Protein sorting motifs for the packaging of therapeutic proteins into yeast extracellular vesicles for targeted delivery to cells and blood

Abstract

Methods, systems, and compositions for the selective loading of proteins containing one or more engineered protein sorting motifs (p-ESMs) into yeast-generated extracellular vesicles (EVs) and their methods of use in treating one or more diseases or conditions.

Description

[0001] PROTEIN SORTING MOTIFS FOR THE PACKAGING OF THERAPEUTIC PROTEINS INTO YEAST EXTRACELLULAR VESICLES FOR TARGETED DELIVERY TO CELLS AND BLOOD CROSS-REFERENCE TO RELATED APPLICATIONS This International PCT Application claims the benefit of and priority to U.S. Provisional Application No.63 / 536,478, filed September 4, 2023, the specification, Tables, sequences, claims and drawings of which are incorporated herein by reference in their entirety. SEQUENCE LISTING The instant application contains contents of the electronic sequence listing (90355_00141_SL.xml; Size: 12495000 bytes; and Date of Creation: August 30, 2024) is herein incorporated by reference in its entirety. TECHNICAL FIELD The present invention is directed to novel methods, systems, and compositions for the selective loading of proteins into yeast-generated extracellular vesicles (EVs) and their methods of use in treating one or more diseases or conditions. BACKGROUND One of the major challenges facing the use of therapeutic molecules and compounds, also sometimes referred to as a drugs, therapeutics, or therapeutic compositions, to control diseases and genetic disorders is an effective means to deliver the drug to a targeted tissue while avoiding degradation or its elimination from the body. Recently, lipid-encased nanovesicles have proven to be an effective means to deliver therapeutic molecules to human cells while protecting their cargo from degradation. The use of lipid nanovesicles to deliver mRNA to cells has proven to be an efficient means to vaccinate humans against SARS-CoV-2. However, the artificial lipids frequently used to make these nanovesicles are not tissue or cell specific and are not well tolerated by humans limiting their applications to non-therapeutic purposes. Furthermore, they have limited access to most organs other than liver and kidneys. Another natural lipid nanovesicle that has also been shown to package RNA, proteins, and other small molecules are extracellular vesicles (EVs) or exosomes produced and released from the surface of human cells. Most eukaryotic EVs are well tolerated but have limitations due to the heterogeneity of cell types from which they originate resulting in broad distributions of particle size and heterogeneous cargo. Furthermore, EVs or exosomes produced in human cell lines can potentially be contaminated by non-target proteins, RNAs and human pathogens such as viruses. An alternative platform for producing more uniform EVs that package therapeutic molecules is the production of EVs in single celled eukaryotic organisms (non-human) having uniform genetic traits. Examples of single-celled organisms that can be engineered to deliver therapeutic molecules include yeast such as Saccharomyces cerevisiae and S. boulardii. Significantly, these yeast strains are well tolerated by humans being used in the manufacture of foods and beverages consumed by humans. In addition, S. boulardii has been safely used as human probiotic for over 65 years. More specifically, S. boulardii has many traits that are ideal to produce EVs for delivery of therapeutics to humans. For example, S. boulardii produces EVs that package RNA, proteins, and other small molecules (Durmusoglu et al., 2020; Kulig et al., 2023). In addition, the genome of S. boulardii is available and can be genetically engineered and / or have its genome edited for the purpose of modifying EV targeting or cargo (Grant et al., 1984; Benatuil et al., 2010). Interestingly, S. boulardii and S. cerevisiae lack the RNA-induced silencing complex (RISC) machinery necessary to generate small interfering RNA populations and thus endogenous yeast EV small RNAs cannot silence human genes. A comparative analysis of the RNAs present in yeast to the human genome and transcriptome indicates that yeast EVs have no RNA sequences that are similar to human transcripts at nucleotide lengths greater than 11 bases and no complementary RNA sequences to human genes with the exception of a single 28 nucleotide stretch that is complimentary to one human non-coding RNA species. Thus, yeast RNAs are unlikely to have any impact on human cells. Furthermore, yeast EVs delivered by injection or orally have access to all organs in the mouse body unlike lipid nanovesicles or human exosomes (See i.e., Figure 1). In addition, S. boulardii can also be grown at low cost in large scale fermenters. Both yeast and yeast EVs also have a long shelf life of over a year for cells at room temperature when freeze-dried. These traits open the possibility of using S. boulardii as a complete, single celled system for the large-scale production and packaging of therapeutic molecules into EVs for delivery to humans. Several traditional methods have been developed to load therapeutic drug compositions into EVs for delivery to target cells or tissues. These methods for encapsulating protein cargo into EVs can be roughly divided into two types: cell-based protein expression and EV packaging methods and exogenous EV loading methods. In the cell-based loading approach, therapeutic proteins are expressed in the donor cells followed by random or active sorting of the protein into EVs. After being packaged into EVs, the EVs are used for therapeutic use. Non-cell-based loading approach involves directly loading biomolecules into isolated EVs through electroporation, sonication, incubation, and / or transfection. However, each of these methods involve specific technical and commercial limitations. For example, passive incubation loading typically includes low loading efficiencies, while transfection relies on transfection efficiency, which is always variable, and may further alter the structure of the EVs. Methods such as electroporation, sonication, and freeze thaw disrupt the EVs structure and increase EV instability and can further result in undesired EV aggregation, add additional steps and costs to the development of therapeutic EVs, and substantially increase costs of production. As such, there exists a long-felt need for an efficient, and commercially viable method to load and enrich therapeutic protein compositions precisely, and uniformly into EVs structures, all while causing minimal disruption to their structure and aggregation patterns. SUMMARY OF THE INVENTION The present invention is directed to novel methods, systems, and composition for actively sorting proteins, and preferably therapeutic proteins into an EV, wherein the EV can deliver the protein to a targeted cell or release the therapeutic protein cargo into the bloodstream thereby treating a disease or condition or generating some physiological or other effect. The present invention is further directed to novel yeast EV protein sorting motifs, also referred to herein as p- ESMs. Exemplary motifs provided in SEQ ID NOs: 5-458, and 638-6666. In a preferred embodiment, the yeast EV protein sorting motifs, or sorting motifs, the present invention can be used to form novel expression constructs configured to express recombinant protein molecules having one or more yeast EV protein sorting motifs incorporated into their sequence thereof. In another embodiment, the invention includes novel protein molecules, and preferably therapeutic protein molecules including one or more EV protein sorting motifs. In one preferred aspect, the current invention includes novel constructs for the heterologous expression of fusion proteins in yeast adapted to facilitate their sorting into yeast EVs as either soluble or membrane protein anchored proteins. The recombinant proteins can be configured to include one or more p-ESMs linked by a protease recognition sequence, such as a TEVp protease recognition sequence (See Figure 11, Table 2) and are expressed in cells which have a TEVp protease is linked to the yeast EV membrane protein anchor Sur7 so that the protease is displayed in the EV lumen and processes the p-ESM – heterologous protein fusion in the EV. In another embodiment, the designed protein populations can be configured to disrupt or delete whole cell or counter sorting protein motifs, also referred to herein as p-CSMs that enrich the protein molecules in whole cells versus EVs. In a preferred embodiment, the p-CSMs can be selected from the amino acid sequences according to SEQ ID NOs: 459-611, 618-637, and 6667-14762. In another embodiment the recombinant proteins can be configured to include a p-ESM, a cell penetrating peptide (CCP) and a protein-of-interest (POI) with associated proteolytic cleavage sites separating the p-ESM, CCP, and POI domains (Fig.5). Cell-penetrating peptides (CCPs) are short peptides that direct the uptake of cargo molecules across the plasma membrane. Exemplary CCPs can be selected from the amino acid sequences according to SEQ ID NOs: 612-617. Cargoes may include other polypeptides, nucleic acids, chemical compounds, or combinations of the same, among other compositions of matter. Three types of CCPs have been identified based on amino acid composition: polycationic (containing a high proportion of lysine and / or arginine); amphipathic (containing alternating charged and hydrophobic amino acids); and hydrophobic (Figures 3 and 4). Three translocation mechanisms have been described: direct penetration of plasma (or organellar) membranes (Figure 4); uptake by endocytosis; and translocation through a transient membrane-associated structure. For the purposes of cargo release from y-EVs, direct translocation by polycationic CCPs (which is independent of biochemical energy) can be applicable. Proteolytic processing by a yeast EV sorted protease, such as BACE1, containing a p-ESM results in removal of the p-ESM from the adjacent CCP and allows for the CCP-POI protein fusion to be released from the yeast EV into the blood. Premature release of the CCP-POI fusion is prevented by production and incubation of yeast EVs in the presence of a competitive, cell permeant protease inhibitor, for example, Verubecestat, which diffuses out of the yeast EVs upon delivery to the human. Additional aspects of the invention will be evident from the specification, figures, tables, and claims provided herein. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Distribution of yeast EVs in mouse after oral delivery.1012 yEVs labeled with fluorescent CellMask dye were administrated to mice by oral gavage. The biodistribution of yEVs was accessed by IVIS imaging from 1h to 24h after the procedure. Figure 2A-B. Sorting recombinant proteins into yeast EVs for delivery to human cells. (A) show exemplary strategy for yEV-targeted soluble proteins: Soluble fusion proteins containing a p-ESM are processed by a sequence-specific protease linked to a yEV membrane protein, i.e., Sur7, to remove the p- ESM and regenerate the protein of interest. (B) show exemplary strategy for yEV targeted membrane-bound proteins: Proteins linked to Sur7 and containing a protease recognition site at the Sur7 junction are proteolytically released by a sequence-specific soluble protease targeted to the yEV by a p-ESM Figure 3. Translocation of a polycationic CCP across a cellular membrane. The CCP is covalently attached to a therapeutic molecule (i.e., for a polypeptide therapeutic, by translational fusion). Membrane integrity is restored after translocation. The residues in green comprise a linker (SEQ ID NO.14763) and / or protease cleavage site. Figure 4. Translocation of a fusion polypeptide comprising a p-ESM (black), a polycationic CCP (red), a BACE1 recognition peptide (green), and a Bradykinin peptide (blue) across the y-EV membrane. Proteolysis by BACE1 liberates active BK, which can then potentiate vasodilation. Figure 5. Model for generating yeast EV permeant proteins in the blood to interact with cognate cell surface receptors. Removal of competitive, cell permeant protease A inhibitor leads to removal of p-ESM in yEVs and release of CCP-POI into blood. Optional protease B processing to remove CCP by an endogenous blood protease Figure 6. Example cassette for expression of TEVp-Sur7 fusion protein Figure 7. 3D rendering of CRH1 showing four p-ESMs in dispersed locations in unstructured regions of the protein. Figure 8.3D rendering of SUN4 showing four p-ESMs in two dispersed locations and in two adjacent locations in unstructured regions of the protein. Figure 9. 