASP1, or amino acids 1-10 of BASP1 were fused to the endogenous transmembrane region of CD40L expressed as a homotrimer. Constructs were prepared for both human and mouse sequences of CD40L because the ligands do not cross-react with the cognate receptor on the other species (
Cell lines from different tissues of origin (HEK293, kidney; HT1080, connective tissue; K562, bone marrow; MDA-MB-231, breast; Raji, lymphoblast) were grown to logarithmic phase and transferred to media supplemented with exosome-depleted serum for ˜6 days except for the HEK293 cells, which were grown in chemically-defined media. Bone marrow-derived mesenchymal stem cells (MSC) were grown on 3D microcarriers for five days and supplemented in serum-free media for three days. Supernatant from each cell line culture was isolated, and exosomes were purified using the Optiprep™ density-gradient ultracentrifugation method described above. Each of the purified exosome preparations was analyzed by LC-MS/MS as described above, and the number of peptide spectrum matches (PSMs) was quantified for BASP1, MARCKS, and MARCKSL1 and two widely studied exosome proteins (CD81 and CD9). The tetraspanins CD81 and CD9 were detectable in most purified exosome populations, but were, in some cases, equal to or lower than the luminal exosome proteins (e.g., compare CD9 to BASP1 or MARCKSL1) (
Luminally Engineered Exosomes
The results in Example 8 demonstrate that numerous human-derived cells naturally express BASP1 and the other novel exosome proteins identified in Example 1. To determine whether BASP1 can be used as a universal exosome scaffold protein, Chinese hamster ovary (CHO) cells were stably transfected with either a plasmid expressing full-length BASP1 fused to a FLAG tag and GFP (“BASP1-GFP-FLAG”), a plasmid expressing amino acids 1-30 of BASP1 fused to a FLAG tag and GFP (“BASP1(1-30)-GFP-FLAG”) or a plasmid expressing amino acids 1-8 of BASP1 fused to a FLAG tag and GFP (“BASP1(1-8)-GFP-FLAG”). Exosomes were purified from wild-type CHO cells and CHO cells transfected with one of the three BASP1 plasmids using the method described in Example 1. As shown in
A producer cell generating lumen-engineered exosomes is made by introducing an exogenous sequence encoding an exosome protein or a modification or a fragment of the exosome protein. The exosome protein is a fusion protein comprising the BASP1 fragments disclosed in Example 4 above, and a cargo protein. A plasmid encoding an exosome protein is transiently transfected to induce high-level expression of the exosome protein in the exosome lumen.
A polynucleotide encoding an exosome protein, a modification or a fragment of an exosome protein, or an exogenous sequence encoding a therapeutic peptide, cargo peptide, or a targeting moiety is stably transformed into a producer cell to produce lumen-engineered exosomes. The exogenous sequence encoding a therapeutic peptide, cargo peptide, or a targeting moiety is inserted into a genomic site encoding an exosome protein to generate a fusion protein comprising the therapeutic peptide or cargo peptide attached to the exosome protein. A polynucleotide encoding a modified exosome protein is knocked in to a genomic site encoding an exosome protein.
A producer cell line is generated by stably transfecting at least two polynucleotides, each encoding an exosome protein, a modification or a fragment of an exosome protein, or an exogenous peptide (e.g., targeting moiety, therapeutic peptide). Two or more exogenous sequences are inserted into multiple genomic sites, within or closed to the genomic sequence encoding an exosome protein, to generate a lumen-engineered exosome comprising multiple modified exosome proteins. Each of the plurality of modified exosome proteins is targeted to the lumen of exosomes.
All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.
The present disclosure provides, inter alia, compositions of exosomes containing modified exogenous proteins and peptides for use in enrichment of exogenous proteins in exosomes. The present disclosure also provides method of and methods of producing enriched exosomes. While various specific embodiments have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s). Many variations will become apparent to those skilled in the art upon review of this specification.
This application claims the benefit of U.S. Provisional Patent Application 62/587,767 filed Nov. 17, 2017, and U.S. Provisional Patent Application 62/634,750, filed Feb. 23, 2018, the disclosures of which are hereby incorporated in their entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 62587767 | Nov 2017 | US | |
| 62634750 | Feb 2018 | US |
