Production and delivery of therapeutic nucleic acids packaged in yeast extracellular vesicles embedded in biocompatible implant matrices

Abstract

The present invention relates to a biocompatible matrix material embedded with a quantity of yeast-generated extracellular vesicles (yEVs) configured for the in-situ delivery of one or more therapeutic polynucleotides contained within the yEV, preferably to a diseased or injured tissue or organ.

Description

[0001] PRODUCTION AND DELIVERY OF THERAPEUTIC NUCLEIC ACIDS PACKAGED IN YEAST EXTRACELLULAR VESICLES EMBEDDED IN BIOCOMPATIBLE IMPLANT MATRICES CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of and priority to U.S. Provisional Application No. 63 / 528,123, filed July 21, 2023, the specification, 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 (90245.01041- Sequence-Listing.xml; Size: 31,767 bytes; and Date of Creation: July 18, 2024) which is herein incorporated by reference in its entirety. TECHNICAL FIELD The present invention is directed to novel methods, systems, and compositions to facilitate the active delivery of nucleic acids, and preferably therapeutic human bone morphogenesis mRNAs contained in yeast-generated extracellular vesicles (EVs) which are embedded into a matrix material that is directly applied to a bone surface to stimulate in situ bone growth and repair at the site of application of the EV impregnated matrix material. BACKGROUND One of the major challenges facing the use of therapeutic molecules and compounds, also sometimes referred to as 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 its degradation or 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. An alternative delivery vehicle natural lipid nanovesicles have also been shown to package RNA, proteins, and other small molecules. One example is extracellular vesicles (EVs) or exosomes produced and released from the surface of eukaryotic 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, composition, and heterogeneous cargo. Furthermore, EVs or exosomes produced in human cell lines can potentially be contaminated by human pathogens such as viruses, carry endogenous off-target RNA species, and are expensive to produce in culture. 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 EVs 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 yEVs 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. 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. Significantly, S. boulardii has been safely used as human probiotic for over 65 years. More specifically, S. boulardii has several traits that are ideal to produce EVs for delivery of therapeutics to humans. S. boulardii produces EVs that package RNA, proteins, and other small molecules. In addition, the genome of S. boulardii is available and S. boulardii can be genetically engineered and / or have its genome edited for the purpose of modifying EV targeting or cargo. S. boulardii can also be grown at large scale in fermenters and has a shelf life of over a year 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 composition into EVs for delivery to target cells or tissues. These methods for encapsulating cargo into EVs can be roughly divided into two types: cell-based loading methods and non-cell-based loading methods. In the cell-based loading are typically produced in the donor cells first. After being packaged into EVs, the cargo can be secreted and collected in an EV-carrying manner for therapeutic use. Non- cell-based loading approach involves directly loading chemical or 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 including the topological orientation of cell-specific targeting molecules, increase EV instability and can further result in undesired EV aggregation. Finally, current cell and non-cell-based methods of EV loading lack the ability to precisely, selectively, and uniformly load each EV so that it can be more effectively dosed for therapeutic applications. Another limitation in the delivery of therapeutic molecules includes the ability to target EVs to specific tissues. Certain attempts have been made to engineer EV surface displayed receptor-specific ligands that recognize and bind to specific cell receptors. However, certain diseases and conditions may necessitate more directed delivery EVs containing therapeutic mRNAs to a specific tissue, or site of disease or injury. In other instances, certain diseases and conditions may necessitate higher localized concentrations of EVs containing therapeutic RNAs, or continual localized dosing of the same. As such, there exists a long-felt need for an efficient, and commercially viable method to load therapeutic compositions precisely and uniformly into EVs structures, all while causing minimal disruption to their structure and aggregation patterns, and further administering EVs containing therapeutic RNAs to localized tissues or structures for continual and / or higher concentrated doses as compared to systemic dosing regimens. SUMMARY OF THE INVENTION The present invention is directed to novel methods, systems, and compositions for actively loading nucleic acids, such as RNA oligonucleotides, and in particular therapeutic RNA oligonucleotides into an EV, wherein the EV can deliver the therapeutic RNAs to a target cell thereby treating a disease or condition or generating some physiological or other effect. In a preferred aspect, the EVs containing mRNAs encoding one or more therapeutic proteins can be embedded in a tissue-specific matrix and applied to a localized site of injury or disease. In another aspect, the localized delivery of EVs can include higher localized concentrations of therapeutic RNAs, while in alternative embodiments, the matrix can be configured to provide a time-dependent, or continuous delivery of therapeutic RNAs to a localized tissue or structure, and preferably bone tissue. Additional aspects of the invention will be evident from the specification, figures, and claims provided herein. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Exemplary expression cassette for in vitro synthesis of mRNA-BMP in one embodiment thereof. Figure 2: 3T3 cells incubated for 20 h with hyEVs loaded with Alexa488-labeled mRNA. Green: mRNA-Alexa488; Blue: nucleus, Hoechst staining. Figure 3: Activity of nanoluciferase enzyme in 3T3 cells treated with hyEVs loaded with NLuc-mRNA Figure 4A-B. Expression profile of BMP9-responsive genes (A) Smad7 and (B) Hey1 in MC3T3 cells treated with hyEVs carrying BMP9 mRNA. Figure 5. The loading of HA bone cement disk with CellMask labeled yeast EVs. Figure 6: mRNA-BMP9 expression in Sb-mBMP9 cells and EVs. 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. For the purposes of the present invention, the following terms are defined above. In another aspect, the present invention includes systems to apply EVs containing polynucleotides, such as therapeutic mRNAs encoding a protein directed to treat one or more bone conditions embedded in a matrix material configured to be biologically compatible with bone tissue. As used herein, a therapeutic RNA can include any RNA polynucleotide that can be used to treat a disease or condition, and preferably a disease or condition affect bone cells / tissue in a subject. In one example, a therapeutic RNA can include a mRNA polynucleotide encoding one or more human bone morphogenesis proteins that can treat a bone-related disease or condition, for example by promoting bone formation and regulating osteoclast homeostasis. As used herein, “bone morphogenetic proteins” or BMPs are multi-functional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. BMPs and related genes have shown that BMP signaling plays critical roles in heart, neural, and cartilage development. BMPs also play an important role in postnatal bone formation. BMP activities are regulated at different molecular levels. Preclinical and clinical studies have shown that, for example BMP-2 can be utilized in various therapeutic interventions such as bone defects, bone injuries, arthritis, non-union fractures, spinal fusion, osteoporosis, and root canal surgery, among others. More specifically, the term “bone disease,” or “bone condition” as used herein means a bone disease, disorder or a condition that is either primarily or secondarily associated with bone loss, a therapy that causes or is associated with bone loss, failure of bone to regenerate, osteoporosis, osteopenia, bone fractures, delayed fracture healing, non-union fracture, demineralization of bone (low bone mineral density), osteomyelitis, osteonecrosis, subclinical low bone density or decreased bone strength, tooth extraction tooth socket damage, inflammatory bone loss, bone marrow depression, bone marrow transplantation, peri-tooth implant bone damage, any infectious lesion affecting bone density / mass, chronic periodontitis, bone implants, and bone grafts. Also included are traumatic injury or non-traumatic necrosis associated with e.g., Gaucher' s disease, sickle cell anemia, systemic lupus erythematosus and other conditions. In a preferred embodiment, EVs can be derived, or isolated from yeast cells, preferably Saccharomyces yeast cells growing in culture. As understood by the disclosure herein, Saccharomyces is a single-celled organism, but the term “extracellular vesicle,” as