Compositions and methods for exosome-mediated delivery of mRNA agents

Abstract

Compositions and methods are provided for delivering an mRNA agent to a subject, the mRNA agent being encapsulated within exosomes prepared from human stem or progenitor cells, such as human mesenchymal stem cells, human embryonic stem cells, or human cardiac progenitor cells. The compositions and methods can be used to deliver an mRNA agent encoding a therapeutic agent, such as an enzyme (e.g., metabolic enzyme), cytokine, growth factor, antigen, antibody, or immunomodulator, by administering the compositions to a subject. Methods for preparing compositions comprising exosomes encapsulating an mRNA agent are also provided.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 331,532, filed April 15, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The success of COVID-19 mRNA vaccines has established mRNA agents as viable drugs for human use, thus opening up a new biotechnology platform for a wide variety of preventive and therapeutic purposes. Current mRNA vaccines utilize modified mRNA (mmRNA) agents and incorporate the mmRNA into lipid nanoparticles (LNPs) for delivery in vivo. Although this has proven successful, there are potential limitations to this current approach.

[0003] Due to the inherent instability of mRNA, a delivery system is needed that protects it from degradation by nucleases and allows cellular uptake during in vivo administration. The current approach using LNPs was first used clinically to allow in vivo delivery of siRNA (Coelho et al. (2013) New Eng. J. Med. 369:819-829, Adams et al. (2018) New Eng. J. Med. 379:11-21). LNPs protect the RNA cargo and are taken up via the endosomal pathway, where a portion of the RNA cargo is released from the endosome and ultimately translated. Early versions of lipid nanoparticles containing ionizable amino lipids used to encapsulate siRNA are described, for example, in Jayarama et al. (2012) Angew Chem. Int. Ed. Engl. 51:8529-8533. This original version of LNPs enabled hepatic uptake and ultimately led to the approval of the first siRNA therapeutics, but they were associated with significant side effects (Coelho et al. (2013) New Eng. J. Med. 369:819-829; Adams et al. (2018) New Eng. J. Med. 379:11-21). However, a new generation of ionizable LNPs has been designed that enhances the release of RNA cargo and significantly improves safety and efficacy (Cheng et al. (2020) Nature Nanotech. 15:313-320). These newer versions allow for large-scale administration, have relatively rare severe side effects, and are being utilized to address new therapeutic candidates.

[0004] Although certain improvements have been made to LNP technology, it still has significant potential limitations, especially with regard to repeated administration, which is often required in treating chronic diseases with mRNA agents. Even with chemical modification of mRNA and more advanced packaging into lipid nanoparticles, the level of protein expression is attenuated by long-term repeated administration. Furthermore, the inability to target mRNA delivery to specific tissues is a challenge when applied to solid organ diseases. Apart from the liver, where intravenous (IV) delivery of LNPs can reach most of the organ, highly efficient in vivo delivery of mRNA agents to other solid organs remains challenging.

[0005] Thus, while there have been various advances in the use of mRNA agents in humans, there remains a need in the art for additional methods and approaches, particularly methods and approaches that provide alternatives to LNPs for delivering mRNA agents in vivo. Summary of the Invention

[0006] The present disclosure provides methods and compositions for delivery of an mRNA agent, where the mRNA agent is encapsulated in an extracellular vesicle (EV), such as an exosome, derived from stem or progenitor cells, e.g., mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, and progenitor cells along various lineages, such as cardiac or pancreatic progenitor cells. The use of stem or progenitor cells as a source of EVs, e.g., exosomes, has advantages including rapid proliferation of stem cells, the possibility of preparing large amounts of encapsulated mRNA agents, and the ability to control the differentiation of stem and progenitor cells, thereby allowing modification of the contents of the EVs. Furthermore, the use of low immunogenic stem or progenitor cells as a source of EVs, e.g., exosomes, allows for the preparation of low immunogenic EVs, e.g., exosomes, encapsulated mRNA agents that are less likely to stimulate immune responsiveness in vivo. Still further, stem or progenitor cell-derived EVs, e.g., exosomes, for delivery of an mRNA agent can be prepared by various approaches described herein, as described herein, and can be applied to different types of mRNA agents for various purposes.

[0007] Thus, in one aspect, the present disclosure relates to a method of delivering an mRNA agent to a subject, the method comprising administering to the subject a composition comprising extracellular vesicles, e.g., exosomes, prepared from human stem cells or human progenitor cells, where the EVs, e.g., exosomes, encapsulate the mRNA agent. For example, the mRNA agent can be encapsulated in the EVs by introducing the mRNA agent into human stem cells or human progenitor cells, preparing EVs from the human stem cells or progenitor cells, and thereby encapsulating the mRNA agent in the EVs.

[0008] In another aspect, the disclosure relates to a method of preparing a composition comprising an mRNA agent, the method comprising encapsulating the mRNA agent in an extracellular vesicle, e.g., an exosome, by: (a) introducing an mRNA agent into human stem cells or human progenitor cells and preparing EVs, e.g., exosomes, from the human stem cells or progenitor cells, thereby encapsulating the mRNA agent in the EVs, e.g., exosomes; or (b) preparing EVs, e.g., exosomes, from human stem cells or human progenitor cells, and introducing an mRNA agent into the EVs, e.g., exosomes, thereby encapsulating the mRNA agent in the EVs, e.g., exosomes.

[0009] In one embodiment, the mRNA agent is introduced into human stem cell or human progenitor cell or EV derived therefrom, such as exosome, by electroporation.In another embodiment, the mRNA agent is introduced into human stem cell or human progenitor cell or EV derived therefrom, such as exosome, by lipid nanoparticle-mediated transfection.

[0010] In yet another aspect, the disclosure relates to a method of delivering an mRNA agent to a subject, the method comprising: a) preparing a composition comprising EVs, e.g., exosomes, that encapsulate the mRNA agent, where the composition comprises: (i) introducing the mRNA agent into human stem cells or human progenitor cells and preparing EVs, e.g., exosomes, from the human stem cells or progenitor cells, thereby encapsulating the mRNA agent in the EVs, e.g., exosomes; or (ii) preparing EVs, e.g., exosomes, from human stem cells or human progenitor cells, and introducing an mRNA agent into the EVs, e.g., exosomes, thereby encapsulating the mRNA agent in the EVs, e.g., exosomes, which method also includes: b) administering the composition to a subject.

[0011] In yet another aspect, the present disclosure relates to a composition comprising EVs, e.g., exosomes, prepared from human stem cells or human progenitor cells, where the EVs, e.g., exosomes, encapsulate an mRNA agent.

[0012] In one embodiment, the mRNA agent comprises at least one modified nucleotide base. In another embodiment, the mRNA agent comprises all unmodified nucleotide bases. Various mRNA modifications are further described herein.

[0013] In one embodiment, the EVs, e.g., exosomes, are prepared from human mesenchymal stem cells (MSCs). In one embodiment, the human mesenchymal stem cells are induced mesenchymal stem cells (iMSCs). In one embodiment, the EVs, e.g., exosomes, are prepared from human embryonic stem (ES) cells. In one embodiment, the EVs, e.g., exosomes, are prepared from human induced pluripotent stem cells (iPSCs). In one embodiment, the EVs, e.g., exosomes, are prepared from human cardiac progenitor cells, such as human ventricular progenitor cells. In one embodiment, the EVs, e.g., exosomes, are prepared from human pancreatic progenitor cells, such as human beta islet progenitor cells.

[0014] In one embodiment, the stem cells from which the EVs, e.g., exosomes, are prepared are hypoimmunogenic, i.e., they have been modified to reduce their immunogenicity in a human subject. In one embodiment, the stem cells have been modified to inactivate major histocompatibility complex (MHC) class I and / or class II genes. In another embodiment, the stem cells have been modified to inactivate MHC class I and / or class II genes, as well as at least one additional gene involved in immune regulation.

[0015] The mRNA agent can encode a therapeutic or prophylactic agent of interest for administration to a subject, for example, based on the subject's condition to be treated or prevented. For example, in one embodiment, the mRNA agent encodes a metabolic enzyme (e.g., for treating a subject with a metabolic disorder). In one embodiment, the mRNA agent encodes an antigen (e.g., for use as a vaccine in a subject). In one embodiment, the mRNA agent encodes an immunomodulatory agent (e.g., for treating a subject with an autoimmune disorder, cancer, or other disease that would benefit from immunomodulation). In various embodiments, the mRNA agent encodes an enzyme, a cytokine, a growth factor, an antigen, an antibody, or an immunomodulatory protein.

[0016] The composition comprising stem cell-derived or progenitor cell-derived EVs, such as exosomes, encapsulating mRNA agents can be administered to a subject by a suitable route for desired effect.In one embodiment, the composition is administered to an intra-organ site of the subject.In one embodiment, the intra-organ site is in the heart.In another embodiment, the intra-organ site is in the kidney, pancreas, liver, lung, or brain.In another embodiment, the composition is administered to an extravascular site of the subject.In another embodiment, the composition is administered to the subject intramuscularly.Various means for delivering the composition are further described herein.

[0017] In another aspect, the present disclosure relates to a method for delivering a functional macromolecule to a cell, the method comprising: The method includes encapsulating an mRNA encoding a functional macromolecule in an extracellular vesicle (EV) prepared from a human stem cell or a human progenitor cell, where the mRNA is, for example, at least 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, or 1 kilobase in length, and the method also includes contacting a cell with the EV, thereby delivering the functional macromolecule to the cell.

[0018] In other embodiments, the mRNA is at least 2 kilobases, 3 kilobases, 4 kilobases, 5 kilobases, 6 kilobases, 7 kilobases, 8 kilobases, 9 kilobases, or 10 kilobases in length.

[0019] In one embodiment, the mRNA encodes a Cre recombinase. In one embodiment, the mRNA encodes a CRISPR Cas9 protein. In other embodiments, the mRNA encodes a CRISPR Cas12, Cas13, or Cas14 protein. In other embodiments, the mRNA encodes VEGF or phospholamban (PLN). In various embodiments, the mRNA agent encodes an enzyme (e.g., a metabolic enzyme), a cytokine, a growth factor, an antigen, an antibody, or an immunomodulatory protein.

[0020] In one embodiment, the EVs are administered to a subject, thereby delivering functional macromolecules to cells in vivo. In various embodiments, the EVs are exosomes, e.g., exosomes derived from induced mesenchymal stem cells (iMSCs).

[0021] In one embodiment, EVs are administered to the subject's intraorgan site, such as the site in the heart, or the site in the kidney, pancreas, liver, lung, or brain.In one embodiment, EVs are administered to the subject's extravascular site.In one embodiment, EVs are administered using an intraluminal delivery device.

[0022] In another aspect, the disclosure relates to a method of expressing a protein in a cell, the method comprising: and encapsulating an mRNA agent encoding the protein in an extracellular vesicle (EV) prepared from a human stem cell or a human progenitor cell, where the mRNA agent is, for example, at least 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, or 1 kilobase in length, and the method also includes: This involves transfecting cells with the EVs so that the mRNA agent expresses the protein within the cells.

