RNA sorting motifs for the packaging of recombinant rnas into yeast extracellular vesicles

Abstract

Methods and compositions for actively sorting RNAs, and preferably therapeutic RNAs into yeast extracellular vesicles (yEV).

Description

[0001] RNA SORTING MOTIFS FOR THE PACKAGING OF RECOMBINANT RNAS INTO YEAST EXTRACELLULAR VESICLES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This International PCT Application claims the benefit of and priority to U.S. Provisional Application No. 63 / 521,078 filed June 14, 2023, the specification, claims, tables, sequences, and drawings of which are incorporated herein by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains contents of the electronic sequence listing (90355.00121- Sequence-Listing.xml; Size: 517,085 bytes; and Date of Creation: June 14, 2024) is herein incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] The present invention is directed to novel methods, systems, and compositions for the selective loading of RNAs into yeast-generated extracellular vesicles (EVs) and their methods of use in treating one or more diseases or conditions.

[0008] BACKGROUND

[0009] 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. 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 human pathogens such as viruses.

[0010] 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 people. 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. In addition, the genome sequence of S. boulardii is available and the organism can be genetically engineered and / or have its genome edited for the purpose of modifying EV targeting or cargo. In addition, S. boulardii can also be grown at large scale in fermenters and has a shelflife 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.

[0011] Several traditional methods have been developed to load therapeutic drug compositions into EVs for delivery to target cells or tissues. These methods for encapsulating RNA cargo into EVs can be roughly divided into two types: cell-based RNA expression and EV packaging methods and exogenous EV loading methods. In the cell-based loading approach, therapeutic RNAs are expressed in the donor cells followed by random or active sorting of the RNAs into EVs. After being packaged into EVs, the EVs are used for therapeutic use. Non-cell-based loading approach involves directly loading RNAs 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 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 RNA compositions precisely, and uniformly into EVs structures, all while causing minimal disruption to their structure and aggregation patterns.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention is directed to novel methods, systems, and composition for actively sorting RNAs, and preferably therapeutic RNAs into an EV, wherein the EV can deliver the RNA to a targeted cell thereby treating a disease or condition or generating some physiological or other effect. The present invention is further directed to novel EV RNA sorting motifs, also referred to herein as ESMs. In a preferred embodiment, the present invention EV RNA sorting motifs, or sorting motifs, can be used to form novel expression constructs configured to express RNA molecules having one or more EV RNA sorting motifs incorporated into their sequence thereof. In another embodiment, the invention includes novel RNA molecules, and preferably therapeutic RNA molecules containing one or more EV RNA sorting motifs.

[0014] In one preferred aspect, the current invention includes novel constructs for the heterologous expression of RNA polynucleotides, including, but not limited to short hairpin RNAs (shRNA), mRNAs, micro RNA, and long non-coding RNAs (IncRNAs) in yeast adapted to facilitate their sorting into yeast EVs. The shRNA and mRNA populations can be configured to include one or more EV-RNA sorting motifs. In another embodiment, the design of RNA molecules, such as preferably shRNA and mRNA populations can be configured to disrupt or delete whole cell or counter sorting RNA motifs, also referred to herein as CSMs that enrich the RNA molecules in whole cells versus EVs.

[0015] Additional aspects of the invention will be evident from the specification, figures, and claims provided herein.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1. The distribution of expression biases of genes having any 5’ UTR EV RNA sorting motif (Table A) plus any 3’ UTR EV RNA sorting motif (Motif combination 1), alongside the distribution of expression biases of an equal number of randomly chosen genes that lack any of these motifs in their UTRs (Motif combination 0). Genes are represented as blue dots, overlaid on box plots indicating the quantiles of the data distributions. The ¥ axis shows the expression bias of each gene indicated as the log2 of the EV versus Cell difference in normalized expression levels. Positive values indicate expression bias towards the EVs. Negative values indicate expression bias towards Cells. The p-values from the two statistical tests of significance are indicated in the figure

[0018] Figure 2. The distribution of expression biases of genes having at least one 5’ UTR motif Genes are represented as blue dots, overlaid on box plots indicating the quantiles of the data distributions. The ¥ axis shows the expression bias of each gene indicated as the log2 of the EV versus Cell difference in normalized expression levels. Positive values indicate expression bias towards the EVs. Negative values indicate expression bias towards Cells. The X axis indicates the number of 5’ UTR motifs in each gene.

[0019] Figure 3. Distribution of the distances between adjacent pairs of 5’ UTR motifs. A: The X axis indicates distances separating pairs of adjacent 5’ UTR motifs in 10 bp bins. The Y-axis indicates the number of genes in each bin. B: The X axis indicates distances separating pairs of adjacent 5’ UTR motifs. The Y-axis indicates the density.

[0020] Figure 4A-B. Cassette for expression of shRNAs. (A), shRNA-ETM; (B), shRNA-C.

[0021] Figure 5. Northern blot analysis of shRNA presence in cells and EVs of shRNA-expressing yeast strains. A, Sb-shRNA-ESM; B, Sb-shRNA-C

[0022] Figure 6. EVs loading coefficient (EV / whole cell ratio) for engineered S. boulardii strains with mRNA expressed with or without the ESM loop in the 3’ UTR.

[0023] Figure 7. yEV partitioning coefficients for engineered S. boulardii (Sb) strains expressing mRNA with or without an ESM loop in their 3’ UTRs.

[0024] Figure 8A-B. Cassette for expression of mRNA with an ESM. (A), mRNA-NLuc including a 5’ ESM or 3’ ESM; (B), control mRNA-NLuc without ESM.

[0025] Figure 9: Normalized yEVs loading coefficient for engineered S. boulardii strains with mRNA expressed with or without the ESM at 5’ UTR and / or 3’ UTR.

[0026] Figure 10: shows Table 6 yEV RNA sorting motif sequences.

[0027] Figure 11: shows Table 7 yEV RNA sorting sequence 4-mer sequences.

[0028] Figure 12: shows Table 8 yEV RNA sorting sequence 5-mer sequences.

[0029] Figure 13: shows Table 9 yEV RNA sorting sequence 6-mer sequences.

[0030] Figure 14: shows Table 10 yEV RNA sorting sequence 7-mers sequences.

[0031] Figure 15: shows Table 11 yEV RNA sorting sequence 8-mers sequences.

[0032] Figure 16: shows Table 12 yEV RNA sorting motif sequences.

[0033] Figure 17: shows Table 13 yEV RNA sorting sequence 4-mer sequences.

[0034] Figure 18: shows Table 14 yEV RNA sorting sequence 5-mer sequences.

[0035] Figure 19: shows Table 15 yEV RNA sorting sequence 6-mer sequences.

[0036] Figure 20: shows Table 16 yEV RNA sorting sequence 7-mer sequences.

[0037] Figure 21: shows Table 17 yEV RNA sorting sequence 8-mer sequences.

[0038] Figure 22: shows Table 18 yEV RNA sorting motifs found to occur in pairs, one in the 5’ UTR and one on the 3’ UTR sequences.

[0039] Figure 23: shows Table 19 cell RNA sorting motif sequences. Figure 24: shows Table 20 cell sorting sequence 4-mer sequences.

[0040] Figure 25: shows Table 21 cell sorting sequence 5-mer sequences.

[0041] Figure 26: shows Table 22 cell sorting sequence 6-mer sequences.