it relates to the EVs, refers to vesicles that are secreted from Saccharomyces into the local environment, such as, but not limited to cell culture medium. In one embodiment, the EVs are secreted from Saccharomyces cerevisiae or Saccharomyces boulardii. In another preferred aspect, the mRNA encoding a therapeutic protein can include one or more extracellular vesicle-sorting motifs (ESM). In another aspect, the present invention includes systems to apply EVs containing polynucleotides, such as mRNAs encoding human bone morphogenesis proteins (BMP2, BMP9) embedded in a matrix material configured to be biologically compatible with bone tissue. In another aspect, the present invention includes systems and methods of embedding yeast EVs carrying bone morphogenesis proteins, such as BMP2 or BMP9) in a hydroxyapatite (HA) matrix for direct application to bone wounds and delivery of EVs in situ by diffusion from the HA matrix. In another preferred aspect, the mRNAs encoding human bone morphogenesis proteins (BMP2, BMP9) can include one or more extracellular vesicle-sorting motifs (ESM). As used herein, the term “matrix material” or “matrix” means a material configured to be embedded with EV that can be applied to a biocompatible cell or tissue and allow in situ delivery of the EVs cargo, which is preferably a therapeutic polynucleotide. In some embodiment, the matrix material is a one bone biocompatible matrix material as generally described herein. In a preferred embodiment, the in-situ delivery of the EVs cargo can be by diffusion from the matrix material and can further be configured to be dose- and time-release controlled. As used herein, the term “biocompatible” means “that no serious systemic toxicity is caused by the presence of the material in a living system. It is contemplated that biocompatible materials may cause some clinically acceptable amounts of toxicity including irritation and / or other adverse reactions in certain individuals. In one preferred aspect, biocompatible matrix material of the invention can include, but not be limited to a biocompatible matrix containing: hydroxy apatite, hyaluronic acid, guar gum, xanthan gum, among other porous bio-compatible matrix materials known in the art. Alternative bone tissue biocompatible matrix material can further include, but not be limited to: soft tissue particles, inorganic polymers or a combination thereof; the soft tissue particles comprises cartilage particles; inorganic particles comprising hydroxyapatite, calcium HA, carbonated calcium HA, beta-tricalcium phosphate (beta-TCP), alpha-tricalcium phosphate (alpha-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), tetracalcium phosphate, biphasic calcium phosphate (BCP), anhydrous dicalcium phosphate (DCPA), dicalcium phosphate dihydrate (DCPD), anhydrous monocalcium phosphate (MCPA), monocalcium phosphate monohydrate (MCPM), and combinations thereof. In another aspect, the present invention includes systems to apply EVs containing polynucleotides configured to be therapeutic for one or more bone disease or conditions. In this preferred aspect, EVs containing polynucleotides, encoding therapeutic proteins directed to treat one or more bone conditions can be embedded in a matrix material configured to be biologically compatible with bone tissue and contacted with or grafted to the bone of a subject in need thereof. In another preferred aspect, EVs containing polynucleotides, encoding therapeutic proteins directed to treat one or more bone conditions can be embedded in a matrix material configured to be biologically compatible with bone tissue and contacted with or grafted to the bone of a subject in need thereof. In another preferred aspect, EVs containing polynucleotides, encoding therapeutic polynucleotides, such as mRNAs encoding human bone morphogenesis proteins (BMP2, BMP9), can be embedded in a matrix material configured to be biologically compatible with bone tissue and contacted with or grafted to the bone of a subject in need thereof. In this preferred aspect, the mRNAs encoding human bone morphogenesis proteins can stimulate site-directed bone development in the subject. The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” “prevent” and grammatical equivalents (including “lower,” “smaller,” etc.) when in reference to the expression of any symptom in an untreated subject relative to a treated subject, mean that the quantity and / or magnitude of the symptoms in the treated subject is lower than in the untreated subject by any amount that is recognized as clinically relevant by any medically trained personnel. In one embodiment, the quantity and / or magnitude of the symptoms in the treated subject is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and / or at least 90% lower than the quantity and / or magnitude of the symptoms in the untreated subject. As used herein, “pharmaceutical compositions” are compositions that include an amount (for example, a unit dosage) of the disclosed VEDs together with one or more non-toxic pharmaceutically acceptable carriers, being additives, including carriers, diluents, and / or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (19th Edition). Pharmaceutical formulations and delivery systems appropriate for the compositions and methods of the disclosure are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20.sup.th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18.sup.th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12.sup.th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11.sup.th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp.253-315). Pharmaceutical compositions / formulations are useful for administration to a subject, in vivo or ex vivo. Pharmaceutical compositions and formulations include carriers or excipients for administration to a subject. As used herein the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically compatible formulation, gaseous, liquid, or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery, or contact. 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 disclosure. A pharmaceutically 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 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 (e.g., 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,” 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA), “Remington's Pharmaceutical Sciences”, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and “Handbook of Pharmaceutical Excipients”, 2nd edition, 1994. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration. Thus, pharmaceutical compositions include carriers (excipients, diluents, vehicles, or filling agents) suitable for administration to any cell, tissue, or organ, in vivo, ex vivo (e.g., tissue or organ transplant) or in vitro, by various routes and delivery, locally, regionally, or systemically. In one embodiment, a biocompatible bone matrix material can be a pharmaceutically acceptable carrier. In a preferred embodiment, the biocompatible bone matrix material is administered to the bone of a subject via grafting. Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen of a pharmaceutical composition described herein is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a subject in practicing the present invention. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, or diagnostic evaluation required, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. Thus, the present invention encompasses intra-patient dose-escalation as determined by the skilled artisan. The term “introducing,” “administered” or “administering”, as used herein, refers to any method of providing “therapeutically effective amount” of a composition of EVs containing one or more therapeutic polynucleotides to a patient such that the composition has its intended effect on the patient. In one embodiment, EVs may be introduced to a patient as part of a biocompatible matrix material that is contacted with, or grafted to a target tissue or structure, such as an bone or other organ of the subject. The term “patient,” or “subject” as used herein, is a human or animal, and preferably a human having a disease or condition that can be treated by administration of the EVs of the invention. A “therapeutically effective amount” of a composition of EVs containing one or more therapeutic polynucleotides is an amount sufficient to provide a therapeutic benefit in the treatment of a disease or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces, or avoids symptoms or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent. A “therapeutically effective amount” may also mean “prophylactically effective amount” of a compound of the present invention, such as EVs containing one or more therapeutic polynucleotides in an amount sufficient to prevent a disease or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. In another aspect, the EVs of the invention may be modified to display surface ligands that target delivery of the EVs to unique receptors displayed on the surfaces of targeted cell types, and preferably bone cells / tissue. The receptor-specific ligands may be fused or anchored to EV membrane proteins and displayed on the EV exterior surface. Examples of EVs displaying surface ligands for directed transport via target cell receptors is described