[0023] In certain embodiments, an mRNA agent is at least 2 kilobases, 3 kilobases, 4 kilobases, 5 kilobases, 6 kilobases, 7 kilobases, 8 kilobases, 9 kilobases, or 10 kilobases in length.

[0024] In some embodiments, the EVs are exosomes, for example exosomes prepared from human mesenchymal stem cells (MSCs). In some embodiments, the MSCs are induced MSCs (iMSCs).

[0025] In some embodiments, the mRNA agent encodes, for example, an enzyme, an antigen, or an immunomodulatory protein. In some embodiments, the protein encoded by the mRNA agent is, for example, Cre recombinase, CRISPR Cas9 protein, VEGF, or phospholamban (PLN).

[0026] In one embodiment, EVs are administered to a subject, thereby delivering proteins to cells of the subject in vivo. In one embodiment, EVs are administered to an intra-organ site of the subject. In one embodiment, the intra-organ site is in the heart. In another embodiment, the intra-organ site is in the kidney, pancreas, liver, lung, or brain. In another embodiment, EVs are administered to an extravascular site of the subject. In one embodiment, EVs are administered using an intraluminal delivery device.

[0027] In another aspect, the present disclosure relates to a composition comprising an exosome prepared from human induced mesenchymal stem cells (iMSCs), wherein the exosome encapsulates an mRNA agent at least 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, or 1 kilobase in length. In an embodiment, the mRNA agent is at least 2 kilobases, 3 kilobases, 4 kilobases, 5 kilobases, 6 kilobases, 7 kilobases, 8 kilobases, 9 kilobases, or 10 kilobases in length. In an embodiment, the mRNA agent comprises at least one modified nucleotide base. In another embodiment, the mRNA agent comprises all unmodified nucleotide bases. The mRNA agent may, for example, code for an enzyme (e.g., a metabolic enzyme), a cytokine, a growth factor, an antigen, an antibody, or an immunomodulatory agent. In an embodiment, the mRNA agent codes for a Cre recombinase, a CRISPR Cas9 protein, a VEGF, or a phospholamban (PLN).

[0028] These and other aspects of the disclosure are described in further detail herein. [Brief description of the drawings]

[0029] [Figure 1]1 is a bar graph showing in vitro expression of modified mRNA (mmRNA) by mesenchymal stem cells (MSCs) after electroporation or LNP-mediated transfection (RNAiMAX). A shows the relative fluorescence intensity of MSCs treated with mmRNA encoding mCherry. B shows VEGF secretion in MSCs treated with mmRNA encoding VEGF at 24, 48, and 72 hours. [Diagram 2] Graph showing in vivo expression of luciferase in mice treated with MSCs electroporated with modified mRNA (mmRNA) encoding luciferase or treated with luciferase-encoding mmRNA complexed with RNAiMAX. [Diagram 3] Representative images are shown demonstrating that iMSC TSPAN markers CD9, CD63, CD81 localize on tomographic bright field visible intra / extracellular vesicles. [Figure 4] 1 is a bar graph showing representative flow cytometry data illustrating the supernatant concentrations of TSPAN-containing exosomes under different density and freeze-thaw conditions. [Diagram 5] Representative images are shown using Nanolive 3D Cell Player-Fluo to visualize transfected mRNA-594-GFP contained in tomographic bright-field visible vesicles within intracellular compartments. [Figure 6] Representative images are shown using Nanolive 3D Cell Explorer-Fluo to visualize transfected mRNA-594-GFP contained in tomographic brightfield visible vesicles within intracellular compartments that are also TSPAN (CD9 / 63 / 81) positive. [Figure 7] Representative images are shown using Nanolive 3D Cell Explorer-Fluo to visualize transfected mRNA-594-GFP contained in tomographic brightfield visible vesicles within the extracellular compartment, which are also TSPAN (CD9 / 63 / 81) positive. [Figure 8]Representative images using Nanolive 3D Cell Explorer-Fluo to visualize translated GFP protein contained in tomographic brightfield visible vesicles (which are also TSPAN (CD9 / 63 / 81) positive) within intra- / extracellular compartments are shown. [Figure 9] Representative images are shown visualizing translated GFP protein expressed within beating cardiomyocytes using standard bright field and fluorescence microscopy techniques. [Figure 10] Representative images are shown in which translated TSPAN-GFP protein contained in tomographic bright-field visible vesicles was visualized using Nanolive 3D Cell Explorer-Fluo, and further visualization of whole TSPAN using a TSPAN (CD9 / 63 / 81)-conjugated antibody. [Figure 11] Representative images of recipient mouse cells after Cre mRNA delivery using iMSC EVs are shown. A shows Cre recombinase expression detected in recipient cells using immunofluorescence staining with anti-Cre antibody. B shows expression of tdTomato reporter gene. C shows control stained with DAPI. [Figure 12] 1 is a bar graph showing the expression of Cas9 mRNA in donor cells electroporated with Cas9 mRNA and in recipient cells treated with iMSC-EVs recovered from the supernatant of donor cells. [Figure 13] 1 is a bar graph showing CD63 expression in the indicated organs from mice injected under the kidney capsule with iMSCs transfected with CD63-GFP mRNA. Quantitative PCR results show the relative expression of exogenous CD63 mRNA in tissues of the indicated organs on days 1, 3, or 7 in the treatment group (CD63-GFP mRNA group) compared to the control group (GFP mRNA group). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present disclosure relates to the use of extracellular vesicles (EVs), such as stem cell or progenitor cell derived exosomes, to deliver mRNA agents to cells. In certain embodiments, the EVs, e.g., exosomes, are derived from mesenchymal stem cells, e.g., induced mesenchymal stem cells (iMSCs). As shown in the examples, iMSC-derived exosomes loaded with mRNA agents can be obtained by several different approaches, and the loaded exosomes can be used to deliver mRNA cargo to cells, such as excitable cardiomyocytes. Additionally, iMSCs can be modified to enhance expression of tetraspanins in iMSCs, thereby promoting exosome formation by iMSCs. Cargo-loaded exosomes (e.g., derived from iMSCs) can be used to deliver mRNA agents to cells, tissues, organs, or body locations of interest, as described herein, for example, directly to the heart in vivo, or to the extravascular space, using a catheter or intraluminal delivery cannula, as described herein. It has been shown that local administration of mRNA agents in vivo can result in systemic distribution of expression of the mRNA agents.

[0031] As used herein, the term "extracellular vesicles" or "EVs" refers to lipid bilayer-encapsulated particles that are naturally released from almost all cell types but cannot replicate. EVs include exosomes, vesicles, and apoptotic bodies. As used herein, "exosomes" refers to a type of extracellular vesicle that is derived from endosomes and is usually approximately 30-120 nm in size, whereas vesicles are usually approximately 100-1000 nm in size and are primarily derived from outward budding of the plasma membrane. As used herein, "loaded" EVs or exosomes refers to vesicles that carry a cargo, such as an mRNA cargo, that has been introduced into the vesicle. Means of loading cargo into EVs and exosomes are further described herein. Exosomes can be detected based on detection of one or more exosome markers, non-limiting examples of which include the tetraspanin proteins CD9, CD63, CD81, CD82, and CD151.

[0032] Various aspects of the disclosure are described in the following subsections.

[0033] I. Stem and Progenitor Cells The methods and compositions of the disclosure utilize EVs, such as exosomes, derived from (i.e., prepared from) stem or progenitor cells, such as human stem cells or human progenitor cells.

[0034] As used herein, the term "stem cell" is used in a broad sense and includes conventional stem cells, precursor cells, pre-progenitor cells, reserve cells, and the like. As used herein, the terms "stem cell" or "progenitor cell" are used interchangeably and refer to undifferentiated cells that can proliferate and give rise to more precursor cells that have the ability to generate a large number of mother cells that can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and generate progeny, which then differentiate into one or more mature cell types, while also retaining one or more cells that have the developmental potential of the parent cell. The term "stem cell" refers to a cell that has the ability or potential to differentiate into a further specialized or differentiated phenotype under certain circumstances, and that retains the ability to proliferate without substantial differentiation under certain circumstances. In one embodiment, the term progenitor cell or stem cell refers to a general mother cell whose progeny (progeny) often specialize in different directions by differentiation, for example, by acquiring completely individual characteristics, as occurs in the progressive diversification of embryonic cells and tissues. Cell differentiation is a complex process that usually occurs through many cell divisions. Differentiated cells may be derived from pluripotent cells that are themselves derived from pluripotent cells. Each of these pluripotent cells may be considered a stem cell, although the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the ability to give rise to cells with higher developmental potential. Such ability may be natural or may be artificially induced upon treatment with various factors. In many biological cases, stem cells are also "pluripotent" because they can generate progeny of multiple different cell types, but this is not a requirement for "stemness". Self-renewal is another classical part of the definition of stem cells and is essential as used herein. In theory, self-renewal can occur by either of two main mechanisms. Stem cells may divide asymmetrically, with one daughter cell retaining the stem cell state and the other daughter cell expressing some other different specific function and phenotype. Alternatively, some of the stem cells in a population may divide symmetrically into two stem cells, thus retaining some stem cells in the population as a whole, while other cells in the population give rise only to differentiated progeny.Formally, cells that begin as stem cells can progress to a differentiated phenotype, but can then "reverse" and re-express the stem cell phenotype, a term often referred to as "dedifferentiation."

[0035] The term "progenitor cell" is used herein to refer to a cell that has a more primitive cellular phenotype (e.g., at an earlier step along a developmental pathway or progression than a fully differentiated cell) compared to the cell that it can give rise to by differentiation. In many cases, progenitor cells also have significant or very high proliferation potential. Depending on the developmental pathway and the environment in which the cell develops and differentiates, progenitor cells can give rise to multiple different differentiated cell types or a single differentiated cell type.

[0036] A. Induced pluripotent stem cells In some embodiments, the stem or progenitor cells used in the methods of the present disclosure are pluripotent or exhibit a pluripotent or pluripotent state. As used herein, the term "pluripotent" refers to cells that have the ability to differentiate into cell types characteristic of all three germ layers (endoderm, mesoderm, and ectoderm) under different conditions. Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers, for example, using nude mice and teratoma formation assays. Although pluripotency is demonstrated by the expression of embryonic stem cell (ES cell) markers, the preferred test of pluripotency is to demonstrate the ability to differentiate into each of the three germ layers. In some embodiments, the pluripotent cells are undifferentiated cells. As used herein, the term "pluripotency" or "pluripotent state" refers to cells that have the capacity to differentiate into all three embryonic germ layers: endoderm (gut tissue), mesoderm (containing blood, muscle, and blood vessels), and ectoderm (such as skin and nerves), and have the potential to divide in vitro for extended periods of time, typically, for example, more than one year or more than 30 passages.