3D rendering of DSE4 showing four p-ESMs in two adjacent locations and in two locations separated by 5 amino acid residues in unstructured regions of the protein. Figure 10A-B. Exemplary constructs for co-expression of TEVp_p-ESM fusion protein (A) and BMP9_Sur7 fusion protein (B)b. Figure 11: Exemplary representation of enzyme-substrate complex with eight binding sites. Positions Pn to Pm’ in the substrate are counter from the bond between P1 and P1’, where the cleavage occurs. Figure 12: Model of therapeutic protein sorting via KISA motif inclusion in opposite termini of an engineered therapeutic protein (opposite termini represented by green and red boxes). The yeast extracellular vesicle more effectively receives engineered heterologous proteins via inclusion of designed protein sorting motifs, whereas the heterologous proteins without motifs would result in retention in intracellular organelles, the cytosol, or cellular membranes and exclusion from the yEV contents. Adapted from “Sumoylated protein” and “squeezing spheroidal cell”, by BioRender.com (2024). Figure 13: Inclusion of the KISA motif at the N-terminus of the transmembrane low CO2- inducible protein A confers sorting to the yEV. A faint cellular signal is visible in the cell of LCIA with no KISA motif just above the 33 kDa marker, however none was evident in the cell when KISA was included at the N-terminus. This suggests effective clearance of translated heterologous protein from the cellular volume. GST-FLAG included for semi-quantitative reference. Figure 14A-B: SDS-PAGE Western blot performed on Luc2 with and without KISA motif in S. boulardii yEV fractions (A) and cell fractions (B). The use of KISA at either termini resulted in sorting to the yEV, however Luc2 displayed a strong preference for the C-terminus. In the presence of the denaturing conditions imparted by SDS, no Luc2 signal was detected in the cell. Figure 15A-B: Native PAGE Western blot performed on Luc2 with and without KISA motif in S. boulardii yEV fractions (A) and cell fractions (B). Native PAGE of yEV extracts followed by Western blot confirms the increased presence of Luc2 with C-terminus motif inclusion. Cellular fractions illustrate that increased sorting to the yEV with KISA at the C- terminus is not negatively correlated with cell content, suggesting alternative mechanisms at play in comparison to low CO2-inducible protein A. Figure 16: SDS-PAGE Western blot performed on NBE with and without KISA motif in S. boulardii yEV fraction. The inclusion of KISA at both the N- and C-termini did not result in an additive effect compared to either alone. Despite being a soluble protein like Luc2, NBE was not more effectively sorted by inclusion of KISA at the C-terminus. However, any placement of the KISA motif resulted in sorting to the yEV. Figure 17A-B: Predicted structure of low CO2-inducible protein A in the absence (A) and presence (B) of KISA at the N-terminus. Hydrophobicity is displayed via the color_h script in PyMOL, based on Eisenberg et al. (1984). The KISA motif is outlined in yellow in (B). The structure was predicted via DeepFold (Lee et al., 2023; Pearce et al., 2022). Figure 18: Predicted structure of Luc2 in the presence of the KISA motif (outlined in blue) at the C-terminus. The yellow box indicates the structured hydrophobic region on the C-terminus which is potentially blocked by KISA. The last residue of Luc2 prior to the KISA motif is highlighted in yellow. Figure 19: In vitro fluorescence assay of EGFP-enhancing nanobodies from lysed yEVs. A) NBE activity at 75 ng of EGFP. B) NBE activity at 100 ng of EGFP, study 1, C) NBE activity at 100 ng of EGFP, study 2. All experiments included 3 replicates trials and were repeated once. For 75 ng EGFP, experiment by treatment interactions were not detected (p = 0.120), so experiments are presented together. For 100 ng of EGFP, experiment by treatment interactions were detected (p = 0.0000890), thus results are presented for each experiment separately. DETAILED DESCRIPTION OF THE INVENTION While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims. All references cited herein are incorporated in their entirety by reference. The terminology used herein is for describing particular embodiments and is not intended to be limiting. As used herein, the singular forms “a,” “and” and “the” include plural referents unless the content and context clearly dictate otherwise. Thus, for example, a reference to “a” or “the” marker may include a combination of two or more such markers. Unless defined otherwise, all scientific and technical terms are to be understood as having the same meaning as commonly used in the art to which they pertain. In one aspect the present invention includes novel polypeptides containing one or more heterologous protein EV-sorting motifs (p-ESMs), wherein the polypeptide is preferentially packaged when introduced to a yeast-derived EV. In a preferred embodiment, the step of introduction can occur in vivo or in vitro. In a preferred embodiment, the p-ESM of the invention can be selected from the amino acid sequences according to SEQ ID NOs: 5-458, and 638-6666, or a fragment or amino acid variant thereof. In a preferred embodiment, the invention comprises a nucleotide sequence, operably linked to a promoter, encoding a polypeptide containing one or more heterologous protein EV-sorting motifs (p-ESMs), wherein the polypeptide when introduced to a yeast-derived EV is preferentially packaged in the EV. In a preferred embodiment, the step of introduction can occur in vivo or in vitro. In one embodiment, the p-ESM of the invention can be selected from a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 5-458, and 638-6666, or a fragment or amino acid variant thereof. In another embodiment, the novel protein molecule of the invention can include one or more novel p-ESMs typically located in unstructured and surface exposed portions of the protein either adjacent to each other or distributed throughout the protein sequence. In another embodiment, the novel protein molecule of the invention can include a protease with a p-ESM designed to release a therapeutic protein fused to the lumenal side of a EV membrane protein and containing a unique protease cleavage recognition sequence, while in an alternative embodiment the novel protein molecule is linked to a cell permeant peptide (CCP) having a p- ESM for controlled release or secretion of the protein of interest from the EV initiated by a proteolytic cleavage event that removes the p-ESM from the CPP in the EV (Boon et al., 2020; Albert et al., 2009; Li et a., 2008; Lin et al., 1999; Yi et al., 2013; Nielsen et al., 2014; Futaki et al., 2001; Baoum et al., 2012); De coupade et al., 2005)). This proteolytic cleavage event is triggered by the loss of cell permeant competitive inhibitor of the protease catalytic activity. An example of a protease and its associated inhibitor can include the BACE1 protease and Verubecestat (Albert et al., 2009; Das et al., 2019; FeFranc-Jullien et al., 2005; Lin et al., 1999; Stachel et al., 2004). Exemplary proteases and associated peptide cleavage sites are provided at web.expasy.org / peptide_cutter / peptidecutter_enzymes.html#exceptions (which is incorporated herein by reference). In an alternative aspect, a novel polypeptide of the invention can be modified to include a p-ESM as described herein and can further be modified such that one or more sequences / motifs that encode a whole cell or counter sorting protein motifs (p-CSM) are disrupted or removed such that the polypeptide is preferentially packaged, in vivo or in vitro, in a yeast-derived EV. Notably, methods for disrupting a protein sequence or motif are known in the art and would be generally understood by one of ordinary skill. In a preferred embodiment. In one embodiment, the p-ESM of the invention can be selected from an amino acid sequence according to SEQ ID NOs: 5-458, and 638-6666, and in a preferred embodiment SEQ ID NOs: 5-24, and a p-CSM of the invention can be selected from an amino acid sequence according to SEQ ID NOs: 459-611, 618-637, and 6667-14762. In a preferred embodiment, the invention comprises a nucleotide sequence, operably linked to a promoter, encoding a polypeptide containing one or more heterologous protein EV-sorting motifs (p-ESMs), wherein the protein molecules is further modified such that one or more sequences / motifs that encode a whole cell or counter sorting protein motifs (p-CSM) are disrupted or removed. In this embodiment, the polypeptide is preferentially packaged, in vivo or in vitro, in a yeast-derived EV. In one embodiment, the p-ESM of the invention can be selected from an p- ESM sequence selected from SEQ ID NOs: 5-458, and 638-6666, and in a preferred embodiment SEQ ID NOs: 5-24, and a p-CSM of the invention can be selected from an amino acid sequence according to SEQ ID NOs: 459-611, 618-637, and 6667-14762. In another embodiment, the invention includes an expression vector comprising the nucleotide encoding a heterologous polypeptide having one or more p-ESMs, and wherein said nucleotide sequence is operably linked to a promoter. In another embodiment, the invention includes an expression vector comprising the nucleotide sequence encoding a heterologous polypeptide having one or more p-CSMs that have been disrupted or removed, and wherein said nucleotide sequence is operably linked to a promoter. In another embodiment, the invention includes an expression vector comprising a nucleotide encoding a heterologous protein molecule having one or more p-ESMs and having one or more p-CSMs that have been disrupted or removed, and wherein said nucleotide sequence is operably linked to a promoter. In another embodiment, the invention includes a cell, and preferably a yeast cell having an expression vector comprising the nucleotide encoding a heterologous polypeptide having one or more p-ESMs, and wherein said nucleotide sequence is operably linked to a promoter. In this preferred embodiment, the cell expresses the heterologous expression vector comprising the nucleotide encoding a heterologous polypeptide having one or more p-ESMs, wherein the polypeptide is preferentially loaded into an EV, which can further be isolated. In another embodiment, the invention includes a cell, and preferably a yeast cell an expression vector comprising the nucleotide sequence encoding a heterologous polypeptide having one or more p-CSMs that have been disrupted or removed, and wherein said nucleotide sequence is operably linked to a promoter. In this preferred embodiment, the cell expresses the heterologous expression vector comprising the nucleotide encoding a heterologous polypeptide having one or more p-CSMs that have been disrupted or removed, wherein the polypeptide is loaded into an EV, which can further be isolated. In another embodiment, the invention includes a cell, and preferably a yeast cell having an expression vector comprising a nucleotide encoding a heterologous protein molecule having one or more p-ESMs and having one or more p-CSMs that have been disrupted or removed, and wherein said nucleotide sequence is operably linked to a promoter. In this preferred embodiment, the cell expresses the heterologous expression vector comprising the nucleotide encoding a heterologous polypeptide having one or more p-ESMs and having one or more p-CSMs that have been disrupted or removed, wherein the polypeptide is preferentially loaded into an EV, which can further be isolated. Any of the expression vectors of the invention can be used to transform a yeast cell, and preferably a yeast cell is selected from: Saccharomyces cerevisiae, or Saccharomyces boulardii. The transformed yeast cell can be cultured and produce a novel polypeptide of the invention having one or more p-ESMs and / or having one or more p-CSMs that have been disrupted or removed, which as noted above are preferentially loaded into an EV produced by the cultured yeast cells. These EVs can be isolated and combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition. Examples of categories of therapeutic peptides include; Antibodies (intracellular and extracellular), Antibody Fragments, sometimes referred to as Nanobodies or a Nanobody, Anticoagulants, Antidiabetics, Anti-Infectives, Anti-Inflammatory, Antineoplastic, Anti-psoriatic, Antirheumatic, Antithrombins. Antiviral, Bone Density Conservation, Coagulants and Thrombotic, Blood Coagulation, Diagnostic, Enzyme Replacement, Enzymes, Fibrinolytic, Hormone Replacement, Hormones, Hypoglycemic, Immunologic Factors, Immunosuppressive, Platelet Aggregation Inhibitors, and Thrombolytics. In a preferred embodiment, a pharmaceutical composition containing a quantity of isolated yeast derived EVs containing a population of therapeutic proteins that have been engineered to be preferentially loaded into the EVs of the invention, can be administered, in a therapeutically effective amount, to a subject in need thereof to treat a disease or condition. (Lau et al., 2018; Usmani et al., 2017; Gaston et al., 2019; Niamsuphap et al., 2020; Slastnikova et al., 2018; Soetens etr al., 2020). In another aspect the present invention includes a nucleotide sequence encoding a novel polypeptide, operably linked to a promoter, containing one or more heterologous EV protein- sorting motifs (p-ESMs), wherein the protein molecules when introduced to a yeast-derived EV, whether in vivo or in vitro are preferentially packaged in the EV, which can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition. The nucleotide sequence of the invention can form an expression vector, having an expression cassette, operably linked to a promoter, encoding a polypeptide containing one or more heterologous p-ESMs. (Grant et al., 1984; Benatuil et al., 2010). In another preferred aspect, the expression vector of the invention can be used to transform a yeast cell to express the heterologous polynucleotide containing one or more heterologous p-ESMs which can be preferentially packaged into an EV in vivo, which can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition. In an alternative aspect, the amino acid sequence of the invention can be further modified such that one or more sequences / motifs that encode a whole p-CSM are disrupted or removed, which can further be introduced to an EV, preferably in vivo, which can further be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition. In another aspect the present invention includes a yeast-generated EV containing a therapeutic polypeptide having one or more p-ESMs. In an alternative aspect, the protein molecule associated with the yeast-generated EV of the invention can further be modified such that one or more sequences / motifs that encode a p-CSM are disrupted or removed. In this preferred aspect, the yeast-generated EV can be derived from a Saccharomyces yeast cell, and preferably Saccharomyces cerevisiae, or Saccharomyces boulardii. The yeast generated EVs of the invention can be isolated and incorporated with a pharmaceutically acceptable carrier forming a pharmaceutical composition. In another aspect the present invention includes a method of treating a disease or condition comprising the step of administering a therapeutically effective amount of a pharmaceutical composition containing a yeast-generated EV containing a therapeutic protein molecule having one or more p-ESMs and a pharmaceutically acceptable carrier. In an alternative aspect, the protein molecule of the pharmaceutical composition can further be modified such that one or more sequences / motifs that encode a p-CSM are disrupted or removed. The present invention includes a fusion peptide configured to be selectively loaded into a yeast derived EV. In this preferred