by Sayre et al., in PCT / US2022 / 014958, which is incorporated herein by reference. In another aspect, the invention includes an expression vector having an expression cassette including a nucleotide sequence, operably linked to a promoter, encoding a modified mRNA encoding a bone morphogenesis protein 9 (BMP9) (SEQ ID NO. 2) containing yeast EV RNA sorting motifs (ESM) (GAAGACAC, or SEQ ID NOS.4-5) in its 3’ UTR (See e.g., SEQ ID NO. 26). In another preferred aspect, the invention further includes a yeast cell, and preferably a S. boulardii cell expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a modified mRNA encoding a bone morphogenesis protein 9 (BMP9) (SEQ ID NO.2) containing one or more yeast EV RNA sorting motifs (ESM) (GAAGACAC, or SEQ ID NOS.4- 5) in its 3’ UTR (See e.g., SEQ ID NO. 1). In another embodiment, the invention includes an expression vector having an expression cassette including a nucleotide sequence, operably linked to a promoter, encoding a modified mRNA encoding a bone morphogenesis protein 9 (BMP9) containing 3’ and 5’ untranslated regions (UTRs), such as 3’ and 5’ UTRs from human hemoglobin subunit beta (hHBB) (SEQ ID NO.1), that can further in some embodiments contain one or more yeast EV RNA sorting motifs (ESM) (GAAGACAC, or SEQ ID NOS.4-5) in one or both UTRs. In another preferred aspect, the invention further includes an engineered EV isolated from a yeast cell, and preferably a S. boulardii cell containing a quantity of modified mRNA encoding a bone morphogenesis protein 9 (BMP9) (SEQ ID NO. 1-2) containing yeast EV RNA sorting motifs (ESM) in its 3’ UTR (SEQ ID NO.26). In a preferred embodiment, the EV of the invention is embedded in a matrix material that is biocompatible with bone cells / tissue which can further be contacted with or grafted to the bone of a subject in need thereof. In another preferred embodiment, the embedded EV of the invention includes a human bone receptor ligand anchored to the to an EV-membrane protein. In this embodiment, the EV-membrane protein can be selected from: EVP1, Sur7, C-terminal domain of LAMP2C, or a fragment or variant thereof, while the human bone receptor ligand is selected from: CCR1, CCR7, CCR9, CXCR4, CXCR5, CXCR6, or a fragment or variant thereof. 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” includes 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, (e.g., 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. As described herein, an “expression vector” refers to a recombinant DNA or RNA construct, such as a plasmid 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 may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. As used herein, a “promoter region,” or “promoter” 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. As used herein, “operably linked” refers to a functional arrangement of elements. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter effects the transcription or expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence, and the promoter can still be considered “operably linked” to the coding sequence. Further, 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” encompasses a nucleotide sequence which includes only coding sequences for the polypeptide, e.g., 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, e.g., a heterologous protein, or for a peptide having a prosequence or for a protein having both a prosequence and presequence. In some instances, a nucleotide sequence encodes a gene, or fragment or variant thereof. 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 (e.g., a polypeptide or a functional RNA) in some manner. A gene includes untranslated regulatory regions of DNA (e.g., 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 (e.g., 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 (e.g., 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) (e.g., 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 (e.g., 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 e.g., 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. A “fusion” or “chimera” protein is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame. As used herein, a “functional” polypeptide or “fragment” is one that substantially retains at least one biological activity normally associated with that polypeptide (e.g., nucleosome formation). In particular embodiments, the “functional” polypeptide or “fragment” substantially retains all of the activities possessed by the unmodified peptide. By “substantially retains” biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide). Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. The terms “comprises,” or “comprising” are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean “includes”, “including” and the like. The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. EXAMPLES Example 1: Uptake of LF-hyEVs by model osteoblasts culture 3T3. To evaluate the ability of yEVs to enter into osteoblast cells we labeled mRNA with Alexa488 and loaded it into hyEVs containing lipofectamine-3000. Alexa488-hyEVs were added to common osteoblasts cell model MC3T3 mouse cells and images were taken 20 h after the lipofectamine hyEVs application. Accumulation of green Alexa488 signal was observed in perinuclear area of MC3T3 cells indicating cellular uptake and center-directed transport of hyEVs in the cytoplasm (Figure 2). Example 2: Translation of mRNA delivered by hybrid yEVs in 3T3 osteoblasts cells. To demonstrate that mRNA delivered to MC3T3 cells by hyEVs is functional and could be translated by cells into protein, we loaded hyEVs with mRNA encoding nanoluciferase enzyme whose activity could be easily detected by standard assay (NanoGlow, Promega). NLuc-mRNA (SEQ ID NO.19) was transcribed in vitro and loaded into yEVs isolated via liposomes composed of a sphingosine-EPC lipid mixture (LSE-hyEVs).1010hyEVs in 100 uL PBS were added to wells in 24-well plate seeded with 3T3 cells. Nanoluciferase activity was measured 24h after hyEV application. High levels of NLuc activity were detected in MC3T3 cells treated with hyEVs but not in negative control PBS samples (Figure 3). Example 3: Changes in gene expression profile of MC3T3 cells in response to treatment with hyEVs loaded with mRNA encoding BMP9. Next, we examined the effect of BMP9 mRNA delivered by hyEVs on the expression profile of BMP-responsive genes Smad7 and Hey1 Both Smad7 and Hey1 proteins are involved in regulation of osteogenesis (Nan et al, 2014) and BMP-dependent signaling cascade. It has been shown that their expression is upregulated by BMP9 (Sharff et al, 2009; Eiraku et al, 2019). MC3T3 cells were placed in low serum conditions (2% serum) and simultaneously supplemented with hyEVs-mBMP9. PBS supplementation was used as negative control, and as positive control we used MC3T3 cells transfected with BMP9 mRNA using Lipofectamine 3000. Transcriptional activation of Smad7 and Hey1 expression was assessed 36 h after hyEVs supplementation by RT- qPCR. A 3.5-fold increase in Smad7 expression was observed in cells treated with hyEVs-mBMP9 compared to cells supplemented with PBS. Expression of Hey1 was approximately twice that of cells treated with PBS sample in cells where BMP9 mRNA was delivered via hyEVs similar to cells transfected with BMP9 mRNA by the lipofectamine method (Fig4). It was previously shown that direct stimulation of osteogenesis in MC3T3 cells by BMP9 protein leads to fast elevation of Smad7 and Hey1 expression in the first 6 h after the addition of protein to the cells. In our case, the gene expression response of 3T3 cells to hyEVs carrying mRNA encoding BMP took 36 hours. The observed delay in gene activation response, however, is not surprising taking in account all the additional events related to delivery of not protein itself but its mRNA. Indeed, hyEV uptake by MC3T3 cells, hyEVs cargo release, BMP9 mRNA translation followed by BMP9 protein maturation and secretion should happen for hyEVs-dependent activation of osteogenesis. Example 4: Loading of HA bone cement with fluorescently labeled EVs. Engineered yeast EVs were embedded into a biocompatible matrix material. As shown in Figure 5, the present inventors embedded a hydroxyapatite (HA) bone cement disk with CellMask labeled yeast EVs (red fluorescence). To verify if EVs solution indeed could penetrate the HA disc, we placed the solution of EVs labeled by red lipophilic dye CellMask onto the top surface of HA disk and applied the vacuum to the bottom surface of the disk. After 3 h of vacuum application the red fluorescent signal was detected throughout the disk, demonstrating the penetration of EVs through the porous structure of pre-casted cement disk. Example 5: Co-expression of bone mesenchymal stem cell receptor-specific ligands anchored to EV membrane proteins. In another embodiment, the invention is directed to genetically modified yeast cells adapted to heterologously express and present on the surface of EVs receptor-specific ligands anchored to EV membrane proteins as a fusion peptide. In a preferred embodiment, the anchor portion of the fusion peptide can include one of several EV membrane localized proteins including EVP1 (multi- pass membrane protein), SEQ ID NO.11, Sur7 (multi-pass membrane protein) (SEQ ID NO.10) or the C-terminal