[0042] Figure 27: shows Table 23 cell sorting sequence 7-mer sequences.

[0043] Figure 28: shows Table 24 cell sorting sequence 8-mer sequences.

[0044] Figure 29: shows Table 25 cell RNA sorting motif sequences.

[0045] Figure 30: shows Table 26 cell sorting sequence 4-mer sequences.

[0046] Figure 31: shows Table 27 cell sorting sequence 5-mer sequences.

[0047] Figure 32: shows Table 28 cell sorting sequence 6-mer sequences.

[0048] Figure 33: shows Table 29 cell sorting sequence 7-mer sequences.

[0049] Figure 34: shows Table 30 cell sorting sequence 8-mer sequences.

[0050] Figure 35: shows Table 31 sequences having overlaps between motifs detected by the first method (STREME), and stem-loop structures predicted by RNAfold were computed in order to establish if each motif tends to be located in stems or loops of RNA secondary structures, or neither.

[0051] Figure 36: shows Table 32 structures having overlaps between motifs detected by the first method (STREME), and stem-loop structures predicted by RNAfold were computed in order to establish if each motif tends to be located in stems or loops of RNA secondary structures, or neither.

[0052] Figure 37: shows Table 33 yEV RNA sorting motif sequences.

[0053] Figure 38: shows Table 34 cell sorting motif sequences.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] 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.

[0056] In one aspect the present invention includes novel ribonucleic acid (RNA) molecules containing one or more heterologous EV RNA-sorting motifs (ESMs), wherein the RNA molecules when introduced to a yeast-derived EV, whether in vivo or in vitro are preferentially packaged in the EV. In a preferred embodiment, the ESM of the invention can be selected from the nucleotide sequences selected from: GAAGACAC, TTGTATAA, TATGTATA, AAAATTTT, SEQ ID NO. 29, SEQ ID NO. 560, SEQ ID NO. 561, SEQ ID NO. 107, and / or SEQ ID NO. 108, or a fragment thereof. In a preferred embodiment, the ESM of the invention can be selected from any ESM sequence identified in Tables 6-18, and 31-33 (See Figures 10-22, and 35-37).

[0057] In another embodiment, the novel RNA molecule of the invention can include one or more novel ESMs positioned with a translated, or more preferably an untranslated region (UTR) and / or an intron region of the RNA polynucleotide. In certain other embodiment, the novel RNA molecules of the invention can include one or more novel ESMs positioned at the 3’ end of the RNA polynucleotide, while in alternative embodiments, the one or more novel ESMs can be positioned at the 5’ end of the RNA polynucleotide. In still further embodiments, novel RNA molecule of the invention can include a plurality of ESMs positioned at the 3’ end and / or the 5’ end of the RNA polynucleotide. In embodiments were the RNA polynucleotide of the invention include a plurality of ESMs, such motif sequences can be separate by between 20-30 base-pairs (bp), and preferably 25 bp.

[0058] In another embodiment, the novel RNA molecule of the invention can include an mRNA, while alternative embodiment the novel RNA molecule of the invention can include small RNA

[0059] (smRNA) or short hairpin RNA (shRNA). In certain embodiment, the ESM of the invention can be a positioned within a secondary structure of an RNA molecules, for example, one or more ESMs of the invention positioned within a loop secondary structure of a shRNA. This loop structure can be endogenous to the RNA polynucleotide, or it can be engineered, for example in the 3’ or 5’ end of the polynucleotide, as well as within an intron region of the RNA at either the 5’ or 3’ end.

[0060] In another embodiment, the novel RNA molecule of the invention can include a therapeutic RNA molecule having one or more novel ESMs. As used herein, a therapeutic RNA can include any RNA polynucleotide that can be used to treat a disease or condition, or perform a diagnostic analysis or assay. In one embodiment, a therapeutic RNA of the invention can include coding RNA such as mRNA as well as short or long non-coding RNAs such as small interfering RNAs (siRNA), micro RNAs, antisense RNA oligonucleotides (ASO) to target mRNA, aptamers, ribozymes, and clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR / Cas) endonuclease to target proteins and DNA.

[0061] In an alternative aspect, the novel RNA molecules of the invention can be further modified such that one or more sequences / motifs that encode a whole cell or counter sorting RNA motifs (CSM) are disrupted or removed such that the RNA molecules are preferentially packaged in vivo or in vitro, in a yeast-derived EV. Notably, method of disrupting an RNA sequence or motif are known in the art and would be generally understood by one or ordinary skill. In a preferred embodiment, the CSM of the invention can be selected from a CSM sequence identified in Tables 19-30, and 34 (See Figures 23-34, and 38).

[0062] In another embodiment, the invention includes an expression vector comprising the nucleotide encoding an RNA molecules having one or more ESMs, and wherein said nucleotide sequence is operably linked to a promoter. In another embodiment, the invention includes an expression vector comprising the nucleotide encoding an RNA molecules having one or more 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 the nucleotide encoding an RNA molecules having one or more ESMs, and having one or more CSMs that have been disrupted or removed, and wherein said nucleotide sequence is operably linked to a promoter.

[0063] Any of the aforementioned 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 the novel RNA molecules of the invention, 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.

[0064] In a preferred embodiment, a pharmaceutical composition containing a quantity of isolated yeast-derived EVs containing a population of therapeutic RNAs 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.

[0065] In another aspect the present invention includes a nucleotide sequence encoding a novel RNA molecule containing one or more heterologous EV RNA-sorting motifs (ESMs), wherein the RNA molecules when introduced to a yeast-derived EV, whether in vivo or in vitro are preferentially packaged in the EV. The nucleotide sequence of the invention can form an expression vector, having an expression cassette, operably linked to a promoter, encoding an RNA molecule containing one or more heterologous ESMs. In another preferred aspect, the expression vector of the invention can be used to transform a yeast cell to express the heterologous RNA molecule containing one or more heterologous ESMs which can be preferentially packaged into an EV in vivo. In an alternative aspect, the nucleotide sequence of the invention can be further modified such that one or more sequences / motifs that encode a whole CSM are disrupted or removed.

[0066] In another aspect the present invention includes a yeast-generated EV containing an RNA molecule having one or more ESMs. In an alternative aspect, the RNA molecule associated with the yeast-generated EV of the invention can further be modified such that one or more sequences / motifs that encode a CSM are disrupted or removed. In this preferred aspect, the yeastgenerated 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.

[0067] 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 RNA molecule having one or more ESMs and a pharmaceutically acceptable carrier. In an alternative aspect, the RNA molecule of the pharmaceutical composition can further be modified such that one or more sequences / motifs that encode a CSM are disrupted or removed.

[0068] In another aspect, the present invention includes systems and compositions to genetically modify yeast cells, and preferably a Saccharomyces yeast cell to express one or more heterologous RNAs configured to facilitate their sorting and loading into an EVs derived from said yeast cell. In one preferred aspect the current invention includes novel constructs for the heterologous expression of short hairpin RNAs (shRNA) and mRNAs in yeast adapted to facilitate their sorting into yeast EVs. The shRNA and mRNA populations are designed to include one or more EV-RNA sorting motifs and designed to avoid the presence of whole cell or counter sorting RNA motifs that enrich the RNA molecules in whole cells versus EVs. The shRNA and mRNA populations can further be designed such that one or more sequences / motifs that encode a CSM are disrupted or removed such that the RNA molecules will be preferentially enriched in the cell’s EVs. 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 RNAs 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 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.