embodiment, the fusion peptide of the invention includes a first and second domain, the first domain comprising a protein of interest (POI), such as a therapeutic or diagnostic peptide or fragment thereof, and the second domain comprising a protein extracellular vesicle sorting motif (p-ESM). In some embodiment the fusion peptide of the invention include a one or more p-ESMs, at preferably the N- or C- terminus of the fusion peptide. The p-ESM of the invention can further be selected from the amino acid sequence according to SEQ ID NOs: 5-458, and 638-6666, or a fragment or variant of any of the same. The fusion peptide of the invention can further include a third domain which can include a cell-penetrating peptide (CPP). In a preferred example, the CPP is positioned between the p-EMS and the POI and can preferably be selected from the amino acid sequence according to SEQ ID NOs: 612-617, or a fragment or variant thereof. Each of the aforementioned domains can be separated by a cleavage site, such as a cleavage site that is targeted by a heterologous or endogenous protease enzyme, such as those provided in Table 2. In a preferred embodiment, a first cleavage site can be positioned between the p-EMS and the (CPP), which optionally a second cleavage site can be positioned between the CPP and POI. As noted previously, the POI may contain one or more protein cell sorting motif (p-CSM). To facilitate selective loading of the fusion peptide into a yeast EV, a p-CSM present in a POI can be disrupted or removed, for example through site directed mutation, or gene editing techniques known in the art. In a preferred embodiment, the p-CSMs of the invention is selected from the amino acid sequences according to SEQ ID NOs: 459-611, 618-637, and 6667-14762, or a fragment or variant thereof. In another preferred embodiment, the p-CSMs of the invention is selected from the amino acid sequences identified in SEQ ID NO.6667-14762, or a fragment or variant of the same. The present invention includes methods, systems, and compositions for selectively loading soluble and membrane-bound peptides in a yeast-derived EV. In one embodiment, the invention includes a yeast-generated extracellular vesicle (EV) expressing a nucleotide sequence, operably linked to a promoter, encoding a first fusion peptide comprising a first and second domain, the first domain comprising a protein of interest (POI), such as a therapeutic or diagnostic peptide or fragment thereof, and the second domain comprising a first protein extracellular vesicle sorting motif (p-ESM), and a third domain comprising a cell-penetrating peptide (CCP), such as a CCP domain selected from SEQ ID NOs: 612-617, or a fragment or variant thereof. In this preferred embodiment, a first cleavage site is positioned between the p-EMS and the CCP, and optionally a second cleavage site is positioned between the CCP and POI. A yeast-generated extracellular vesicle (EV) of the invention can further include a nucleotide sequence encoding a second fusion peptide containing a protease enzyme directed to the first and optionally the second cleavage site coupled with a second p-ESM. In this embodiment, the protease enzyme cleaves the first fusion peptide at the first cleavage site such that the CCP-POI portion of the fusion protein is released from the yeast EV. Notably, a protease inhibitor can be contacted with the yeast EV during incubation to inhibit the action of the protease enzyme and premature cleave at the cleavage site. The released portion of the CCP-POI portion of the fusion peptide can be delivered to a target cell, receptor or tissue where an endogenous protease, and preferably an endogenous protease of a human subject can cleave the second cleavage site construction the POI at the cellular target. In another embodiment, the invention includes a yeast-generated extracellular vesicle (EV) expressing a nucleotide sequence, operably linked to a promoter, encoding a first fusion peptide comprising a first and second domain, the first domain comprising a protein of interest (POI), such as a therapeutic or diagnostic peptide, or fragment thereof, and the second domain comprising a first protein extracellular vesicle sorting motif (p-ESM), and a third domain, the third comprising a yeast EV anchoring membrane protein, such as Sur7 peptide, or fragment thereof. In this configuration, a first cleavage site is positioned between the POI and the EV anchoring membrane protein domains. The yeast-generated extracellular vesicle (EV) can further include a nucleotide sequence (co- expressed by the same or separate expression vector as the first fusion peptide) encoding second fusion peptide containing a protease enzyme, such as a TEV protease or a protease identified in Table 2, directed to the first cleavage site coupled with a second p-ESM in this embodiment, when co-expressed, the protease cleaves the first fusion peptide at the first cleavage site such that the POI portion of the fusion protein is released from the yeast EV. As noted below, the domains of the fusion peptides in this embodiment can be separated by a linker, such as a peptide linker (SEQ ID NO: 14763) as describe herein. The p-ESM of the fusion peptides described above can further be selected from the amino acid sequence according to SEQ ID NOs: 5-458, and 638-6666, or a fragment or variant of any of the same. The p-ESM of the invention can further be selected from one or more of the of the p- ESMs according to SEQ ID NO.’s 5-458, and 638-6666, or a fragment or variant of any of the same. To further facilitate selective loading of the fusion peptide into a yeast EV, a p-CSM present in a POI can be disrupted or removed, for example through site directed mutation, or gene editing techniques known in the art. In a preferred embodiment, the p-CSMs of the invention is selected from the amino acid sequences according to SEQ ID NOs: 459-611, 618-637, and 6667-14762, or a fragment or variant thereof. In another preferred embodiment, the p-CSMs of the invention is selected from the amino acid sequences according to SEQ ID NO.6667-14762, or a fragment or variant of the same. The present invention further includes a nucleotide sequence encoding the fusion proteins of the invention as described above. This nucleotide sequences can be incorporated into an expression vector forming an expression cassette operably linked to a promoter. In another preferred embodiment a yeast cell, such as Saccharomyces cerevisiae, or Saccharomyces boulardii yest cell can be transformed by the expression vector of the invention. The transformed call can further be cultured and express the fusion peptide of the invention as described above, which is further selectively loaded into a yeast-derived EV. The yeast-derived EV containing the fusion peptide of the invention can be isolated and combined with a pharmaceutically acceptable carrier forming a pharmaceutical composition configured to deliver the POI to a cell or blood of a subject in need thereof. This pharmaceutical composition of the invention can be used for treating a disease or condition to a subject in need thereof. In this method, a comprising a therapeutically effective amount of the pharmaceutical composition of the invention can be administered to a subject in need thereof. It will be appreciated that appropriate dosages of the pharmaceutical compositions comprising the active fusion peptides of the invention, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and / or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects. Administration in vivo can be effected in one dose, continuously or intermittently (i.e., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. A “pharmaceutical composition” or “pharmaceutical composition of the invention” refers to an active ingredient, such as a quantity of yeast derived EVs containing a population of therapeutic proteins or antibodies that have been engineered to be preferentially loaded into the EVs of the invention, and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises two or more pharmaceutically acceptable carriers and / or excipients. In other embodiments, the pharmaceutical composition further comprises at least one additional antibiotic, such as through a co-treatment. As used herein, a “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered composition of the invention. The pharmaceutical acceptable carrier may comprise any conventional pharmaceutical carrier or excipient. The choice of carrier and / or excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the carrier or excipient on solubility and stability, and the nature of the dosage form. The term “pharmaceutically acceptable carrier” as used herein further pertains to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (i.e., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, “Handbook of Pharmaceutical Additives,” 2ndEdition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA), “Remington’s Pharmaceutical Sciences”, 20thedition, pub. Lippincott, Williams & Wilkins, 2000; and “Handbook of Pharmaceutical Excipients”, 2ndedition, 1994. Suitable pharmaceutically acceptable carriers include inert diluents or fillers, water, and various organic solvents (such as hydrates and solvates). The pharmaceutical compositions may, if desired, contain additional ingredients such as flavorings, binders, excipients, and the like. Thus, for oral administration, tablets containing various excipients, such as citric acid may be employed together with various disintegrants such as starch, alginic acid and certain complex silicates and with binding agents such as sucrose, gelatin, and acacia. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules. Non-limiting examples of materials, therefore, include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration the active compound therein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof. The pharmaceutical composition of the invention may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution suspension, for parenteral injection as a sterile solution, suspension, or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. Exemplary parenteral administration forms include solutions or suspensions of active compounds in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if desired. A pharmaceutical composition of the invention may be administered as single or multiple agents, for example a pharmaceutical composition of the compound of the invention, or a pharmaceutical composition of the compound of the invention and a second therapeutic compound or agent. In some embodiments, the methods the pharmaceutical composition of the invention can be used to treat a disease or condition, or one or more of its symptoms. Pharmaceutical compositions suitable for the delivery of the compound of the invention as described herein, and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in ‘Remington’s Pharmaceutical Sciences’, 19thEdition (Mack Publishing Company, 1995), the disclosure of which is incorporated herein by reference in its entirety. The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, i.e., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, i.e., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (i.e., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, Nucleotides may be referred to by their commonly accepted single-letter codes. “Therapeutic peptide” means a pharmaceutically active peptide (or analog or conjugate comprising a therapeutic peptide), that can be used to treat or diagnose a disease or condition in a subject. “Diagnostic peptide” means a diagnostically active peptide (or analog or conjugate comprising a Diagnostic peptide) that can be used to identify or diagnose a disease or condition in a subject. The term “expression,” as used herein, or “expression of a coding sequence” (for example, a gene or a transgene) refer to the process by which the coded information of a nucleic acid transcriptional unit (including, i.e., genomic DNA or cDNA) is converted into an operational, non- operational, or structural part of a cell, often including the synthesis of a protein. Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s). As used herein, a promoter region or promoter element refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. The term “nucleic acid” or “nucleic acid molecules” include single- and double-stranded forms of DNA; single-stranded forms of RNA; and double-stranded forms of RNA (dsRNA). The term “nucleotide sequence” or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex. The term “ribonucleic acid” (RNA) is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), tRNA (transfer RNA), whether charged or discharged with a corresponding acetylated amino acid), and cRNA (complementary RNA). The term “deoxyribonucleic acid” (DNA) is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids. The terms “nucleic acid segment” and “nucleotide sequence segment,” or more generally “segment,” will be understood by those in the art as a functional term that includes both genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller engineered nucleotide sequences that encoded or may be adapted to encode, peptides, polypeptides, or proteins. The term “fusion protein” as used herein is used as it is in the art. Namely, the fusion proteins used in the methods and compositions of the present invention involve two separate proteins or protein domains that are linked by a covalent bond. In one embodiment, the covalent bond linking the two domains is an amine bond. In more specific embodiments, the anchor protein and the foreign protein is a fusion protein comprising a single-chain polypeptide. In even more specific embodiments, the single-chain polypeptide comprising the anchor protein, and the foreign protein further comprises a linker peptide sequence. Any linker sequence can be used to covalently link the anchor protein and the foreign protein. As used herein, the term “peptide linker(s),” “linker(s),” or “linker moiety” refers to a peptide or polypeptide sequence, i.e., a synthetic peptide or polypeptide sequence, which connects two domains in a linear amino acid sequence of a polypeptide chain. In one embodiment, the polypeptides of invention are encoded by nucleic acid molecules that encode peptide linkers which either directly or indirectly connect the anchor protein and foreign protein which make up the construct. These linkers may be interposed between the anchor protein and foreign protein. If the linker connects two protein moieties contiguously in the linear polypeptide sequence, it is referred to as a “direct” linkage. In contrast, the linkers may link the first protein moiety, i.e., anchor protein or foreign protein, to a binding moiety which is, in turn, linked to the second protein moiety, i.e., anchor protein or foreign protein, thereby forming an indirect linkage. Linkers are typically located at the N or C terminus of the protein moieties. In one embodiment, the linker linking the anchor protein, and the foreign protein is a peptide comprised of glycine (Gly)n, wherein n is an integer that is the same or higher than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In another embodiment, the linker linking the anchor protein, and the foreign protein is a gly-ser linker. As used herein, the term “gly-ser peptide linker” (GS) refers to a peptide comprising or consisting of glycine (G or Gly) and serine (Sor Ser) residues. Exemplary gly-ser peptide linkers comprise the amino acid sequence (Gly4 Ser)n or (Gly3 Ser)n. Another exemplary gly-ser peptide linker comprises the amino acid sequence S(Gly4 Ser)n wherein n is an integer that is the same or higher than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. As used herein, a “fragment” of a polypeptide refers to a single amino acid or a plurality of amino acid residues comprising an amino acid sequence that has at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 20 contiguous amino acid residues or at least 30 contiguous amino acid residues of a sequence of the polypeptide. A “variant” include a peptide having the same function as an identified peptide, while having a different sequence or being derived from a different organism than an identified peptide. As used herein, a “fragment” of poly- or oligonucleotide refers to a single nucleic acid or to a polymer of nucleic acid residues comprising a nucleic acid sequence that has at least 15 contiguous nucleic acid residues, at least 30 contiguous nucleic acid residues, at least 60 contiguous nucleic acid residues, or at least 90% of a sequence of the polynucleotide. In some embodiment, the fragment is an antigenic fragment, and the size of the fragment will depend upon factors such as whether the epitope recognized by an antibody is a linear epitope or a conformational epitope. Thus, some antigenic fragments will consist of longer segments while others will consist of shorter segments, (i.e., 5, 6, 7, 8, 9, 10, 11 or 12 or more amino acids long, including each integer up to the full length of the polypeptide). Those skilled in the art are well versed in methods for selecting antigenic fragments of proteins. The term “endogenous” gene or protein means that said gene or protein is expressed from a gene naturally found in the genome of a eukaryotic cell. The term “heterologous” gene or protein means that said gene or protein is not expressed from a gene naturally found in the genome of a eukaryotic cell. The term “gene” or “sequence” refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (i.e., a polypeptide or a functional RNA) in some manner. A gene includes untranslated regulatory regions of DNA (i.e., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (down-stream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). The term “structural gene” as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide. It should be noted that any reference to a SEQ ID, or sequence specifically encompasses that sequence, as well as all corresponding sequences that correspond to that first sequence. For example, for any amino acid sequence identified, the specific specifically includes all compatible nucleotide (DNA and RNA) sequences that give rise to that amino acid sequence or protein, and vice versa. A “polynucleotide” refers to a single nucleotide or a polymer of nucleic acid residues of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides, and / or their analogs and may be double-stranded or single stranded. A polynucleotide can comprise modified nucleic acids (i.e., methylated), nucleic acid analogs or non-naturally occurring nucleic acids and can be interrupted by non-nucleic acid residues. For example, a polynucleotide includes a gene, a gene fragment, cDNA, isolated DNA, mRNA, tRNA, rRNA, isolated RNA of any sequence, recombinant polynucleotides, primers, probes, plasmids, and vectors. Included within the definition are nucleic acid polymers that have been modified, whether naturally or by intervention. A nucleic acid molecule may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (i.e., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties: for example, peptides; intercalators: for example, acridine, psoralen, etc.; chelators; alkylators; and modified linkages: for example, alpha anomeric nucleic acids, etc.). The term “nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hair-pinned, circular, and padlocked conformations. A polynucleotide sequence is operably linked to an expression control sequence(s) (i.e., a promoter and, optionally, an enhancer) when the expression control sequence controls and regulates the transcription and / or translation of that polynucleotide sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (i.e., degenerate codon substitutions), the complementary (or complement) sequence, and the reverse complement sequence, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see i.e., Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy of nucleic acid codons, one can use various different polynucleotides to encode identical polypeptides. An expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. As used herein, “expression cassette” refers to a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The peptides of the invention of the present invention may be fusion peptides also referred to as chimeric. The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti- sense) strand. The coding sequence which encodes the peptides may be identical to the coding sequence shown in the sequence listing, or that of any of the deposited clones, or may be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same fusion proteins as shown in the sequence listing. The term “nucleotide sequence encoding a peptide” encompasses a nucleotide sequence which includes only coding sequences for the polypeptide, i.e., heterologous protein, as well as a polynucleotide which includes additional coding and / or non-coding sequences. Thus, for example, the polynucleotides of the present invention may encode for a peptide, i.e., a heterologous protein, or for a peptide having a prosequence or for a protein having both a prosequence and presequence. The polynucleotides of the present invention may also have the coding sequence fused in frame to, for example, a marker sequence which allows for identification of the polypeptide of the present invention. The marker sequence may be a GFP protein, a hexa-histidine tag to provide for purification of the fusion protein is used. The invention also relates to vectors, including but not limited to, expression vectors comprising the polynucleotides encoding the fusion proteins of the present invention. Types of vectors for expression for proteins and fusion proteins are well known in the art. In one embodiment, the vector is an expression vector for protein expression in Saccharomyces. Yeast expression vectors are commercially available from manufacturers. To accomplish delivery of therapeutic compositions, such as nucleic acids to target cells, the methods and compositions of the present invention comprises extracellular vesicles (EVs), and preferably EV generated from Saccharomyces, such as S. boulardii or S. cerevisiae. EVs generated from Saccharomyces spp. Sometimes are also interchangeably referred to as Saccharomyces- generated extracellular vesicles, sometimes also referred to as SGEVs. The term extracellular vesicles are membranous vesicles released from cells. The extracellular vesicles of the methods and compositions of the invention are composed of lipid bilayers that can envelope and carry cargo, and preferably preferentially loaded fusion peptides containing a p-ESM as described herein in its interior. The lipid bilayer of the EVs may also include proteins embedded therein. In some embodiments, the SGEVs of the compositions and methods of the present invention can be exosomes or ectosomes. As is well-known, exosomes are generally formed upon the endocytosis of multivesicular endosomes (MVEs) to form intraluminal vesicles (ILVs) which are subsequently released into the extracellular environment as exosomes, whereas ectosomes are assembled and released from the plasma membrane. Often, the primary structural feature distinguishing ectosomes and ectosomes is diameter. In some embodiments, the diameter of the SGEVs are between about 30 nm to about 180 nm, between about 50 nm to about 200 nm, between about 75 nm to about 250 nm, between about 100 nm to about 300 nm, between about 125 nm to about 350 nm, between about 150 nm to about 400 nm, between about 175 nm to about 450 nm, between about 200 nm to about 500 nm, between about 250 nm to about 550 nm, between about 300 nm to about 600 nm, between about 350 nm to about between about 650 nm, between about 400 nm to about 700 nm, between about 450 nm to about 750 nm, between about 500 to about 800 nm, between about 550 nm to about 850 nm, between about 600 nm to about 900 nm, between about 650 nm to about 950 nm, between about 700 nm to about 1000 nm, between about 750 nm to about 1050 nm, between about 800 nm to about 1100 nm, between about 850 nm to about 1150 nm or between about 900 nm to about 1200 nm. Thus, exosomes may comprise components on their membrane surface, including but not limited to proteins, glycoproteins, proteoglycans, carbohydrates and lipids, which may be used to direct cargo into to exosome. As understood by the disclosure herein, Saccharomyces is a single-celled organism, but the term “extracellular vesicle,” as it relates to the SGEVs, refers to vesicles that are secreted from Saccharomyces into the local environment, such as, but not limited to cell culture medium and organisms that may have ingested or consumed or been administered the Saccharomyces secreting the vesicles containing a therapeutic composition. In one embodiment, the SGEVs are secreted from Saccharomyces cerevisiae or Saccharomyces boulardii. The disclosure also relates to methods of making and using these Saccharomyces- generated EVs. In one embodiment, the methods of making the SGEVs of the present invention comprises introducing into the Saccharomyces the expression vector encoding one or more novel peptides of the present invention to generate a host Saccharomyces cell. The host cell is then cultured under conditions to permit peptide production from the vector encoding the heterologous nucleotide. In one embodiment, the host cells of the present invention Saccharomyces cerevisiae or Saccharomyces boulardii. Culture conditions for culturing yeast host cells are well-known in the art. The continued culture of the host cell will permit production and secretion of the SGEVs into the cell culture environment, where they can be isolated from culture. Methods of isolating extracellular vesicles, such as exosomes, from cell culture media are well- known in the art and are reviewed in Li, P. et al., Theranostics, 7(3):789-804 (2017), which is incorporated by reference herein. Generally speaking, methods of isolating the SGEVs from culture include but are not limited to ultracentrifugation methods, size-based exclusion methods, immunoaffinity capture-based methods, precipitation methods, microfluidics-based methods or some combination thereof. The route of administration of the SGEVs includes, but is not limited to, topical, Transdermal, intranasal, rectal, oral, subcutaneous, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal as disclosed herein. In one example SGEV’s may be derived or isolated from a GRAS and / or probiotic yeast cell, such as Saccharomyces cerevisiae, and preferably Saccharomyces boulardii. For example, Saccharomyces boulardii probiotics, releasing wild type exosomes, have been shown to diminish disease severity by reducing the expression of inflammatory cytokines and stimulating the expression of anti- inflammatory cytokines in multiple organs including the lungs and cardiovascular system. Saccharomyces boulardii cells also have low immunogenicity and positively modulate host immune response in the presence of additional antigens. Sb is well established for to allows the present inventors to engineer the Saccharomyces boulardii strain for expression and loading of specific xenobiotics in exosomes. Cultivation of Saccharomyces boulardii is fast, low-cost, and easy to scale up using established procedures. Finally, the lipids present in EVs are natural and thus not likely to be cytotoxic when used therapeutically unlike artificial lipids frequently used to package vaccines. In specific embodiments, the oral administration of the SGEVs include administering engineered yeast, producing the SGEVs, as a probiotic. As used herein, a probiotic is a microorganism, such as a bacteria or yeast, generally recognized as safe for human or animal consumption. The probiotics of the resent invention may or may not have additional health benefits to the consumer. In specific embodiments of the present invention, the probiotics is a Saccharomyces cerevisiae or a Saccharomyces boulardii. For example, Saccharomyces boulardii probiotics, releasing wild type exosomes, have been shown to diminish disease severity by reducing the expression of inflammatory cytokines and stimulating the expression of anti- inflammatory cytokines in multiple organs including the lungs and cardiovascular system. Saccharomyces boulardii cells also have low immunogenicity and positively modulate host immune response in the presence of additional antigens. Saccharomyces boulardii is well established for genetic manipulation which allows the present inventors to engineer the Saccharomyces boulardii strain for expression and loading of specific therapeutic compositions in exosomes. Finally, cultivation of Saccharomyces boulardii is fast, low-cost, and easy to scale up using established procedures. The probiotic used in the methods of administering will be engineered to produce the SGEVs of the present invention. The term “introducing,” “administered” or “administering”, as used herein, refers to any method of providing a composition of EVs to a patient such that the composition has its intended effect on the patient. In one embodiment, EVs may be introduced to a patient in vivo, while in other alternative embodiments, EVs may be introduced to subject cells in vitro which may then be administered to a patient in vivo. The term “patient,” or “subject” as used herein, is a human or animal and need not be hospitalized. For example, out-patients, persons in nursing homes are “patients.” A patient may comprise any age of a human or non-human animal and therefore includes both adult and juveniles (i.e., children). It is not intended that the term “patient” connote a need for medical treatment, therefore, a patient may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies. Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc.. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters unless otherwise noted. EXAMPLES Example 1: Materials and Methods for identification of protein EV motifs. Saccharomyces boulardii (ATCC MYA-796) was cultivated overnight in YNBD medium. Triplicate cultures were made at 30°C and at 37°C. Approximately 108cells were recovered by centrifugation from each culture. Extracellular vesicles (EVs) were isolated by 1) low-speed centrifugation, 2) 0.45 um filtration of supernatant, 3) tangential flow filtration of filtrate from (2), and 4) dialfiltration and elution of retentate from (3) as EV suspension fraction. Cell pellets and EV fractions were submitted to Purdue Proteomics Facility (PFF) for Global Quantification (LC- MS / MS) using a Q-Exactive Orbitrap HF instrument. Initial analysis of mass spectrophotometric datasets was performed by PFF using MaxQuant (maxquant.org)(a quantitative proteomics software package designed for analyzing large mass-spectrometric data sets). Data files from PFF included raw MS data; also, peptide and protein group lists and iBAQ (intensity-based absolute quantitation; intensities normalized by the size of protein in the list (within sample normalization)) and LFQ (label-free quantitation; the intensities normalized across all data) measurements. The LFQ values were analyzed using the R package DEP (26ioconductor.org / packages / release / bioc / html / DEP.html), which imputes missing values and compares the levels of the same protein under different conditions, indicating whether it has higher expression in Cells or EVs, and provides a statistical measure (p-value) indicating the probability that the observation is a false positive result. These results provided two sets of proteins having significantly greater expression in EVs and Cells, respectively. Differentially expressed proteins were then grouped into the sets shown in Table 4A. Table 4A. Differentially expressed sets of proteins used for motif discovery. Sequence file name Set description ev_biased_prot_sig_2_fold.faa Proteins significantly biased towards the EV fraction having a fold-difference greater than 2 cell_biased_prot_sig_2_fold.faa Proteins significantly biased towards the Cell fraction having a fold-difference greater than 2 Applicants then undertook to identify short sequence features that were significantly over- represented in EV-biased proteins versus cell-biased proteins. As used herein, these sequence features are generally referred to herein protein EV-sorting motifs (p-ESMs). Such p-ESMs can overrepresented in EVs can be determinants of protein sorting into the EV compartment. Applicant’s first approach to motif discovery was to use the STREME program (Bailey TL.2021. STREME: accurate and versatile sequence motif discovery. Bioinformatics.37(18):2834–2840.), which is part of the MEME suite of algorithms (meme-suite.org / meme / ). Applicants employed the parameters: streme –verbosity 1 –oc . –protein –totallength 4000000 –time 14400 –minw 8 – maxw 15 –thresh 0.05 –align center –p motif_file –n control_file In the above command, motif_file refers to the sequence file in which we are searching for overrepresented motifs, and control_file refers to a set of sequences in which we expect diminished presence of such motifs. The pairs of motif_file and control_file that we compared for p-ESM discovery are shown in Table 5. Table 5. The sequence sets used for p-ESM discovery Motif sequences Control sequences ev_biased_prot_sig_2_fold.faa cell_biased_prot_sig_2_fold.faa A second method was applied to discover motifs overrepresented in EV-biased proteins. For the pair of sequence sets to be compared, Applicants extracted every kmer from 4-12 amino acid residues long, counted the sequences in each set containing the kmer, and those lacking the kmer. For each kmer Applicants computed a p-value using Fisher’s exact test, the null hypothesis being that the presence of the kmer and the compartment (Cells versus EVs) were independent of one another. A p-value of less than or equal to 0.05 caused Applicants to reject the null hypothesis and conclude that the presence of the kmer and the compartment were interdependent. The sequence sets compared with this approach are shown in Table 4A. Analysis of multiple motifs occurring in EV-biased proteins Proteins with significantly EV-biased expression with a 2-fold or greater difference were scanned to identify the locations of all p-ESMs. Analysis of multiple motifs occurring in Cell-biased proteins Proteins with significantly Cell-biased expression with a 2-fold or greater difference were scanned to identify the locations of all counter sorting motifs. Discovery of counter-sorting motifs To identify protein sequence motifs that may prevent entry of protein molecules into EVs, we repeated the run of STREME using sets of cell-biased proteins as the sequences in which to find enriched motifs, and the sets of EV-biased proteins as the control sequences, as shown in Table 6. Table 6. Sequence set pairs used for counter-sorting motif discovery. Motif sequences Control sequences cell_biased_prot_sig_2_fold.faa ev_biased_prot_sig_2_fold.faa A second method was applied to discover motifs overrepresented in Cell-biased proteins. For a pair of sequence sets to be compared, Applicants extracted every kmer from 4-12 amino acid residues long, counted the sequences in each set containing the kmer, and those lacking the kmer. For each kmer we computed a p-value using Fisher’s exact test, the null hypothesis being that the presence of the kmer and the compartment (Cells versus EVs) were independent of one another. A p-value of less than or equal to 0.05 caused Applicants to reject the null hypothesis and conclude that the presence of the kmer and the compartment were interdependent. The sequence sets compared with this approach were all significantly Cell biased proteins and all significantly EV biased proteins each having at least a 2-fold difference between the two samples (Table 6). Locations of p-ESMs with respect to 3D protein structure The AlphaFold predicted 3D structures of every available S. boulardii protein were obtained from the European Bioinformatics Institute AlphaFold database web site (alphafold.ebi.ac.uk; Senior et al., 2020). The coordinates of the 20 most EV-biased p-ESMs longer than 8 amino acid residues discovered using the first approach (STREME) were mapped onto their cognate 3D structures and visualized using the Open Source version of the programme PyMOL (github.com / schrodinger / pymol-open-source). Example 2: Protein EV-sorting motif (p-ESM) identification. Protein motifs enriched in sequences biased in their expression towards EVs Proteins having expression levels biased in favor of EVs are shown in Sequence file 1. The motifs shown in Table 1A (incorporated by reference from U.S. Provisional Application No. 63 / 536,478) were found to be significantly enriched in proteins having expression levels biased in favor of EVs using the first detection method (STREME). The motifs shown in Table 7 (incorporated by reference from U.S. Provisional Application No.63 / 536,478) were found to be significantly enriched in proteins having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test). The motifs shown in Table 8 (matching_kmers column) (incorporated by reference from U.S. Provisional Application No.63 / 536,478) are variants of the Table 1A consensus motifs that were identified by STREME in individual proteins. Analysis of multiple motifs occurring in EV-biased proteins The top 25 EV-biased proteins and the p-ESMs they contain are indicated in Table 1 (incorporated by reference from U.S. Provisional Application No. 63 / 536,478) and identified herein according to the amino acid sequences selected from SEQ ID NOs: 5-24. Analysis of multiple motifs occurring in Cell-biased proteins The top 25 Cell-biased proteins (identified by gene name) and the counter sorting motifs (p-CSMs) they contain are indicated in Table 9 (SEQ ID NOs: 618-637). Counter sorting protein motifs Proteins having expression levels biased in favor of Cells are shown in Sequence File 2. The motifs shown in Table 3 (incorporated by reference from U.S. Provisional Application No. 63 / 536,478) were found to be significantly enriched in proteins having expression levels biased in favor of Cells using the first detection method (STREME). No motifs significantly enriched in proteins having expression levels biased in favor of Cells were discovered using the second method. The motifs shown in Table 10 (matching_kmers column) (incorporated by reference from U.S. Provisional Application No.63 / 536,478) are variants of the Table consensus motifs that were identified by STREME in individual proteins. Locations of p-ESMs with respect to 3d protein structure Representative images of three protein structures having identified p-ESMs are shown in Figures 7, 8 , and 9 (Table 12). Table 12. Proteins structures shown in Figures 7, 8, and 9. Protein ID Gene ID Uniprot ID Gene symbol KOH50507.1 KO01_01750 A0A0L8VQQ6 CRH1 KOH48328.1 KO01_04327 A0A0L8VIZ4 SUN4 KOH48453.1 KO01_04453 A0A0L8VJB5 DSE4 Example 3: Engineering S. boulardii to express EVs containing peptides of interest S. boulardii strain design and construction: To create S. boulardii strains expressing recombinant proteins, wild-type S. boulardii was transformed by dsDNA segments including the protein-of-interest expressing cassettes and geneticin-resistance gene flanked on 5’ and 3’ ends by integration sequences homological to sequences from YPRCt3 locus on XVI chromosome. Transformation was performed by electroporation following the protocol described by Benatuil et al (2010). mRNA sequences in all constructs were screened against the presence of potential cell- sorting motifs. EV isolation: Overnight cultures of Saccharomyces boulardii were diluted 100-fold (volumetric) with YPD medium. Cultures were then incubated for 24 h at 30 °C with shaking (200 rpm). For EVs isolation, cells and debris were removed by centrifugation at 3500 × g for 35 min. EVs were concentrated from supernatant using tangential flow filtration device (Pall) with 300 kD membrane. Isolated EVs (retentate) were aliquoted and stored at −80 °C. Delivery of Bradykinin via oral or injected yeast extracellular vesicles: A gene construct encoding multiple yeast p-ESMs, a cell penetrating peptide (CCP) and a protein-of- interest (POI=bradykinin) with associated proteolytic BACE1 and thrombin cleavage sites separating the p-ESM, CCP, and POI domains, respectively (Fig. 4, SEQ ID NO. 1) can be expressed in yeast as described above (Davenport et al., 2005; Yin et al., 2021). A separate gene construct encoding BACE1 with a C-terminal p-ESM (SEQ ID NO.2) can be co-expressed in the yeast strain expressing SEQ ID NO. 1). Proteolytic processing by a yeast EV sorted BACE1 protease containing a p-ESM results in removal of the p-ESM from the adjacent CCP and allows for the CCP-POI (Bradykinin) protein fusion to be released from the yeast EV into the blood. Proteolytic cleavage by BRACE1 is inhibited in yeast and in isolated EVs due to the presence of the cell-permeant, BACE1 competitive inhibitor, Verubecestat. Once the EVs are taken orally or by injection the BACE1 inhibitor diffuses out of the EV allowing BACE1 to cleave off the p-ESM from the CCP releasing the CCP-Bradykinin fusion peptide. Bradykinin is released from the CCP- Bradykinin fusion protein in the blood by cleavage at the thrombin protease recognition site. Delivery of bone morphogenesis protein 9 via oral or injected yeast extracellular vesicles: Gene constructs encoding the TEV protease fused with multiple yeast p-ESMs is expressed in yeast as described above (Fig 10, SEQ ID NO.3). A separate gene construct encoding a protein of interest (POI=BMP9) fused with anchoring yEVs membrane protein (AEMP=Sur7) through peptide linker (SEQ ID NO.14763) and carrying a TEV protease-specific recognition site ENLYFQ(G / S) separating AEMP and POI domain (SEQ ID NO. 4) can be co-expressed in the yeast strain expressing the nucleotide sequence according to SEQ ID NO. 3 (Draebing et al., Yerneni et al., 2021). Proteolytic processing by yeast EVs sorted TEV-protease results in cleavage of POI from anchoring EVs membrane protein. Applicants selected TEV protease (TEVp) (Tobacco Etch Virus nuclear-inclusion-a endopeptidase) as an example of protease suitable for the solubilization of membrane-bound proteins by site-specific cleavage. TEV protease has highly site-specific mode of action and cleaves protein at ENLYFQ(G / S) site. TEVp sequence has internal recognition site for self- cleavage so the protein is slowly losing proteolytic activity and deactivates itself (Yi et al., 2013). As example of protein target for the cleavage of TEV protease Applicants chose bone morphogenetic protein 9 (BMP9) which is inductor and regulator of bone