domain of LAMP2C (SEQ ID NO. 12) or fragments or variants thereof. In another embodiment, enriched human bone mesenchymal stem cells receptor ligands can be anchored to the EV membrane protein C- or N-terminus so that the ligands are displayed on the surface of the EV. More specifically surface displayed ligands to target EVs to bone cell specific receptors potentially including one or more CC chemokine receptors (CCR1 (SEQ ID NO. 13), CCR7 (SEQ ID NO.14), and CCR9 (SEQ ID NO.15)) and one or more CXC chemokine receptors (CXCR4 (SEQ ID NO.16), CXCR5 (SEQ ID NO.17), and CXCR6 (SEQ ID NO.18)). Example 6: Expression of modified bone morphogenesis protein 9 mRNA and packaging in yeast EVs. For improved sorting of mRNA-BMP9 into extracellular vesicles (EVs), 3 extracellular vesicle-sorting motifs (ESM) (GAAGACAC, or SEQ ID NOS.4-5) were included in the 3’ UTR of mRNA. This expression cassette was integrated into the YPRCt3 locus on XVI chromosome of S. boulardii yielding strain Sb-mBMP9. It was previously shown that integration at this locus does not affect the cell growth and gene expression in S. boulardii. In one embodiment a modified mRNA encoding a bone morphogenesis protein 9 (BMP9) (SEQ ID NO.2) containing yeast EV RNA sorting motifs (ESM) (GAAGACAC, or SEQ ID NOS.4-5) in its 3’ UTR (See e.g., SEQ ID NO. 26) was expressed in engineered S. boulardii cells. The BMP9 containing yeast EVs were isolated from cell-free yeast supernatants by tangential flow filtration using a 300 kD cutoff filter and stored in PBS buffer. As shown in Figure 6, to confirm the expression of mRNA-BMP9 and to evaluate the loading of the mRNA-BMP9 into EVs the present inventors extracted total RNA from yeast cells and EVs from strain Sb-mBMP9 and performed qPCR analysis to measure the comparative level of mRNA-BMP9 in cells and EVs. As a housekeeping gene for the qPCR measurements, DAD3 mRNA was used which is present in both wild type S. boulardii cells and EVs. It was found that while mRNA-NMP9 was present in both cells and EVs from engineered strain, the amount of mRNA-BMP9 was substantially (>20X) higher in EVs than yeast cells. Example 7: Materials and Methods. S. boulardii strains design and construction: To create S. boulardii strains expressing mRNA-NMP9, wild-type S. boulardii was transformed using linear dsDNA fragments including the RNA expressing cassette and geneticin-resistance gene flanked on 5’ and 3’ ends by integrating sequences homologous to sequences from the YPRCt3 locus on XVI chromosome (sequence ID CP046096.1). Transformation was performed by electroporation following the protocol described by Benatuil et al (2010) mRNA synthesis: mRNA was synthesized in vitro using HiScribe T7 High Yield RNA Synthesis kit (NEB). Plasmids for template for mRNA synthesis were created by subcloning genes encoding appropriate mRNA sequences under the control of a T7 promoter into pUC57 plasmid. For improved mRNA stability 5’ and 3’ UTRs of mRNA were stabilized by the addition of hHBB UTRs sequences (Leppek et al, 2022) (Fig1).5’ cap-1 structure and 3’ polyA tail were added by HiScribe kit according to the manufacturer’s instructions. mRNA loading into yEVs: To load mRNA into yEVs we first loaded mRNA into cationic liposomes made from an EPC-sphingosine mixture or from lipofectamine 3000 (Invitrogene) mixture, and then liposomes-mRNA were fused with yEVs yielding hybrid yEVs (referred to as LSE-hyEVs or LF-hyEVs, or hyEVs respectively). EPC (1,2-dioleoyl-sn-glycero-3- ethylphosphocholine, Broadfarm) and sphingosine (SP, Avanti lipids) were dissolved in 1 mL chloroform in 2:1 molar ratio and dried under nitrogen flow to form a thin lipid film. Lipid films were further dried in Labconco FreeZone 2.5 liter benchtop freeze dryer. Then lipid films were hydrated in 1 mL H20, sonicated in a Co-Z digital ultrasonic cleaner for 10 minutes and extruded through 0.1 μm pore size polycarbonate membrane (Cytiva's Whatman) to form liposomes with diameters ranging from 110-130 nm.1011liposomes were incubated with 10 ug mRNA for 60 min. Liposomes loaded with mRNA were mixed with 1011yEVs in 1 ml H2O, incubated for 30 min at 30 C and freeze-thawed 3 times by placing tubes in liquid nitrogen and then in water bath at 37oC. HyEVs made by fusing liposomes with yEVs were extruded through 0.1 μm pore size polycarbonate membrane and purified on PD SpinTrap G-25 column. Cell Culture: The MC3T3-E1 subclone 14 (ATCC CRL-2594) murine preosteoblast cell line obtained from the American Tissue Culture Collection (ATCC) was used in all experiments and maintained in Alpha-Minimum Essential Medium ( ^-MEM) (A1049001 Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS) (ATCC 30-2020), penicillin (100 UI / mL) and streptomycin(100 ^g / mL) (P / S) (15140148 Gibco) at 37 ^C, 5% CO2with humidity. For serum starvation experiments, MC3T3 cells were plated in a 12-well plate (142475 Thermo Scientific) at a density of 1.5 x 105cells per well. Sixteen hours after plating, the media was replaced with ^-MEM supplemented with 2% (FBS) and P / S. In duplicate, cells were treated with LF- hyEV, LF-H2O (control made by performing mock loading in absence of yEVs), LSE- yEV, LSE-H2O, Lipofectamine 3000 (L3000001 Invitrogen), PBS or BMP9 protein (5566-BP- 010 / CF R & D Systems). For mineralization experiments, MC3T3 cells were plated in a 24-well plate (P24-1.5H-N Cellvis) at a density of 2 x 104cells per well. Sixteen hours after plating, the media was replaced with DMEM / F12 (10565018 Gibco) supplemented with 10% (FBS) and P / S. To each well 50 ^g / mL or 60 ^g / mL of L-Ascorbic Acid 2-Phosphate (A25215G Fisher Scientific) was added, in addition to beta-Glycerophosphate (J62121.AD Thermo Fisher) (final concentration 2 mM) and HEPES (H0887 Millipore Sigma) (final concentration 20 mM) EV isolation: Overnight cultures of Saccharomyces boulardii were diluted 100-fold with YPD medium and then incubated for 24 h at 30 °C with shaking (200 rpm). For yEV isolation, cells and debris were removed by centrifugation at 3500 × g for 35 min. yEVs were concentrated from supernatant using tangential flow filtration device (Pall) with a 300 kD cutoff membrane. Isolated yEVs (retentate) were aliquoted and stored at −80 °C. qPCR: Relative BMP9 mRNA expression was measured by quantitative real-time PCR (qRT-PCR). Total RNA was isolated using mirvana kit (ThermoFisher®). Real-time PCR amplification was performed by using a Mx3000P QPCR system (Agilent technologies). A Luna® Universal One-Step RT-qPCR Kit (NEB) was used to perform one step RT-PCR. Oligonucleotides concentration and cycling conditions used were used according to manufacturer recommendations. Gene specific primers are listed in Table 2. Approximately 50 ng of total RNA was used in each reaction. Relative expression level of the specific transcripts was calculated using the native S. boulardii DAD3 mRNA as the internal reference for normalization. Relative Smad7 or Hey1 gene expression in 3T3 cells treated with LSE-WT or LSE hyEVs carrying BMP9 mRNA was measured by quantitative real-time PCR (qRT-PCR). Total RNA was isolated using RNA plus Nucleospin kit (TakaraBio®). A Luna® Universal One-Step RT-qPCR Kit (NEB) was used to perform one step RT-PCR. Real-time PCR amplification was performed using a Mx3000P QPCR system (Agilent technologies®). Oligonucleotide concentrations and cycling conditions used were according manufacturer recommendations. Gene specific primers are listed in Table 4. Approximately 25 ng of total RNA was used in each reaction. Relative expression levels of the specific transcripts were calculated using the Geneticin resistance gene mRNA expression level as the internal reference for normalization. Measurements of Nanoluciferase activity: MC3T3 cells were treated with 100 uL containing 1010hyEVs-mBMP9 or 100 ul PBS, and the activity of nanoluciferase was measured 24 h post-treatment using a NanoGlow kit (Promega) according manufacturer protocol. Loading of EVs to HA disk: 5 mL of yEVs solution having a yEV concentration 1011yEVs / mL were stained using CellMaskTMlipid dye (Molecular probes) according to manufacturer’s protocol. After staining the yEVs solution was diluted in 20 mL of PBS and concentrated to 1 mL in 300 kD cutoff spin filtration unit (Sartorius). The sample was rinsed with PBS 3 times, and yEVs were resuspended in final volume of 1 mL. The pre-cast hydroxyapatite disk was pre-soaked in PBS for 3 h, then 50 uL of labeled fluorescent yEVs was applied at the top surface, and vacuum force was applied to bottom surface. As yEVs infiltrated into the disk, new drops of yEV solution were applied to the top of the disk, until the total volume of 200 µL of yEVs was absorbed by the disk. At this point, the red fluorescent dye was apparent on the bottom surface of the disk, marking the penetration of EVs through the disk. The image of the cross-section of the disk was taken on an inverted fluorescent microscope. TABLES Table 1. Strains and cells lines used in this work Strains Genotype OriginSb-BMP9-C BMP9-C This invention Oligo Sequence descriptionqDAD3-F AACTATTCGCAACACAGGCAAC (SEQ ID NO.5) Primer for qPCR for DAD3 3 9 9 name SequenceSec2GAAGACAC Oligos (gene) Sequence Description qSmad7-F TTTCTCAAACCAACTGCAGGC (SEQ ID NO.20) Primer used for qPCR