[0069] 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 (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.

[0070] 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.

[0071] 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.

[0072] 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', 19th Edition (Mack Publishing Company, 1995), the disclosure of which is incorporated herein by reference in its entirety.

[0073] 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, e.g., hydroxyproline, y- 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 a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., 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. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0074] 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, e.g., 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, such as sequencing, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).

[0075] 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.

[0076] ??

[0077] 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 NO., 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.

[0078] As used herein, a nucleotide or other sequence is “engineered” when it contains a non- naturally occurring or heterologous coding sequence, such as a heterologous ESM, or where I has been modified from its naturally occurring sequences, such as when a CSM coding sequence has been destroyed or disrupted. An engineered cell includes a cell that has been genetically modified and express a heterologous nucleotide sequence.

[0079] 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, intemucleotide 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 molecules” also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hair-pinned, circular, and padlocked conformations.

[0080] 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.

[0081] 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.

[0082] 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 RNAs of the invention of the present invention may be chimeric.

[0083] To accomplish delivery of therapeutic compositions, such as nucleic acids to target cells, the methods and compositions of the present invention comprise 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 or yEVs. 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 RNAs 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.

[0084] The present invention 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 comprise introducing into the Saccharomyces the expression vector encoding one or more novel RNAs of the present invention to generate a host Saccharomyces cell. The host cell is then cultured under conditions to permit RNA production from the vector encoding the heterologous RNA. In one embodiment, the host cells of the present invention Saccharomyces cerevisiae or Saccharomyces boulardii.

[0085] 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.

[0086] 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 antiinflammatory cytokines in multiple organs including the lungs and cardiovascular system. Saccharomyces boulardii (Sb) 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 Sb strain for expression and loading of specific xenobiotics in exosomes. Cultivation of Sb 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 mRNA for vaccines.

[0087] 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 present 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. The probiotic used in the methods of administering will be engineered to produce the SGEVs of the present invention.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] EXAMPLES

[0092] Example 1. Identification of EV and Whole Cell RNA Sorting Motifs from the Small RNA (smRNA) Fraction of EVs and Whole Cells.

[0093] Applicants used bioinformatics analyses of the small RNA and mRNA fractions isolated from whole cells and EVs to determine if there are unique RNAs and associated RNA signature sequences or motifs that are statistically more abundant in EVs than in whole cells. The hypothesis being that if there are RNA sequences that are statistically more enriched in EVs than in whole cells that these RNA molecules may contain unique RNA sequences or motifs involved in the sorting or targeting of unique RNA sequences into EVs relative to whole cells. EV RNA sorting and / or avoidance (prefers whole cells) motifs then could be incorporated or excluded from, respectively, recombinant RNA sequences that are to be targeted for enhanced loading into EVs for therapeutic application.

[0094] To determine if there were RNA sorting motifs present in EV enriched RNA species, triplicate RNA samples were derived from purified whole cells (Cells) and extracellular vesicles (EVs). The total RNA was divided into two separate fractions, small RNA = < 500 nucleotides and mRNA. The small RNA population was sequenced in paired end 150 bp configuration using the Takara SMART er® smRNA-Seq Kit for Illumina®. This protocol preserves the strandedness of the RNA such that the first read in each pair represents the 5’ to 3’ RNA sequence. Only the first read of each pair was studied further. Adapter sequences were removed using the program Cutadapt (V4.1). The trimmed reads were aligned to the Saccharomyces boulardii genome using the hisat2 algorithm (V2.2.1) and reads not aligning were discarded. The reads aligning to the genome were then pooled and assembled into their cognate RNA molecules using Trinity (V2.13.2). Any remaining redundancy among these sequences was eliminated using cd-hit-est (V4.8.1). The resulting set of sequences were treated as the canonical set of small RNA molecules represented in any sample. The levels of expression of these RNA molecules in each sample were then evaluated by counting the number of sequence reads belonging to each one using salmon (VI.9.0). Expression bias towards EVs or Cells was evaluated using the program DESeq2 (VI.38), which assigns a normalized level of expression to each molecule, indicates 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 RNA molecules having significantly greater expression in EVs and Cells, respectively.

[0095] Differentially expressed molecules were then grouped into the sets shown in Table 1.

[0096] Table 1. Differentially expressed sets of small RNA molecules used for motif discovery.

[0097] Applicants then undertook to identify short sequence features that were significantly overrepresented in EV samples versus Cell samples. Applicants call these sequence features motifs, our hypothesis being that motifs overrepresented in EVs may be determinants of RNA sorting into the EV compartment. Our first approach to motif discovery was to use the STREME program, which is part of the MEME suite of algorithms (https: / / meme-suite.org / meme / ). We employed the parameters: streme —verbosity 1 — oc . —totallength 4000000 —time 14400 — minw 8 — maxw 15 —thresh 0.05 —align center — p motif file — n control file

[0098] In the above command, motif file refers to the sequence file in which Applicants searched for overrepresented motifs, and control file refers to a set of sequences in which Applicants expect diminished presence of such motifs. The pairs of motif file and control file that Applicants compared for small RNA motif discovery are shown in Table 2.

[0099] Table 2. Sequence set pairs used for small RNA motif discovery

[0100] A second method was applied to discover motifs overrepresented in EV-biased small RNA molecules. For a pair of sequence sets to be compared, Applicants extracted every kmer from 4-8 nucleotides 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 were All significantly EV biased RNA and All significantly Cell biased RNA (Table 1).

[0101] Secondary structures (principally stem-loop structures) were predicted in every sequence in the canonical set of small RNA molecules using RNAfold (V2.5.1) resulting in a file containing dot-bracket notation describing the secondary structure for each sequence. The locations of every stem-loop structure and every predicted motif in each of the canonical set of small RNA sequences were compared with one another in order to establish if each motif tends to be located in stems or loops of RNA secondary structures, or neither.

[0102] Example 2: Identification of Messenger RNA (mRNA) EV and Whole Cell RNA Sorting Motifs.

[0103] Triplicate poly-A+ RNA samples derived from purified whole cells (Cells) and purified extracellular vesicles (EVs) were sequenced in paired end 150 bp configuration. Adapters and low- quality sequences were removed using Trimmomatic (V0.39). The trimmed reads were aligned to the Saccharomyces boulardii genome using the hisat2 algorithm (V2.2.1). Only paired reads aligning in the correct orientation were retained. The reads aligning to each S. boulardii proteincoding gene were counted using htseq-count (V2.0.2), and these counts were used as input for DESeq2 (VI.38), which assigns a normalized level of expression to each gene, indicates 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 genes having significantly greater expression in EVs and Cells, respectively. The locations of the protein coding genes in the S. boulardii genome were used to extract the coding sequences (CDS), the 5’ untranslated regions (UTRs) and the 3’ UTRs. For Applicants purposes the UTRs were defined as 150 base pairs upstream and downstream of each CDS. The CDS, 3’ UTR and 5’ UTR sequences were grouped into files as shown in Table 3.

[0104] Table 3. Differentially expressed sets of mRNA molecules used for motif discovery.

