Treatment of ehlers-danlos syndrome with extracellular vesicle composition

Abstract

Disclosed are methods of treating Ehlers-Danlos Syndrome in a subject by administering a therapeutic MSC secretome product made by a method comprising culturing bone marrow-derived MSCs under conditions that include oxygen tension below 5% and a culture media with a pH below 7.

Description

TREATMENT OF EHLERS-DANLOS SYNDROME WITH EXTRACELLULAR VESICLE COMPOSITIONCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 568,282 filed on March 21, 2024, and U.S. Provisional Application No. 63 / 705,416 filed on October 9, 2024, the entirety of each is hereby incorporated by reference herein.BACKGROUND

[0002] Of the several types of Ehlers-Danlos Syndrome (EDS) the most common type is hypermobile EDS (hEDS). Hypermobile EDS appears to be a multisystem disorder with unknown pathophysiology. It affects tens of thousands of people in the United States alone. Common characteristics include persistently painful hyper-mobile joints, joint dislocations, GI disturbances, headaches, orthostatic tachycardia, fatigue, and brain fog with difficulty concentrating. Diagnosis is by clinical means only. There is no genetic or other diagnostic laboratory test. There exists a need for a safe and effective treatment for Ehlers-Danlos Syndrome.SUMMARY

[0003] This Summary introduces a selection of concepts that are described further below in the Detailed Description. This Summary is not intended to limit the scope of the claimed subject matter.