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[0003] SEQUENCE LISTING SEQ ID NO.1 DNA mRNA-BMP9* Homo sapiens acatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccATGTGTCCTGGTGCCTTGTG GGTTGCGCTGCCACTCCTGAGTCTGCTGGCTGGGTCACTGCAGGGGAAGCCCCTGCAAAGCTGGGGCCGC GGAAGCGCTGGTGGTAATGCTCATAGTCCTCTGGGAGTACCAGGTGGCGGACTTCCCGAGCACACCTTCA ACCTCAAAATGTTCCTTGAGAATGTCAAAGTCGATTTCCTTAGGAGCCTCAATTTGAGTGGAGTGCCCTC ACAGGACAAAACTAGGGTAGAACCTCCCCAATACATGATAGACCTTTACAATCGATATACTTCAGACAAA TCCACGACTCCGGCAAGTAACATAGTGAGGTCCTTTTCCATGGAGGACGCTATAAGCATTACTGCAACTG AGGACTTTCCATTCCAGAAGCACATCCTGTTGTTTAATATTAGCATCCCTCGCCATGAGCAGATTACTCG CGCCGAGTTGAGGCTTTATGTAAGCTGTCAGAACCATGTAGACCCTTCTCATGATCTGAAGGGGTCCGTA GTAATTTATGATGTTTTGGACGGAACTGATGCGTGGGATAGTGCCACTGAAACAAAAACtTTCCTGGTGT CCCAGGACATCCAAGACGAAGGCTGGGAAACgCTTGAAGTGAGCAGCGCTGTCAAACGCTGGGTGCGATC AGATTCTACAAAAAGTAAGAATAAGTTGGAGGTCACAGTCGAGTCACACAGGAAGGGTTGTGACACCCTG GACATTTCCGTGCCTCCAGGGAGCAGGAACTTGCCATTCTTTGTAGTGTTTTCCAACGACCATAGTTCAG GTACGAAAGAAACACGCCTGGAGTTGCGCGAGATGATCTCACATGAGCAAGAGAGCGTGCTGAAAAAATT GTCTAAGGACGGCTCCACGGAAGCTGGAGAATCTTCCCATGAGGAGGACACCGATGGCCATGTAGCAGCG GGGAGTACGCTCGCCCGCCGAAAAAGGAGCGCTGGCGCCGGAAGTCATTGTCAAAAAACGTCTCTGCGCG TTAATTTTGAAGATATCGGGTGGGATTCATGGATTATCGCTCCAAAAGAATATGAAGCCTACGAGTGTAA AGGTGGCTGCTTCTTTCCCCTTGCTGACGACGTCACTCCTACCAAACATGCAATCGTTCAGACTCTTGTT CATCTGAAGTTCCCCACAAAGGTTGGAAAAGCGTGTTGTGTACCCACCAAGCTTTCCCCTATCAGCGTAC TGTACAAAGACGATATGGGGGTTCCTACCTTGAAATACCACTATGAAGGTATGTCCGTAGCGGAGTGTGG CTGTCGGTGAgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactac taaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcatt gcaa *5’ and 3’hHBB UTRs are in low case, BMP9 CDS is in capital case SEQ ID NO.2 DNA mRNA-BMP9 Homo sapiens ATGTGTCCTGGTGCCTTGTGGGTTGCGCTGCCACTCCTGAGTCTGCTGGCTGGGTCACTGCAGGGGAAGC CCCTGCAAAGCTGGGGCCGCGGAAGCGCTGGTGGTAATGCTCATAGTCCTCTGGGAGTACCAGGTGGCGG ACTTCCCGAGCACACCTTCAACCTCAAAATGTTCCTTGAGAATGTCAAAGTCGATTTCCTTAGGAGCCTC AATTTGAGTGGAGTGCCCTCACAGGACAAAACTAGGGTAGAACCTCCCCAATACATGATAGACCTTTACA ATCGATATACTTCAGACAAATCCACGACTCCGGCAAGTAACATAGTGAGGTCCTTTTCCATGGAGGACGC TATAAGCATTACTGCAACTGAGGACTTTCCATTCCAGAAGCACATCCTGTTGTTTAATATTAGCATCCCT CGCCATGAGCAGATTACTCGCGCCGAGTTGAGGCTTTATGTAAGCTGTCAGAACCATGTAGACCCTTCTC ATGATCTGAAGGGGTCCGTAGTAATTTATGATGTTTTGGACGGAACTGATGCGTGGGATAGTGCCACTGA AACAAAAACtTTCCTGGTGTCCCAGGACATCCAAGACGAAGGCTGGGAAACgCTTGAAGTGAGCAGCGCT GTCAAACGCTGGGTGCGATCAGATTCTACAAAAAGTAAGAATAAGTTGGAGGTCACAGTCGAGTCACACA GGAAGGGTTGTGACACCCTGGACATTTCCGTGCCTCCAGGGAGCAGGAACTTGCCATTCTTTGTAGTGTT TTCCAACGACCATAGTTCAGGTACGAAAGAAACACGCCTGGAGTTGCGCGAGATGATCTCACATGAGCAA GAGAGCGTGCTGAAAAAATTGTCTAAGGACGGCTCCACGGAAGCTGGAGAATCTTCCCATGAGGAGGACA CCGATGGCCATGTAGCAGCGGGGAGTACGCTCGCCCGCCGAAAAAGGAGCGCTGGCGCCGGAAGTCATTG TCAAAAAACGTCTCTGCGCGTTAATTTTGAAGATATCGGGTGGGATTCATGGATTATCGCTCCAAAAGAA TATGAAGCCTACGAGTGTAAAGGTGGCTGCTTCTTTCCCCTTGCTGACGACGTCACTCCTACCAAACATG CAATCGTTCAGACTCTTGTTCATCTGAAGTTCCCCACAAAGGTTGGAAAAGCGTGTTGTGTACCCACCAA GCTTTCCCCTATCAGCGTACTGTACAAAGACGATATGGGGGTTCCTACCTTGAAATACCACTATGAAGGT ATGTCCGTAGCGGAGTGTGGCTGTCGGTGA SEQ ID NO.3 DNA Sec4 Artificial CAACAGCAACAAC SEQ ID NO.4 DNA Sec8 – RNA Artificial GAAGAAGAAGAAG SEQ ID NO.5 DNA qDAD3-F AACTATTCGCAACACAGGCAAC SEQ ID NO.6 DNA qDAD3-R ACCTCTATGTCCCTCATCTCCT SEQ ID NO.7 DNA qBMP9-F CAGCCTTAACCTGAGTGGGG SEQ ID NO.8 DNA qBMP9-R TCTTCCATGCTGAAGCTCCG SEQ ID NO.9 Amino Acid DAD3 S. boulardii MEHNLSPLQQEVLDKYKQLSLDLKALDETIKELNYSQHRQQHSQQETVSPDEILQEMRDIEVKIGLVGTL LKGSVYSLILQRKQEQESLGSNSK SEQ ID NO.10 Amino Acid SUR7 Saccharomyces cerevisiae MVKVWNIVLRLVVLLFLAGNTLLLILMIISGATDHYPVNRFYWVQGNTTGIPNAGDETRWTFWGACLQDK DGSDTCTSNLAPAYPISPVDNFNTHINVPHQFISKRDAFYYLTRFSFCFFWIALAFVGVSFILYVLTWCS KMLSEMVLILMSFGFVFNTAAVVLQTAASAMAKNAFHDDHRSAQLGASMMGMAWASVFLCIVEFILLVFW SVRARLASTYSIDNSRYRTSSRWNPFHREKEQATDPILTATGPEDMQQSASIVGPSSNANPVTATAATEN QPKGINFFTIRKSHERPDDVSV SEQ ID NO.11 Amino Acid EVP1 Candida albicans MCCILEIFAVTGSYDNKKYLTDTYLINFHIDSLDLTKLIDSSRITKRDGVGSKMVSINPRQGTPTKRAPA ESGWYYVPGGSNTQSGPSATASSGGNAVASGSAINWWSPVESGSATSSGSGSGSTSSGSSGSDSSNSGST SGGYYTTVSQAVHSLLSNVNPQVLGMAQVYSVGFWGYCRGYVIQDDSKKTKKFDNSNVNYTWCSEPKVSF FFNPVEIFKKEMNNTLYGIQVDSQAAGIGQLSYTQKSELKVLIDHLDIDELNLPGNINGKLQQLHNLTNA SFGLLMAVAVLSFVSVLIQMLAFCFSPEKCCLSFLNFLFECIIGLIAFVGAVVVTATYSYVKAQVNGNSD TFGVKSFLSINFYAFVWSAVIVCILVVFFNLLGHCCGLFGTRRHYRTVRNPQPDAEQHKEETESD SEQ ID NO.12 Amino Acid LAMP2C fragment Mus musculus MSADSDLNFLIPVAVGVALGFLIIVVFISYMIGRRKSRTGYQS SEQ ID NO.13 Amino Acid CCR1 Homo sapiens METPNTTEDYDTTTEFDYGDATPCQKVNERAFGAQLLPPLYSLVFVIGLVGNILVVLVLVQYKRLKNMTS IYLLNLAISDLLFLFTLPFWIDYKLKDDWVFGDAMCKILSGFYYTGLYSEIFFIILLTIDRYLAIVHAVF ALRARTVTFGVITSIIIWALAILASMPGLYFSKTQWEFTHHTCSLHFPHESLREWKLFQALKLNLFGLVL PLLVMIICYTGIIKILLRRPNEKKSKAVRLIFVIMIIFFLFWTPYNLTILISVFQDFLFTHECEQSRHLD LAVQVTEVIAYTHCCVNPVIYAFVGERFRKYLRQLFHRRVAVHLVKWLPFLSVDRLERVSSTSPSTGEHE LSAGF SEQ ID NO.14 Amino Acid CCR7 Homo sapiens