[0105] Applicants then undertook to identify short sequence features (motifs) that were significantly overrepresented in EV samples versus Cell samples. Applicants called these sequence features motifs, our hypothesis being that motifs overrepresented in EVs may be determinants of RNA sorting into the EV compartment. Our first approach to motif discovery was to use the STREME program, which is part of the MEME suite of algorithms (meme-suite.org / meme / ). We employed the parameters: streme —verbosity 1 — oc . —totallength 4000000 —time 14400 — minw 8 — maxw 15 —thresh 0.05 —align center — p motif file — n control file

[0106] In the above command, motif file refers to the sequence file in which Applicants searched for overrepresented motifs, and control file refers to a set of sequences in which Applicants expected diminished presence of such motifs. The pairs of motif file and control file that Applicants compared for mRNA motif discovery are shown in Table 4. Table 4. Sequence set pairs used for mRNA motif discovery.

[0107] A second method was applied to discover motifs overrepresented in EV-biased mRNA molecules. For a pair of sequence sets to be compared, Applicants extracted every kmer from 4-8 nucleotides 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 us 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 EV biased genes and all significantly Cell biased genes (Table 4), for CDS, 5’ UTR and 3’ UTR sequences.

[0108] Secondary structures (principally stem-loop structures) were predicted in every sequence in the CDS, 5’ UTR and 3’ UTR sequences using RNAfold (V2.5.1) resulting in a file containing dot-bracket notation describing the secondary structure for each sequence. The locations of every stem-loop structure and every predicted motif in the CDS, 5’ UTR and 3’ UTR sequences were compared with one another to establish if each motif tends to be located in stems or loops of RNA secondary structures, or neither.

[0109] Example 3: Analysis of EV RNA sorting motifs occurring in both 5’ and 3’ UTRs of mRNA sequences. Genes were examined to identify those having pairs of motifs, one occurring in the 5’ UTR and one occurring in the 3’ UTR. Genes having such pairs of motifs were counted and ranked by frequency. The 50 most abundant pairs of motifs were selected and their non-redundant sets of 5’ UTR and 3’ UTR motifs were identified. Genes were then selected having any of the unique 5’ motif set and any of the 3’ motif set. The expression biases of these genes, expressed as the log2 of the EV versus Cell difference in normalized expression levels (means of three samples) were computed. The same biases were computed for an equally-sized random set of genes having none of these motifs. The expression biases of these two groups of genes were compared to one another using the t-test and the Wilcoxon Rank Sum test to establish whether having these pairs of motifs was associated with a significant expression bias towards EVs versus Cells.

[0110] Example 4: Analysis of multiple EV RNA sorting motifs occurring in the 5’ UTR of mRNA sequences.

[0111] Genes were identified having at least one 5’ UTR motif identified as being enriched in genes with expression significantly biased towards EVs versus cells. These genes were grouped into sets having 1, 2, 3, or 4 five-prime UTR motifs. The expression biases of these genes, expressed as the log2 of the EV versus Cell difference in normalized expression levels (means of three samples) were computed, and compared to one another to establish whether the number of 5’ UTR motifs present was associated with the level of expression bias towards EVs versus Cells. Example 5: Discovery of EV counter-sorting RNA motifs.

[0112] To identify RNA sequence motifs that may prevent entry of RNA molecules into EVs, we repeated certain runs of STREME using sets of cell-biased RNAs as the sequences in which to find enriched motifs, and the sets of EV-biased RNAs as the control sequences, as shown in Table 5.

[0113] Table 5. Sequence set pairs used for counter-sorting motif discovery.

[0114] A second method was applied to discover motifs overrepresented in Cell-biased RNA molecules. For a pair of sequence sets to be compared, we extracted every kmer from 4-8 nucleotides 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 us 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 RNA sequences and all significantly EV biased RNA sequences (Table 5).

[0115] Example 6: Identification of sorting motif sequences.

[0116] In the following tables and throughout this document motif sequences are written using IUPAC ambiguity codes (bioinformatics.org / sms / iupac.html).

[0117] Small RNA motifs enriched in sequences biased in their expression towards EVs

[0118] The RNA sorting motifs shown in Table 6 (See Figure 10, incorporated herein by reference) were found to be enriched in small RNA molecules having expression levels biased in favor of EVs using the first detection method (STREME).

[0119] The RNA sorting sequence 4-mers shown in Table 7 (See Figure 10, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test).

[0120] The RNA sorting sequence 5-mers shown in Table 8 (See Figure 12, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test).

[0121] The RNA sorting sequence 6-mers shown in Table 9 (See Figure 13, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test).

[0122] The RNA sorting sequence 7-mers shown in Table 10 (See Figure 14, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test). The RNA sorting sequence 8-mers shown in Table 11 (See Figure 15, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test). mRNA motifs enriched in sequences biased in their expression towards EVs

[0123] The RNA sorting motifs shown in Table 12 (See Figure 16, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of EVs using the first detection method (STREME).

[0124] The RNA sorting 4-mers shown in Table 13 (See Figure 17, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test).

[0125] The RNA sorting 5-mers shown in Table 14 (See Figure 18, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test).

[0126] The RNA sorting 6-mers shown in Table 15 (See Figure 19, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test).

[0127] The RNA sorting 7-mers shown in Table 16 (See Figure 20, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test).

[0128] The RNA sorting 8-mers shown in Table 17 (See Figure 21, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of EVs using the second detection method (Fisher’s exact test).

[0129] Motifs occurring in both 5’ and 3’ UTRs of mRNA sequences

[0130] The most abundant EV RNA sorting motifs found to occur in pairs, one in the 5’ UTR and one on the 3’ UTR are shown in Table 18. (See Figure 22, incorporated herein by reference) The distribution of expression biases of genes having any 5’ UTR motif (Table 18) plus any 3’ UTR motif (Table 18) are shown in Figure 1, alongside the distribution of expression biases of an equal number of randomly chosen genes that lack any of these motifs in their UTRs. 178 out of 305 genes having a motif combination demonstrated an expression bias towards the EVs.

[0131] Multiple EV RNA sorting motifs occurring in the 5’ UTR of mRNA sequences

[0132] The distribution of expression biases of genes having at least one 5’ UTR motif are shown in Figure 2. A t-test comparing the first two groups (1 versus 2 motifs) yielded a p-value of 0.033, causing us to reject the null hypothesis and conclude that having 2 versus 1 motifs is associated with a significantly greater expression bias towards EVs. The distributions of the distances between adjacent pairs of 5’ UTR motifs (measured from the start position of one motif to the start position of the other) are shown in Figure 3. The mode is close to 25 base pairs.

[0133] Counter sorting or whole cell sorting motifs

[0134] The RNA sorting motifs shown in Table 19 (See Figure 23, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of Cells using the first detection method (STREME).

[0135] The RNA sorting 4-mers shown in Table 20 (See Figure 24, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of Cells using the second detection method (Fisher’s exact test).

[0136] The RNA sorting 5-mers shown in Table 21 (See Figure 25, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of Cells using the second detection method (Fisher’s exact test).

[0137] The RNA sorting 6-mers shown in Table 22 (See Figure 26, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of Cells using the second detection method (Fisher’s exact test).

[0138] The RNA sorting 7-mers shown in Table 23 (See Figure 27, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of Cells using the second detection method (Fisher’s exact test).

[0139] The RNA sorting 8-mers shown in Table 24 (See Figure 28, incorporated herein by reference) were found to be enriched in small RNA sequences having expression levels biased in favor of Cells using the second detection method (Fisher’s exact test).