[0037] In one embodiment, the disclosed methods and compositions utilize exosomes derived from (i.e., prepared from) embryonic stem cells, e.g., human embryonic stem cells. The terms "embryonic stem cells," "ES cells," and "ESCs" are used interchangeably herein and refer to pluripotent stem cells of the inner cell mass of blastocysts (see, e.g., U.S. Patent Nos. 5,843,780 and 6,200,806, which are incorporated herein by reference). Such cells can be similarly obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, e.g., U.S. Patent Nos. 5,945,577, 5,994,619, and 6,235,970, which are incorporated herein by reference). The distinguishing characteristics of embryonic stem cells define the phenotype of the embryonic stem cells. Thus, a cell has the phenotype of an embryonic stem cell if it has one or more of the unique characteristics of embryonic stem cells such that the cell can be distinguished from other cells. Exemplary distinguishing embryonic stem cell characteristics include, but are not limited to, gene expression characteristics, proliferation capacity, differentiation capacity, karyotype, responsiveness to specific culture conditions, and the like. In some embodiments, ES cells can be obtained without destroying an embryo, e.g., without destroying a human embryo. Numerous embryonic stem cell lines are well established and available in the art, non-limiting examples of which include ES03 cells (WiCell Research Institute) and H9 cells (Thomson, JA et al. (1998) Science 282:1145-1147). Media and culture conditions for maintaining and growing ES cell lines are also well established in the art and commercially available. Preparation of extracellular vesicles, e.g., exosomes, derived from ES cells has been described in the art (see, e.g., Ke et al. (2021) Stem Cell Res. & Therap. 12:21).

[0038] In one embodiment, the methods and compositions of the present disclosure utilize exosomes derived from (i.e., prepared from) induced pluripotent stem cells (iPSCs), e.g., human induced pluripotent stem cells. As used herein, "induced pluripotent stem cells" refers to a type of pluripotent stem cell that is derived from an adult somatic cell but has been reprogrammed to become pluripotent through the induction of certain genes and factors. Numerous human iPSC lines are well established and available in the art, including, but not limited to, 19-11-1, 19-9-7, or 6-9-9 cells (e.g., as described in Yu, J. et al. (2009) Science 324:797-801). Media and culture conditions for maintaining and expanding iPSCs are also well established in the art and commercially available. Preparation of extracellular vesicles, such as exosomes, from iPSCs has been described in the art (see, e.g., Jeske et al. (2020) Tissue Eng. Part B: Reviews 26:129-144).

[0039] In certain embodiments, pluripotent stem cells are identified or indicated by expression of one or more pluripotent stem cell markers, non-limiting examples of which include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, and / or SOX2.

[0040] In one embodiment, the methods and compositions of the present disclosure utilize exosomes derived from (i.e., prepared from) adult stem cells, such as human adult stem cells. The term "adult stem cell" or "ASC" is used to refer to any multipotent stem cell derived from non-embryonic tissues, including fetal tissues, juvenile tissues, and adult tissues. Stem cells have been isolated from a wide variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells.

[0041] B. Mesenchymal stem cells In one embodiment, the disclosed methods and compositions utilize exosomes derived from (i.e., prepared from) mesenchymal stem cells (MSCs), such as induced mesenchymal stem cells (iMSCs), which can be prepared from pluripotent stem cells. As used herein, the term "mesenchymal stem cells" refers to multipotent adult stem cells that can self-renew by dividing and differentiate into multiple tissues, including bone, cartilage, muscle cells, adipocytes, and connective tissue. Mesenchymal stem cells naturally occur in multiple tissues, including umbilical cord, bone marrow, adipose tissue, and peripheral blood. Thus, in one embodiment, the MSCs used to prepare exosomes are MSCs isolated from a subject to which the mRNA-loaded exosomes (prepared as described herein) are administered (i.e., the MSCs are isolated from the same subject to be treated with the MSC-derived exosomes).

[0042] In one embodiment, MSCs are bone marrow mesenchymal stem cells (BMSCs), which can be directly isolated from a subject. US Patent Application US2008 / 0279828A1 discloses a method for mobilizing bone marrow stem cells into the peripheral blood of a donor to harvest bone marrow stem cells, which is incorporated herein by reference in its entirety. The method includes administering an effective amount of at least one chelated copper to a donor, thereby expanding bone marrow stem cells in vivo while reversibly inhibiting the differentiation of bone marrow stem cells, and harvesting bone marrow stem cells by white matter removal.

[0043] Alternatively, cells that differentiate into BMSCs ("BMSC precursors") can be isolated from a subject and then exposed to one or more chemical or biological agents in culture to differentiate into BMSCs. U.S. Patent No. 5,486,359 describes the isolation of human mesenchymal stem cells that can differentiate into multiple tissue types (e.g., bone, cartilage, muscle, or bone marrow stroma), as well as methods for isolating, purifying, and culturing human mesenchymal stem cells.

[0044] Additional sources of MSCs from the adult niche include adipose / adipose-derived MSCs and peripheral blood-derived MSCs. Additionally, MSCs from the prenatal / neonatal environment, including umbilical cord- and placenta-derived MSCs, can be used in the methods described herein. Human umbilical cord- and placenta-derived MSCs, as well as peripheral blood-derived MSCs, can be isolated from patients using methods known in the art, for example, by a combination of tissue explant culture and / or by density gradient separation by centrifugation (Beeravolu et al. (2017) J. Vis. Exp, 122; Chong et al. (2012) J Orthop Res., 30(4): 634-42. For the isolation of adipose / adipose-derived MSCs, cells can be first isolated, for example, using a method involving liposuction and excision (Schneider et al. (2017) Eur. J. Med. Res. 22(1): 17. Although some degree of functional diversity exists among mesenchymal stem cells derived from different patients and / or different tissue sources, for mesenchymal stem cells to maintain their identity, they should possess three functional attributes: 1) the potential for self-renewal, 2) the ability to grow on plastic, and 3) the ability to differentiate into three major cell types, including osteoblasts (bone), chondrocytes (cartilage), and adipocytes (fat). Furthermore, regardless of the source of MSCs, they should possess identifying markers such as CD73, CD90, and the absence of CD14, CD34, and CD45 (Ullah et al. (2015) Biosci. Rep., 35(2); Fitzsimmons et al. (2018) Stem Cells Int. 2018:8031718). In one embodiment, the MSCs are induced MSCs (iMSCs) prepared from pluripotent stem cells, such as human embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs). Methods for preparing iMSCs from pluripotent stem cells have been described in the art (see, for example, Soontararak et al. (2018) Stem Cells Transl. Med. 7:456-467; Yang et al. (2019) Cell Death and Disease 10:718; Xu et al. (2019) Stem Cells 37:754-765). Culture protocols for differentiation of iMSCs from pluripotent stem cells are also described in detail in U.S. Provisional Patent Application No. 63 / 307,368, filed February 7, 2022, the entire contents of which are specifically incorporated herein by reference.

[0045] As shown in Example 3, iMSCs express tetraspanins such as CD9, CD63, and CD81. Tetraspanins are a protein superfamily that form clusters and organize membrane microdomains called tetraspanin-enriched microdomains (TEMs) by interacting with various transmembrane and cytosolic signaling proteins (see, e.g., Hemler et al. (2005) Nat. Rev. Mol. Cell. Biol. 6:801-811). Tetraspanins are expressed on various types of inner cell membranes, and therefore they are used in the art as exosome markers. Non-limiting examples of tetraspanins include CD9, CD63, CD81, CD82, and CD151. In one embodiment, iMSCs express at least one, and preferably multiple (e.g., two, three, four, or five) tetraspanins selected from the group consisting of CD9, CD63, CD81, CD82, and CD151. Tetraspanin expression on cells can be measured by methods well established in the art, such as using anti-tetraspanin antibodies for immunodetection.

[0046] As shown in Example 6, transfection of iMSCs with a nucleic acid construct(s) encoding tetraspanin(s) (e.g., mRNA encoding a tetraspanin) promotes the formation of iMSC-derived exosomes that are tetraspanin positive. Thus, in one embodiment, iMSCs are modified (e.g., genetically modified) to express one or more tetraspanins, such as one or more selected from the group consisting of CD9, CD63, CD81, CD82, and CD151. Regardless of whether iMSCs endogenously express tetraspanin(s), the cells can be modified to enhance expression of tetraspanins, thereby promoting exosome formation. In one embodiment, the cells are modified with one or more mRNA constructs encoding tetraspanin(s). In one embodiment, the cells are modified with one or more DNA constructs encoding tetraspanin(s).

[0047] C. Cardiac progenitor cells.

[0048] In one embodiment, the methods and compositions of the present disclosure utilize exosomes derived from (i.e., prepared from) cardiac progenitor cells, e.g., human cardiac progenitor cells. As used herein, the term "cardiac progenitor cells" refers to progenitor cells that are committed to the cardiac lineage and have the ability to differentiate into all three cardiac lineage cells (cardiomyocytes, endothelial cells, and smooth muscle cells). Cultures of human cardiac progenitor cells can be obtained, for example, by culturing human stem cells under conditions that bias the stem cells toward differentiation into the cardiac lineage. In certain embodiments, the stem cells cultured to generate human cardiac progenitor cells are human embryonic stem cells or human induced pluripotent cells. Various methods for differentiating pluripotent stem cells along the cardiac lineage, thereby generating cardiac progenitor cells, are well established in the art. Additionally, preparation of extracellular vesicles, e.g., exosomes, derived from cardiac progenitor cells has been described in the art (see, e.g., Wang et al. (2019) J. Cell Mol. Med. 23:7124-7131).

[0049] In one embodiment, the cardiac progenitor cells from which the exosomes are derived are ventricular progenitor cells, e.g., human ventricular progenitor cells. As used herein, the terms "ventricular progenitor cells", "human ventricular progenitor cells", and "HVP" refer to progenitor cells that are committed to the cardiac lineage and differentiate primarily into ventricular myocytes (i.e., more than 50% of the differentiated cells derived from the progenitor cells, preferably more than 60%, more than 70%, more than 80%, or more than 90% of the differentiated cells are ventricular myocytes). Methods for differentiating pluripotent stem cells along the ventricular lineage, thereby generating ventricular progenitor cells, are well established in the art. For example, methods for generating human ventricular progenitor cells (HVPs) are described in detail in U.S. Patent Publication Nos. 2016 / 0053229, 2016 / 0108363, 2018 / 0148691, and 2019 / 0062696. Non-limiting examples of HVP markers include ISL1, JAG1, FZD4, LIFR, FGFR3, TNFSF9, PDGFRA, and NRP-1.

[0050] D. Pancreatic progenitor cells In one embodiment, the disclosed methods and compositions utilize exosomes derived from (i.e., prepared from) pancreatic progenitor cells, e.g., human pancreatic progenitor cells. As used herein, the term "pancreatic progenitor cells" refers to multipotent progenitor cells derived from the developing foregut endoderm that have the ability to differentiate into lineage-specific precursors committed to the developing pancreas, including both endocrine and exocrine cells. Cultures of human pancreatic progenitor cells can be obtained, for example, by culturing human stem cells under conditions that bias the stem cells toward differentiation into pancreatic lineages. In certain embodiments, the stem cells cultured to generate human pancreatic progenitor cells are human embryonic stem cells or human induced pluripotent cells. Various methods for differentiating pluripotent stem cells along pancreatic lineages, thereby generating pancreatic progenitor cells, are well established in the art. Furthermore, preparation of extracellular vesicles, such as exosomes, from pancreatic progenitor cells has been described in the art (see, e.g., Figliolini et al. (2014) PLoS ONE 9(7):e102521; Guay et al. (2015) Cell Commun. Signal. 13:17).