[0004] Disclosed herein are methods and compositions for treating Ehlers-Danlos Syndrome (EDS) or symptoms thereof in a subject. Disclosed herein are methods and compositions for treating Mast Cell Activation Syndrome (MCAS) or symptoms thereof in a subject. Disclosed herein are methods and compositions for treating Hypermobile Spectrum Disorder (HSD) or symptoms thereof in a subject. Disclosed herein are methods and compositions for reducing pain in a subject. Disclosed herein are methods and compositions for reducing sleeplessness, headaches, brain fog, concentration, fatigue, joint pain, overall pain, joint looseness, joint dislocations, gastrointestinal function, gastrointestinal mobility, CBD or THC use, NSAID use, use of pain medication, and overall feeling of well-being in a subject. In some embodiments, the methods disclosed herein comprise administering to the subject a composition comprising one or more extracellular vesicles (EVs), wherein the composition comprises NUP85, DAPP1, PDGF R alpha, SLITRK5, FAP, Artemin, DPPII, cIAP-1, Pentraxin 3, Visfatin, Neprilysin, Albumin, Galec-tin-1, UNC5H3, IL-20 R beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midkine, Calreticulin, Osteoactivin,Legumain, TAZ, Cathepsin L, RBP4, Serpin A4, JAM- A, MCSF, LIMPII, OPG, IL-22, Ga- lectin-3, MOG, Trypsin 3, SIRP alpha, and Syndecan-4, GSTM1, NUP85, MeprinA, IL-1 F10, TGFbl, Ephrin-A4, Aminopeptidase LRAP, GDF-9, PDGF R alpha, PAPP -A, Aryl-sulfatase A, LAIR2, ULBP-4, TFPI, SOX2, SLITRK5, Spinesin, ENPP-2, CD97, CTACK, Integrin alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin DI, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angio-tensinogen, Follistatin, CF VII, Persephin, TRAIL Rl, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Nepri-lysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mcl-1, DPPIV, SREC-II, Nor-rin, JAM-C, Bcl-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gpl3O, C-myc, LILRB4, BMP-2,, MIA, CD34, IFNab R2, Glypican 2, MSP R, DSCAM, Matriptase, KIR2DL3, CD30, Sigi ec- 10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nidogen-1, CD2, Kallikrein 1, TSLP R, TROY, VCAM-1, Siglec-11, S100A1, PARI, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R beta, Throm-bospondin-2, SMPD1, B2M, MFRP, LRP-6„ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD- ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 alpha, 0X40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, Semaphorin 4D, ROBO2, PDX-1, APRIL, Neurturin, Kremen-2, EMMPRIN, Activin RIB, Neuroligin 2, Epiregulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF Rl, DSPG3, SorCSl, Matrilin-2, sFRP-3, EphB3, NCK1, Prolactin, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Pappalysin-2, Syndecan-3, Jag-ged 1, AKR1C4, Olfactomedin-2, Osteoadherin, NKp44, Thyroglobulin, IL- 21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA125, HAO-1, PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, R0R1, CMG-2, 4-1BB Ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Leg-umain, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 beta, TAFA1, Podocalyxin, Rai A, CRELD2, GRAP2, SP-D, BID, GFR alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Con- tactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan,Neudesin, ILT4, uPAR, Axl, WIF-1, IL-7 R alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Cochlin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL- 17 RC, MIP-3b, Cy statin C, Cy statin D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathep-sin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, ADAM23, NOV, Galectin-2, Neu-rexin 3 beta, TLR3, Sirtuin 2, Numb, IL-28 R alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM- A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA I3, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-Selectin, Hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, LTAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-lra, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsin-ogen II, Desmoglein-3, Nectin-4, SCF, Serpin A5, PTH, FGF-19, MSP, IL-28 A, FGF-12, METAP2, AS AHL, EDIL3, NT AL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, 0X40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, R0R2, EphB4, Glypican 1, LAP(TGFbl), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, Nestin, TPO, ErbB3, Kirrel3, FLRT1, Galec-tin-3, CXCL16, JAM-B, DR6,Nogo Receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R alpha 1, AMIG02, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM- 1, Inhibin A, Cadherin-4, P1GF-2, Neurogranin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-Spondin 2, CK19, Cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombos- pondin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL- 1, ROBO3, RANTES, Endocan, Granulysin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin 3, ARSB, LIF R alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP- 3a, SDF-lb, NKp46, MCP-3, IL-32 alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Syntaxin 6, GRO, Bcl-w, Lipocalin-2, PDGF-AA, IL-2 Ra, Angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.l, EDAR, ADAMTS13, Kynureninase, PTH1R, IFN-gamma Rl, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalci-tonin, SOX15, Kallikrein 11, BCMA, Dectin-2, EpCAM,HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecan-4, IL-2 Rg, MICA, Dopa Decarboxylase, NPDC-1, MCP-4, EG-VEGF, Glycoprotein V, Semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, Endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, Galectin-8, CA4, Cystatin E M, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, Kallikrein 7, or IL-13, or a combination of two or more thereof. In some embodiments, the methods disclosed herein comprise administering to the subject a composition comprising one or more extracellular vesicles (EVs), wherein the composition comprises TIMP-1 (tissue inhibitor of metalloproteinases 1), TEMP -2 (tissue inhibitor of metalloproteinases 2), CD63 antigen, Ferritin, IGFBP-4 (Insulin-like growth factor binding protein-4), MIF (Macrophage migration inhibitory factor), LAMB 1 (Laminin Subunit Beta 1), FBN1 (Fibrillin 1), HSPG2 (Heparin Sulphate Proteoglycan 2), BGN (Biglycan), or FN1 (Fibronectin 1), or a combination of two or more thereof. In some embodiments, the composition further comprises beta-IG-H3 (Transforming growth factor-beta-induced protein ig- H3), CA2 (Carbonic anhydrase 2), Cathepsin B, CD81 antigen, CD9 antigen, CD44 antigen, CD99 antigen, CD 109 antigen, CNTF (Ciliary neurotrophic factor), Decorin, DcR3 (Tumor necrosis factor receptor superfamily member 6B), Dkk-3 (Dickkopf-related protein 3), Endoglycan (podocalyxin-like protein 2), Furin, GM-CSF Ra (Granulocyte-macrophage colonystimulating factor receptor subunit alpha), GPR115 (Adhesion G protein-coupled receptor F4), GP73 (Golgi membrane protein 1), HS3ST4 (Heparan sulfate glucosamine 3-O-sulfotransferase 4), IGF -2 (Insulin-like growth factor-2), IGFBP-2 (Insulin-like growth factor binding protein-2), IGFBP-3 (Insulin-like binding protein-3), IGFBP-6 (Insulin-like growth factor binding protein- 6), IL18BP (Interleukin- 18 Binding Protein), IL-1 R6 (Interleukin 1 Receptor 6), ILFBP-4 (Insulin-like growth factor binding protein-4), LAMP2 (Lysosome-associated membrane glycoprotein 2), Lumican, OPN (Osteopontin), PALI (Plasminogen activator inhibitor- 1 or SERPINE 1), PCK1 (Phosphoenol pyruvate carboxykinase, cytosolic), PF4 (Platelet Factor 4), Periostin, PRDX4 (Peroxidredoxin-4), RGM-C (Hemojuvelin), Semaphorin 6C, Serpin B6, Serpin Fl (Pigment epithelium-derived factor), Sortilin, TGM4 (Protein-glutamine gammaglutamyltransferase 4), Thrombomodulin, TSP-1 (Thrombospondin 1), TSP-2 (Thrombospondin 2), CAD11 (Cadherin 11), JAM3 (Junctional Adhesion Molecule 3), VIM (Vimentin), FBLN5 (Fibulin 5), FBN2 (Fibrillin 2), Hyaluronic Acid, COL1 Al (Collagen Type 1 Alpha 1), COL1 A2 (Collagen Type 1 Alpha 2), or Transferrin, or a combination of two or more thereof. In some embodiments, the composition further comprises one or more of COL2A1, COL3A1, COL5A1, COL5A2, COL6A1, COL11 Al, or COL14A1, or a combination of two or more thereof. In some embodiments, the methods comprise administering to the subject a composition comprising one or more extracellular vesicles (EVs), wherein the composition comprises hsa-miR-21-5p, miR-24-3p, hsa-miR-222-3p, hsa-miR-27b-3p, or let-7, or a combination of two or more thereof. In some embodiments, the composition further comprises hsa-miR-125b-5p, hsa- miR-132-3p, hsa-miR-145-5p, hsa-miR-191-5p, hsa-miR-199a-3p, hsa-miR-221-3p, hsa-miR- 22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-miR-27a-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-31-5p, hsa-miR-320a, hsa-miR-34a-5p, hsa-miR-423-3p, hsa-miR-424-5p, or hsa-miR- 940, or a combination of two or more thereof. In some embodiments, the methods comprise administering to the subject a composition comprising one or more extracellular vesicles (EVs), wherein at least 80% of the one or more EVs are CD63+, CD9-, CD81-. In some embodiments, the EDS is hypermobile EDS. In some embodiments, the EDS is vascular EDS. In some embodiments, the subject is (i) positive or negative for human leukocyte antigen B27 (HLA- B27), (ii) has, or does not have, a mutation in COL1 Al and COL3A1 collagen genes, (iii) has, or does not have, a mutation in the MTHFR gene, (iv) is positive or negative for ANA, or (v) a combination of two or more of (i) to (iv). In some embodiments, the symptoms comprise sleeplessness, headaches, brain fog, diminished concentration, fatigue, joint pain, overall pain, joint looseness, joint dislocations, gastrointestinal function, gastrointestinal mobility, CBD or THC use, NSAID use, use of pain medication, or overall feeling of well-being, or a combination of two or more thereof. In some embodiments, the subject has a collagen synthesis disorder. In some embodiments, the subject has hypermobile joints. In some embodiments, the subject has painful joints. In some embodiments, the administering comprises administering 15 mL of the composition. In some embodiments, the composition further comprises 30 mL of saline. In some embodiments, four doses of the composition are administered. In some embodiments, three doses of the composition are administered. In some embodiments, two doses of the composition are administered. In some embodiments, the second dose is administered 3 to 4 months after administration of the first dose. In some embodiments, the second dose is administered 24 hours after administration of the first dose. In some embodiments, the second dose of the composition is administered 3 to 4 months after the administration of the first dose, wherein the third dose is administered 24 hours after the administration of the second dose, and wherein the fourth dose is administered 2 months after the administration of the third dose. In some embodiments, the second dose of the composition is administered 24 hours after the administration of the first dose, and wherein the third dose is administered 2 months after the administration of the second dose. In some embodiments, the administering comprises intravenous administration. In some embodiments, the intravenous administration is carried out over the course of 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the composition is prepared by a process comprising: (a) culturing bone marrow mesenchymal stem cells (BM-MSCs) under the following conditions to produce an MSC conditioned media: (i) oxygen tension below 5%; and (ii) culture mediahaving a pH below 7; (b) harvesting the MSC conditioned media; and (c) formulating the MSC conditioned media to produce the composition. In some embodiments, the methods disclosed herein comprise preparing the composition prior to the administering, wherein the preparing the composition occurs by a process comprising: (a) culturing bone marrow mesenchymal stem cells (BM-MSCs) under the following conditions to produce an MSC conditioned media: (i) oxygen tension below 5%; and (ii) culture media having a pH below 7; (b) harvesting the MSC conditioned media; and (c) formulating the MSC conditioned media to produce the composition. In some embodiments, the culture media is serum-free. In some embodiments, the culture media has a glucose concentration below 4.5 g / L. In some embodiments, formulating the MSC conditioned media comprises exchanging the conditioned media for a pharmaceutically acceptable formulation. In some embodiments, the pharmaceutically acceptable formulation comprises saline. In some embodiments, the composition comprises at least 6xlO10to 8xlO10extracellular vesicles per mL and is administered at a dose of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml. In some embodiments, the dose is administered in combination with normal saline at a final volume of 100 mL. In some embodiments, the composition comprises saline (0.9% sodium chloride). In some embodiments, the saline is present in the composition at about 80% to about 95% saline. In some embodiments, the composition comprises sodium chloride, sodium lactate, potassium chloride, and / or calcium chloride. In some embodiments, the molecular weight of any non-excipient component of the composition is greater than about 2 kDa (kilodaltons), 8 kDa, 10 kDa, 50 kDa, or 100 kDa. In some embodiments, the composition comprises an oligosaccharide. In some embodiments, the oligosaccharide is present in the composition at from about 0.2 M to about 0.6 M. In some embodiments, any non-excipient component of the composi tion has a size of less than about 0.2 microns. In some embodiments, the composition is sterile by USP <71>. In some embodiments, the composition is endotoxin USP <85> free. In some embodiments, the composition is negative for mycoplasma DNA. In some embodiments, the composition is cell-free. In some embodiments, the composition is stored between -80 °C and -60 °C. In some embodiments, the composition is administered within 6 hours of thaw when maintained at ambient temperature. In some embodiments, the composition is present in a glass vial. In some embodiments, the composition is freeze-dried and stored at room temperature. In some embodiments, the composition is formulated by reconstitution of a powdered cake with sterile water into a solution. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition has a pH of about 6 to about 7.5. In some embodiments, the BM-MSCs are negative for CD14, CD31, CD34, and CD45. In some embodiments, the BM-MSCs are positive for CD73, CD105, CD166, and CD90. In some embodiments, the BM-MSCs are capable ofundergoing trilineage differentiation in vitro toward adipocyte, osteoblast, and chondrocyte phenotypes. In some embodiments, the BM-MSCs are obtained from an iliac crest aspiration of a single donor. In some embodiments, a concentration of the one or more EVs in the composition is measured by nanoparticle tracking analysis (NTA). In some embodiments, the NTA comprises light scatter and fluorescence evaluation. In some embodiments, the concentration of the one or more EVs is about 10 billion to about 250 billion EVs per mL of the composition. In some embodiments, the concentration of the one or more EVs is at about 1 billion to about 40 billion EVs per ml of the composition. In some embodiments, the one or more EVs have an average diameter of about 30 nm to about 170 nm. In some embodiments, a total protein concentration of the composition is about 10 to about 40 pg per ml of the composition. In some embodiments, a total protein concentration of the composition is about 1.5 to about 6 pg per ml of the composition. In some embodiments, the total protein concentration is measured by ELISA. In some embodiments, the composition comprises TIMP-1 (tissue inhibitor of metalloproteinases 1), TIMP-2 (tissue inhibitor of metalloproteinases 2), CD63 antigen, Ferritin, IGFBP-4 (Insulin-like growth factor binding protein-4), MIF (Macrophage migration inhibitory factor), LAMB1 (Laminin Subunit Beta 1), FBN1 (Fibrillin 1), HSPG2 (Heparin Sulphate Proteoglycan 2), BGN (Biglycan), or FN1 (Fibronectin 1), or a combination of two or more thereof. In some embodiments, the composition further comprises beta-IG-H3 (Transforming growth factor-beta-induced protein ig-H3), CA2 (Carbonic anhydrase 2), Cathepsin B, CD81 antigen, CD9 antigen, CD44 antigen, CD99 antigen, CD 109 antigen, CNTF (Ciliary neurotrophic factor), Decorin, DcR3 (Tumor necrosis factor receptor superfamily member 6B), Dkk-3 (Dickkopf-related protein 3), Endoglycan (podocalyxin-like protein 2), Furin, GM-CSF Ra (Granulocyte-macrophage colony-stimulating factor receptor subunit alpha), GPR115 (Adhesion G protein-coupled receptor F4), GP73 (Golgi membrane protein 1), HS3ST4 (Heparan sulfate glucosamine 3-O-sulfotransferase 4), IGF-2 (Insulin-like growth factor-2), IGFBP-2 (Insulin-like growth factor binding protein-2), IGFBP-3 (Insulin-like binding protein- 3), IGFBP-6 (Insulin-like growth factor binding protein-6), IL18BP (Interleukin- 18 Binding Protein), IL-1 R6 (Interleukin 1 Receptor 6), ILFBP-4 (Insulin-like growth factor binding protein-4), LAMP2 (Lysosome-associated membrane glycoprotein 2), Lumican, OPN (Osteopontin), PAI-1 (Plasminogen activator inhibitor- 1 or SERPINE 1), PCK1 (Phosphoenolpyruvate carboxykinase, cytosolic), PF4 (Platelet Factor 4), Periostin, PRDX4 (Peroxi dredoxin-4), RGM-C (Hemojuvelin), Semaphorin 6C, Serpin B6, Serpin Fl (Pigment epithelium-derived factor), Sortilin, TGM4 (Protein-glutamine gamma-glutamyltransferase 4), Thrombomodulin, TSP-1 (Thrombospondin 1), TSP-2 (Thrombospondin 2), CAD11 (Cadherin 11), JAM3 (Junctional Adhesion Molecule 3), VIM (Vimentin), FBLN5 (Fibulin 5), FBN2(Fibrillin 2), Hyaluronic Acid, COL1A1 (Collagen Type 1 Alpha 1), COL1A2 (Collagen Type 1 Alpha 2), or Transferrin, or a combination of two or more thereof. In some embodiments, the composition further comprises one or more of COL2A1, COL3A1, COL5A1, COL5A2, COL6A1, COL11 Al, or COL14A1, or a combination of two or more thereof. In some embodiments, the composition comprises hsa-miR-21-5p, miR-24-3p, hsa-miR-222-3p, hsa- miR-27b-3p, or let-7, or a combination of two or more thereof. In some embodiments, the composition further comprises hsa-miR-125b-5p, hsa-miR-132-3p, hsa-miR-145-5p, hsa-miR- 191-5p, hsa-miR-199a-3p, hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-miR-27a-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-31-5p, hsa-miR-320a, hsa-miR- 34a-5p, hsa-miR-423-3p, hsa-miR-424-5p, or hsa-miR-940, or a combination of two or more thereof. In some embodiments, at least 80% of the one or more EVs are CD63+, CD9-, CD81-.

[0005] Described herein is the use of a composition comprising a mesenchymal stem cell (MSC) secretome in treating Ehlers-Danlos Syndrome (EDS).BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0007] FIG. 1 shows a graph of symptom intensity over time in a typical responder receiving the therapeutic MSC secretome product of the present disclosure. Each line represents one patient.

[0008] FIG 2 shows questionnaire elements used to assess individual subject’s parameter severity on six different dates. The questionnaire also included space (not shown) to capture patient identification information and other clinical notes.