[0140] The RNA sorting motifs shown in Table 25 (See Figure 29, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of Cells using the first detection method (STREME).

[0141] The RNA sorting 4-mers shown in Table 26 (See Figure 30, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of Cells using the second detection method (Fisher’s exact test).

[0142] The RNA sorting 5-mers shown in Table 27 (See Figure 31, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of Cells using the second detection method (Fisher’s exact test). The RNA sorting 6-mers shown in Table 28 (See Figure 32, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of Cells using the second detection method (Fisher’s exact test).

[0143] The RNA sorting 7-mers shown in Table 29 (See Figure 33, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of Cells using the second detection method (Fisher’s exact test).

[0144] The RNA sorting 8-mers shown in Table 30 (See Figure 34, incorporated herein by reference) were found to be enriched in mRNA sequences having expression levels biased in favor of Cells using the second detection method (Fisher’s exact test).

[0145] Secondary structures associated with EV-biased mRNA sorting motifs

[0146] The overlaps between motifs detected by the first method (STREME), and stem-loop structures predicted by RNAfold were computed in order to establish if each motif tends to be located in stems or loops of RNA secondary structures, or neither. Table 31 (See Figure 35, incorporated herein by reference) shows the results of this analysis.

[0147] Secondary structures associated with EV-biased small RNA sorting motifs

[0148] The overlaps between motifs detected by the first method (STREME), and stem-loop structures predicted by RNAfold were computed in order to establish if each motif tends to be located in stems or loops of RNA secondary structures, or neither. Table 32 (See Figure 36, incorporated herein by reference) shows the results of this analysis.

[0149] Below shows the column definitions used in Tables 31 and 32.

[0150] Column definitions for Tables 31 and 32

[0151] Example 7: Experimental Validation of EV RNA Sorting Motifs

[0152] Targeting shRNA into yeast extracellular vesicles (EV): For expression of short hairpin RNA (shRNA) in yeast Applicants designed RNA expression cassettes in which complementary sense and antisense dsRNA strands were separated by 180 bp long loop (Fig 4A). This construct was expressed under the control of TDH3 promoter and CYC1 terminator (Fig. 4A). Three potential EV RNA-sorting motifs (ESM) designated Sec2, Sec4 and Sec8 were included in the loop sequence of shRNA. Random RNA sequences of equivalent lengths to the Sec2, Sec4, and Sec8 ESM targeting sequences were used for the control shRNA (shRNA-C) (Fig IB). All expression cassettes were genome integrated into the YPRCt3 locus on XVI chromosome of yeast yielding strains Sb-shRNA-ESM and Sb-shRNA-C. It was previously shown that integration at this locus does not affect cell growth and gene expression in S. boulardii.

[0153] Evaluation of shRNA sorting and loading into EVs: To evaluate the relative abundance of shRNA-ESM and shRNA-C in yeast EVs, Applicants extracted miRNA from yeast cells and EVs fractions and performed northern blot analyses on equal amount of RNA using probes specific to the loop of shRNA-ESM and shRNA-C lacking an EV RNA sorting motif (Fig 5). Applicants found that while both engineered strains expressed similar amounts of shRNA, EVs extracted from Sb-shRNA-C strain had extremely low levels of shRNA-C (Fig 5b). In contrast, EVs extracted from Sb-shRNA-ESM had substantial levels of shRNA-ESM in EVs. The abundance of shRNA-

[0154] ESM in EVs was substantially greater than that present in equal numbers of cells (Fig 5a). This data demonstrates that the inclusion of specific EV RNA sorting motifs in the loop of shRNA can enhance the sorting of shRNAs into EVs.

[0155] Targeting of mRNA into EVs using shRNA EV targeting motifs: To determine if short RNA (<500 nt) EV RNA sorting motifs could sort mRNAs into EVs Applicants added EV RNA sorting motifs to the untranslated regions of a specific mRNA. For this purpose, Applicants placed the loop sequence from shRNA-ESM into the 3’ UTR of mRNA encoding the enhanced green fluorescent protein (EGFP) or bone morphogenetic protein 9 (BMP9). The sorting loop was placed in the beginning of 3” UTR 50 bp after the mRNA stop codon. As a control, Applicants used corresponding mRNAs without modification of 3’ UTR. Similar to the previously described shRNA constructs, all mRNA sequences were expressed under the control of a yeast TDH3 promoter and CYC1 terminator. Total RNA was extracted from cells and EVs fractions of both engineered strains for qPCR analysis to measure the comparative level of mRNA-gfp in the cells and EVs. As housekeeping gene, Applicants used DAD3 mRNA which is present in both wildtype S. boulardii cells and EVs. To evaluate the efficiency of mRNA sorting into EVs (mRNA loading coefficient), Applicants calculated the ratio of mRNA present in EVs to that present in cells in each engineered strain. Applicants found that the loading coefficient was significantly higher for both strains where mRNA was fused with ESM loop compared to strains expressing mRNA without the loop (Fig. 6). Taken together with results from shRNA loading analysis, this data indicates that the inclusion of ESMs in the UTRs of mRNAs strongly enhances mRNA partitioning into EVs.

[0156] Similarly, in one embodiment, EV mRNA sorting motifs when incorporated into the 5’, 3’ or introns of recombinant mRNA(s) designed to sort into yeast EVs can be similarly as effective in targeting mRNAs into EVs as were the short RNA sorting motifs.

[0157] Example 8: Targeting shRNA into yeast extracellular vesicles.

[0158] To determine if short ESM motifs identified above are responsible for targeting the RNA into yEVs, Applicants compared the abundance of shRNA sequences with or without ESMs in whole yeast cells and yEVs. For these studies Applicants selected 3 exemplary ESMs identified as top candidates for sorting RNAs into yEVs designated Sec2, Seed and Sec8 and included them in a shRNA sequence. For expression of short hairpin RNA (shRNA) in yeast we designed RNA expression cassettes in which the complementary sense and sense dsRNA strands are separated by 180 bp long loop (Fig 1 A). This construct was expressed under the control of TDH3 promoter and CYC1 terminator (Grant et al, 1984) (Fig. 4A). Sec2, Sec4 and Sec8 were placed in the loop sequence of the shRNA (SEQ ID NO. 545). Random RNA sequences of equivalent lengths to the Sec2 (GAAGACAC), Sec4 (SEQ ID NO. 545-550), and Sec8 (SEQ ID NO. 573) ESM targeting sequences were used as negative controls shRNA-C, (SEQ ID NO. 546)) (Fig 4B). All expression cassettes were integrated into the YPRCt3 locus on XVI chromosome of the yeast genome generating the yeast strains Sb-shRNA-ESM and Sb-shRNA-C. It was previously shown that integration at this locus does not affect cell growth and gene expression in S. boulardii. Example 9: Evaluation of shRNA loading into yEVs.

[0159] To assess the relative abundance of shRNA-ESM and shRNA-C in yeast EVs and whole cells, Applicants extracted miRNA from yeast cells and yEVs fractions and performed northern blot analyses on equal amounts of RNA using probes specific to the loop of shRNA-ESM (SEQ ID NO. 545) and shRNA-C (SEQ ID NO. 546) (Figure 5). Applicants found that while both engineered strains expressed similar amounts of shRNA in whole cells, yEVs extracted from Sb- shRNA-C strain had roughly 4 times less shRNA-C compared to same amount of total RNA analyzed from whole cells (Figure 5b). In contrast, in the S. boulardii-shRN A-ESM strain the abundance of shRNA-ESM in yEVs fraction was approximately 5 times greater than for whole cells. (Figure 5a). This data demonstrates that the inclusion of specific yEV RNA targeting motifs in the loop of shRNA enhances the sorting of shRNAs into EVs.