[0051] In one embodiment, the pancreatic progenitor cells from which the exosomes are derived are beta islet progenitor cells, e.g., human beta islet progenitor cells. As used herein, the term "beta islet progenitor cells" refers to progenitor cells that are committed to the pancreatic lineage and differentiate primarily into beta islet cells of the pancreas. Beta islet progenitor cells include beta cell progenitor cells that are MafB+ / Pdx1+ / Nkx2.2+ cells, and beta cell progenitor cells that express Pax1. Methods for differentiating pluripotent stem cells along the pancreatic lineage, thereby generating beta islet progenitor cells, are well established in the art. For example, methods for generating human islet progenitor cells are described in Pagliuca and Melton (2013) Development 140:2472-2483; Zhou and Melton (2018) Nature 557:351-358; Ma et al. (2013 ... al. (2018) Proc. Natl. Acad. Sci. USA 115:3924-3929; U.S. Patent Publication 20130344594; U.S. Patent Publication 20150231181; U.S. Patent Publication 20160326494; U.S. Patent Publication 20160175363; U.S. Patent Publication 20161777267; U.S. Patent Publication 20161777268; U.S. Patent Publication 20161777269; U.S. Patent Publication 20170029778; U.S. Patent Publication 20200199539; and U.S. Patent Publication 202000347358.

[0052] E. Low immunogenic cells In one embodiment, the disclosed methods and compositions utilize exosomes derived from (i.e., prepared from) stem or progenitor cells that are hypoimmunogenic. As used herein, the term "hypoimmunogenic" refers to modification of stem or progenitor cells to reduce their immunogenicity in vivo (e.g., to reduce their ability to stimulate an immune response in a human subject). Typically, cells are made hypoimmunogenic by disabling one or more genes involved in the recognition of stem / progenitor cells by the immune system and / or activation of the immune system by stem / progenitor cells. Genes can be disabled by standard recombinant DNA techniques that are well established in the art, including numerous approaches for gene "knockout." In one embodiment, the cells are modified to lack expression of major histocompatibility complex (MHC) genes. In one embodiment, the cells lack expression of MHC class I and / or class II genes. In another embodiment, the cells lack expression of one or more additional genes involved in immune recognition or activation, such as minor histocompatibility genes. In one embodiment, the cells lack expression of MHC class I and / or class II and also lack expression of CD47. In another embodiment, the cells lack expression of MHC class I and / or class II and also lack expression of CD47, PD-L1, and HLAG. Low immunogenic human pluripotent stem cells and methods for their preparation are well known in the art (see, e.g., Han et al. (2019) Proc. Natl. Acad. Sci. USA 116:10441-10446; Deuse et al. (2019) Nature 37:252-258; Deuse et al. (2019) Nature Biotechnology 37:252-258; Zhao et al. (2020) iScience 23:101162; Ye et al. (2020) Cell Prolif. 53:e12946; U.S. Patent Publication 2019 / 0309259; and U.S. Patent Publication 2021 / 0261916).

[0053] II. mRNA Agents The mRNA agent used in the disclosed methods and compositions may be natural or non-natural mRNA. In one embodiment, the mRNA comprises natural nucleobases, nucleosides, or nucleotides (i.e., all nucleobases, nucleosides, or nucleotides in the mRNA are natural). In another embodiment, the mRNA comprises one or more modified nucleobases, nucleosides, or nucleotides as described below, in which case the mRNA may be referred to as "modified mRNA" or "mmRNA". As described herein, a "nucleoside" is defined as a compound that comprises a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). As described herein, a "nucleotide" is defined as a nucleoside that comprises a phosphate group.

[0054] An mRNA agent may include a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and / or a coding region (e.g., an open reading frame). An mRNA may include any suitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900), or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) of base pairs. Any number (e.g., all, some, or none) of the nucleic acid bases, nucleosides, or nucleotides may be analogs, substituted, modified, or non-naturally occurring versions of standard species. In certain embodiments, all of a particular nucleobase type may be modified.

[0055] In some embodiments, an mRNA agent can include a 5' cap structure, a chain-terminating nucleotide, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem loop, a polyA sequence, and / or a polyadenylation signal.

[0056] A 5' cap structure or cap species is a compound that includes two nucleoside moieties joined by a linker and can be selected from a natural cap, a non-natural cap or cap analog, or an anti-reverse cap analog (ARCA). A cap species can include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap is a guanine nucleotide and a guanine (G) nucleotide linked at the 5' position by a triphosphate bond and methylated at the 7 position, typically m 7 It is written as GpppG, m 7 It may contain G(5')ppp(5')G.

[0057] In some embodiments, an mRNA agent is an unmodified mRNA that does not use chemically modified nucleosides but still includes a 5' cap structure or cap species described above.

[0058] The mRNA agent may include a chain-terminating nucleoside. For example, the chain-terminating nucleoside may include nucleosides that are deoxygenated at the 2' and / or 3' positions of their sugar groups. Such species may include 3'-deoxyadenosine (chodycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, and 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, and 2',3'-dideoxythymine. In some embodiments, the incorporation of a chain-terminating nucleotide at, for example, the 3' end of an mRNA may result in stabilization of the mRNA, as described, for example, in International Patent Publication No. WO2013 / 103659.

[0059] The mRNA may include a polyA sequence and / or a polyadenylation signal. The polyA sequence may be composed entirely or mainly of adenine nucleotides or analogs or derivatives thereof. The polyA sequence may be a tail located adjacent to the 3' untranslated region of the mRNA. In some embodiments, the polyA sequence may affect the nuclear export, translation, and / or stability of the mRNA.

[0060] The mRNA agent can include a microRNA binding site. Numerous microRNA binding site sequences are known in the art.

[0061] In some embodiments, an mRNA agent comprises one or more modified nucleic acid bases, nucleosides, or nucleotides (referred to as "modified mRNA" or "mmRNA"). In some embodiments, modified mRNAs may have useful properties compared to a reference unmodified mRNA, including improved stability, improved intracellular retention, improved translation, and / or lack of substantial induction of an innate immune response in a cell into which the mRNA is introduced. Thus, the use of modified mRNAs may have enhanced efficiency of protein production, intracellular retention of nucleic acids, as well as reduced immunogenicity.

[0062] In some embodiments, the mRNA comprises one or more (e.g., 1, 2, 3, or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the mRNA comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the modified mRNA has reduced degradation in a cell into which the mRNA is introduced, compared to the corresponding unmodified mRNA. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the modified nucleobase is a modified cytosine. In some embodiments, the modified nucleobase is a modified adenine. In some embodiments, the modified nucleobase is a modified guanine. In some embodiments, the mRNA agent comprises a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0063] In certain embodiments, the mRNA agent is uniformly modified (i.e., fully modified, modified throughout the entire sequence) for a particular modification. For example, the mRNA is modified with N1-methylpseudouridine (m 1 Ψ) or 5-methyl-cytidine (m 5 C), which means that all uridine or all cytosine nucleosides in the mRNA sequence are N1-methylpseudouridine (mC). 1 Ψ) or 5-methyl-cytidine (m 5 C). Similarly, an mRNA agent can be uniformly modified for any type of nucleoside residue present in the sequence by substitution with a modified residue, such as those described above.

[0064] In some embodiments, an mRNA agent is modified in a coding region (e.g., an open reading frame encoding a polypeptide). In other embodiments, an mRNA agent is modified in a region other than the coding region. For example, in some embodiments, the 5'-UTR and / or 3'-UTR are used, either or both of which may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the coding region.

[0065] Non-limiting examples of nucleoside modifications and combinations thereof that may be present in an mmRNA agent include, but are not limited to, those described in PCT Patent Application Publications: WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.

[0066] In some embodiments, the mRNA agent may be codon-optimized. Codon optimization methods are known in the art and can be useful for a variety of purposes, namely, matching the codon frequency in the host organism to ensure proper folding, biasing the GC content to increase mRNA stability or reduce secondary structure, minimizing tandem repeat codons or base runs that may impair gene assembly or expression, customizing transcriptional and translational control regions, inserting or removing protein trafficking sequences, removing / adding post-translational modification sites (e.g., glycosylation sites) of the encoded protein, adding, removing, or shuffling protein domains, inserting or deleting restriction enzyme sites, modifying ribosome binding sites and mRNA degradation sites, adjusting the translation rate so that various domains of the protein can fold properly, or reducing or eliminating problematic secondary structures in polynucleotides. Codon optimization tools, algorithms, and services are known in the art. Non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, Calif.), and / or proprietary methods. In one embodiment, an optimization algorithm is used to optimize the mRNA sequence, for example, to optimize expression in mammalian cells or to enhance mRNA stability.

[0067] In some embodiments, the mRNA agent is a "large" mRNA that is at least 1 kilobase long. In some embodiments, the mRNA agent is at least 1 kilobase long, at least 1.5 kilobase long, at least 2 kilobase long, at least 2.5 kilobase long, at least 3 kilobase long, at least 3.5 kilobase long, at least 4 kilobase long, at least 4.5 kilobase long, at least 5 kilobase long, at least 5.5 kilobase long, at least 6 kilobase long, at least 6.5 kilobase long, at least 7 kilobase long, at least 7.5 kilobase long, at least 8 kilobase long, at least 8.5 kilobase long, at least 9 kilobase long, at least 9.5 kilobase long, or at least 10 kilobase long. In some embodiments, the large mRNA encodes a functional protein as described herein.

[0068] The mRNA agent may be produced by means available in the art, including but not limited to in vitro transcription (IVT) and in vitro synthesis. Enzyme (IVT), solid phase, liquid phase, combinatorial synthesis, small area synthesis, and ligation methods may be utilized. In one embodiment, the mRNA is produced using IVT enzymatic synthesis. Methods for producing polynucleotides by IVT are known in the art and are described in International Application PCT / US2013 / 30062, the contents of which are incorporated herein by reference in their entirety.

[0069] Non-natural modified nucleobases may be introduced into polynucleotides, such as mRNA, during or after synthesis. In certain embodiments, the modifications may be on the internucleoside linkage, the purine or pyrimidine base, or the sugar. In certain embodiments, the modifications may be introduced at the end of the polynucleotide chain or elsewhere within the polynucleotide chain using chemical synthesis or polymerase enzymes. Examples of modified nucleic acids and their synthesis are disclosed in PCT Application No. PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0070] III. Encapsulation of mRNA Agents into Extracellular Vesicles By different means, as described below, mRNA agent can be encapsulated in stem cell-derived EV, for example, exosome.In one embodiment, mRNA agent is introduced into stem cell, and then EV, for example, exosome is prepared from the cell.In another embodiment, EV, for example, exosome is prepared from stem cell, and then mRNA agent is introduced into EV, for example, exosome.

[0071] A. Delivery of mRNA agents into cells In one embodiment, an mRNA agent is introduced into stem or progenitor cells, and then EVs, e.g., exosomes, are prepared from the mRNA-loaded cells, such that the EVs, e.g., exosomes, encapsulate the loaded mRNA within the cells.