formation. Example 4: Validation of p-ESMs Summary To validate exemplary pESM, a representative transmembrane protein, namely a chloroplast envelope membrane targeted low CO2-inducible protein A (LCIA) (SEQ ID NO. 14765), was reliably sorted into yeast extracellular vesicles via the addition of a protein sorting motif (p-ESM) called KISA which was inserted at the N-terminus of the LCIA membrane protein. Furthermore, no LCIA protein was detected in the cell when KISA (SEQ ID NO. 6464) was included as opposed to the absence of KISA, demonstrating that the KISA p-ESM sorted the LCIA protein to the yEV membrane. The soluble protein Luc2 (SEQ ID NO. 14764) was also sorted into the yEV with KISA incorporated at either the N- or C-terminus of Luc2, however it was more effectively sorted into yEVs with the addition of the KISA motif at the C-terminus of Luc2, indicating a position-specific mechanism. Proportionately, cellular content of Luc2 was higher with the C-terminus motif but was also noted with N-terminus addition. A small (13 kDa) single-domain nanobody enhancer of EGFP, NBE (SEQ ID NO.14766), was also only sorted to the yEV with KISA and was undetected without any motif addition in a Western blot. An in vitro assay confirmed the Western blot findings: the attachment of KISA at the N- or C-terminus resulted in the ability of NBE to enhance EGFP upon lysing. In summary, the positional specificity of the exemplary pESM KISA demonstrates the ability to fine-tune sorting of different protein types into yEVs and maintain proper function. Example 5: Validation of p-ESMs Results. Results indicated that each protein type had a clear preference to maximize sorting to the yEV. For LCIA, the membrane protein, inclusion of KISA at the N-terminus alone was enough for sorting and detection in the yEV, with no protein detection in yeast cell itself (Fig.13). However, the soluble Luc2 displayed different trends, indicating that protein localization in the host organism can coding sequence position specific. Luc2 was detected in yEVs at a higher concentration when the C-terminus was the location of KISA, the opposite of LCIA which was modified by the addition of KISA to the N-terminus. Nonetheless, Luc2 was similar to LCIA in that no heterologous protein was detected in the yEV when no p-ESM was included (Fig.14). Luc2 was detected in the cells when using a non-denaturing PAGE gel, and Luc2 containing the C-terminus p-ESM motif also resulted in a higher detection of protein in the cell (Fig. 15). Only a modest signal was observed in the cell for Luc2 when no p-ESM was included. NBE was detected in the yEV only when p-ESM motifs were included in the coding sequence. Since NBE is expected to be soluble like Luc2, the results obtained in Fig. 16 were unexpected, indicating that the p-ESM motif location is a major factor impacting subcellular localization in yEVs, but other factors of the protein structure such as size or sequence also potentially impact protein localization in yEVs C-terminus p-ESM motif insertion resulted in a low detection of NBE in the yEV; however, when the p-ESM was included at the N-terminus in addition to the C-terminus or alone, a significant improvement was detected in sorting the modified protein into the yEV (Fig. 16). These results illustrate the possibility that specific termini for sorting are potentially independent of any sorting effect at the opposite end of the protein. Importantly, the insertion in NBE of either the N- or N- and C- termini placement of KISA resulted in functionality of the protein, i.e. enhanced EGFP fluorescence in the presence of the nanobody, consistent with the signal intensities observed in the Western blot (Figure 19, Table A). Such a finding is critical; as long as the KISA motif was present at the N-terminus, functionality didn’t depend on whether the C-terminus was modified with KISA or a FLAG-tag. Thisdemonstrates the feasibility of not only sorting a protein into yEVs but sorting functionally active proteins (nanobodies) in yEVs for intracellular delivery. Why Luc2 was sorted more effectively to yEVs, and present in the cell, at higher concentrations with the C-terminus placement of KISA may be due to a different mechanism. Presumably, if the C-terminus motif was responsible for endoplasmic retention (i.e., cellular) retention, the amount of Luc2 in the yEV would be reduced in comparison to Luc2 with no motif (which in our case wasn’t detected at all). Hydrophobicity at the C-terminus has been associated with extracellular secretion (Lai et al., 2008), which may be a contributing factor, although in our findings the N-terminus placement resulted in an improvement in yEV protein sorting as well. The C-terminus placement clearly displayed enhanced total protein content overall in both the cell and yEV (Figs. 14 & 15). Luc2 contains a highly-structured and hydrophobic region which was rendered potentially inaccessible via the addition of KISA. Example 6: Materials and Methods Construct design: Independent gene constructs were used for production of a) Luc2 (SEQ ID NO. 14765), b) low CO2-inducible protein A (LCIA) (SEQ ID NO. 14765), or c) an EGFP- enhancing nanobody (NBE) (SEQ ID NO.14766) in S. boulardii for sorting into yeast extracellular vesicles (yEVs). Luc2 and LCIA served as full-length soluble and membrane proteins, respectively, while NBE represented an example of a very small therapeutic protein (13.07 kDa), all to test with different insertion locations of a single protein sorting motif (p-ESM) termed the “KISA” motif (SEQ ID NO.6464) into the coding sequence of the gene-of-interest. The purpose of inserting the p-ESM into the test protein to be sorted into the yEV was to direct the produced heterologous protein to the yEV from the cell following translation (Fig.12). The KISA motif was a k-mer match of a consensus motif detected via STREME in yEV- biased proteins, TSSSSSSSSSSSS (SEQ ID NO.6), and the KISA motif itself was present in the NCA3p protein (GenBank: KOH49714.1) of S. boulardii. KISA fit several criteria that were desirable for sorting: a) it was present in a highly-expressed (439 TPM) gene, b) was within the most terminal 15% of the amino acid sequence (KISA was within the most N-terminal 1% of the amino acid sequence), c) was a biologically-present motif in S. boulardii as opposed to the pattern consensus motif, TSSSSSSSSSSSS (SEQ ID NO.6), and d) was predicted with high probability via DeepLoc (Thumuluri et al. 2022) to target its associated protein to the extracellular space (96.4%). For all tested proteins, a base construct was made in which the protein had no p-ESM in its amino acid sequence, and one in which the heterologous protein had the KISA motif located on the N-terminus after the start methionine. For both Luc2 and NBE, the KISA motif was also tested at the C-terminus, while for NBE the motif was also tested in combination at both the N- and C- termini. All constructs had a FLAG tag placed at their C-terminus (SEQ ID NOs: 14764-14766). Transformation of S. boulardii: Genes for Luc2, LCIA, and NBE were transformed into E. coli (DH5α, New England Biolabs, Item No. C2987) via the addition of 4 ng of the respective designed pUC57 (GenScript, Item No. SD1176) plasmid (SEQ ID NO.14768) including the gene- of-interest followed by a 30 min incubation on ice and a 40 sec heat shock at 42 °C. Transformed E. coli were grown for 2 hr, following which selection was carried out for 12 hours on LB agar with 50 μg mL-1kanamycin. Positive colonies were grown at 37 °C for 18 hr in LB broth with 50 μg mL-1kanamycin. Plasmid DNA was extracted from positive colony cultures via the EZNA Plasmid DNA Mini Kit I (Omega Bio-Tek, Item No. D6942) and linearized via BamHI and EcoRI. After purification of linearized DNA (PureLink PCR Purification Kit, Thermo Fisher Scientific, Item No. K310001), it was precipitated with 300 mM sodium acetate and ice-cold absolute ethanol (final concentration of 71%) for 18 hr at -20 °C. Precipitated DNA was washed with 70% ethanol then dried and resuspended in nuclease-free water. The processed plasmid DNA was transformed into S. boulardii yeast via electroporation as follows. A culture of wild-type S. boulardii yeast was grown in YPD media to an OD600 of 1.0, after which cells were rinsed in ice-cold water twice. This was followed by a wash in ice-cold electroporation buffer (1 M sorbitol, 1 mM MgCl2) and a 30 min culture in conditioning buffer (100 mM LiAc, 10 mM dithiothreitol) at 30 °C. Cells were electroporated in a 0.2 cm gap electroporation cuvette at 2.5 kV and 25 mcF prior to recovery for 2 hr in YPD media containing 1 M sorbitol. Following recovery, cells were pelleted at 3,000 rcf for 5 min and selected on a YPD agar plate containing 100 μg mL-1G418. For preparation of yEVs, transformed colonies were grown in four 2 L shaker flasks (500 mL total culture in each) of YPD containing 100 μg mL-1G418 for 22 hr at 30 °C prior to yEV extraction for each construct. After the 22 hr culture, the suspension was pelleted at 3,000 rcf for 10 min at 4 °C and supernatant was filtered through 0.45 μm PES filter under vacuum. This supernatant was then subjected to tangential flow filtration at ~83 kPa with a 0.02 m2filtration area and 300 kDa cutoff (Centramate T-Series Cassette, Pall Corp., Item No. OS300T02) and rinsed with PBS prior to elution in a 5 mL volume and a successive 0.22 μm vacuum filtration. Filtered yEV extract was finally concentrated to 1 × 1011– 1 × 1012particles mL-1using a 300 kDa cutoff PES filter (Vivaspin 20, Sartorius, Item No. VS2051) and yEVs were quantified via a nanoparticle tracking analyzer (ZetaView, Particle Metrix). Cell protein extraction: Total protein was extracted from cells to confirm the presence of the translated, heterologous protein. Briefly, cells from each yeast transformant were grown in YPD at 30 °C for 22 hr and normalized to an OD600of 5.0 with extra media. Following this, 15 mL were centrifuged at 3,000 rcf and 4 °C for 5 min prior to three successive washes with 3 mL of ice-cold TBS. The pellet was then re-centrifuged and vortexed for 5 seconds with 500 uL of yeast protein extraction reagent (YPER, Thermo Fisher Scientific, Item No.78991). The mixture was then agitated on a tilting platform for 20 min prior to centrifugation at 14,000 rcf for 10 min. Supernatant was collected and frozen at -20 °C until further processing. Each cell protein extract contained the equivalent lysate of ~1.12 × 109S. boulardii cells. Western blots: Protein from yEVs was not subjected to protein extraction as mentioned above but directly added to an equal volume of the PAGE sample buffer (65.8 mM Tris-HCl, pH = 6.8, 26.3% (w / v) glycerol, and 0.01% bromophenol blue). For denaturing gels, 2.1% SDS was also included in the sample buffer. Protein extracts were run using a 4-20% polyacrylamide gradient PAGE gel (Mini-PROTEAN® TGX™ Precast Protein Gels, Item No.456109). For LCIA and NBE, denatured cell and yEV protein was separated using a running buffer consisting of 25 mM Tris, 192 mM glycine, and 0.1% SDS at pH = 8.3Tris / Gly / SDS buffer, while for Luc2, yEVs and cells were separated using both the aforementioned buffer as well as a native PAGE Tris / Gly buffer with the same components except the 0.1% SDS, separately. LCIA sample separation was carried out at 250 V for 20 min, NBE was run at 150 V for 20 min, and for Luc2, SDS- and native PAGE gels were run at 150 V and 200 V for 30 min, respectively. Transferring and blotting of SDS gels were carried out in accordance with antibody manufacturer’s instructions and Chen et al. (2023), with minor modifications. Gels were transferred to PVDF membranes which were pre-soaked in methanol. A methanol-free transfer buffer was used with ice packs both in and outside the transfer apparatus. For LCIA and NBE detection, samples were transferred for 1.5 hr and 30 min, respectively, at 300 mA; both were probed at a 1:2,500X concentration of mouse anti-FLAG-tag, Abcam, Item No. ab125243). For Luc2 detection, SDS-PAGE gels were transferred at 300 mA for 1.5 hr while native PAGE gels were transferred at 250 mA for 70 min. Both of the Luc2 gel types were probed at 1:2,500X of mouse anti-firefly luciferase (Abcam, Item No. ab16466). All membranes were then secondarily probed and developed via the Pierce™ Fast Western Kit, SuperSignal™ West Pico Kit (anti- mouse, Thermo Fisher Scientific, Item No.35065). Protein structure prediction: Protein structures were predicted using the Deepfold v1 model in AlphaFold2 (Jumper et al., 2021; Lee et al., 2023; Mirdita et al., 2022; Pearce et al., 2022). First rank models were chosen for all figures. PyMOL (Schrödinger, LLC) was used for display of protein structures, with hydrophobicity illustrated as described by Eisenberg et al. (1984). In vitro fluorescence assay: A total of either 75 or 100 ng of recombinant EGFP (Cell Biolabs, Item No. STA-201) was added to 100 uL wells in a 96-well plate. A total of 1.75 × 1010yEVs of each type of NBE strain were added to each EGFP concentration, then incubated in the presence of a final concentration of 1% Triton X-100 (Sigma-Aldrich, Item No. X100). EGFP fluorescence levels were compared to that observed in EGFP-containing wells with only Triton X- 100 and no yEVs. For each EGFP concentration, wild-type S. boulardii yEVs were also added that contained no engineered proteins in order to serve as a control. A full control consisted of 1% Triton X-100 along with PBS. Fluorescence was taken from the bottom of the plate at an excitation of 485 nm and emission of 520 nm following a 5 second shake. For EGFP standards, the fluorescence of the full control was subtracted as background, while for each yEV-treated EGFP well, the fluorescence observed when that yEV was incubated in only 1% Triton X-100 without EGFP was subtracted as background. Analysis of variance was carried out in R (R Core Team, 2024) using the readr (Wickham et al. 2024), multcompView (https: / / lselzer.r- universe.dev / multcompView), and dplyr (Wickham et al. 2023) libraries in R. Means were separated via Tukey’s HSD at α = 0.05. TABLES Table 2: Exemplary Cleavage Enzymes TABLE 4B: Representative Polycationic Cell-Penetrating Peptides (SEQ ID NOs: 612-617)