[0009] FIGs. 3A-3I are graphs showing baseline and final scores for nine assessed parameters. The Human Bone Marrow Mesenchymal Stem Cell Extracellular Vesicle IP (“IP”) causes statistically significant improvements in multiple parameters. Box and whisker plots are shown with symbols representing individual subjects, box height indicating the mean and whiskers showing 95% confidence intervals. Baseline parameter scores captured by the questionnaire one to three days before the first infusion of IP are shown on the left of each plot for the nine parameters. Scores obtained from the final assessment of each subject’s parameters are shown on the right of each plot. Assessed parameters were: A) Overall Feelings of Well Being (FIG. 3A); B) Headaches (FIG. 3B); C) Brain Fog / Concentration (FIG. 3C); D) Fatigue (FIG. 3D);E) Joint Pain (FIG. 3E); F) Joint Looseness / Dislocation (FIG. 3F); G) GI Function / Motility (FIG. 3G); H) CBD / THC Use (FIG. 3H); I) NSAID Use (FIG. 31). Statistical analysis by t-test where *p<0.05, **p<0.01, ***p<0.001, ns: not significant.

[0010] FIGs. 4A-4I are graphs showing baseline and final scores for nine assessed parameters. Subject responses to IP treatment indicate the existence of “responders” and “non-responders”. Baseline scores (left in each plot) for the nine assessed parameters (FIGs. 4A-4I) are plotted for each subject and connected by a solid line to the final score for the same subject. Data is identical to that in FIGs. 3A-3I. Assessed parameters were: A) Overall Feelings of Well Being (FIG. 4A); B) Headaches (FIG. 4B); C) Brain Fog / Concentration (FIG. 4C); D) Fatigue (FIG. 4D); E) Joint Pain (FIG. 4E); F) Joint Looseness / Dislocation (FIG. 4F); G) GI Function / Motility (FIG. 4G); H) CBD / THC Use (FIG. 4H); I) NSAID Use (FIG. 41). The parameter “Other Pain Medication Use” is not included as cited in FIGs. 3A-3I.

[0011] FIGs. 5A-5D are graphs demonstrating that a single initial dose of IP is sufficient to identify patients that are responsive to the IP. FIGs. 5A-5B: Six subjects were provided a single dose of IP as Dose 1, followed by a back-to-back (B:B) double dose 24 hours apart as Doses 2 & 3, and then followed by a single fourth and final Dose. Post-dose scores show results taken two weeks after each specified dosage. Scores for subjects 6-H, 5-J, and 2-B are identified in FIG. 5B to clarify the trends for these subjects. FIGs. 5C-5D: Four subjects were provided a back-to- back (B:B) double dose of IP as Dose 1 & 2, followed by a third and final single dose (Dose 3). Subject 8-1 is also identified for clarity in FIG. 5D.

[0012] FIGs. 6A-6D are graphs of responders and non-responders from each dosing regimen showing response heterogeneity to the initial dose. FIGs. 6A-6B show score results obtained at baseline (black) and at two weeks (gray) after the single initial dose from a responder subject (FIG. 6A) and a non-responder subject (FIG. 6B). FIGs. 6C-6D show score results from the back-to-back double initial dose from a different responder subject (FIG. 6C) and a different non-responder subject (FIG. 6D).DETAILED DESCRIPTION

[0013] Provided herein are compositions and methods for treating and managing symptoms of Ehlers-Danlos Syndrome (EDS).

[0014] Example compositions herein comprise an extracellular vesicle (EV) and / or a protein. The EV may originate from a mesenchymal stem cell (MSC). The protein may originate from a MSC. In an exemplary embodiment, the MSC is a bone marrow MSC (BM-MSC). The EV and / or protein may be purified or otherwise separated from the MSC growth and / or culturing condition from which the EV and / or protein was secreted into. Purified may include partiallypurified, such that some of the MSC growth and / or culturing condition is present in the composition. The composition may be formulated into an aqueous solution for intravenous administration. The composition may be referred to as a therapeutic composition.I. Therapeutic CompositionsA. Extracellular Vesicles

[0015] Extracellular vesicles (EV) are small membrane bound spheres containing proteins and RNA (of which exosomes are a subset). Exosomes are small lipid bilayer vesicles secreted by cells that lack a nucleus and cannot replicate. Other EV populations are derived directly from the plasma membrane or are formed during apoptosis (apoptotic bodies). Disclosed herein are compositions comprising an EV. In example embodiments, the EV is an exosome. Embodiments of an EV herein have a diameter of about 20 nm to about 200 nm. In some embodiments, the diameter is measured by nanoparticle tracking analysis (NTA).

[0016] The number of EVs within a composition may be about 10 billion to about 250 billion EVs per mL when suspended. The suspension may be diluted for intravenous administration, wherein the EVs within the composition may be about 1 billion to about 40 billion EVs per mL.

[0017] In some embodiments, the EV has a phenotype of CD63+CD9 and CD81 . In some embodiments, at least 70, 75, 80, 85, 90, 91, 92, 93, 94, or 95% of the EVs are CD63+CD9 and CD81 . In some embodiments, at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, or 95% of the EVs are CD9 . In some embodiments, at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, or 95% of the EVs are CD81 .

[0018] In some embodiments, the EV is produced from a MSC. The MSC may be a bone marrow MSC. The MSC may be a human MSC. In some embodiments, the EV is produced from a MSC that has the capacity to undergo trilineage differentiation in vitro toward adipocyte, osteoblast, and chondrocyte phenotypes. In some embodiments, the MSC is positive for CD73, CD105, CD166, and CD90 and is negative for CD14, CD31, CD34, and CD45.

[0019] In some embodiments, EVs are analyzed via light scatter and fluorescence evaluation (e.g., NanoSight, Malvern Panalytical Ltd., United Kingdom). In some embodiments, the EVs are characterized by single particle interferometric reflectance imaging sensor technology to visualize and quantify fluorescent antibody-labeled particles (e.g., Nano View Biosciences, Boston, MA).

[0020] In some embodiments, the EV comprises a peptide or protein. In some embodiments, the peptide or protein is within the EV. In some embodiments, the peptide or protein is outside the EV. In some embodiments, the peptide or protein is embedded in the EV. In some embodiments, the peptide or protein is attached to the EV. In some embodiments, the EV comprises a nucleicacid. Nucleic acids include ribonucleic acids (RNA), such as siRNA, shRNA, and microRNA (miRNA). In some embodiments, the nucleic acid is within the EV. In some embodiments, the nucleic acid is outside the EV. In some embodiments, the nucleic acid is embedded in the EV. In some embodiments, the nucleic acid is attached to the EV.

[0021] In some embodiments, the EV comprises one or more proteins (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100). Non-limiting examples of the one or more proteins include: beta-IG-H3 (Transforming growth factor-beta-induced protein ig-H3), CA2 (Carbonic anhydrase 2), Cathepsin B, CD63 antigen, CD81 antigen, CD9 antigen, CD44 antigen, CD99 antigen, CD 109 antigen, CNTF (Ciliary neurotrophic factor), Decorin, DcR3 (Tumor necrosis factor receptor superfamily member 6B), Dkk-3 (Dickkopf-related protein 3), Endoglycan (podocalyxin-like protein 2), Ferritin, Furin, GM-CSF Ra (Granulocyte-macrophage colony-stimulating factor receptor subunit alpha), GPR115 (Adhesion G protein-coupled receptor F4), GP73 (Golgi membrane protein 1), HS3ST4 (Heparan sulfate glucosamine 3-O- sulfotransferase 4), IGF-2 (Insulin-like growth factor-2), IGFBP-2 (Insulin-like growth factor binding protein-2), IGFBP-3 (Insulin-like binding protein-3), IGFBP-4 (Insulin-like growth factor binding protein-4), IGFBP-6 (Insulin-like growth factor binding protein-6), IL-1 R6 (Interleukin 1 Receptor 6), IL18BP (Interleukin- 18 Binding Protein), ILFBP-4 (Insulin-like growth factor binding protein-4), LAMP2 (Lysosome-associated membrane glycoprotein 2), Lumican, MIF (Macrophage migration inhibitory factor), OPN (Osteopontin), PALI (Plasminogen activator inhibitor- 1 or SERPINE 1), PCK1 (Phosphoenolpyruvate carboxykinase, cytosolic), PF4 (Platelet Factor 4), Periostin, PRDX4 (Peroxidredoxin-4), RGM-C (Hemojuvelin), Semaphorin 6C, Serpin B6, Serpin Fl (Pigment epithelium-derived factor), Sortilin, TGM4 (Protein-glutamine gamma-glutamyltransferase 4), Thrombomodulin, TSP-1 (Thrombospondin 1), TIMP-1 (tissue inhibitor of metalloproteinases 1), TIMP-2 (tissue inhibitor of metalloproteinases 2), TSP-2 (Thrombospondin 2), CAD11 (Cadherin 11), JAM3 (Junctional Adhesion Molecule 3), VIM (Vimentin), FBLN5 (Fibulin 5), FBN2 (Fibrillin 2), Hyaluronic Acid, COL1A1 (Collagen Type 1 Alpha 1), COL1A2 (Collagen Type 1 Alpha 2), Transferrin, LAMB1 (Laminin Subunit Beta 1), FBN1 (Fibrillin 1), HSPG2 (Heparin Sulphate Proteoglycan 2), BGN (Biglycan), and FN1 (Fibronectin 1). An EV that comprises the one or more of the proteins may include the one or more proteins within the EV. An EV that comprises one or more of the proteins may include the one or more proteins anchored within the EV. An EV that comprises the one or more of the proteins may be associated with the outside of the EV.