MDLGKPMKSVLVVALLVIFQVCLCQDEVTDDYIGDNTTVDYTLFESLCSKKDVRNFKAWFLPIMYSIICF VGLLGNGLVVLTYIYFKRLKTMTDTYLLNLAVADILFLLTLPFWAYSAAKSWVFGVHFCKLIFAIYKMSF FSGMLLLLCISIDRYVAIVQAVSAHRHRARVLLISKLSCVGIWILATVLSIPELLYSDLQRSSSEQAMRC SLITEHVEAFITIQVAQMVIGFLVPLLAMSFCYLVIIRTLLQARNFERNKAIKVIIAVVVVFIVFQLPYN GVVLAQTVANFNITSSTCELSKQLNIAYDVTYSLACVRCCVNPFLYAFIGVKFRNDLFKLFKDLGCLSQE QLRQWSSCRHIRRSSMSVEAETTTTFSP SEQ ID NO.15 Amino Acid CCR9 Homo sapiens MTPTDFTSPIPNMADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFASHFLPPLYWLVFIVGALGNSLVI LVYWYCTRVKTMTDMFLLNLAIADLLFLVTLPFWAIAAADQWKFQTFMCKVVNSMYKMNFYSCVLLIMCI SVDRYIAIAQAMRAHTWREKRLLYSKMVCFTIWVLAAALCIPEILYSQIKEESGIAICTMVYPSDESTKL KSAVLTLKVILGFFLPFVVMACCYTIIIHTLIQAKKSSKHKALKVTITVLTVFVLSQFPYNCILLVQTID AYAMFISNCAVSTNIDICFQVTQTIAFFHSCLNPVLYVFVGERFRRDLVKTLKNLGCISQAQWVSFTRRE GSLKLSSMLLETTSGALSL SEQ ID NO.16 Amino Acid CXC4 Homo sapiens MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLR SMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVH ATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLIL PGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTV HKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFH SS SEQ ID NO.17 Amino Acid CXCR5 Homo sapiens MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSLIFLLGVIGNV LVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLL ACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEILFAKVSQGHHNNSLPRCTFSQENQA ETHAWFTSRFLYHVAGFLLPMLVMGWCYVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIF LDTLARLKAVDNTCKLNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQ LFPSWRRSSLSESENATSLTTF SEQ ID NO.18 Amino Acid CXCR6 Homo sapiens MAEHDYHEDYGFSSFNDSSQEEHQDFLQFSKVFLPCMYLVVFVCGLVGNSLVLVISIFYHKLQSLTDVFL VNLPLADLVFVCTLPFWAYAGIHEWVFGQVMCKSLLGIYTINFYTSMLILTCITVDRFIVVVKATKAYNQ QAKRMTWGKVTSLLIWVISLLVSLPQIIYGNVFNLDKLICGYHDEAISTVVLATQMTLGFFLPLLTMIVC YSVIIKTLLHAGGFQKHRSLKIIFLVMAVFLLTQMPFNLMKFIRSTHWEYYAMTSFHYTIMVTEAIAYLR ACLNPVLYAFVSLKFRKNFWKLVKDIGCLPYLGVSHQWKSSEDNSKTFSASHNVEATSMFQL SEQ ID NO.19 DNA NLuc mRNA* Artificial gacccaagcttggtaccgagctcggatccgccaccatgaagaccttaattcttgccgttgcattagtcta ctgcgccactgttcattgccagATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCC GGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCG TAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCC GTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGAT GATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCG ACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCT GTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACC ATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAAggacaagcatcaagcacgcagc aacaagcacgagcaatgagccagcctagccacacccccacgggaaacagcagtgattaacctttagcaat aaacgaaagtttaaccaagctatactaaccccagggttggtcaatttcgtgccagccacaccgtcacacg attaacccaagtc *5’ UTR is in low case, NLuc CDS is in capital case, 3’ mtRNR1 UTR is in lower italic case. SEQ ID NO.20 DNA qSmad7-F Artifical TTTCTCAAACCAACTGCAGGC SEQ ID NO.21 DNA qSmad7-R Artifical GACACAGTAGAGCCTCCCCA SEQ ID NO.22 DNA qHey1-F Artifical GCCTTTGAGAAGCAGGGATCT SEQ ID NO.23 DNA qHey1-R Artifical GGCATTCCCGAAACCCCAAA SEQ ID NO.24 DNA qGAPDH-F Artifical AACTTTGGCATTGTGGAAGG SEQ ID NO.25 DNA qGAPDH-R Artifical CACATTGGGGGTAGGAACAC SEQ ID NO.26 DNA mRNA-BMP9 + 3’ URT (lower letters) Homo sapiens ATGTGTCCTGGGGCACTGTGGGTGGCCCTGCCCCTGCTGTCCCTGCTGGCTGGCTCCCTACAGGGGAAGCCACTGCA GAGCTGGGGACGAGGGTCTGCTGGGGGAAACGCCCACAGCCCACTGGGGGTGCCTGGAGGTGGGCTGCCTGAGCACA CCTTCAACCTGAAGATGTTTCTGGAGAACGTGAAGGTGGATTTCCTGCGCAGCCTTAACCTGAGTGGGGTCCCTTCG CAGGACAAAACCAGGGTGGAGCCGCCGCAGTACATGATTGACCTGTACAACAGGTACACGTCCGATAAGTCGACTAC GCCAGCGTCCAACATTGTGCGGAGCTTCAGCATGGAAGATGCCATCTCCATAACTGCCACAGAGGACTTCCCCTTCC AGAAGCACATCTTGCTCTTCAACATCTCCATTCCTAGGCATGAGCAGATCACCAGAGCTGAGCTCCGACTCTATGTC TCCTGTCAAAATCACGTGGACCCCTCTCATGACCTGAAAGGAAGCGTGGTCATTTATGATGTTCTGGATGGAACAGA TGCCTGGGATAGTGCTACAGAGACCAAGACCTTCCTGGTGTCCCAGGACATTCAGGATGAGGGCTGGGAGACCTTGG AAGTGTCCAGCGCCGTGAAGCGCTGGGTCCGGTCCGACTCCACCAAGAGCAAAAATAAGCTGGAAGTGACTGTGGAG AGCCACAGGAAGGGCTGCGACACGCTGGACATCAGTGTCCCCCCAGGTTCCAGAAACCTGCCCTTCTTTGTTGTCTT CTCCAATGACCACAGCAGTGGGACCAAGGAGACCAGGCTGGAGCTGAGGGAGATGATCAGCCATGAACAAGAGAGCG TGCTCAAGAAGCTGTCCAAGGACGGCTCCACAGAGGCAGGTGAGAGCAGTCACGAGGAGGACACGGATGGCCACGTG GCTGCGGGGTCGACTTTAGCCAGGCGGAAAAGGAGCGCCGGGGCTGGCAGCCACTGTCAAAAGACCTCCCTGCGGGT AAACTTCGAGGACATCGGCTGGGACAGCTGGATCATTGCACCCAAGGAGTATGAAGCCTACGAGTGTAAGGGCGGCT GCTTCTTCCCCTTGGCTGACGATGTGACGCCGACGAAACACGCTATCGTGCAGACCCTGGTGCATCTCAAGTTCCCC ACAAAGGTGGGCAAGGCCTGCTGTGTGCCCACCAAACTGAGCCCCATCTCCGTCCTCTACAAGGATGACATGGGGGT GCCCACCCTCAAGTACCATTACGAGGGCATGAGCGTGGCAGAGTGTGGGTGCAGGTAGactagttacggtcgtatag cagcacagtgccaatcagtgaagaagaagaagcttagtagaagttgaaaaaggcgttttgcctcaacttgaacagcc ctatgtgttcatcaaacgttcggatgctcgaactgcacaacagcaacaaccctcatggtcatgttatggttgagctg gtagcagaactcgaaggcattcagtacggtcgtagtggtgagacacgaagacacccgcgtggcacacatagcgtata cgaccgtaagttcttaattaaattt