[0002] TABLE 9: p-CSMs Motifs Gene TDH1 CHC1 HSP26 RPL4A LEU4 SSC1 RPL6B TSL1 RPL35B HTA2 RPS1B HIS4 BMH1 BAT1 HTB2 ACC1 YMR090W MDH3 RPL9B HSP82 GLR1 PST2 PMA1 RPS19B SUI2 TABLE 9 (Cont.) (SEQ ID NO.618-637) SEQ ID NO. p-CSMs Motifs 618 ALSKALKE 619 GETFGVSLLDJ 620 KKLKLKKEKVEKEL 621 LIQFFEGE 622 RLIGDAAK 623 ANAQGNRTTIN 624 DEDELMDD 625 EEIPRLVPVIEVKED 626 ELLEKLYE 627 GAAGGARGG 628 INEPTAAA 629 KKEEEEKAKEKVLKI 630 LFRSTLDP 631 LIRGIVKQIV 632 TRIPKVQK 633 VAPLSLGIETAG 634 VLVPAIFNDELR 635 LJKRNSTIPTKK 636 KAIALDLTLGKE 637 EGVVTPQE Table B. Analysis of variance determining effects of NBEs on 75 ng and 100 ng EGFP. 75 ng 100 ng

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Claims

CLAIMS What is claimed is:

1. A fusion peptide having a first and second domain, the first domain comprising a protein of interest (POI), or fragment thereof, and the second domain comprising a protein extracellular vesicle sorting motif (p-ESM).

2. The peptide of claim 1, further comprising a third domain, the third comprising a cell-penetrating peptide (CCP).

3. The peptide of any of claims 1-2, wherein the CCP is positioned between the p-EMS and the POI.

4. The peptide of any of claims 1-3, further comprising a first cleavage site positioned between the p-EMS and the (CCP).

5. The peptide of any of claims 1-3, further comprising a second cleavage site positioned between the CCP and POI.

6. The peptide of any of claims 4-5, wherein the cleavage site comprises a protease cleavage site.

7. The peptide of any of claims 1-6, wherein said POI comprises a therapeutic peptide, or fragment thereof.

8. The peptide of any of claims 1-7, wherein said POI comprises a diagnostic peptide, or fragment thereof.

9. The peptide of any of claims 1-8, whereinp-ESM is selected from SEQ ID NOs: 5-458, and 638-6666, or a fragment or variant thereof.

10. The peptide of any of claims 1-9, wherein said p-ESM is selected from SEQ ID NOs.25-458, or a fragment or variant thereof.

11. The peptide of any of claims 1-10, wherein said p-ESM is selected from SEQ ID NOs.638- 5524, or a fragment or variant thereof.

12. The peptide of any of claims 1-11, wherein said p-ESM is selected from the amino acid sequences according to SEQ ID NOs.5525-6666, or a fragment or variant thereof.

13. The peptide of any of claims 1-12, wherein said cleavage site is targeted by a protease identified in Table 2.

14. The peptide of any of claims 1-13, wherein said CCP is selected from SEQ ID NOs: 612-617, or a fragment or variant thereof.

15. The peptide of any of claims 1-14, wherein said POI further comprises a protein cell sorting motif (p-CSM) that has been disrupted or removed.

16. The peptide of any of claims 1-15, wherein said p-CSM comprises an endogenous p-CSM within the POI of the first domain.

17. The peptide of any of claims 1-16, wherein said p-CSM is selected from SEQ ID NOs: 459- 611, 618-637, and 6667-14762, or a fragment or variant thereof.

18. The peptide of any of claims 1-17, wherein said p-CSM is selected from SEQ ID NO.6667- 14762, or a fragment or variant thereof.

19. A polynucleotide sequence encoding the fusion peptide of any of claims 1-18.

20. An expression vector comprising the nucleotide sequence of claim 19, wherein said nucleotide sequence is operably linked to a promoter.

21. A yeast cell transformed to express the expression vector of claim 20.

22. The yeast cell of claim 21, wherein said yeast cell is selected from: Saccharomyces cerevisiae, or Saccharomyces boulardii.

23. An extracellular vesicle (EV) isolated from the transformed yeast cell of any of claims 21-22.

24. A pharmaceutical composition comprising the EV of claim 23, and a pharmaceutically acceptable carrier.

25. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 24, to a subject in need thereof.

26. A fusion peptide isolated from the transformed yeast cell or EV of any of claims 21-23.

27. A nucleotide sequence encoding a fusion peptide comprising a first and second domain, the first domain comprising a protein of interest (POI), or fragment thereof, and the second domain comprising a protein extracellular vesicle sorting motif (p-ESM).

28. The nucleotide of claim 27, further comprising a third domain, the third comprising a cell- penetrating peptide (CCP).

29. The nucleotide of any of claims 27-28, wherein the CCP is positioned between the p-EMS and the POI.

30. The nucleotide of any of claims 27-28, further comprising a first cleavage site positioned between the p-EMS and the (CCP).