[0022] In some embodiments, the EV comprises one or more nucleic acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100). Non-limiting examples of the one or more nucleic acids include: hsa-miR-21-5p, miR-24-3p, hsa-miR-222-3p, hsa-miR-27b-3p, let-7, hsa-miR-125b-5p, hsa-miR-132-3p, hsa-miR-145-5p, hsa-miR-191-5p, hsa-miR-199a-3p, hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-miR-27a-3p, hsa-miR- 29a-3p, hsa-miR-29c-3p, hsa-miR-31-5p, hsa-miR-320a, hsa-miR-34a-5p, hsa-miR-423-3p, hsa- miR-424-5p, and hsa-miR-940. miRNA sequences may be obtained from www.mirbase.org. An EV that comprises the one or more of the nucleic acids may include the one or more nucleic acids within the EV.B. Proteins

[0023] Disclosed herein are compositions comprising a protein. The protein may be independent of an EV. For instance, the protein may not be present within the EV or within the membrane of the EV. The composition may comprise one or more proteins (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100). The protein may be present as a monomer or multimer. The protein may have a molecular weight (as a monomer or multimer as applicable) of at least about 10 kDa (kilodalton). Non-limiting examples of the one or more proteins include: LAMB1 (Laminin Subunit Beta 1), FBN1 (Fibrillin 1), HSPG2 (Heparin Sulphate Proteoglycan 2), BGN (Biglycan), FN1 (Fibronectin 1), IL18BP (Interleukin- 18 Binding Protein), TSP-2 (Thrombospondin 2), CAD11 (Cadherin 11), JAM3 (Junctional Adhesion Molecule 3), VIM (Vimentin), FBLN5 (Fibulin 5), FBN2 (Fibrillin 2), Hyaluronic Acid, COL1A1 (Collagen Type 1 Alpha 1), COL1A2 (Collagen Type 1 Alpha 2), beta-IG-H3 (Transforming growth factor-beta-induced protein ig-H3), CA2 (Carbonic anhydrase 2), Cathepsin B, CD63 antigen, CD81 antigen, CD9 antigen, CD44 antigen, CD99 antigen, CD 109 antigen, CNTF (Ciliary neurotrophic factor), Decorin, DcR3 (Tumor necrosis factor receptor superfamily member 6B), Dkk-3 (Dickkopf-related protein 3), Endoglycan (podocalyxin-like protein 2), Ferritin, Furin, GM-CSF Ra (Granulocyte-macrophage colony-stimulating factor receptor subunit alpha), GPR115 (Adhesion G protein-coupled receptor F4), GP73 (Golgi membrane protein 1), HS3ST4 (Heparan sulfate glucosamine 3-O-sulfotransferase 4), IGF-2 (Insulin-like growth factor-2), IGFBP-2 (Insulin-like growth factor binding protein-2), IGFBP-3 (Insulin-like binding protein-3), IGFBP-4 (Insulin-like growth factor binding protein-4), IGFBP- 6 (Insulin-like growth factor binding protein-6), IL-1 R6 (Interleukin 1 Receptor 6), ILFBP-4 (Insulin-like growth factor binding protein-4), LAMP2 (Lysosome-associated membrane glycoprotein 2), Lumican, MIF (Macrophage migration inhibitory factor), OPN (Osteopontin), PAI-1 (Plasminogen activator inhibitor- 1 or SERPINE 1), PCK1 (Phosphoenolpyruvatecarboxykinase, cytosolic), PF4 (Platelet Factor 4), Periostin, PRDX4 (Peroxidredoxin-4), RGM- C (Hemojuvelin), Semaphorin 6C, Serpin B6, Serpin Fl (Pigment epithelium-derived factor), Sortilin, TGM4 (Protein-glutamine gamma-glutamyltransferase 4), Thrombomodulin, TSP-1 (Thrombospondin 1), TIMP-1 (tissue inhibitor of metalloproteinases 1), TIMP-2 (tissue inhibitor of metalloproteinases 2), and Transferrin. Additional non-limiting examples of the one or more proteins include: NUP85, DAPP1, PDGF R alpha, SLITRK5, FAP, Artemin, DPPII, cIAP-1, Pentraxin 3, Visfatin, Neprilysin, Albumin, Galec-tin-1, UNC5H3, IL-20 R beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7 A, NKp80, Cy statin B, Osteoadherin, Midkine, Calreticulin, Osteoactivin, Legumain, TAZ, Cathepsin L, RBP4, Serpin A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Ga-lectin-3, MOG, Trypsin 3, SIRP alpha, and Syndecan-4, GSTM1, NUP85, MeprinA, IL-1 F10, TGFbl, Ephrin-A4, Aminopeptidase LRAP, GDF-9, PDGF R alpha, PAPP-A, Aryl-sulfatase A, LAIR2, ULBP-4, TFPI, SOX2, SLITRK5, Spinesin, ENPP-2, CD97, CTACK, Integrin alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin DI, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angio-tensinogen, Follistatin, CF VII, Persephin, TRAIL Rl, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, P0N1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Nepri-lysin, Stabilin-2, IL-17 RD, Albumin, Folli statin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mcl-1, DPPIV, SREC-II, Nor-rin, JAM-C, Bcl-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gpl30, C-myc, LILRB4, BMP-2,, MIA, CD34, IFNab R2, Glypican 2, MSP R, DSCAM, Matriptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nidogen-1, CD2, Kallikrein 1, TSLP R, TROY, VCAM-1, Siglec-11, S100A1, PARI, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R beta, Throm-bospondin-2, SMPD1, B2M, MFRP, LRP-6„ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF- 23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 alpha, 0X40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, Semaphorin 4D, R0B02, PDX-1, APRIL, Neurturin, Kremen-2, EMMPRIN, Activin RIB, Neuroligin 2, Epiregulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF Rl, DSPG3, SorCSl, Matrilin-2, sFRP-3, EphB3, NCK1, Prolactin, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Pappalysin-2, Syndecan-3, Jag-ged 1, AKR1C4, Olfactomedin-2,Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA125, HAO-1, PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, R0R1, CMG-2, 4-1BB Ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Leg-umain, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 beta, TAFA1, Podocalyxin, RalA, CRELD2, GRAP2, SP-D, BID, GFR alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Con-tactin-2, CD83, IL-1 R3, SALM4, GBA3, R0B04, OSCAR, VEGF, IGSF3, Biglycan, Neudesin, ILT4, uPAR, Axl, WIF-1, IL-7 R alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Cochlin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR- Al, TAFA2, N-Cadherin, IL-17B, IL- 17 RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, Prolactin R, FGF-20, CD28, Nogo- A, HSD17B1, IL- 19, Enteropeptidase, Cathep-sin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, ADAM23, NOV, Galectin-2, Neu-rexin 3 beta, TLR3, Sirtuin 2, Numb, IL-28 R alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL- 27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha- 1, Smad4, ICAM-1, MEF2C, TREM-1, L- Selectin, Hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, LTAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-lra, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsin-ogen II, Desmoglein-3, Nectin-4, SCF, Serpin A5, PTH, FGF-19, MSP, IL-28A, FGF- 12, METAP2, AS AHL, EDIL3, NT AL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, 0X40, IL- 13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, R0R2, EphB4, Glypican 1, LAP(TGFbl), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL- 10 Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, Nestin, TPO, ErbB3, Kirrel3, FLRT1, Galec-tin-3, CXCL16, JAM- B, DR6,Nogo Receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R alpha 1, AMIG02, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurogranin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-Spondin 2, CK19, Cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII,PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombos-pondin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, R0B03, RANTES, Endocan, Granulysin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin 3, ARSB, LIF R alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-lb, NKp46, MCP-3, IL-32 alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, Mer, TREM- 2, Flt-3L, CDS, IL-6, CD229, Insulin, Syntaxin 6, GRO, Bcl-w, Lipocalin-2, PDGF-AA, IL-2 Ra, Angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.l, ED AR, ADAMTS13, Kynureninase, PTH1R, IFN-gamma Rl, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein 11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP- 1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecan-4, IL-2 Rg, MICA, Dopa Decarboxylase, NPDC-1, MCP-4, EG-VEGF, Glycoprotein V, Semaphorin 4G, IL-12p40, PSA- total, IL-15, MAP1D, Clq, TNF4, Dtk, Endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL- 8, Galectin-8, CA4, Cystatin E M, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, Kallikrein 7, and IL-13. The one or more proteins may comprise a collagen protein.

[0024] In some embodiments, the composition may comprise one or more collagen proteins. The one or more collagen proteins may comprise but are not limited to: COL1A1, COL1 A2, COL2A1, COL3A1, COL5A1, COL5A2, COL6A1, COL11A1, or COL14A1, or a combination of two or more thereof.

[0025] In some embodiments, the one or more proteins comprises TEMPI. In some embodiments, the TEMPI is present at about 200 pg / mL to about 80 ng / mL of the composition or about 30 pg / mL to about 12 ng / mL. In some embodiments, the one or more proteins comprises OPN. In some embodiments, the OPN is present at about 200 pg / mL to about 80 ng / mL of the composition or about 30 pg / mL to about 12 ng / mL. In some embodiments, the one or more proteins comprises IGFBP4. In some embodiments, the IGFBP4 is present at about 200 pg / mL to about 80 ng / mL of the composition or about 30 pg / mL to about 12 ng / mL. In some embodiments, the one or more proteins comprises osteonectin. In some embodiments, the osteonectin is present at about 200 pg / mL to about 80 ng / mL of the composition or about 30 pg / mL to about 12 ng / mL. In some embodiments, the concentration of the one or more proteins is measured by ELISA.

[0026] In some embodiments, the total protein concentration of the one or more proteins is about 10 to about 40 pg per ml of the composition. For example, the concentration in frozen product. In some embodiments, the total protein concentration of the one or more proteins is about 1.5 to about 6 pg per ml of the composition. For example, the concentration in an intravenous solution for administration.C. Pharmaceutical Applications

[0027] Disclosed herein are compositions for use in pharmaceutical applications. In some embodiments, compositions described herein can safely target one or more (e.g., more than hundreds) different biomolecular interactions or signaling pathways. In some embodiments, compositions described herein can treat one or more injuries or diseases caused by multiple etiologies. In some embodiments, compositions described herein can be used for treating a disease or condition without identifying the pathogen underlying the disease or condition, thus offering an advantage for treating a disease or condition caused by an emerging or previously unknown pathogen.

[0028] Compositions described herein may be administered in vivo in a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may biologically suitable, z.e., the material may be administered to a subject, along with the EV and / or protein, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the therapeutic composition in which it is contained. As a non-limiting example, the pharmaceutically acceptable carrier may comprise sodium chloride.

[0029] Compositions described herein may be administered to a patient. Administration may comprise parenteral, oral, intravenous, intramuscular, subcutaneous, inhalation, topical, sublingual, rectal, and / or transdermal administration. Compositions described herein may be administered via a variety of routes and formulations including but not limited to: oral, intravenous, intramuscular, subcutaneous, inhalation, topical, sublingual, rectal, transdermal, sublingual, buccal, intranasal, intrathecal, epidural, ocular, otic, vaginal, transbuccal, intraocular, intraperitoneal, intravesical, intradermal, intraventricular, intracardiac, intrapulmonary, sublingual tablet, oral solution, oral suspension, effervescent tablets, oral film, oral powder, chewable tablet, extended-release tablet, enteric-coated tablet, intramuscular injection, intravenous infusion, inhaler, nebulizer, transdermal patch, buccal tablet, film strip, lozenge, suppository, cream, ointment, gel, aerosol, nasal spray, enema, eye drop, ear drop, and implant. Parenteral administration of a composition, if used, is generally characterized by injection. The injection may be an intravenous injection. The injection may involve administration of the composition over a period of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.