Claims

CLAIMS What is claimed is:

1. A biocompatible composition comprising a matrix material embedded with a therapeutically effective amount of isolated yeast-generated extracellular vesicle (EVs) containing at least one heterologous therapeutic polynucleotide, wherein when said matrix material is contacted with the tissue of a subject, the EVs are delivered to the tissue wherein the therapeutic polynucleotides are translated in-situ.

2. The composition of claim 1, wherein said matrix material comprises a matrix material that is biocompatible with bone tissue.

3. The composition of claim 1, wherein said bone tissue comprises human bone tissue.

4. The composition of any of claims 1-3, wherein the matrix material is selected from: hydroxy apatite, hyaluronic acid, guar gum, xanthan gum, or a combination of the same.

5. The composition of claim 1, wherein the matrix material is selected from: hydroxy apatite, hyaluronic acid, guar gum, xanthan gum, inorganic polymers, soft tissue particles, cartilage particles; hydroxyapatite, calcium HA, carbonated calcium HA, beta-tricalcium phosphate (beta- TCP), alpha-tricalcium phosphate (alpha-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), tetracalcium phosphate, biphasic calcium phosphate (BCP), anhydrous dicalcium phosphate (DCPA), dicalcium phosphate dihydrate (DCPD), anhydrous monocalcium phosphate (MCPA), monocalcium phosphate monohydrate (MCPM), or a combination of the same.

6. The composition of claim 1, wherein the EVs comprise EVs generated from Saccharomyces cerevisiae, or Saccharomyces boulardii.

7. The composition of claim 1, therapeutic polynucleotide comprises a therapeutic messenger ribonucleic acid (mRNA).

8. The composition of claim 7, wherein said therapeutic mRNA has at least one yeast yEV RNA sorting motifs (ESM).

9. The composition of claim 8, wherein said ESM is positioned at the 3’ end of said therapeutic mRNA.

10. The composition of claim 9, ESM is positioned at the 3’ end of said therapeutic mRNA is selected from: GAAGACAC, or SEQ ID NO’s.4-5, or a combination of the same.

11. The composition of any of claims 1-10, wherein said heterologous therapeutic polynucleotide comprises a polynucleotide encoding a human bone morphogenesis protein (BMP), or a fragment or variant thereof.

12. The composition of claim 11, wherein said BMP is selected from: BMP2, BMP9 or a combination of the same.

13. The composition of claim 1, wherein said BMP comprises SEQ ID NO.1, 2 or 26, or a fragment or variant thereof.

14. The composition of claim 1, wherein said heterologous therapeutic polynucleotide comprises a polynucleotide encoding a human bone morphogenesis protein (BMP), or a fragment or variant thereof, having at least one ESM.

15. The composition of claim 14, wherein the BMP having at least one ESM comprises SEQ ID NO.26, or a fragment or variant thereof.

16. The composition of claim 1, further comprising a fusion peptide having a human bone receptor ligand anchored to the to an EV-membrane protein.

17. The composition of claim 16, wherein said EV-membrane protein is selected from: EVP1, Sur7, C-terminal domain of LAMP2C, or a fragment or variant thereof.

18. The composition of claim 16, wherein said EV-membrane protein is selected from: SEQ ID NO.11-12, or a fragment or variant thereof.

19. The composition of claim 16, wherein said human bone receptor ligand is selected from: CCR1, CCR7, CCR9, CXCR4, CXCR5, CXCR6, or a fragment or variant thereof.

20. The composition of claim 19, wherein said human bone receptor ligand is selected from: SEQ ID NO.13-18, or a fragment or variant thereof.