31. The nucleotide of any of claims 27-30, comprising a second cleavage site positioned between the CCP and POI.

32. The nucleotide of any of claims 27-31, wherein the cleavage site comprises a protease cleavage site.

33. The nucleotide of any of claims 27-32, wherein said POI comprises a therapeutic peptide, or fragment thereof.

34. The nucleotide of any of claims 27-33, wherein said POI comprises a diagnostic peptide, or fragment thereof.

35. The nucleotide of any of claims 27-34, wherein said p-ESM is selected from a nucleotide sequence encoding an amino acid sequence according to SEQ ID NOs: 5-458, and 638-6666, or a fragment or variant thereof.

36. The nucleotide of any of claims 27-35, wherein said p-ESM is selected from a nucleotide sequence encoding a p-ESM selected from SEQ ID NO.25-458, or a fragment or variant thereof.

37. The nucleotide of any of claims 27-36, wherein said p-ESM is selected from a nucleotide sequence encoding a p-ESM selected from SEQ ID NOs. 638-5524, or a fragment or variant thereof.

38. The nucleotide of any of claims 27-37, wherein said p-ESM is selected from a nucleotide sequence encoding a p-ESM selected from SEQ ID NOs. 5525-6666, or a fragment or variant thereof.

39. The nucleotide of any of claims 27-38, wherein said cleavage site is targeted by a protease identified in Table 2.

40. The nucleotide of any of claims 27-39, wherein said CCP is selected from a nucleotide sequence encoding a CCP selected from SEQ ID NOs: 612-617, or a fragment or variant thereof.

41. The nucleotide of any of claims 27-40, wherein said POI further comprises a protein cell sorting motif (p-CSM) that has been disrupted or removed.

42. The nucleotide of any of claims 27-41, wherein said p-CSM comprises an endogenous p-CSM within the POI of the first domain.

43. The nucleotide of any of claims 27-42, wherein said p-CSM is selected from a nucleotide sequence encoding a CSM selected from SEQ ID NOs: 459-611, 618-637, and 6667-14762, or a fragment or variant thereof.

44. The nucleotide of any of claims 27-43, wherein said p-CSM is selected from a nucleotide sequence encoding a p-CSM selected from SEQ ID NO. 6667-14762, or a fragment or variant thereof.

45. An expression vector comprising the nucleotide sequence of any of claims 27-44, wherein said nucleotide sequence is operably linked to a promoter.

46. A yeast cell transformed to express the expression vector of claim 45.

47. The yeast cell of claim 46, wherein said yeast cell is selected from: Saccharomyces cerevisiae, or Saccharomyces boulardii.

48. An extracellular vesicle (EV) isolated from the transformed yeast cell of any of claims 46-47.

49. A pharmaceutical composition comprising the EV of claim 48, and a pharmaceutically acceptable carrier.

50. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 49, to a subject in need thereof.

51. A fusion peptide isolated from the transformed yeast cell or EV of any of claims 46-48.

52. A yeast-generated extracellular vesicle (EV) containing a fusion peptide comprising a first and second domain, the first domain comprising a protein of interest (POI), or fragment thereof, and the second domain comprising a protein extracellular vesicle sorting motif (p-ESM).

53. The EV of claim 52, further comprising a third domain, the third comprising a cell-penetrating peptide (CCP).

54. The EV of any of claims 52-53, wherein the CCP is positioned between the p-EMS and the POI.

55. The EV of any of claims 52-54, further comprising a cleavage site positioned between the p- EMS and the (CCP).

56. The EV of any of claims 52-55, further comprising a cleavage site positioned between the CCP and POI.

57. The EV of any of claims 52- 56, wherein said cleavage site comprises a protease cleavage site.

58. The EV of any of claims 52-57, wherein said POI comprises a therapeutic peptide, or fragment thereof.

59. The EV of any of claims 52-58, wherein said POI comprises a diagnostic peptide, or fragment thereof.

60. The EV of any of claims 52-59, wherein said p-ESM is selected from SEQ ID NOs: 5-458, and 638-6666, or a fragment or variant thereof.

61. The EV of any of claims 52-60, wherein said p-ESM is selected from SEQ ID NO.25-458, or a fragment or variant thereof.

62. The EV of any of claims 52-61, whereinp-ESM is selected from SEQ ID NOs.638-5524, or a fragment or variant thereof.

63. The EV of any of claims 52-62, wherein said p-ESM is selected from SEQ ID NOs. 5525- 6666, or a fragment or variant thereof.

64. The EV of any of claims 52-63, wherein said cleavage site is targeted by a protease identified in Table 2.

65. The EV of any of claims 52-64, wherein said CCP is selected from SEQ ID NOs: 612-617, or a fragment or variant thereof.

66. The EV of any of claims 52-65, wherein said POI further comprises a protein cell sorting motif (p-CSM) that has been disrupted or removed.

67. The EV of any of claims 52-66, wherein said p-CSM comprises an endogenous p-CSM within the POI of the first domain.

68. The EV of any of claims 52-67, wherein said p-CSM is selected from SEQ ID NOs: 459-611, 618-637, and 6667-14762, or a fragment or variant thereof.

69. The EV of any of claims 52-68, wherein said p-CSM is selected from SEQ ID NO. 6667- 14762, or a fragment or variant thereof.

70. A pharmaceutical composition comprising the EV of any of claims 52-69, and a pharmaceutically acceptable carrier.

71. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 70, to a subject in need thereof.

72. A yeast-generated extracellular vesicle (EV) expressing a nucleotide sequence, operably linked to a promoter, encoding:^ a first fusion peptide comprising a first and second domain, the first domain comprising a protein of interest (POI), or fragment thereof, and the second domain comprising a first protein extracellular vesicle sorting motif (p-ESM), and a third domain, the third comprising a cell-penetrating peptide (CCP), wherein a first cleavage site is positionedbetween the p-EMS and the CCP, and optionally a second cleavage site is positioned between the CCP and POI; ^ a second fusion peptide containing a protease enzyme directed to the first and optionally the second cleavage site coupled with a second p-ESM, wherein said protease cleaves the first fusion peptide at the first cleavage site such that the CCP-POI portion of the fusion protein is released from the yeast EV.

73. The EV of claim 72, wherein said first, and second cleavage sites are targeted by different protease enzymes.

74. The EV of any of claims 72-73, wherein said second cleavage site is targeted by a heterologous protease.

75. The EV of any of claims 72-74, wherein said heterologous protease comprises a human protease expressed in a human subject.

76. The EV of any of claims 72-75, wherein said p-ESM is positioned at the c-terminus of the first fusion peptide.

77. The EV of any of claims 72-76, further comprising a protease inhibitor, wherein the inhibitor is contacted with the yeast EV during incubation to inhibit the action of the protease enzyme.

78. The EV of any of claims 72-77, wherein said POI comprises a therapeutic peptide, or fragment thereof.

79. The EV of any of claims 72-78, wherein said POI comprises a diagnostic peptide, or fragment thereof.

80. The EV of any of claims 72-79, wherein said p-ESM is selected from SEQ ID NOs: 5-458, and 638-6666, or a fragment or variant thereof.

81. The EV of any of claims 72-80, wherein said p-ESM is selected from SEQ ID NO.25-458, or a fragment or variant thereof.

82. The EV of any of claims 72-81, wherein said p-ESM is selected from SEQ ID NOs.638-5524, or a fragment or variant thereof.

83. The EV of any of claims 72-82, wherein said p-ESM is selected from SEQ ID NOs. 5525- 6666, or a fragment or variant thereof.

84. The EV of any of claims 72-83, wherein the cleavage site is responsive to a protease identified in SEQ ID NO.459-611.

85. The EV of any of claims 7-84, further comprising a CCP domain selected from SEQ ID NOs: 612-617, or a fragment or variant thereof.

86. The EV of any of claims 72-85, wherein said POI further comprises a protein cell sorting motif (p-CSM) that has been disrupted or removed.

87. The EV of any of claims 72-86, wherein said p-CSM comprises an endogenous p-CSM within the POI of the first domain.

88. The EV of any of claims 72-87, wherein said p-CSM is selected from SEQ ID NOs: 459-611, 618-637, and 6667-14762, or a fragment or variant thereof.

89. The EV of any of claims 72-88, wherein said p-CSM is selected from SEQ ID NO. 6667- 14762, or a fragment or variant thereof.

90. A pharmaceutical compositionthe EV of any of claims 72-89, and a pharmaceutically acceptable carrier.

91. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 90, to a subject in need thereof.

92. A yeast-generated extracellular vesicle (EV) expressing a nucleotide sequence, operably linked to a promoter, encoding: ^ a first fusion peptide comprising a first and second domain, the first domain comprising a protein of interest (POI), or fragment thereof, and the second domain comprising a first protein extracellular vesicle sorting motif (p-ESM), and a third domain, the third comprising a yeast EV anchoring membrane protein, or fragment thereof, wherein a first cleavage site is positioned between the POI and the EV anchoring membrane protein; ^ a second fusion peptide containing a protease enzyme directed to the first cleavage site coupled with a second p-ESM, wherein said protease cleaves the first fusion peptide at the first cleavage site such that the POI portion of the fusion protein is released from the yeast EV.

93. The EV of claim 92, wherein said first and said second domain are separated by a linker.

94. The EV of any of claims 92-93, wherein said yeast EV anchoring membrane protein comprises SUR7, or a fragment thereof.

95. The EV of any of claims 92-94, wherein said protease comprises TEV protease, or a fragment thereof.

96. The EV of any of claims 92-95, wherein said POI comprises a therapeutic peptide, or fragment thereof.

97. The EV of any of claims 92-96, whereinPOI comprises a diagnostic peptide, or fragment thereof.

98. The EV of any of claims 92-97, wherein said p-ESM is selected from SEQ ID NOs: 5-458, and 638-6666, or a fragment or variant thereof.

99. The EV of any of claims 92-98, wherein said p-ESM is selected from SEQ ID NO.25-458, or a fragment or variant thereof.

100. The EV any of claims 92-99, wherein said p-ESM is selected from SEQ ID NOs.638-5524, or a fragment or variant thereof.

101. The EV any of claims 92-100, wherein said p-ESM is selected from SEQ ID NOs. 5525- 6666, or a fragment or variant thereof.

102. The EV of any of claims 92-101, wherein said cleavage site is responsive to a protease identified in Table 2.

103. The EV of any of claims 92-102, and further including CCP domain selected from SEQ ID NOs: 612-617, or a fragment or variant thereof.

104. The EV of any of claims 92-103, wherein said POI further comprises a protein cell sorting motif (p-CSM) that has been disrupted or removed.

105. The EV of any of claims 92-104, wherein said p-CSM comprises an endogenous p-CSM within the POI of the first domain.

106. The EV of any of claims 92-105, wherein said p-CSM is selected from SEQ ID NOs: 459- 611, 618-637, and 6667-14762, or a fragment or variant thereof.

107. The EV of any of claims 92-106, wherein said p-CSM is selected from SEQ ID NO.6667- 14762, or a fragment or variant thereof.

108. A pharmaceutical composition comprising the EV of any of claims 92-107, and a pharmaceutically acceptable carrier.

109. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 107, to a subject in need thereof.

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