[0072] The mRNA agent can be introduced into stem cells or progenitor cells by methods known in the art.Methods include, but are not limited to, electroporation, transfection (e.g., using cationic lipid transfection reagents), and lipid nanoparticles that encapsulate the mRNA agent.The loading of the mRNA agent into stem cells is also described in detail in Example 1.

[0073] In a non-limiting exemplary embodiment, lipid-mediated transfection is used to introduce an mRNA agent into stem or progenitor cells (e.g., MSCs). For example, bone marrow derived mesenchymal stem cells (BMSCs) are grown in culture (e.g., 20-30 x 10 cells in a 6-well plate or flask). 4 cells / well) and 0.2–0.4 mL / cm 2 Approximately 4,000-6,000 cells / cm in culture medium 2 For example, MSCs grown in T-75 flasks are typically seeded at 300,000 cells / flask in 15 mL of medium. An exemplary mRNA agent is modified mRNA (mmRNA), and mmRNA complexes are formed with cationic-lipid transfection reagents and incubated with BMSCs in culture. For example, mmRNA complexes can be formed by using, for example, 2.5 μl of Lipofectamine™ MessengerMAX™ reagent per μg of mmRNA (RNAiMax reagent and Lipofectamine 2,000 and 3,000 reagents are also effective). Calculations are performed to transfect BMSCs with a dose of 10 pg / cell of mRNA (e.g., reporter mRNA encoding luciferase, GFP, or mCherry). The ratio of modified mRNA to cells can range from 1 pg / cell to 100 pg / cell.

[0074] In another non-limiting exemplary embodiment, the mRNA agent is introduced into stem or progenitor cells (e.g., MSCs) using electroporation. For example, MSCs can be grown in culture and electroporated with a specific dose of the mRNA agent, e.g., mmRNA. In some embodiments, human MSCs, e.g., hBMSCs, are transfected with the mRNA agent using a Nucleofector™ 2b device and hMSC Nucleofector™ kit (Lonza) according to the manufacturer's instructions. Briefly, cells are resuspended in 100 μL of Nucleofector™ solution, mixed with modified mRNA (e.g., 100 ng to 100 μg per million cells), transferred to a cuvette, and electroporated using program U-23 of the Nucleofector™ device. Nucleofected samples can be placed in pre-warmed medium for resuspension in low glucose DMEM solution supplemented with FBS and Pen / strep (such as Lonza hMSC-GM™) and 10% DMSO and frozen at -80°C (can be stored in a liquid nitrogen tank at -180°C) until further use.

[0075] After the introduction of the mRNA agent into the stem or progenitor cells, exosomes are then prepared from the cells, thereby obtaining exosomes loaded with mRNA. As used herein, the term "exosome" refers to small endosome-derived lipid particles (usually 30-120 nm in diameter) that are actively secreted by exocytosis in most living cells. Thus, exosomes are naturally secreted from stem or progenitor cells in culture. Thus, the first step in exosome preparation is to collect the culture supernatant from stem or progenitor cells loaded with the mRNA agent. The supernatant (also referred to as conditioned medium) can be collected, for example, daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every week. This conditioned medium is usually pre-cleaned of dead cells and cell debris by differential centrifugation, and then subjected to further processing to collect the exosomes.

[0076] For example, in one embodiment, the previously removed medium is subjected to ultracentrifugation on a sucrose buffer, followed by a washing step, to recover the exosomes (e.g., as described in Faruqu et al. (2018) J. Vis. Exp. 142:10.3791). Alternative methods known in the art for recovering exosomes include microfiltration centrifugation, gradient centrifugation, and size exclusion chromatography. The recovered exosomes can be further analyzed, for example, for yield, morphology, and exosomal marker expression. Suitable methodologies known in the art for analyzing exosomes include nanoparticle tracking analysis, protein quantification, electron microscopy, and flow cytometry. Various methods for isolating and analyzing exosomes are reviewed in Doyle and Wang (2019) Cells 8:727 and Familtseva et al. (2019) Mol. Cell. Biochem. 459:1-6.

[0077] B. Delivery of mRNA Agents into Exosomes In one embodiment, exosomes are prepared from stem or progenitor cells and then the mRNA agent is introduced into the EVs, e.g., exosomes, such that the EVs, e.g., exosomes, encapsulate the mRNA agent.

[0078] First, EVs, e.g., exosomes, are prepared from stem or progenitor cells as described above in subsection IIIA (unless the cells have already been loaded with an mRNA agent). The EVs, e.g., exosomes, thus obtained are then used for loading with mRNA as follows.

[0079] The mRNA agent can be introduced into the EVs, e.g., exosomes, by methods known in the art, including, but not limited to, electroporation, transfection, and cell nanoporation.

[0080] Introduction of nucleic acids into EVs, e.g., exosomes, has been described in the art. In one embodiment, the mRNA agent is introduced into EVs, e.g., exosomes, by lipid-mediated transfection, such as using lipofectamine. In one embodiment, the mRNA agent is introduced into EVs, e.g., exosomes, by calcium chloride-mediated transfection (e.g., as described in Zhang et al. (2017) Am. J. Physiol. Lung 312:L110-L121). In one embodiment, the mRNA agent is introduced into EVs, e.g., exosomes, by cell nanoporation (e.g., as described in Yang et al. (2019) Nature Biomed. Eng. 4:69-83). In one embodiment, the mRNA agent is introduced into EVs, e.g., exosomes, by electroporation, e.g., using a Nucleofector™ 2b device (Lonza). In one embodiment, the mRNA agent is introduced using a commercially available kit for transfection of EVs, e.g., exosomes, e.g., Exo-Fect™ Exosome Transfection kit (System Biosciences Inc.). Additional descriptions of methods for introducing nucleic acids into EVs, e.g., exosomes, are available in the art, non-limiting examples of which include Lamichhane et al. (2015) Mol. Pharmaceutics 12(10):3650-3657; Usman et al. (2018) Nature Commun. 9:2359; Yang et al. (2019) Nature Biomed. Eng. 4:69-83; and Piffoux et al. (2021) Adv. Drug Deliv. Rev. 178:113972.

[0081] IV. Extracellular vesicle delivery The composition comprising the EVs, e.g., exosomes, loaded with the mRNA agent can be delivered to the subject by any means that delivers the composition to its desired location in vivo.Non-limiting examples of routes of administration of the composition include parenteral (e.g., subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intraarticular, intra-arterial, intracerebrospinal, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable injection technique), oral, transdermal or intradermal, subcutaneous, rectal, intravaginal, topical (e.g., by powder, ointment, cream, gel, lotion, and / or drop), mucosal, nasal, buccal, intestinal, intravitreal, intratumoral, sublingual, intranasal; intratracheal instillation, bronchial instillation, and / or by inhalation; as oral spray and / or powder, nasal spray, and / or aerosol, and / or via portal vein catheter.In some embodiments, the composition may be administered intravenously, intramuscularly, intradermal, intraarterial, intratumoral, subcutaneously, or by inhalation. In some embodiments, the composition is administered intramuscularly.

[0082] In one embodiment, the composition is administered locally. In one embodiment, the composition is administered systemically. In one embodiment, the composition is administered by intraorgan delivery.

[0083] In some embodiments, the composition is administered directly into a solid organ (intracerebrovascular delivery). Non-limiting examples of organs to which the composition can be delivered directly include the heart, kidney, liver, pancreas, stomach, spleen, lung, brain, bladder, and uterus.

[0084] The composition can be administered by any means appropriate for the route of administration. In one embodiment, the composition can be administered by injection using a syringe, such as for intramuscular, intravenous, or intra-arterial injection. In one embodiment, the composition is administered using a catheter or intraluminal delivery cannula, such as for delivery into an organ or to an extravascular site.

[0085] Suitable intraluminal delivery cannulas are generally described, for example, in PCT Publication WO2009 / 124990, PCT Publication WO2012 / 004165, EP Patent 2291213B, and U.S. Patent 8,876,792, as well as Grankvist et al. (2019) J. Int. Med. 285:398-406, the contents of each of which are specifically incorporated herein by reference.

[0086] Additionally, an intraluminal delivery device, referred to as the "Extroducer," is described in detail in U.S. Provisional Patent Application No. 63 / 216,348, filed June 29, 2021, the entire contents of which are specifically incorporated herein by reference. Use of the Extroducer to deliver compositions in vivo directly into the heart of a pig, as compared to use of a 26G needle, is described in further detail in Example 2.

[0087] In one embodiment, EVs, e.g., exosomes, are delivered to cardiomyocytes. As shown in Example 5, iMSC-derived exosomes can effectively deliver mRNA cargo to cardiomyocytes during exercise. In one embodiment, EVs, e.g., exosomes, are delivered to cardiomyocytes in vitro. In one embodiment, EVs, e.g., exosomes, are delivered to cardiomyocytes in vivo. In one embodiment, EVs, e.g., exosomes, are delivered to cardiomyocytes in vivo, for example, by using the above-mentioned catheter or intraluminal delivery cannula to deliver to heart.

[0088] V. Use The mRNA-loaded exosome compositions of the present disclosure can be used for a variety of prophylactic and / or therapeutic purposes, with the particular mRNA agent being selected based on the needs of the subject being treated.

[0089] As shown in Example 7, the iMSC-derived EVs, e.g., exosomes, of the present disclosure can be used to deliver large mRNAs (e.g., 1 kb or larger) encoding functional macromolecules to recipient cells. In an embodiment, the mRNA is at least 1 kilobase in length, at least 1.5 kilobase in length, at least 2 kilobase in length, at least 2.5 kilobase in length, at least 3 kilobase in length, at least 3.5 kilobase in length, at least 4 kilobase in length, at least 4.5 kilobase in length, at least 5 kilobase in length, at least 5.5 kilobase in length, at least 6 kilobase in length, at least 6.5 kilobase in length, at least 7 kilobase in length, at least 7.5 kilobase in length, at least 8 kilobase in length, at least 8.5 kilobase in length, at least 9 kilobase in length, at least 9.5 kilobase in length, or at least 10 kilobase in length.

[0090] Also, as shown in Example 7, the iMSC-derived EVs, e.g., exosomes, of the present disclosure can be used to deliver mRNA to recipient cells for extended periods of time, e.g., at least 1 day, at least 2 days, at least 3 days, or more. The ability to retain mRNA delivered by EVs, e.g., exosomes, for extended periods of time allows the mRNA delivery system to be used to treat chronic disorders (e.g., enzyme deficiency disorders, chronic autoimmune disorders, etc.) by allowing for the continuous delivery of therapeutic agents.

[0091] In one embodiment, the mRNA agent is encoded as an antigen, and the mRNA-loaded exosomes can be used to induce an immune response to the antigen in a subject (e.g., for vaccination). In various embodiments, the antigen is derived from a pathogen, such as a bacterium, a virus, a yeast, a parasite, or a fungus.

[0092] In one embodiment, the mRNA agent encodes an antibody (e.g., a therapeutic antibody), and the mRNA-loaded exosomes can be used for immunotherapy in any clinical setting where therapeutic antibodies have been shown to be beneficial (e.g., autoimmune diseases, cancer). Non-limiting examples of antibodies include monoclonal antibodies, human and humanized antibodies, bispecific antibodies, intrabodies, and related agents that contain immunoglobulin VH and VL regions, or binding portions thereof for binding to targets.