[0030] In some embodiments, the composition for administration may comprise an excipient. In some embodiments, the excipient may comprise saline. The composition for injection may comprise saline, e.g., 0.9% saline (NaCl). The 0.9% saline may be about 85% to about 99% or more of the composition by volume. For instance, the composition comprises about 85% of 0.9% saline and about 15% of an EV and / or protein component. The EV and / or protein component may be formulated in an isotonic infusion solution. The EV and / or proteincomponent may comprise sodium chloride. The EV and / or protein component may comprise sodium chloride, sodium lactate, potassium chloride, and calcium chloride. The EV and / or protein component may comprise sodium chloride (NaCl) at 100-150 mEq / L, sodium lactate (CsEENaCh) at 25-35 mEq / L, potassium chloride (KC1) at 2-6 mEq / L, calcium chloride (CaCL) at 1-5 mEq / L, and chloride ions (CL) at 80-130 mEq / L. The EV and / or protein component may comprise 0.1 M to 0.8 M oligosaccharide. Non-limiting example oligosaccharides include, but are not limited to, glucose, aldose (D-allose, D-altrose, D-mannose, etc.), glucopyranose, pentahydroxyhexanal, alpha-D-glucopyranoside dihydrate, a-D-glucopyranosyl-D-glucose, a-D- glucopyranosyl-dihydrate, polymer of P-D-glycopyranosyl units, P-D-fructofuranosyl a-D- glucopyranoside (anhydrous / dihydrate), P-D-galactopyranosyl-D-glucose, a-D-glucopyranosyl- a-D-glucopyranoside (anhydrous / dihydrate), galactose, pentoses (ribose, xylose, lyxose), dextrose, dodecacarbon monodecahydrate, fructose, sucrose, lactose, maltose, trehalose, agarose, D-galactosyl-0-(l-4)-anhydro-L-galactosyl, cellulose, starch, polyhydric alcohol, polyalcohol, alditol, erythritol, glycitol, glycerol, xylitol, and sorbitol. In some embodiments, the composition has a pH of from about 6.0 to about 7.5. In some embodiments, the composition has a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. In some embodiments, the molecular weight of any non-excipient component of the composition may be greater than about 2 kDa (kilodaltons), 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or 100 kDa. In some embodiments, the molecular weight of any non-excipient component of the composition is greater than about 2 kDa (kilodaltons), 8 kDa, 10 kDa, 50kDa, or lOOkDa.

[0031] In some embodiments, the composition is sterile per USP <71>. In some embodiments, the composition is endotoxin free per USP <85>. In some embodiments, the composition is negative for mycoplasma DNA. In some embodiments, the composition is cell-free. In some embodiments, the composition is stored between -80 °C and -60 °C. In some embodiments, the composition is freeze-dried into a powder cake and stored at room temperature. In some embodiments, the composition will have the powdered cake reconstituted with water prior to administration. In some embodiments, the composition is administered within 6 hours of thaw when maintained at ambient temperature. In some embodiments, the composition is stored in a glass vial. In some embodiments, the composition comprises a certain % of water as determined by Karl Fisher measurement. In some embodiments, the composition comprises less than 2.0%, less than 2.5%, less than 3.0%, less than 3.5%, less than 4.0%, less than 4.5%, or less than 5.0% water as determined by Karl Fisher measurement.D. Methods of Composition Production

[0032] Compositions herein comprising an EV and a protein may include components produced from a MSC. The EV of the composition may be produced from a MSC. The protein of the composition may be produced from a MSC. The EV and / or protein may be produced from a MSC cultured at one or more of the following conditions: about 0.1% to about 5% oxygen, reduced or no serum, pH of about 5-7.5, reduced glucose, increased temperature, or any of these elements in various combinations. The MSC may be cultured under the aforementioned one or more conditions after the cell achieves confluency. The MSC may be cultured under the aforementioned one or more conditions after the cell is cultured at 37°C and 5% CO2. The EV and / or protein of the composition may be obtained from a method comprising: (1) growing the MSCs to 50-80% confluency, then (2) culturing the MSCs at one or more of the following conditions: about 1% to about 5% oxygen, reduced or no serum, reduced glucose, or increased temperature. The growing at step (1) may be performed at about 37°C and about 5% CO2. Culturing the MSCs may comprise introducing the cells to a culture media. The culture media may comprise basal media. For instance, basal media comprising amino acids, vitamins, and inorganic salts, and optionally a carbon source such as glucose. The culture media may comprise an isotonic infusion solution. In a non-limiting example embodiment, the culture media comprises a basal media and an isotonic infusion solution. The culture media may comprise basal media and sodium chloride. The culture media may comprise basal media, sodium chloride, sodium lactate, potassium chloride, and calcium chloride. The culture media may comprise a basal media and an isotonic infusion solution. The culture media may comprise basal media and sodium chloride, without serum. The culture media may comprise basal media, sodium chloride, sodium lactate, potassium chloride, and calcium chloride, without serum. The culturing at step (2) may occur over a period of 1, 2, 3, 4, 5, 6, or 7 days, wherein the oxygen and pH may change over time. At any time during the culturing step, the pH may be about 6.5 to about 7. At any time during the culturing step, the oxygen may be at about 0.5%, 1%, 1.5%, or 2%.

[0033] The EV and / or protein produced in the second culturing step (2) may be purified (e.g., partially or entirely), from the culture media of the second culturing step (2). The purification may comprise formulating the EV and / or the protein into an EV and / or protein component. The EV and / or protein component may comprise an isotonic infusion solution. The EV and / or protein component may comprise sodium chloride. The EV and / or protein component may comprise sodium chloride, sodium lactate, potassium chloride, and calcium chloride. The EV and / or protein component may comprise sodium chloride (NaCl) at 100-150 mEq / L, sodium lactate (CsHsNaCh) at 25-35 mEq / L, potassium chloride (KC1) at 2-6 mEq / L, calcium chloride(CaCh) at 1-5 mEq / L, and chloride ions (CL) at 80-130 mEq / L. The EV and / or protein component may comprise an oligosaccharide, e.g., 0.1 M to 0.8 M oligosaccharide. Non-limiting example oligosaccharides include, but are not limited to, glucose, aldose (D-allose, D-altrose, D- mannose, etc.), glucopyranose, pentahydroxyhexanal, alpha-D-glucopyranoside dihydrate, a-D- glucopyranosyl-D-glucose, a-D-glucopyranosyl-dihydrate, polymer of P-D-glycopyranosyl units, P-D-fructofuranosyl a-D-glucopyranoside (anhydrous / dihydrate), P-D-galactopyranosyl-D- glucose, a-D-glucopyranosyl-a-D-glucopyranoside (anhydrous / dihydrate), galactose, pentoses (ribose, xylose, lyxose), dextrose, dodecacarbon monodecahydrate, fructose, sucrose, lactose, maltose, trehalose, agarose, D-galactosyl-0-(l-4)-anhydro-L-galactosyl, cellulose, starch, polyhydric alcohol, polyalcohol, alditol, erythritol, glycitol, glycerol, xylitol, and sorbitol. The amount of oligosaccharide in the EV and / or protein component may be about 0.2 M to about 0.6 M, or about 0.4 M. In some embodiments, the amount of oligosaccharide in the compositions disclosed herein may be about 0.10 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M, 0.35 M, 0.40 M, 0.45 M, 0.50 M, 0.55 M, 0.60 M, 0.65 M, or 0.70 M. In some embodiments, the EV and / or protein component is frozen. The frozen material may be thawed and combined with saline (e.g., 0.9% saline or sodium chloride) to generate an IV formulation for IV administration. The amount of oligosaccharide in the IV formulation may be about 40 mM to about 80 mM, or about 60 mM.

[0034] The EV and / or protein component may be filter-sterilized. The EV and / or protein component may be filter sterilized after and / or during formulation from the culture media into the EV and / or protein component. The EV and / or protein may be concentrated. The concentration may occur during and / or after purification and / or exchange from the culture media into the EV and / or protein component. The EV and / or protein may be frozen, e.g., after and / or during purification. If frozen, the EV and / or protein may be formulated with a cryoprotectant prior to freezing. The cryoprotectant may comprises an oligosaccharide, e.g., as described above.

[0035] In some embodiments, reduced glucose is less than a normal control e.g., 4.5 g / L). For example, the glucose reduction may be about 5% to about 15%, from about 10% to about 20%, from about 15% to about 25%, from about 20% to about 30%, or from about 25% to about 35% of the glucose in a normal control. In some embodiments, the reduced glucose is present in the MSC culture media (e.g., at step (2) as noted above) at a concentration of about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0 g / L, or a range between any two of these values. In some embodiments, glucose is present at a concentration of less than or no more than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5 g / L.

[0036] In some embodiments, the MSC is cultured, e.g., at step (2) at about 0.1% to about 5% oxygen, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5% oxygen. In an example embodiment, the oxygen is about 0.5% to about 1.5%, or about 1%.

[0037] The pH at which the MSC is cultured, e.g., at step (2), can be from about 6.0 to about 7.4, for example, from 6.5 to about 7, or about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, or 7.4.

[0038] The temperature of the culture environment, e.g., at step (2), may be raised relative to physiologic homeostasis temperature (e.g., 37°C). In one aspect, the temperature of the culture can be 35.0, 35.1, 35.2, 35.3, 36.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36.0, 36.1, 36.2, 36.3, 36.4, 36.5, 36.6, 36.7, 36.8, 36.9, 37.0, .37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38.0, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39.0, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, or 40.0°C.