[0160] Example 10: Sorting of mRNA into EVs using shRNA EV targeting motifs.

[0161] To determine if short RNA (<500 nt) ESMs could sort mRNAs into yEVs Applicants incorporated yEV RNA sorting motifs into the untranslated regions of a specific mRNA. For this purpose, Applicants placed the loop sequence from the previously described shRNA-ESM into the 3’ UTR of mRNA encoding the enhanced green fluorescent protein (EGFP, SEQ ID NO. 547) or bone morphogenetic protein 9 (BMP9, SEQ ID NO. 549). The sorting loop was placed at the beginning of 3’ UTR 40 bp after stop codon. As a control, Applicants used identical mRNAs but without the ESM modification of the 3’ UTR (SEQ ID NO. 548 and SEQ ID NO. 550, respectively).

[0162] Similar to the shRNA expression constructs, all mRNA sequences were expressed under the control of a yeast TDH3 promoter and CYC1 terminator as described above. Total RNA was extracted from cells and yEVs fractions from all engineered strains for qPCR analysis to measure the comparative level of mRNA-gfp or mRNA-BMP9 in the cells and EVs. The length of coding sequence of eGFP mRNA was 717 bp and total mRNA length 1027 nt, while the length of BMP9 coding sequence and total mRNA were 1290 and 1597 correspondingly. As housekeeping gene for qPCR analysis, Applicants used DAD3 mRNA which is present in both wild-type S. boulardii cells and EVs.

[0163] To evaluate the efficiency of mRNA sorting into yEVs (mRNA loading coefficient), Applicants calculated the ratio of mRNA in EVs to that present in cells in each engineered strain. Applicants found that the yEV vs whole cell partitioning coefficient was significantly higher for both strains where mRNA was fused with ESM loop compared to strains expressing mRNA without the loop. The qPCR normalized (housekeeping gene) yEV / whole cell partitioning ratio of mRNAs containing ESMs was 3.5 for eGFP mRNA and 11.5 for the BMP9 mRNA (Figure 7). Since the eGFP mRNA was smaller than the BMP9 mRNA it suggests that smaller mRNAs (eGFP = 1,027 nt) do not have a preference for sorting into yEVs versus larger mRNAs (BMP9 = 1,597 nts) Taken together with results from the impact of incorporating ESMs into shRNA and its effects on yEV partitioning compared to whole cells, this data indicates that the inclusion of ESMs in the UTRs of RNAs strongly enhances RNA partitioning into EVs.

[0164] Example 11 : Sorting of mRNA into EVs using mRNA-specific EVs targeting motifs.

[0165] As described above, 2 different sets of yEV ESM motifs were identified by RNAseq analysis of the transcriptome of yEVs and yeast for mRNAs larger 500 nt. These sets include short (4-8 nt) sequences overrepresented at 5’ and 3’ RNA regions. The most common ESMs are indicated in Table A below. To determine if inclusion of these ESMs, either singly or in multiple combinations in either the 5’ or 3’ UTR would impact mRNA sorting into yEVs Applicants engineered an mRNA encoding Nanoluciferase (NLuc) to include different ESMs incorporated into the 5’ and / or 3’ UTR. As a control Applicants used an S. boulardii strain expressing NLuc mRNA without modification of the UTRs (Table B). Notably, the length of NLuc mRNA is 516 nt and total mRNA length if around 790-800 nt for different constructs. The mRNA expression cassettes were integrated into the S. boulardii genome as described above for the shRNA expression cassettes (Figure 8).

[0166] Total RNA was extracted from cells and yEV fractions of engineered S. boulardii strains for qPCR analysis to measure the comparative level of mRNA-NLuc in cells and yEVs. As housekeeping gene, Applicants used TDH3 mRNA which is present in both wild-type S. boulardii cells and yEVs. Again, the ratio of engineered mRNA in yEVs to that present in cells in each engineered strain was used to determine if there was enhanced sorting of mRNA into yEVs. Eight different ESM-NLuc-mRNA constructs were evaluated. Three modified mRNAs had ESMs included in the 5’ UTR LN2, LN4 and LN7, 2 mRNA constructs had ESMs incorporated only into 3’ UTRs, and LN4, and 3 mRNA constructs had ESMs incorporated into both the 5’ and 3’ UTRs LN1, LN6 and LN8 (See Table B). For all constructs 5’ UTR ESMs were located 40 nt upstream of start codon mRNA, and all 3’ UTR ESMs were located 5 nt downstream of stop codon. As shown at Figure 9, the partitioning coefficient of mRNA with an ESM located only in the 5’ or 3’ UTR was only slightly elevated compared to control mRNA lacking an ESM (range 1.0 -1.6x above the control level). All mRNA constructs with ESMs located in both the 5’ and 3’ UTRs had 2.0-2.5 fold greater partitioning of mRNA into yEVs than into whole cells. Taken together, these data demonstrate that the inclusion of ESMs in UTRs enhance the loading of RNA molecules into yEVs.

[0167] Example 10: Additional motif identification methods and results.

[0168] A third motif identification method was applied. This method, implemented in the fmdMotifs.pl script of the HOMER software package, also compares two sequence sets, one expected to contain motifs of interest, and a reference set expected to lack such enrichment. HOMER was applied to the pairs of sequence sets described in Tables 2-5 in order to identify further motifs having statistical bias towards the EV and Cell RNA samples. The HOMER command used was: fmdMotifs.pl motif file fasta . / output folder -fasta control file -len

[0169] 4,6,8,10,12 -rna. In the above command, motif file refers to the sequence file in which Applicants searched for overrepresented motifs, and control file refers to a set of sequences in which

[0170] Applicants expect diminished presence of such motifs. Application of HOMER to these sequence sets yielded additional statistically significant motifs not detected by the other two methods, and identified in Table 33 (See Figures 37: describing additional structures associated with EV-biased mRNA sorting motifs) and Table 34 (See Figures 38: describing additional Secondary structures associated with cell-biased small RNA sorting motifs).

[0171] Example 9: Materials and Methods.

[0172] Table B: mRNA-ESM constructs used in this study

[0173] Table C. Strains and cells lines used in this work

[0174] Table D: Oligonucleotides used in this work:

[0175] Table E: EVs targeting motifs included in the sequence of shRNA ETM and EGFP ETM

[0176] S. boulardii strains design and construction: To create S. boulardii strains expressing shRNAs or mRNAs, wild-type S. boulardii was transformed with dsDNA segments including

[0177] RNA expression cassette and geneticin-resistance gene flanked on 5’ and 3’ ends by integration sequences homologous to DNA sequences from the YPRCt.3 locus on XVI chromosome. Yeast transformation was performed by electroporation following the protocol described by Benatuil et al (2010). All mRNA sequences for all constructs were prescreened for the presence of potential cell-sorting RNA motifs and. none were found to be present.

[0178] 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 x 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.