[0093] In one embodiment, the mRNA agent encodes an enzyme, such as an enzyme that is missing in a lysosomal storage disorder, thereby reconstituting the enzyme in the subject. For example, in one embodiment, the mRNA agent can encode alpha-galactosidase (aGAL) in the treatment of Fabry disease. In another embodiment, the mRNA can encode N-sulfoglucosamine sulfohydrolase in the treatment of Sanfilippo A disease. In another embodiment, the mRNA can encode glucocerebrosidase in the treatment of Gaucher disease.

[0094] In one embodiment, the mRNA agent encodes a growth factor. Numerous growth factors with well-described biological functions are known in the art, non-limiting examples of which include vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and the like.

[0095] In one embodiment, the mRNA agent encodes a factor involved in bone development for the treatment of bone defects. Non-healing bone defects may occur after severe trauma, non-union fracture, tumor resection, or craniofacial surgery. For example, in one embodiment, the mRNA agent(s) encodes vascular endothelial growth factor (VEGF) and / or bone morphogenetic protein (BMP) for the treatment of bone defects.

[0096] In one embodiment, the mRNA agent codes for an immunomodulator, such as a cytokine, chemokine, or immune checkpoint regulator, for the purpose of immunomodulation in a subject.In one embodiment, the mRNA agent stimulates immune responsiveness in a subject, for example, for use in cancer treatment.In one embodiment, the mRNA agent inhibits immune responsiveness in a subject, for example, for use in the treatment of autoimmune disorders.

[0097] In one embodiment, the mRNA agent encodes a cardiac-related drug for use in treating cardiac disorders. In such clinical settings, cardiac progenitor cells (e.g., HVPs) can be used as a source of exosomes.

[0098] In one embodiment, the mRNA agent encodes a pancreas-related drug for use in treating pancreatic disorders. In such clinical settings, pancreatic progenitor cells (e.g., beta islet progenitor cells) can be used as an exosome source.

[0099] In one embodiment, the mRNA agent encodes a functional macromolecule involved in gene modification, such as gene editing. In one embodiment, the mRNA encodes a Cre recombinase, e.g., thereby using the delivery system of the present disclosure with a Cre-Lox system. In another embodiment, the mRNA encodes a CRISPR Cas molecule, e.g., thereby using the delivery system of the present disclosure with a CRISPR gene editing system. In one embodiment, the mRNA encodes a Cas9 molecule. In other embodiments, the mRNA encodes a Cas molecule selected from the group consisting of Cas12, Cas13, Cas14, and subtypes thereof. The CRISPR gene editing system can be used, for example, to correct / edit disease-causing mutations, knock down toxic gene mutations, interrupt tumor-specific genes, and the like.

[0100] In certain embodiments, the mRNA delivered by the iMSC-derived EVs, e.g., exosomes, of the present disclosure is used to treat a particular disease or disorder. In one embodiment, the disease or disorder is a cardiac disease or disorder. In one embodiment, the cardiac disease or disorder is ischemia-related heart failure, such as post-myocardial infarction cardiac dysfunction. In such embodiments, the delivered mRNA can be, for example, any or all of the isoforms from VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF (hereinafter referred to as "VEGF family"), and / or HIF1α, HIF2α, HIF3α, and HIF1β (hereinafter referred to as "HIF1 family").

[0101] In another embodiment, the disease is a cardiomyopathy resulting from a genetic mutation, such as a phospholamban mutation (i.e., R14del), which leads to dilated cardiomyopathy and fibrosis. In such an embodiment, the delivered mRNA may code for, for example, wild-type phospholamban (PLN), a VEGF family member(s), and / or a HIF1 family member(s), and / or a gene editing endonuclease, such as CRISPR / Cas9 (including a guide RNA), and / or a base editing endonuclease, such as CRISPR / Cas13 (including a guide RNA and a deaminase enzyme).

[0102] In another embodiment, the disease is a skin ulcer, including a diabetic ulcer. In such an embodiment, the delivered mRNA can be, for example, a VEGF family member(s) and / or a HIF1 family member(s) and / or an epidermal growth factor (hereinafter referred to as EGF).

[0103] In another embodiment, the disease is peripheral vascular disease (PVD). In such an embodiment, the delivered mRNA can be, for example, a VEGF family member(s) and / or a HIF1 family member(s) and / or an EGF.

[0104] In another embodiment, the disease is critical limb ischemia (CLI). In such an embodiment, the delivered mRNA may be, for example, a VEGF family member(s) and / or a HIF1 family member(s) and / or an EGF.

[0105] In another embodiment, the disease is a respiratory disorder, such as pulmonary arterial hypertension (PAH). In such an embodiment, the delivered mRNA can be, for example, a VEGF family member(s) and / or a HIF1 family member(s) and / or angiotensin converting enzyme(s), such as angiotensin I, angiotensin II, angiotensin III, angiotensin IV (collectively hereinafter referred to as the "ACE family"), and / or endothelial nitric oxide synthase 3 (hereinafter referred to as "eNOS").

[0106] In another embodiment, the disease is pneumonia, e.g., pneumonia caused by COVID 19 infection. In such an embodiment, the delivered mRNA can be, for example, VEGF family member(s) and / or HIF1 family member(s) and / or ACE family member(s), and / or eNOS. EXAMPLES

[0107] Below are examples of specific embodiments for carrying out the present invention. These examples are presented for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0108] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology that are within the skill of one in the art. Such techniques are explained fully in the literature, see, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (Easton, Pennsylvania: Mack Publishing Company, 1992); rd Ed. (Plenum Press) Vols A and B (l992).

[0109] Unless otherwise noted, all reagents and chemicals were obtained from commercial sources and used without further purification.

[0110] Example 1: Stem cell loading with mRNA agents In this example, various types of stem cells (embryonic stem cells and mesenchymal stem cells) were loaded with an mRNA agent as cargo and the levels of protein expression from the mRNA in vitro and in vivo were compared to the equivalent mRNA agent loaded into cells using lipid nanoparticles (LNPs).

[0111] In the first set of experiments, mesenchymal stem cells (MSCs) were loaded with modified mRNA (mmRNA) encoding the fluorescent protein mCherry either by electroporation or by transfection using the LNP reagent Lipofectamine™ RNAiMAX Reagent (ThermoFisher Scientific). The dose of mmRNA used was 5ug per million MSCs.

[0112] Electroporation of mmRNA into MSCs was performed using a Lonza Nucleofector 2b device. Loading of mmRNA into MSCs via electroporation was performed as follows: Supplemented Nucleofector solution was pre-heated at room temperature. While plating the cells, the medium was removed and the cells were washed with PBS. Lonza trypsin solution was used to harvest the cells. Trypsin was inactivated using MSC growth medium. It was suggested that medium containing growth serum may negatively interfere with electroporation efficiency, so from this point onwards, only MSC basal medium was used. After cell counting, the cells were placed in a 15mL Falcon tube at a ratio of 1e^6 cells per mL of medium and pelleted. How to perform nucleofection: After centrifugation, the supernatant was carefully and completely removed. Cells were resuspended in RT Nucleofector solution (Lonza Nucleofector kit) at a ratio of 100uL per 1e^6 cells. The Nucleofector solution contained 82uL of Tube A (from the kit), 18uL of Tube B (from the kit), and the desired amount of mmRNA. The mmRNA was kept on ice and added to the Nuclefector solution only immediately prior to loading into the cuvette for electroporation. The mmRNA can be concentrated to approximately 1ug / uL. More than 30uL of mmRNA (total electroporation volume greater than 130uL) can negatively affect transfection efficiency. The optimal total nucleofection volume for electroporation was approximately 105-110uL. Additionally, the cell-mRNA mixture was never left in the nucleofection solution for a period longer than 15 minutes, as this may also affect cell viability and mRNA integrity.

[0113] The cell-nucleofection solution was transferred to a cuvette (provided with Lonza's nucleofection kit), minimizing air bubbles, and the selected program (U23) was applied. Immediately, 500uL of pre-equilibrated culture medium (MSC-GM) was added per 1e^6 hMSCs and equilibrated for 5-30 minutes in a 37°C incubator. After nucleofection and recovery, cells were re-counted and either frozen in freezing medium or re-seeded onto pre-warmed culture plates containing MSC growth medium. To freeze cells for later use, cells were resuspended in freezing medium consisting of 90% MSC growth medium and 10% DMSO and stored in cryopreservation vials at up to 10e^6 per 1mL cryovial.

[0114] Alternatively, transfection of MSCs with mmRNA was performed using RNAiMAX according to the manufacturer's instructions. Cellular expression of mCherry was assessed by standard flow cytometry.

[0115] Representative results are shown in Figure 1A, which demonstrates that electroporation of mmRNA into MSCs leads to significantly higher protein expression compared to LNP-mediated transfection.

[0116] In a second set of experiments, modified mRNA (mmRNA) encoding vascular endothelial growth factor (VEGF) was loaded into MSCs either by electroporation or by transfection using Lipofectamine™ RNAiMAX reagent as described above. The dose of mmRNA used was 20ug per million MSCs and cells were seeded at 50,000 cells / well. VEGF protein expression (ng / mL) in the supernatant was assessed 24, 48, and 72 hours after loading. Representative results are shown in Figure 1B, which show that electroporation of mmRNA into MSCs leads to significantly higher protein secretion compared to transfection via LNPs.

[0117] Similar electroporation experiments were also performed with unmodified mRNA encoding VEGF using bone marrow-derived MSCs, demonstrating that unmodified mRNA could also be effectively introduced into MSCs by electroporation (data not shown).

[0118] In the third set of experiments, modified mRNA (mmRNA) encoding luciferase was electroporated into MSCs using Lonza Nucleofector™ technology at a dose of 20ug per million MSCs, followed by freezing the cells for storage. The frozen electroporated MSCs were then thawed on the day of use. The thawed electroporated MSCs were delivered under the kidney capsule of mice by syringe injection using a 27G needle. For comparison, modified luciferase mRNA was conjugated with LNPs (RNAiMAX) and the mmRNA-LNP complex was similarly delivered under the kidney capsule. Luciferase expression in vivo was assessed over a 5-day time course. Representative results are shown in Figure 2, which demonstrate that electroporating MSCs with mmRNA in vitro followed by injection of mmRNA-loaded MSCs in vivo leads to significantly higher protein expression in vivo compared to mmRNA delivery via LNPs in vivo.

[0119] In a fourth set of experiments, modified mRNA (mmRNA; 5ug) encoding mCherry was loaded into human embryonic stem (ES) cells either by electroporation as described above or by LNP-mediated transfection using RNAiMAX. Protein expression, assessed by standard fluorescence, showed that both electroporation and LNP-mediated transfection resulted in efficient and comparable expression of mCherry in human ES cells (data not shown).