[0039] In some embodiments, compositions described herein can be lyophilized (freeze-dried) for packaging and storing. In some embodiments, compositions described herein can be stored at ambient or room temperature (e.g., between 60°F and 75°F or between 15°C and 24°C). In some embodiments, the stability of compositions described herein at ambient or room temperature can provide advantages for logistics and / or delivery.II. Methods of treating Ehlers-Danlos Syndrome

[0040] Provided herein in some embodiments are methods and compositions for treating Ehlers- Danlos Syndrome (EDS) in a subject. In some embodiments, the subject has been diagnosed with Ehlers-Danlos Syndrome (EDS). In some embodiments, the EDS is hypermobile type EDS (hEDS). In some embodiments, the EDS is vascular EDS.

[0041] In some embodiments, the subject is a human subject. In some embodiments, the subject is from 21 to 65 years of age. In some embodiments, the subject is positive for human leukocyte antigen B27 (HLA-B27). In some embodiments, the subject does not have an autoimmune disease. In some embodiments, the subject has a negative antinuclear antibody (ANA) panel. In some embodiments, the subject is not taking one or more immunosuppressants. In some embodiments, the subject has not been diagnosed with lupus erythematosus. In some embodiments, the subject has a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene.

[0042] In some embodiments, the subject has a collagen synthesis disorder. In some embodiments, the subject has painful joints. In some embodiments, the subject has hypermobilejoints. In some embodiments, the subject has a Hypermobile Spectrum Disorder (HSD). In some embodiments, the subject has Mast Cell Activation Syndrome (MCAS). In some embodiments, the subject exhibits one or more symptoms including but not limited to headaches, peripheral neuropathy, and / or postural orthostatic tachycardia syndrome (POTS). In some embodiments, the subject exhibits one or more symptoms including but not limited to sleeplessness, headaches, brain fog, diminished concentration, fatigue, joint pain, overall pain, joint looseness, joint dislocations, gastrointestinal discomfort, gastrointestinal disruption, and / or reduced gastrointestinal mobility. In some embodiments, the subject exhibits one or more symptoms including but not limited to hyperflexible joints, joint dislocations, chronic pain, soft and velvety skin, easy bruising, hyper-elastic skin, delayed wound healing, atrophic scars, muscle weakness, internal bleeding, sprains, strains, chronic fatigue, arterial rupture, heart valve problems, gastrointestinal issues, flat feet, scoliosis, fragile blood vessels, tendon or ligament ruptures, hypotonia, frequent sprains, chronic pain syndromes, dysautonomia, visible veins, brittle teeth, frequent headaches, shortness of breath, early osteoarthritis, skin that tears easily, poor proprioception, varicose veins, excessive sweating, increased scarring, uterine rupture or complications in pregnancy, delayed puberty, frequent nosebleeds, hernias, easy tissue damage, restless legs, sleep disturbances, dental crowding, teeth with a translucent appearance, fragile corneas, soft or thin nails, hypermobility of the fingers, corneal rupture or tearing, increased skin infections, reduced range of motion in some joints, irregular heart rhythms, and / or increased risk of blood clots.

[0043] In some embodiments, the subject has a delayed response to treatment. In some embodiments, the subject may experience improvement in one or more of the following symptoms in response to treatment: sleeplessness, headaches, brain fog, diminished concentration, fatigue, joint pain, overall pain, joint looseness, joint dislocations, gastrointestinal function, gastrointestinal mobility, CBD or THC use, NSAID use, use of pain medication, or overall feeling of well-being, or a combination of two or more thereof.

[0044] In some embodiments, the compositions disclosed herein may be used in a method of treating Ehlers-Danlos Syndrome in a human subject. In some embodiments, the compositions disclosed herein may be administered to the human subject. In some embodiments, the administration may be intravenous. In some embodiments, the administration may comprise an intravenous infusion. In some embodiments, the administration may be carried out over the course of several minutes. In some embodiments, the administration may be carried out over the course of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the administration may be carried out over the course of greater than 60 minutes. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 milliliters (mL) of the compositions disclosed hereinmay be administered at a time. In some embodiments, a dose of a composition disclosed herein may be administered. In some embodiments, a dose of a composition disclosed herein may comprise 1, 2, 3, 4, or 5 milliliters. In some embodiments, a dose of a composition disclosed herein may be combined with normal saline. In some embodiments, a dose of a composition disclosed herein may be combined with normal saline for a final volume of 45 milliliters. In some embodiments, a dose of 15 milliliters of a composition disclosed herein may be combined with 30 milliliters of normal saline. In some embodiments, a dose of a composition disclosed herein may be combined with normal saline for a final volume of 100 milliliters. In some embodiments, a dose of 5 milliliters of a composition disclosed herein may be combined with 95 milliliters of normal saline. In some embodiments, the compositions disclosed herein may be administered once. In some embodiments, the compositions disclosed herein may be administered more than once.

[0045] In some embodiments, the compositions disclosed herein may comprise one or more of: beta-IG-H3 (Transforming growth factor-beta-induced protein ig-H3), CA2 (Carbonic anhydrase 2), Cathepsin B, CD63 antigen, CD81 antigen, CD9 antigen, CD99 antigen, CD109 antigen, CNTF (Ciliary neurotrophic factor), Decorin, DcR3 (Tumor necrosis factor receptor superfamily member 6B), Dkk-3 (Dickkopf-related protein 3), Endoglycan (podocalyxin-like protein 2), Ferritin, Furin, GM-CSF Ra (Granulocyte-macrophage colony-stimulating factor receptor subunit alpha), GPR115 (Adhesion G protein-coupled receptor F4), GP73 (Golgi membrane protein 1), HS3ST4 (Heparan sulfate glucosamine 3-O-sulfotransferase 4), IGF-2 (Insulin-like growth factor-2), IGFBP-2 (Insulin-like growth factor binding protein-2), IGFBP-3 (Insulin-like binding protein-3), IGFBP-4 (Insulin-like growth factor binding protein-4), IGFBP-6 (Insulinlike growth factor binding protein-6), IL-1 R6 (Interleukin 1 Receptor 6), ILFBP-4 (Insulin-like growth factor binding protein-4), LAMP2 (Lysosome-associated membrane glycoprotein 2), Lumican, MIF (Macrophage migration inhibitory factor), OPN (Osteopontin), PALI (Plasminogen activator inhibitor- 1 or SERPINE 1), PCK1 (Phosphoenolpyruvate carboxykinase, cytosolic), PF4 (Platelet Factor 4), Periostin, PRDX4 (Peroxidredoxin-4), RGM-C (Hemojuvelin), Semaphorin 6C, Serpin B6, Serpin Fl (Pigment epithelium-derived factor), Sortilin, TGM4 (Protein-glutamine gamma-glutamyltransferase 4), Thrombomodulin, TSP-1 (Thrombospondin 1), TIMP-1 (Tissue inhibitor of MMPs 1), TIMP-2 (tissue inhibitor of metalloproteinases 2), and Transferrin, or a combination of two or more thereof. In some embodiments, the compositions disclosed herein may comprise one or more of: hsa-miR-21-5p, miR-24-3p, hsa-miR-222-3p, hsa-miR-27b-3p, let-7, hsa-miR-125b-5p, hsa-miR-132-3p, hsa- miR-145-5p, hsa-miR-191-5p, hsa-miR-199a-3p, hsa-miR-21-5p,hsa-miR-221-3p, hsa-miR- 222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-miR-27a-3p, hsa-miR-27b-3p,hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-31-5p, hsa-miR-320a, hsa-miR-34a-5p, hsa-miR- 423-3p, hsa-miR-424-5p, and hsa-miR-940, or a combination of two or more thereof.

[0046] In some embodiments, the compositions disclosed herein may comprise one or more of TIMP-1 (tissue inhibitor of metalloproteinases 1), TIMP-2 (tissue inhibitor of metalloproteinases 2), CD63 antigen, Ferritin, IGFBP-4 (Insulin-like growth factor binding protein-4), MIF (Macrophage migration inhibitory factor), LAMB 1 (Laminin Subunit Beta 1), FBN1 (Fibrillin1), HSPG2 (Heparin Sulphate Proteoglycan 2), BGN (Biglycan), or FN1 (Fibronectin 1), or a combination of two or more thereof. In some embodiments, the compositions disclosed herein may comprise one or more of beta-IG-H3 (Transforming growth factor-beta-induced protein ig- H3), CA2 (Carbonic anhydrase 2), Cathepsin B, CD81 antigen, CD9 antigen, CD44 antigen, CD99 antigen, CD 109 antigen, CNTF (Ciliary neurotrophic factor), Decorin, DcR3 (Tumor necrosis factor receptor superfamily member 6B), Dkk-3 (Dickkopf-related protein 3), Endoglycan (podocalyxin-like protein 2), Furin, GM-CSF Ra (Granulocyte-macrophage colonystimulating factor receptor subunit alpha), GPR115 (Adhesion G protein-coupled receptor F4), GP73 (Golgi membrane protein 1), HS3ST4 (Heparan sulfate glucosamine 3-O-sulfotransferase 4), IGF -2 (Insulin-like growth factor-2), IGFBP-2 (Insulin-like growth factor binding protein-2), IGFBP-3 (Insulin-like binding protein-3), IGFBP-6 (Insulin-like growth factor binding protein- 6), IL18BP (Interleukin- 18 Binding Protein), IL-1 R6 (Interleukin 1 Receptor 6), ILFBP-4 (Insulin-like growth factor binding protein-4), LAMP2 (Lysosome-associated membrane glycoprotein 2), Lumican, OPN (Osteopontin), PALI (Plasminogen activator inhibitor- 1 or SERPINE 1), PCK1 (Phosphoenol pyruvate carboxykinase, cytosolic), PF4 (Platelet Factor 4), Periostin, PRDX4 (Peroxidredoxin-4), RGM-C (Hemojuvelin), Semaphorin 6C, Serpin B6, Serpin Fl (Pigment epithelium-derived factor), Sortilin, TGM4 (Protein-glutamine gammaglutamyltransferase 4), Thrombomodulin, TSP-1 (Thrombospondin 1), TSP-2 (Thrombospondin2), CAD11 (Cadherin 11), JAM3 (Junctional Adhesion Molecule 3), VIM (Vimentin), FBLN5 (Fibulin 5), FBN2 (Fibrillin 2), Hyaluronic Acid, COL1 Al (Collagen Type 1 Alpha 1),COL1 A2 (Collagen Type 1 Alpha 2), or Transferrin, or a combination of two or more thereof. In some embodiments, the compositions disclosed herein may comprise one or more of COL 1 Al, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL6A1, COL11A1, or COL14Al, or a combination of two or more thereof.