[0179] Northern blot analysis: Total RNA was extracted from 108yeast cells or 2xl012isolated EVs using a mirVana miRNA isolation kit (Ambion). 6-15 μg of RNA was loaded for separation on Novex™ 15% TBE-Urea gels along with an ssRNA ladder (New England Biolabs). Afterward RNA was transferred onto a positively charged nylon transfer membrane (Whatman Nytran SuPerCharge, GE Helthcare Life Sciences, Germany) (Kim et al, 2010). Chemically synthesized RNA oligonucleotides were obtained from Integrated DNA Technologies, Inc. (USA, San Diego). RNA probes were labeled to high specific activity using a DIG Oligonucleotide 3 ’-End labeling kit, 2 Generation (Roshe Diagnostics GmbH, Germany). After UV cross-linking (UVP HL -2000 HybriLinker) the membranes were prehybridized at 42°C for 30 min in an

[0180] ULTRAhybTM-Oligo Hybridization Buffer (Thermo Fisher Scientific Baltics UAB, Lithuania). After prehybridization, the purified labeled probe was added to the prehybridization buffer and was incubated at 42°C for 14 - 18 h. The membranes were then washed twice with 2x SSC-0.2%

[0181] SDS (20 min at 42°C), 2x SSC-0.2% SDS (20 min at 55°C, twice), and with lx SSC-0.1% SDS (20 min at 55°C, twice, and washed and blocked with DIG Wash and Block Buffer Set, respectively (Roshe Diagnostics GmbH, Germany). DIG-labeled probes were detected using AP-coupled anti- digoxigenin Fab fragments (Roche Applied Science) at 1 : 100 in alkaline phosphatase buffer and photoemission was detected using the ChemiDoc XRS+ Imaging System (Bio-Rad). The signal intensities were quantified by densitometry using the Volume Tools of the Image Lab software, version 6.0.1 build 34 (Bio-Rad). TABLE A

[0182] INCORPORATION BY REFERENCE

[0183] Applicant specifically incorporates specifically by reference the Tables 6 through 32 identified in U.S. Provisional Application No. 63 / 521,078 filed June 14, 2023, including all sequences and additional column descriptions and definitions contained therein, icnluding p-value.

[0184] REFERENCES

[0185] 1. Kim SW, Li Z, Moore PS, Monaghan AP, Chang Y, Nichols M, John B. A sensitive non-radioactive northern blot method to detect small RNAs. Nucleic Acids Res. 2010 Apr;38(7):e98. doi: 10.1093 / nar / gkpl235. Epub 2010 Jan 15. PMID: 20081203; PMCID: PMC2853138.

[0186] 2. Benatuil L, Perez JM, Belk J, Hsieh CM. An improved yeast transformation method for the generation of large human antibody libraries. Protein Eng Des Sei. 2010 Apr;23(4): 155-9. doi: 10.1093 / protein / gzq002. Epub 2010 Feb 3. PMID: 20130105

[0187] 3. Deniz Durmusoglu, et. al., Establishing Probiotic Saccharomyces boulardii as a Model Organism for Synthesis and Delivery of Biomolecules. bioRxiv 2020.01.22.915389.

[0188] 4. Grant A. Bitter, Kevin M. Egan. Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3 -phosphate dehydrogenase gene promoter. Gene, Volume 32, Issue 3,1984.

[0189] 5. Kulig K, Kowalik K, Surowiec M, Karnas E, Barczyk-Woznicka O, Zuba-Surma E, Pyza E, Kozik A, Rapala-Kozik M, and Karkowska-Kuleta J. (2023) Isolation and Characteristics of Extracellular Vesicles Produced by Probiotics: Yeast Saccharomyces boulardii CNCM I-745and Bacterium Streptococcus salivarius K12. Probiotics and Antimicrobial Proteins.

[0190] 6. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, Cheng IX, Murre C, Singh H, Glass CK. 2010. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Molecular cell 38:576-589.

[0191] SEQUENCE LISTING WITH LEGENDS

[0192] SEQ ID NO. 545

[0193] DNA shRNA-ESM*

[0194] Artificial

[0195]

[0196] *5’ UTR and 3’ UTR are in lower case; mRNA is in capital case; ESMs are in capital bold font

[0197]

[0198] *5’ UTR and 3’ UTR are in lower case; mRNA is in capital case; ESMs are in capital bold font

Claims

CLAIMSWhat is claimed is:

1. An isolated polynucleotide encoding an engineered ribonucleic acid (RNA) molecule having a heterologous EV RNA-sorting motifs (ESM).

2. The polynucleotide of claim 1, wherein the RNA comprises a therapeutic RNA.

3. The polynucleotide of claim 1, wherein said ESM is positioned at the 3’ end of the RNA.

4. The polynucleotide of claim 1, wherein said ESM is positioned at the 5’ end of the RNA.

5. The polynucleotide of claim 1, wherein said ESM is positioned within an untranslated region (UTR) of the RNA and / or an intron region of the RNA.

6. The polynucleotide of claim 1, wherein said ESM comprises a plurality of ESMs.

7. The polynucleotide of claim 6, wherein said plurality of said ESMs comprise a first ESM positioned at the 3’ end of the RNA, and a second ESM positioned at the 5’ end of the RNA.

8. The polynucleotide of claim 7, wherein said first and said second ESMs are between 20-30 basepairs apart.

9. The polynucleotide of claim 1, wherein said first and said second ESMs are 25 base-pairs apart.

10. The polynucleotide of any of claims 1- 9, wherein the RNA comprises a small RNA (smRNA).

11. The polynucleotide of any of claims 1- 9, wherein the RNA comprises a short hairpin RNA (shRNA).

12. The polynucleotide of claim 11, wherein the ESM is positioned within a loop secondary structure of the shRNA.

13. The polynucleotide of any of claims 1-13, wherein said ESM comprises a nucleotide sequence selected from: GAAGACAC, TTGTATAA, TATGTATA, AAAATTTT, SEQ ID NO. 29, SEQ ID NO.560, SEQ ID NO. 561, SEQ ID NO. 107, and / or SEQ ID NO. 108, or a fragment thereof.

14. The polynucleotide of any of claims 1-13, wherein said ESM is selected from an ESM sequence identified in Tables 6-18, 31-33, and Table A.

15. The polynucleotide of any of claims 1-14, and further comprising wherein a whole cell or counter sorting RNA motifs (CSM) present in the RNA sequence is disrupted or removed.

16. The polynucleotide of claim 15, wherein the CSM is selected from a CSM sequence identified in Tables 19-30, and 34.

17. An expression vector comprising the nucleotide encoding a sequence of any of claims 1-16 wherein said nucleotide sequence is operably linked to a promoter.

18. A yeast cell transformed to express the expression vector of 17.

19. The yeast cell of claim 18, wherein said yeast cell is selected from: Saccharomyces cerevisiae, or Saccharomyces boulardii.

20. A extracellular vesicle (yEV) isolated from the transformed yeast cell of claim 18.

21. A pharmaceutical composition comprising the yEV of claim 20, and a pharmaceutically acceptable carrier.

22. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 21, to a subject in need thereof.

23. An isolated nucleotide sequence encoding a ribonucleic acid (RNA) molecule and a heterologous an EV RNA-sorting motifs (ESM).