[0120] In a fifth set of experiments, induced mesenchymal stem cells (iMSCs) were electroporated with mRNA constructs encoding either green fluorescent protein (GFP) or CD63-GFP by standard methods described herein. Electroporated iMSCs were injected under the kidney capsule of one of the immunocompromised mice, with the contralateral kidney serving as a non-injected control. Local administration under the kidney capsule area retained the injected cells and ensured that in vivo mRNA delivery was in a cell-independent manner. Samples were collected for analysis from treated and untreated kidneys, as well as liver, spleen, lung, muscle, and heart tissues. Quantitative PCR was performed on extracted RNA to detect expression of non-native exogenous CD63 mRNA, which indicates the distribution of mRNA cargo in vivo. As shown in FIG. 13, exogenous CD63 mRNA could be detected at least at some time points over a 7-day period in all organs tested, and throughout the entire 7-day period in many tissues. This widespread distribution of CD63 mRNA expression indicates that the mRNA cargo is delivered systemically in vivo, despite the local administration of mRNA-loaded cells (i.e., under the kidney capsule). Immunofluorescence staining using an anti-GFP antibody confirmed the expression of GFP (translated from either CD63-GFP or GFP mRNA constructs) in kidney and spleen tissues (data not shown).

[0121] In summary, these experiments show that mRNA agents can be efficiently loaded into stem cells in vitro, and that protein expression from mRNA agents in vitro and in vivo is generally greater when electroporation is used for mRNA loading compared to when LNP-mediated transfection is used for mRNA loading.Furthermore, the experiments show that mRNA agents in delivery vehicles administered locally in vivo, for example under the kidney capsule, can nevertheless exhibit systemic expression of the mRNA, including widespread organ distribution.

[0122] Example 2: Use of the Extroducer for the delivery of stem cells in vivo In this example, human mesenchymal stem cells (MSCs) were delivered into porcine cardiac tissue in vivo using a catheter-based delivery system referred to herein as the Extroducer.

[0123] In the first set of experiments, we investigated the effect of passaging human MSCs through the Extroducer in vitro. First, MSCs were modified by electroporation with modified mRNA encoding green fluorescent protein (GFP) (5 μg) and modified mRNA encoding vascular endothelial growth factor (VEGF) (5 μg) and cryopreserved. After thawing, modified MSCs were either seeded immediately on culture vessels or passed through the Extroducer before seeding. 1 × 10 cells in 100 μl of medium were cultured at 4 °C for 1 h. 6 The cells were passaged through the Extroducer. Test #1, where cells were passed through the Extroducer for 3 minutes and 47 seconds, resulted in 72% cell recovery and 72% cell viability. Test #2, where cells were passed through the Extroducer for 3 minutes and 10 seconds, resulted in 77% cell recovery and 75% cell viability. The results of this first set of experiments indicated that passaging MSCs through the Extroducer did not significantly affect cell viability, as MSCs plated immediately after thawing showed 75% viability.

[0124] We next investigated the engraftment of MSCs into porcine cardiac tissue after in vivo delivery using the Extroducer. Healthy naive pigs without immunosuppression were used as recipients. To evaluate the retention of MSCs in the porcine heart, MSCs radiolabeled with Zr89 were delivered to the apex of healthy pigs using the Extroducer (n=3). For comparison, radiolabeled MSCs were also delivered to the apex of healthy pigs using a 26-gauge needle (n=3). All animals were followed up for 5 days after injection. Gamma counter measurements were performed to determine the retention (%) of the injected dose (ID). The results are summarized in Table 1 below. Radioactivity was measured in megabecquerels (MBq).

[0125] [Table 1] The results show that the Extroducer is significantly better than the 26-gauge needle in delivering MSCs to the apex of the porcine heart, with cell retention at least 3-fold higher and in some cases as much as 8-9-fold higher in animals treated with the Extroducer versus the 26-gauge needle. Furthermore, none of the animals treated with the 26-gauge needle achieved 10% retention of the injected dose of radiolabeled cells, while all of the animals treated with the Extroducer showed retention of more than 10% of the injected dose of radiolabeled cells. Individual Extroducer-treated animals showed retention of more than 15%, more than 35%, and more than 40% of the injected dose of radiolabeled cells, thereby indicating that the Extroducer is capable of delivering cells to the heart, resulting in a significant portion of the delivered cells being retained within the heart.

[0126] Example 3: Release of tetraspanin-positive exosomes by iMSCs In this example, we investigated the expression of tetraspanins by iMSCs and the release of tetraspanin-positive exosomes from iMSCs.

[0127] In the first set of experiments, induced MSCs (iMSCs) were prepared and the expression of tetraspanin (TSPAN) proteins was examined by standard methods using anti-TSPAN antibodies. The experiments showed that iMSCs expressed the TSPAN proteins CD9, CD63, and CD81. Furthermore, tetraspanin-positive exosomes could be visualized by cross-sectional imaging.

[0128] Representative images showing iMSC TSPAN markers CD9, CD63, CD81 localized on tomographic bright field visible intra / extracellular vesicles are shown in Figure 3.

[0129] Next, we investigated the release of TSPAN-positive exosomes from iMSCs. We measured the tetraspanin concentrations in the supernatants of high-density and low-density iMSCs, as well as the concentrations in the supernatants after freeze-thawing of high-density or low-density iMSCs. The results are shown in Figure 4 along with PBS and medium-only controls. The results showed that iMSCs released TSPAN-positive exosomes into the supernatant, with high-density iMSCs that had not been freeze-thawed showing the highest concentrations in the supernatant.

[0130] Thus, this example shows that iMSCs express TSPAN protein and release TSPAN-positive exosomes into the supernatant upon cell culture.

[0131] Example 4: Loading of iMSCs and exosomes with mRNA agents In this example, various approaches were used to load iMSCs and / or exosomes with mRNA agents.

[0132] In the first set of experiments, iMSCs were transfected with a labeled mRNA construct, and the presence of labeled mRNA was detected in the intracellular vesicles of iMSCs after transfection.Furthermore, when the transfected cells were further cultured, labeled mRNA was detected outside the cells in the extracellular vesicles released from the transfected iMSCs.Therefore, extracellular vesicles containing mRNA agents could be obtained by transfecting iMSCs with mRNA.

[0133] Fluorescent nucleotide-modified mRNA-594 (mRNA-594-GFP), encoding the fluorescent protein GFP, was loaded into pluripotent stem cell-derived mesenchymal stem cells (iMSCs) by either electroporation or transfection using the LNP reagent Lipofectamine™ RNAiMAX Reagent (ThermoFisher Scientific). The dose of mRNA-594-GFP used was 5ug per million iMSCs.

[0134] Electroporation of mRNA-594-GFP into iMSCs was performed using a Lonza Nucleofector 2b device. Loading of iMSCs with mRNA-594-GFP via electroporation was performed as follows: Supplemented Nucleofector solution was pre-heated at room temperature while mRNA was kept on ice until transfection mix was made. 70-80% confluent iMSC cultures were aspirated and washed once with PBS followed by enzymatic dissociation using TrypLE (ThermoFisher Scientific) for 5-7 min. Dissociated cells were washed from the wells using mild trituration with basal medium added in a 1:1 ratio (add 1ml of TrypLE:1ml of basal medium). Centrifuge the suspension at 300g or RCF for 5 min and discard the supernatant. Resuspend the cell pellet in a 15ml conical at a ratio of 1e^6 cells per mL. Performing nucleofection: After centrifugation of the aliquoted suspension, the supernatant was carefully and completely removed, and the cells were then resuspended in RT Nucleofector Solution (Lonza Nucleofector kit) at a ratio of 100uL per 1e^6 cells. The Nucleofector solution contained 82uL of solution A (Tube A of the kit), 18uL of solution B (Tube B of the kit), and the desired amount of mRNA-594-GFP.

[0135] It is important to minimize contact between the cell-mRNA and the nucleofection solution for 15 minutes, as this will negatively impact cell viability and mRNA integrity. The cell-nucleofection solution was transferred to a cuvette (provided in the Lonza nucleofection kit), minimizing air bubbles, and the selected program (C-017 or U-020) was applied. Immediately after nucleofection, 500uL of pre-equilibrated iMSC culture medium was added to each electroporated sample, and the cells were plated onto an Ibidi 35mm imaging dish (Ibidi catalog number 88156). The cells were allowed to equilibrate and reattach for 4 hours at 37°C. After nucleofection and recovery, the 35mm Ibidi dish was transferred to the Nanoive 3D-Cell Explorer-Fluo for imaging. The Nanolive stage incubator and gas composition were maintained at 37°C and normoxia during all tomographic imaging experiments.

[0136] Alternatively, transfection of iMSCs with mRNA-594-GFP was performed using RNAiMAX according to the manufacturer's instructions. Expression of 594 fluorescence with simultaneous cross-sectional imaging in iMSCs was also performed using Nanolive 3D-Cell Explorer-Fluo.

[0137] Representative images using Nanolive 3D Cell Explorer-Fluo to visualize transfected mRNA-594-GFP contained within tomographic bright-field visible vesicles within intracellular compartments are shown in Figure 5 .

[0138] Representative images using Nanolive 3D Cell Explorer-Fluo to visualize transfected mRNA-594-GFP contained in tomographic bright-field visible vesicles (also TSPAN (CD9 / 63 / 81) positive) within intracellular compartments are shown in Figure 6 .

[0139] Representative images using Nanolive 3D Cell Explorer-Fluo to visualize transfected mRNA-594-GFP contained in tomographic brightfield visible vesicles (also TSPAN (CD9 / 63 / 81) positive) within the extracellular compartment are shown in Figure 7 .

[0140] In a second set of experiments, we transfected the labeled mRNA construct into iMSCs and detected the presence of the mRNA protein product in iMSC-derived exosomes. These results showed that exosomes derived from mRNA-transfected iMSCs contained detectable mRNA protein products.

[0141] mmRNA-GFP (mRNA-GFP), encoding the fluorescent protein GFP, was loaded into pluripotent stem cell-derived mesenchymal stem cells (iMSCs) by either electroporation or transfection using the LNP reagent Lipofectamine™ RNAiMAX Reagent (ThermoFisher Scientific). The dose of mRNA-GFP used was 5ug per million iMSCs.

[0142] Electroporation of mRNA-GFP into iMSCs was performed using a Lonza Nucleofector 2b device. Loading of iMSCs with mRNA-GFP via electroporation was performed as follows: Supplemented Nucleofector solution was pre-heated at room temperature while mRNA was kept on ice until transfection mix was made. 70-80% confluent iMSC cultures were aspirated and washed once with PBS followed by enzymatic dissociation using TrypLE (ThermoFisher Scientific) for 5-7 min. Dissociated cells were washed from the wells using mild trituration with basal medium added in a 1:1 ratio (add 1ml of TrypLE:1ml of basal medium). Centrifuge the suspension at 300g or RCF for 5 min and discard the supernatant. Resuspend the cell pellet in a 15ml conical at a ratio of 1e^6 cells per mL. Performing nucleofection: After centrifugation of the aliquot suspension, the supernatant was carefully and completely removed, and the cells were then resuspended in RT Nucleofector Solution (Lonza Nucleofector kit) at a ratio of 100uL per 1e^6 cells. The Nucleofector solution contained 82uL of solution A (Tube A of the kit), 18uL of solution B (Tube B of the kit), and the desired amount of mRNA-GFP.