[0047] In some embodiments, the compositions disclosed herein may comprise one or more of hsa-miR-21-5p, miR-24-3p, hsa-miR-222-3p, hsa-miR-27b-3p, or let-7, or a combination of two or more thereof. In some embodiments, the compositions disclosed herein may comprise one or more of hsa-miR-125b-5p, hsa-miR-132-3p, hsa-miR-145-5p, hsa-miR-191-5p, hsa-miR-199a- 3p, hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-miR-27a-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-31-5p, hsa-miR-320a, hsa-miR-34a-5p, hsa-miR-423- 3p, hsa-miR-424-5p, or hsa-miR-940, or a combination of two or more thereof.

[0048] In some embodiments, a cell-equivalent dose of a composition disclosed herein may comprise from 0.7 to 7.0 million cells per kilogram. In some embodiments, a cell-equivalent dose of a composition disclosed herein may comprise 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0 million cells per kilogram. In some embodiments, a dose of a composition disclosed herein provides 9xlOnto 1.2xl012extracellular vesicles or 5xl0nto 1.5xl012, 6xlOnto 1.4xl012, 7xlOnto 1.3xl012, 8xl0nto 1.2xl012, or 8xl0nto 1.3xl012extracellular vesicles. In some embodiments, a composition disclosed herein is administered at a dose that provides at least 5xl0n, 6xlOn, 7xlOn, 8xl0n, 9xlOn, IxlO12, l. lxlO12, 1.2xl012, 1.3xl012, 1.4xl012, or 1.5xl012extracellular vesicles. In some embodiments, a composition disclosed herein comprises 6xlO10to 8xl010extracellular vesicles per mL and is administered at a dose of 5 to 20 mL. In some embodiments, four doses of the composition is administered. In some embodiments, three doses of the composition is administered. In some embodiments, two doses of the composition is administered. In some embodiments, the second dose is administered 3 to 4 months after administration of the first dose. In some embodiments, the second dose is administered 24 hours after administration of the first dose. In some embodiments, the second dose of the composition is administered 3 to 4 months after the administration of the first dose, wherein the third dose is administered 24 hours after the administration of the second dose, and wherein the fourth dose is administered 2 months after the administration of the third dose. In some embodiments, the second dose of the composition is administered 24 hours after the administration of the first dose, and wherein the third dose is administered 2 months after the administration of the second dose.III. Definitions

[0049] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. The terms “and / or”, “a combination of two or more thereof’, and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B andC; A and C; and A, B, and C ” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0050] The term “about” or “approximately” can mean within an acceptable error range for the particular value, which may depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0051] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0052] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0053] Certain specific details of this description are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known techniques or methods have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

[0054] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods, and materials are described below.IV. Examples

[0055] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.A. Example 1 - Production of Therapeutic Composition

[0056] An MSC secretome therapeutic composition (referred to herein as investigational product or “IP” as well as investigational medicinal product or “IMP”) was made by the following method: human bone marrow-derived MSCs were cultured in culture vessels with growth media to expand the MSC population. Growth media was then removed, and the cells were washed with PBS. The MSCs were then cultured at a pH below 7.0, less than 5% oxygen (e.g., to a final concentration of about 1% oxygen), and in a culture media comprising basal media and sodium chloride. The culture media comprising the MSC secretome was formulated into an isotonic infusion solution, such as sodium chloride, and filter sterilized. The production process for the IMP was done under current Good Manufacturing Practices and Current Good Tissue Practices.

[0057] The IMP was manufactured from the banked hBM-MSCs of a single donor under cGMP conditions and according to FDA Master File protocols. Each lot of the IMP was characterized by proteomic and miRNA characterization. Additionally, the size and quantity of EVs and the presence of a specific surface marker expression profile were confirmed.

[0058] The IMP comprises extracellular vesicles (EVs) that are acellular and nonimmunogenic, containing no nucleus or deoxyribonucleic acid (DNA).

[0059] The tetraspanin profile of extracellular vesicles present in the IMP was determined, and it was found that greater than 95% of the extracellular vesicles present in the composition were CD63+CD9 CD8 I . The EVs were measured via Nanoparticle tracking analysis (NT A), having a median diameter of about 100 nm. At least 10 billion EVs per mL were calculated using NTA with fluorescent staining of EV membranes.

[0060] Protein content of the IMP was determined, and the following proteins were found to be present. Proteins were detected using an antibody -based sandwich ELISAs or by Luminex or Nanoview / Unchained Labs-based methods. The total concentration of certain proteins in the IMP was measured via ELISA, with each protein having a concentration of about 200 pg / mL to about 80 ng / mL. For instance, TEMPI, OPN, IGFBP4, and osteonectin were characterized. Nonlimiting examples of proteins present in the IMP are shown in Table 1 below.Table 1

[0061] The nucleic acid content of the IMP was determined, and the following nucleic acids were found to be present: hsa-miR-21-5p, miR-24-3p, hsa-miR-222-3p, hsa-miR-27b-3p, let-7, hsa-miR-125b-5p, hsa-miR-132-3p, hsa-miR-145-5p, hsa-miR-191-5p, hsa-miR-199a-3p, hsa- miR-221-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-miR-27a-3p, hsa-miR-29a- 3p, hsa-miR-29c-3p, hsa-miR-31-5p, hsa-miR-320a, hsa-miR-34a-5p, hsa-miR-423-3p, hsa- miR-424-5p, and hsa-miR-940. RNA were measured using semi-quantitative PCR. Total RNA was analyzed by UV detection using a ClarioStar Plus microplate reader and BMG Labtech LVis plate adapter for nucleic acid quantification. Using this method, the amount of each RNA may be less than the limit of detection, or about 50 ng / mL.

[0062] Additional proteins that may be present in the IMP include: NUP85, DAPP1, PDGF R alpha, SLITRK5, FAP, Artemin, DPPII, cIAP-1, Pentraxin 3, Visfatin, Neprilysin, Albumin, Galectin-1, UNC5H3, IL-20 R beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R,TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midkine, Calreticulin, Osteoactivin, Legumain, TAZ, Cathepsin L, RBP4, Serpin A4, JAM- A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin 3, SIRP alpha, and Syndecan-4, GSTM1, NUP85, MeprinA, IL-1 F10, TGFbl, Ephrin-A4, Aminopeptidase LRAP, GDF-9, PDGF R alpha, PAPP -A, Arylsulfatase A, LAIR2, ULBP-4, TFPI, SOX2, SLITRK5, Spinesin, ENPP-2, CD97, CTACK, Integrin alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF- 2R, ADAMTSL-1, Erythropoietin, Plexin DI, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Persephin, TRAIL Rl, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, P0N1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mcl-1, DPPIV, SREC-II, Norrin, JAM-C, Bcl- 10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gpl30, C- myc, LILRB4, BMP-2,, MIA, CD34, IFNab R2, Glypican 2, MSP R, DSCAM, Matriptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nidogen-1, CD2, Kallikrein 1, TSLP R, TROY, VCAM-1, Siglec-11, S100A1, PARI, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6„ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD- ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 alpha, 0X40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, Semaphorin 4D, ROBO2, PDX-1, APRIL, Neurturin, Kremen-2, EMMPRIN, Activin RIB, Neuroligin 2, Epiregulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF Rl, DSPG3, SorCSl, Matrilin-2, sFRP-3, EphB3, NCK1, Prolactin, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Pappalysin-2, Syndecan-3, Jagged 1, AKR1C4, Olfactomedin-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin- 1, CD51, Notch- 1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA125, HAO-1, PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, R0R1, CMG-2, 4-1BB Ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Legumain, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 beta, TAFA1, Podocalyxin, Rai A, CRELD2, GRAP2, SP-D, BID, GFR alpha-2, Notch-3, VEGF R3, DLL4,TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Neudesin, ILT4, uPAR, Axl, WIF-1, IL-7 R alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Cochlin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL- 17 RC, MIP-3b, Cy statin C, Cy statin D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, ADAM23, NOV, Galectin-2, Neurexin 3 beta, TLR3, Sirtuin 2, Numb, IL-28 R alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM- A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA I3, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-Selectin, Hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, LTAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-lra, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpin A5, PTH, FGF-19, MSP, IL-28 A, FGF-12, METAP2, AS AHL, EDIL3, NT AL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, 0X40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, ROR2, EphB4, Glypican 1, LAP(TGFbl), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, Nestin, TPO, ErbB3, Kirrel3, FLRT1, Galectin-3, CXCL16, JAM-B, DR6,Nogo Receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R alpha 1, AMIG02, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM- 1, Inhibin A, Cadherin-4, P1GF-2, Neurogranin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-Spondin 2, CK19, Cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospondin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan, Granulysin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin 3, ARSB, LIF R alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-lb, NKp46, MCP-3, IL-32 alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Syntaxin 6, GRO, Bcl-w, Lipocalin-2, PDGF-AA, IL-2 Ra, Angiogenin, LYVE-1, CD4, RAGE,CDNF, Brevican, NAP-2, PU.1, ED AR, ADAMTS13, Kynureninase, PTH1R, IFN-gamma Rl, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein 11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP- 10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecan-4, IL-2 Rg, MICA, Dopa Decarboxylase, NPDC-1, MCP-4, EG- VEGF, Glycoprotein V, Semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, Endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, Galectin-8, CA4, Cystatin E M, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, Kallikrein 7, and IL-13, and combinations of two or more thereof.B. Example 2 - Clinical Study of Bone Marrow Derived Mesenchymal Stem Cell Extracellular Vesicle Isolate as a Successful Treatment for Hypermobile Type Ehlers-Danlos Syndrome (hEDS)

[0063] In this Example, the inventors report successful treatment in seven (7) out of ten (10) patients using intravenous bone marrow-derived mesenchymal extracellular vesicle (EV) administration. All study participants were clinically diagnosed by multiple consultants as having Ehlers-Danlos Syndrome, Hypermobility Type (hEDS).