24. The sequence of claim 23, wherein the RNA comprises a therapeutic RNA.

25. The sequence of claim 23, wherein said ESM is positioned at the 3’ end of the RNA.

26. The sequence of claim 23, wherein said ESM is positioned at the 5’ end of the RNA.

27. The sequence of claim 23, wherein said ESM is positioned within an untranslated region (UTR) of the RNA and / or an intron region of the RNA.

28. The sequence of claim 23, wherein said ESM comprises a plurality of ESMs.

29. The sequence of claim 28, wherein said plurality of said ESMs comprise a first ESM positioned at the 3’ end of the RNA, and a second ESM positioned at the 5’ end of the RNA.

30. The sequence of claim 29, wherein said first and said second ESMs are between 20-30 basepairs apart.

31. The sequence of claim 23, wherein said first and said second ESMs are 25 base-pairs apart.

32. The sequence of any of claims 23-31, wherein the RNA comprises a small RNA (smRNA).

33. The sequence of any of claims 23-31, wherein the RNA comprises a shRNA.

34. The sequence of claim 33, wherein the ESM is positioned within a loop secondary structure of the shRNA.

35. The sequence of any of claims 23-34, wherein said ESM comprises a nucleotide sequence selected from: SEQ ID NO’s. GAAGACAC, TTGTATAA, TATGTATA, AAAATTTT, SEQ ID NO.29, SEQ ID NO. 560, SEQ ID NO. 561, SEQ ID NO. 107, and / or SEQ ID NO. 108, or a fragment thereof.

36. The sequence of any of claims 23-34, wherein said ESM is selected from an ESM sequence identified in Tables 6-18, , and Table A.

37. The sequence of any of claims 23-36, and further comprising wherein a whole cell or counter sorting RNA motifs (CSM) present in the RNA sequence is disrupted or removed.

38. The sequence of claim 37, wherein the CSM is selected from a CSM sequence identified in Tables 19-30, and 34.

39. An expression vector comprising the nucleotide sequence of any of claims 23-38, wherein said nucleotide sequence is operably linked to a promoter.

40. A yeast cell transformed to express the expression vector of 39.

41. The yeast cell of claim 40, wherein said yeast cell is selected from: Saccharomyces cerevisiae, or Saccharomyces boulardii.

42. A extracellular vesicle (yEV) isolated from the transformed yeast cell of claim 41.

43. A pharmaceutical composition comprising the yEV of claim 42, and a pharmaceutically acceptable carrier.

44. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 43, to a subject in need thereof.

45. A genetically modified yeast cell expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a ribonucleic acid (RNA) molecule engineered to include an EV RNA-sorting motif (ESM).

46. The sequence of claim 45, wherein said yeast cell is selected from: Saccharomyces cerevisiae, or Saccharomyces boulardii.

47. The yeast cell of claim 45, wherein the RNA comprises a therapeutic RNA.

48. The yeast cell of claim 45, wherein said ESM is positioned at the 3’ end of the RNA.

49. The yeast cell of claim 45, wherein said ESM is positioned at the 5’ end of the RNA.

50. The yeast cell of claim 45, wherein said ESM is positioned within an untranslated region (UTR) of the RNA and / or an intron region of the RNA.

51. The yeast cell of claim 45, wherein said ESM comprises a plurality of ESMs.

52. The yeast cell of claim 51, wherein said plurality of said ESMs comprise a first ESM positioned at the 3’ end of the RNA, and a second ESM positioned at the 5’ end of the RNA.

53. The yeast cell of claim 52, wherein said first and said second ESMs are between 20-30 basepairs apart.

54. The yeast cell of claim 45, wherein said first and said second ESMs are 25 base-pairs apart.

55. The yeast cell of any of claims 45- 54, wherein the RNA comprises a small RNA (smRNA).

56. The yeast cell of any of claims 45- 54, wherein the RNA comprises a shRNA.

57. The yeast cell of claim 56, wherein the ESM is positioned within a loop secondary structure of the shRNA.

58. The yeast cell of any of claims 45-57, wherein said ESM comprises a nucleotide sequence selected from: SEQ ID NO’s. 7 GAAGACAC, TTGTATAA, TATGTATA, AAAATTTT, SEQ ID NO.29, SEQ ID NO. 560, SEQ ID NO. 561, SEQ ID NO. 107, and / or SEQ ID NO. 108, or a fragment thereof.

59. The yeast cell of any of claims 45-57, wherein said ESM is selected from ayn ESM sequence identified in Tables 6-18, 31-33, and Table A.

60. The yeast cell of any of claims 45-59, and further comprising wherein a whole cell or counter sorting RNA motifs (CSM) present in the RNA sequence is disrupted or removed.

61. The yeast cell of claim 60, wherein the CSM is selected from a CSM sequence identified in Tables 19-30, and 34.

62. An EV isolated from the yeast cell of any of claims 45-61, wherein said EV includes an RNA molecule having an ESM.

63. A pharmaceutical composition comprising the EV of claim 62, and a pharmaceutically acceptable carrier.

64. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 63, to a subject in need thereof.

65. A yeast-generated extracellular vesicle (yEV) containing an RNA molecule engineered to include an ESM.

66. The EV of claim 65, wherein said yeast-generated EV is a Saccharomyces cerevisiae generated yEV, or Saccharomyces boulardii generated yEV.

67. The EV of claim 65, wherein the RNA comprises a therapeutic RNA.

68. The EV of claim 65, wherein said ESM is positioned at the 3’ end of the RNA.

69. The EV of claim 65, wherein said ESM is positioned at the 5’ end of the RNA.

70. The EV of claim 65, wherein said ESM is positioned within an untranslated region (UTR) of the RNA and / or an intron region of the RNA.

71. The EV of claim 65, wherein said ESM comprises a plurality of ESMs.

72. The EV of claim 71, wherein said plurality of said ESMs comprise a first ESM positioned at the 3’ end of the RNA, and a second ESM positioned at the 5’ end of the RNA.

73. The EV of claim 72, wherein said first and said second ESMs are between 20-30 base-pairs apart.

74. The EV of claim 65, wherein said first and said second ESMs are 25 base-pairs apart.

75. The EV of any of claims 65- 74, wherein the RNA comprises a small RNA (smRNA).

76. The EV of any of claims 65- 74, wherein the RNA comprises a shRNA.

77. The EV of claim 76, wherein the ESM is positioned within a loop secondary structure of the shRNA.

78. The EV of any of claims 65-77, comprises a nucleotide sequence selected from: SEQ ID NO’s.GAAGACAC, TTGTATAA, TATGTATA, AAAATTTT, SEQ ID NO. 29, SEQ ID NO. 560, SEQ IDNO. 561, SEQ ID NO. 107, and / or SEQ ID NO. 108, or a fragment thereof.

79. The EV of any of claims 65-77, wherein said ESM is selected from an ESM sequence identified in Tables 6-18, 31-33, and Table A.

80. The EV of any of claims 65-79, and further comprising wherein a whole cell or counter sorting RNA motifs (CSM) present in the RNA sequence is disrupted or removed.

81. The EV of claim 80, wherein the CSM is selected from a CSM sequence identified in Tables 19-30, and 34.

82. The EV of any of claims 65-81, wherein the EV is generated in vivo, or in vitro.

83. A pharmaceutical composition comprising the EV of any of claims 65-82, and a pharmaceutically acceptable carrier.

84. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 83, to a subject in need thereof.

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