[0143] It is important to minimize contact between the cell-mRNA and the nucleofection solution for 15 minutes, as this will negatively impact cell viability and mRNA integrity. The cell-nucleofection solution was transferred to a cuvette (provided in the Lonza nucleofection kit), minimizing air bubbles, and the selected program (C-017 or U-020) was applied. Immediately after nucleofection, 500uL of pre-equilibrated iMSC culture medium was added to each electroporated sample, and the cells were plated onto an Ibidi 35mm imaging dish (Ibidi catalog number 88156). The cells were allowed to equilibrate and reattach for 4 hours at 37°C. After nucleofection and recovery, the 35mm Ibidi dish was transferred to the Nanolive 3D-Cell Explorer-Fluo for imaging. The Nanolive stage incubator and gas composition were maintained at 37°C and normoxia during all tomographic imaging experiments.

[0144] Alternatively, transfection of iMSCs with mRNA-GFP was performed using RNAiMAX according to the manufacturer's instructions. Expression of GFP fluorescence with simultaneous cross-sectional imaging in iMSCs was also performed using Nanolive 3D-Cell Explorer-Fluo.

[0145] Representative images using Nanolive 3D Cell Explorer-Fluo to visualize translated GFP protein contained in tomographic brightfield visible vesicles (which are also TSPAN (CD9 / 63 / 81) positive) within the intra- / extracellular compartments are shown in Figure 8 .

[0146] These results indicate that exosomes derived from mRNA-transfected (LNP / electroporated) iMSCs contain detectable mRNA protein products.

[0147] Example 5: Delivery of iMSC-derived exosomes to exercising cardiomyocytes In this example, iMSC-derived exosomes were used to deliver mRNA cargo to beating cardiomyocytes in culture.

[0148] mmRNA-GFP (mRNA-GFP), encoding the fluorescent protein GFP, was loaded into pluripotent stem cell-derived mesenchymal stem cells (iMSCs) via transfection of mRNA-GFP, performed using RNAiMAX according to the manufacturer's instructions. iMSCs were transfected at 10ug per 1e^6 cells. 4 hours after transfection, the medium was replaced at 2ml per sample.

[0149] iMSC EV Isolation: 12ml of iMSC supernatant was harvested 28 hours post-transfection from an 80% confluent 6-well plate (approximately 2 million cells). Following the Izon EV isolation protocol, EVs were spun down once at 200g for 10 minutes and the supernatant was transferred to a new tube and centrifuged at 2000g for 10 minutes. The supernatant was then carefully removed and concentrated using an Amicon filter unit (MWCO=100kDa, Merck Millipore) to reach a final volume of 150ul. The concentrated input was layered onto the column using an Izon Automatic Fraction Collector (AFC1) fitted with an Izon qEV Single Column. After elution and discarding 1ml of buffer, a sample elution of 600uL of exosome isolate was performed, which was fractionated into 4x150ul aliquots based on size ratio using the AFC1. The complete isolate was kept for downstream applications and the four fractions were combined. 600 uL of the complete isolate was further concentrated to a volume of 15 uL using an Amicon filter unit (MWCO=100 kDa, Merck Millipore) before administration to the cardiomyocytes.

[0150] Cardiomyocyte differentiation from pluripotent stem cells was completed after 14 days using an established protocol from Foo et al., 2018 Molecular Therapy. iMSC exosome isolates were added to beating cardiomyocytes on day 15 and imaged on day 16.

[0151] Representative images visualizing translated GFP protein expressed within beating cardiomyocytes using standard bright field and fluorescence microscopy are shown in Figure 9.

[0152] These results demonstrated that exosomes isolated from iMSCs could be successfully delivered to excited cardiomyocytes to deliver a payload of either mRNA or mRNA-protein products.

[0153] Example 6: Modification of iMSCs with TSPAN to enhance exosome formation In this example, iMSCs were modified to express exogenously introduced TSPAN protein and the effect on exosome formation was examined.

[0154] An mRNA construct encoding a labeled TSPAN protein was designed and prepared using green fluorescent protein (GFP) as a label. Constructs encoding CD9-GFP, CD63-GFP, and CD81-GFP were used (5 μg each). The labeled TSPAN constructs were introduced into iMSCs by both LNP transfection and electroporation using the methods previously described in Example 4. iMSCs transfected with the labeled TSPAN constructs were plated on ibidi microscope dishes and imaged using Nanolive 3D Cell Explorer-Fluo as described in Example 4. Expression of the labeled TSPAN protein was detectable in iMSCs 1 and 6 hours after transfection, as evidenced by its displacement of the TSPAN antibody, with an observable increase in labeled vesicles. By 12 hours after transfection, nearly all exosomes visible in tomography were tagged with the labeled TSPAN protein, and the abundance of TSPAN was clearly higher than endogenous expression.

[0155] Representative images of the translated TSPAN-GFP protein contained in tomographic bright-field visible vesicles visualized using Nanolive 3D Cell Explorer-Fluo, as well as the visualization of whole TSPAN using a TSPAN (CD9 / 63 / 81)-conjugated antibody, are shown in Figure 10 .

[0156] These results indicate that modification of iMSCs to express exogenously derived TSPAN protein led to an enhancement of exosomes that carried the introduced TSPAN protein.

[0157] Example 7: Delivery of large mRNA using iMSC-derived extracellular vesicles In this example, extracellular vesicles (EVs) from iMSCs were loaded with large mRNA encoding functional polymers and used to deliver the mRNA to target cells.

[0158] In the first set of experiments, the ability to use iMSC EVs to deliver functional macromolecules was tested using the Cre-LoxP system. In this system, which uses the tdTomato reporter line, Cre recombinase remodels the LoxP locus and initiates the expression of tdTomato fluorescent protein. iMSCs were loaded with Cre recombinase mRNA (10ug per million iMSCs) via electroporation as described in the previous examples. 24 hours after electroporation, the supernatant of the iMSC culture medium was collected, thereby obtaining the mRNA-loaded EVs, which were used to treat the mouse ROSA26:tdTomato reporter cell line. Two days later, Cre recombinase was detected in mouse cells by using immunofluorescence staining with an anti-Cre antibody (mouse cells do not express endogenous Cre) (see Figure 11A). tdTomato-positive mouse cells could also be observed (see FIG. 11B), indicating that the mRNA-encoded Cre recombinase was functional in inducing expression of tdTomato in mouse cells.

[0159] In a second set of experiments, the ability to use iMSC EVs to deliver large mRNAs encoding functional macromolecules was tested using the CRISPR-Cas9 system. iMSCs were loaded with Cas9 mRNA (4.2 kb) via electroporation as described in the previous examples. The level of Cas9 mRNA levels was determined in two donor cells (iMSC-Donor-D1 and iMSC-Donor-D3) and recipient cells. Representative results are shown in Figure 12. The results showed that a large amount of Cas9 mRNA was detectable in both iMSC donor and recipient cells, indicating that iMSC EVs successfully delivered large mRNAs from donor cells to recipient cells. Furthermore, the duration of Cas9 mRNA expression in donor cells was examined, showing that in one of the donor cells, mRNA could still be detected 3 days after electroporation, indicating the feasibility of using this system for chronic treatment in vivo.

[0160] Incorporation by Reference Each patent, publication, and non-patent literature cited in this application is incorporated herein by reference in its entirety, as if each was individually incorporated by reference.

Claims

1. A method for preparing a composition containing an mRNA agent, comprising transfecting the mRNA agent into human stem cells or human progenitor cells, preparing extracellular vesicles (EVs) from the human stem cells or human progenitor cells, and thereby encapsulating the mRNA agent in the EVs, thereby encapsulating the mRNA agent in extracellular vesicles.

2. The method according to claim 1, wherein the mRNA agent comprises at least one modified nucleotide base.

3. The method according to claim 1, wherein the mRNA agent contains all unmodified nucleotide bases.

4. The method according to claim 1, wherein the EV is an exosome prepared from human mesenchymal stem cells (MSCs).

5. The method according to claim 1, wherein the EV is an exosome prepared from a stem cell or progenitor cell selected from the group consisting of human embryonic stem (ES) cells, human induced pluripotent stem cells (iPSCs), human induced mesenchymal stem cells (iMSCs), human cardiac progenitor cells, and human pancreatic progenitor cells.

6. The method according to claim 1, wherein the stem cells or progenitor cells are low immunogenic.

7. The method according to claim 1, wherein the mRNA agent encodes an enzyme, cytokine, growth factor, antigen, antibody, or immunomodulator.

8. The method according to claim 1, wherein the composition is administered to the internal location of the target organ.

9. A method for expressing a protein in a cell ex vivo or in vitro, A step of encapsulating an mRNA agent encoding the protein in an extracellular vesicle (EV) prepared from human stem cells or human progenitor cells, wherein the mRNA agent is encapsulated in the EV by introducing the mRNA agent into the human stem cells or human progenitor cells by transfection, and the mRNA agent is at least 300 base pairs long. The steps include transfecting the cells with the EV so that the cells express the protein encoded by the mRNA agent, Methods that include...

10. The method according to claim 9, wherein the mRNA agent has a length of at least 1 kb.

11. The method according to claim 9, wherein the EV is an exosome.

12. The method according to claim 11, wherein the exosome is prepared from human mesenchymal stem cells (MSCs) or induced MSCs (iMSCs).

13. The method according to claim 9, wherein the mRNA agent encodes an enzyme, cytokine, growth factor, antigen, antibody, or immunomodulatory protein.

14. A pharmaceutical composition comprising an extracellular vesicle (EV), wherein the EV is produced by the method described in any one of claims 9 to 13, and the EV comprises an mRNA agent encoding a protein for in vivo delivery.

15. The pharmaceutical composition according to claim 14, which is suitable for administration to the internal location of the target organ.

16. The EV according to claim 14, which is suitable for administration to an external vascular site of the target.

17. The EV according to claim 14, which is suitable for administration using an intracavitary delivery device.

18. A composition comprising extracellular vesicles (EVs) prepared from human stem cells or human progenitor cells, wherein the EVs encapsulate an mRNA agent having a length of at least 300 bases, and the mRNA agent is encapsulated within the EVs by introducing the mRNA agent into the human stem cells or human progenitor cells by transfection.

19. The composition according to claim 18, wherein the mRNA agent comprises at least one modified nucleotide base.

20. The composition according to claim 18, wherein the mRNA agent comprises all unmodified nucleotide bases.

21. The composition according to claim 18, wherein the mRNA agent encodes an enzyme, cytokine, growth factor, antigen, antibody, or immunomodulatory protein.

22. The composition according to claim 18 for delivering the mRNA agent to cells.

23. The composition according to claim 18 for delivering the mRNA agent to a target.

24. A composition comprising human stem cells or human progenitor cells transfected with the mRNA agent described in any one of claims 18 to 23, wherein the cells produce extracellular vesicles (EVs) containing the mRNA agent.

25. The composition according to claim 24, wherein the EV comprises the mRNA agent and the protein encoded by the mRNA agent.

26. The composition according to claim 24, wherein the cells are mesenchymal stem cells.

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