21. A method of treating a bone disease or condition comprising the step of contacting a therapeutically effective amount of the composition of any of claim 1-20 to the bone of a subject in need thereof.

22. The method of claim 21, wherein said subject is a human. 23 The method of claim 21, wherein said step of contacting comprises grafting the composition to the bone of the subject.

24. The method of claim 21, wherein said bone disease or condition is selected from: bone loss, failure of bone to regenerate, osteoporosis, osteopenia, bone fractures, delayed fracture healing, non-union fracture, demineralization of bone, osteomyelitis, osteonecrosis, subclinical low bone density or decreased bone strength, tooth extraction tooth socket damage, inflammatory bone loss, bone marrow depression, bone marrow transplantation, peri-tooth implant bone damage, any infectious lesion affecting bone density / mass, chronic periodontitis, bone implants, bone grafts, traumatic injury, non-traumatic necrosis associated Gaucher' s disease, sickle cell anemia, systemic lupus erythematosus, or a combination of the same.

25. An isolated yeast-generated extracellular vesicle (yEVs) containing at least one heterologous therapeutic mRNA encoding a human bone morphogenesis protein (BMP), or a fragment or variant thereof.

26. The yeast EV of claim 25, wherein the EVs comprise EVs generated from Saccharomyces cerevisiae, or Saccharomyces boulardii.

27. The yeast EV of claim 25, wherein said therapeutic mRNA has at least one yeast EV RNA sorting motifs (ESM).

28. The yeast EV of claim 27, wherein said ESM is positioned at the 3’ end of said therapeutic mRNA.

29. The yeast EV of claim 28, wherein said ESM is positioned at the 3’ end of said therapeutic mRNA is selected from: GAAGACAC, or SEQ ID NOS.4-5, or a combination of the same.

30. The yeast EV of claim 25, wherein said BMP is selected from: BMP2, BMP9 or a combination of the same.

31. The yeast EV of claim 25, wherein said BMP comprises SEQ ID NO. 26, or a fragment or variant thereof.

32. The yeast EV of claim 25, wherein said heterologous therapeutic polynucleotide comprises a polynucleotide encoding a human bone morphogenesis protein (BMP), or a fragment or variant thereof, having at least one ESM.

33. The yeast EV of claim 32, wherein the BMP having at least one ESM comprises SEQ ID NO. 26, or a fragment or variant thereof.

34. The composition of claim 25, further comprising a fusion peptide having a human bone receptor ligand anchored to the to an EV-membrane protein.

35. The composition of claim 34, wherein said EV-membrane protein is selected from: EVP1, Sur7, C-terminal domain of LAMP2C, or a fragment or variant thereof.

36. The composition of claim 34, wherein said EV-membrane protein is selected from: SEQ ID NO.11-12, or a fragment or variant thereof.

37. The composition of claim 34, wherein said human bone receptor ligand is selected from: CCR1, CCR7, CCR9, CXCR4, CXCR5, CXCR6, or a fragment or variant thereof.

38. The composition of claim 34, wherein said human bone receptor ligand is selected from: SEQ ID NO.13-18, or a fragment or variant thereof.

39. A method of treating a bone disease or condition comprising the step of contacting a therapeutically effective amount of the composition of any of claim 25-38 embedded in a biocompatible bone matrix material to the bone of a subject in need thereof.

40. The method of claim 39, wherein said subject is a human. 41 The method of claim 39, wherein said step of contacting comprises contacting the bone of a subject with a therapeutically effective amount of the pharmaceutical composition.

42. The method of claim 39, wherein said step of contacting comprises grafting the composition to the bone of the subject.

43. The method of claim 39, wherein said bone disease or condition is selected from: bone loss, failure of bone to regenerate, osteoporosis, osteopenia, bone fractures, delayed fracture healing, non-union fracture, demineralization of bone, osteomyelitis, osteonecrosis, subclinical low bone density or decreased bone strength, tooth extraction tooth socket damage, inflammatory bone loss, bone marrow depression, bone marrow transplantation, peri-tooth implant bone damage, any infectious lesion affecting bone density / mass, chronic periodontitis, bone implants, bone grafts, traumatic injury, non-traumatic necrosis associated Gaucher' s disease, sickle cell anemia, systemic lupus erythematosus, or a combination of the same.

44. The method of claim 39, wherein said biocompatible bone matrix material is selected from: hydroxy apatite, hyaluronic acid, guar gum, xanthan gum, inorganic polymers, soft tissue particles, cartilage particles; hydroxyapatite, calcium HA, carbonated calcium HA, beta-tricalcium phosphate (beta-TCP), alpha-tricalcium phosphate (alpha-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), tetracalcium phosphate, biphasic calcium phosphate (BCP), anhydrous dicalcium phosphate (DCPA), dicalcium phosphate dihydrate (DCPD), anhydrous monocalcium phosphate (MCPA), monocalcium phosphate monohydrate (MCPM), or a combination of the same.

45. The method of claim 39, wherein said wherein the EVs comprise EVs generated from Saccharomyces cerevisiae, or Saccharomyces boulardii.

46. An isolated nucleotide sequence according to SEQ ID NO.

1.

47. An isolated nucleotide sequence according to SEQ ID NO.

2.

48. An isolated nucleotide sequence according to SEQ ID NO.26.

49. An expression vector having a nucleotide sequence, operably linked to a promoter, encoding SEQ ID NO.1-2, or 26, or a fragment or variant thereof.

50. A yeast cell transformed to express the expression vector of any of claims 46 to 49.

51. The yeast cell of claim 50, where the yeast cell is a Saccharomyces cerevisiae cell, or Saccharomyces boulardii cell.

52. A yEV isolated from the transformed cell of any of claims 50 to 51, wherein the yEV contains one or more mRNAs according to 1-2, or 26, or a fragment or variant thereof.

53. A pharmaceutical composition comprising a therapeutically effective amount of the yEVs of claim 52, and a pharmaceutically acceptable carrier, 54. The pharmaceutical composition of claim 53, wherein the pharmaceutically acceptable carrier is a biocompatible bone matrix material.

55. The method of claim 54, wherein said biocompatible bone matrix material is selected from: hydroxy apatite, hyaluronic acid, guar gum, xanthan gum, inorganic polymers, soft tissue particles, cartilage particles; hydroxyapatite, calcium HA, carbonated calcium HA, beta-tricalcium phosphate (beta-TCP), alpha-tricalcium phosphate (alpha-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), tetracalcium phosphate, biphasic calcium phosphate (BCP), anhydrous dicalcium phosphate (DCPA), dicalcium phosphate dihydrate (DCPD), anhydrous monocalcium phosphate (MCPA), monocalcium phosphate monohydrate (MCPM), or a combination of the same.

56. A method of treating a bone disease or condition comprising the step of contacting a therapeutically effective amount of the pharmaceutical composition of any of claims 53 to 55 to a subject in need thereof.

57. The method of claim 56, wherein said subject is a human.

58. The method of claim 56, wherein said step of contacting comprises grafting the composition to the bone of the subject.

59. The method of claim 56, wherein said bone disease or condition is selected from: bone loss, failure of bone to regenerate, osteoporosis, osteopenia, bone fractures, delayed fracture healing, non-union fracture, demineralization of bone, osteomyelitis, osteonecrosis, subclinical low bone density or decreased bone strength, tooth extraction tooth socket damage, inflammatory bone loss, bone marrow depression, bone marrow transplantation, peri-tooth implant bone damage, any infectious lesion affecting bone density / mass, chronic periodontitis, bone implants, bone grafts, traumatic injury, non-traumatic necrosis associated Gaucher' s disease, sickle cell anemia, systemic lupus erythematosus, or a combination of the same.

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