[0064] Patient selection: Patients were drawn from a local Ehlers-Danlos Syndrome (EDS) support group, some of whom contacted other non-local candidates. Subjects had all been previously diagnosed with hEDS usually by multiple consultants and were on various treatments. They were instructed to continue whatever prior treatments found helpful during the study. All but one (1) had seen a rheumatologist to exclude autoimmune disease — This patient had a negative ANA. None were on immunosuppressants. None had a diagnosis of lupus erythematosus although one (1) had an ANA of 360 with no other abnormal blood findings. One (1) patient had prior treatment with extracellular vesicle infusion of placental origin without abatement of symptoms. Two (2) were positive for HLA-B27. One (1) had a mutation in the MTHFR gene. All were clinically interviewed in person and found to have physical and historical findings compatible with the diagnosis of hEDS.

[0065] Materials and methods: Extracellular vesicles derived from bone marrow, as described in Example 1 were utilized. Such extracellular vesicles (EVs) may regulate and normalize immune responses.

[0066] Protocol: The study was IRB approved. The format was prospective, open label, and non-randomized conducted at a single site. Bone marrow derived mesenchymal stem cell extracellular vesicles (EVs) prepared according to Example 1 were initially administered as a single dose of 15 cc intravenously to five (5) patients, then repeated in three (3) months as symptoms began to return. The study was then expanded to ten (10) patients; ages 21 to 57, 2male and 8 female, all ten (10) of whom then received two (2) intravenous doses over 24 hours and a follow-up single intravenous dose at two (2) months. 15 ml is a common dose used in prior published studies for adult respiratory distress syndrome (ARDS). No side effects were detected at this dose in these prior studies.

[0067] Results: Within one (1) to two (2) weeks of infusion, six (6) out of the ten (10) patients reported a substantial and statistically significant reduction in symptoms based upon a ten- symptom scale recorded first on a daily, then weekly basis. Notably, the parameter "Over-all Feelings of Well-being" showed substantial improvement and was accompanied with such patient comments as: "Much improved nightly sleep”; "drastic difference in symptoms”; "good experience in clinical trial”; "I am where I want to be physically”; "feel better than I have in years”; "feels like I have hours back in my day”; "better than I ever thought I could be”; "improved outlook on life”; and "a miracle”. For those four (4) among the first group of five (5) that did respond, symptoms began to return by 10 to 12 weeks post infusion thus accounting for the change in protocol to infuse every two (2) months. Cumulative beneficial effects seem to have occurred in responders with each of the subsequent infusions. The reduction in symptom intensity per typical responder over the course of 7 days of treatment is shown in FIG. 1. One patient, initially thought to be a non-responder, began to respond only after several infusions ultimately reaching a statistically significant result. The inventors refer to this patient as a "delayed responder." It is unknown why this patient had such an apparently delayed response. Both HLA-B27 positive patients were responders. No side effects were found in any patient - responder or non-responder.

[0068] Discussion: Disorders in collagen synthesis are documented and genetically identified in other forms of EDS. While it is suspected that a disorder in collagen synthesis may play a role in the pathophysiology of hEDS, no specific genetic or other laboratory abnormality has yet been found. Hypermobile EDS (hEDS) is a clinical diagnosis. Treatment with glycosaminoglycans, NS AIDs, pregabalin, opioids, and other such offer only temporary and partial help in ameliorating the chronic and debilitating joint pain. These patients often cannot hold steady jobs. Their lives are severely limited although their intellect and life span are not, at least in the Hypermobile Type of EDS (hEDS). Hypermobile EDS (hEDS) is not a congenital condition in the sense that it can be diagnosed in early childhood. Except for hypermobile joints, the young child may appear normal. No specific clinical finding or laboratory test is diagnostic. Usually between 10 to 12 years of age there may be complaints of foot, neck, jaw, and joint pain. The middle school child may have trouble keeping up with classmates at gym or on the field. Not uncommonly, the child is accused by teachers and coaches of laziness and lack of toughness. Self-esteem and self-doubt issues may arise. Parents are frequently unaware of issues at schooland only vaguely aware of issues at home. Once addressed as a possible medical issue, misdiagnosis over several years is common. Multiple doctors are usually consulted before a confident diagnosis is proposed. The day of diagnosis is not uncommonly declared as the "best day of my life" by the, usually by now, late-teenage patient. Many are diagnosed in their midtwenties and beyond. There have been proposed relatively specific criteria for the diagnosis of hEDS. The condition is subsumed under the broader heading of Hypermobile Spectrum Disorder (HSD) under which is a broad group of loose-joint conditions. The first usual clue to hEDS, however, is the presence of painful, hypermobile joints. Not all hypermobile joints are painful. Rheumatology evaluation and genetics consultation are usually in order.

[0069] Conclusion: Since there is, to date, no satisfactory treatment, the success of treatment described herein with the use of bone marrow derived extracellular vesicle infusion offers hope to those looking for relief to the painful and other debilitating symptoms of this condition. These findings support a trial by clinicians of intravenous bone marrow derived mesenchymal stem cell EVs as a treatment option for those with the diagnosis of hEDS.

[0070] Prior therapies by study participants: Participants were instructed to continue use of any helpful modality and medication during the study. Most responders voluntarily either reduced or discontinued these as the study progressed. The list of therapies includes: physical therapy and chiropractic, NSAIDS, opioids, ketamine, Flexeril (cyclobenzaprine), pregabalin, gabapentin, trazodone, benzodiazepines, naltrexone, THC and CBD, midodrine, topiramate, and platelet rich plasma (PRP).C. Example 3 - Clinical Study of Bone Marrow Derived Mesenchymal Stem Cell Extracellular Vesicle Isolate as a Successful Treatment for Hypermobile Type Ehlers-Danlos Syndrome (hEDS)

[0071] In this Example, the safety and efficacy of a biologic drug investigational product (IP) comprised of human bone marrow mesenchymal stem cell derived extracellular vesicles (HBM-MSC EV) was evaluated as a treatment option for Hypermobile Type Ehlers-Danlos Syndrome (hEDS). hEDS is a life-long disorder that affects multiple organ systems, accounts for 80-90% of all Ehlers-Danlos cases and is of uncertain pathophysiology. Symptoms include joint looseness, intractable joint pain, hyperextensible skin, chronic fatigue, brain fog, irritable bowel symptoms, and mast cell dysfunction. Ten hEDS patients were recruited and treated with multiple doses of the IP by intravenous infusion for up to five months. Symptoms were assessed using a 10-criteria questionnaire addressing pain, joint dislocations, pain relief medication usage and other common symptoms (FIG. 2). No treatment related adverse events were noted. Statistically significant improvements were observed for seven of the nine symptoms, and 60%of the subjects exhibited rapid and sustained overall improvement of their symptoms within the study time frame. Only one subject failed to show any improvement. These results demonstrate that a regenerative therapy approach using an advanced HBM-MSC EV can safely and effectively improve hEDS symptoms.

[0072] Of the several types of Ehlers-Danlos Syndrome (EDS) the most common type is hypermobile EDS (hEDS). With the current US Census estimating approximately 336,000,000 people living in the US and frequency estimates of 1 in 3,100 to 1 in 5,000. It is estimated that 67,200 to 108,387 persons are afflicted with hEDS in the United States.

[0073] While genetic bases for other forms of EDS have been defined, diagnosis for hEDS is by clinical means only and there is currently no genetic or other diagnostic laboratory test. There are no satisfactory treatment options and care is focused on symptom management. hEDS is a multisystem disorder with unknown pathophysiology. The most common characteristics are persistently painful hyper-mobile joints, joint dislocations, GI disturbances, headaches, orthostatic tachycardia, fatigue, and brain fog with difficulty concentrating. The assembly of symptoms can be related in part to a disorder in mast cells, referred to as Mast Cell Activation Syndrome (MCAS). Many symptoms of hEDS, including headaches, peripheral neuropathy, postural orthostatic tachycardia syndrome (POTS), and gastrointestinal, rheumatologic and urogynecologic conditions, are consistent with MCAS. A disorder in collagen synthesis may also play a role in the pathophysiology of hEDS.

[0074] Treatment with glycosaminoglycans, NSAIDs, pregabalin, opioids, and other methods offer only temporary and partial help in ameliorating chronic and debilitating joint pain. hEDS patients also often turn to complementary and alternative medicine treatment options with some success found in massage and nutritional supplements. These patients often cannot hold steady jobs and their life is severely limited although their intellect and life span are not.

[0075] This study evaluated the safety and therapeutic properties of the IP in altering some or all of the common symptoms associated with hEDS. In summary, this study shows successful treatment in hEDS patients using intravenous (IV) administration and repeated dosing of the IP.

[0076] Materials and Methods

[0077] Patient Selection: Subjects were drawn from a local EDS support group, some of whom contacted other non-local candidates. Subjects had all been previously diagnosed with hEDS usually by multiple consultants and were using various treatments at the start of the study (Table 2). All were clinically interviewed and examined by the PI and found to have physical and historical findings compatible with the diagnosis of hEDS. Participants were instructed to continue use of any helpful modality and medication during the study. All but one had seen a rheumatologist to exclude autoimmune disease. None were on immunosuppressants. None had adiagnosis of lupus erythematosus although one subject had an antinuclear antibody (ANA) of 360 with no other abnormal blood findings. One patient had prior treatment with extracellular vesicle infusion of placental origin without abatement of symptoms. Two were positive for HLA-B27. One had a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene.

[0078] Table 2. Therapies used by study participants prior to entry into this study

[0079] Investigational Product: Briefly, the IP was prepared by the method of Example 1. Briefly, HBM-MSC are cultured and EVs are harvested, sterilized, processed, and bottled under cGMP conditions and according to FDA Master File protocols to provide the IP product. The IP was supplied as frozen 5 mL vials. For a single dose three of the 5 mL vials were thawed at room temperature and immediately combined, diluted, and gently mixed into 30 mL of normal saline and then administered by IV push. Each dose of a total of 15 mL of IP contained approximately 1.2 trillion EV particles.

[0080] Protocol: The patient demographics included two males and eight females, ranging in age from 21 to 57 years old. The format was prospective, open label and non-randomized, and it was conducted at a single site. The IP was initially administered by IV as a single dose (total volume of 45 mL containing 15 mL of IP) to six patients, then repeated three to four months later as a back-to-back double dose (24 hours apart) as symptoms began to return. This was followed 2 months later by a single dose. The study was then expanded to include fouradditional patients, to a total of ten subjects. This second group of four patients received an initial back-to-back dose (24 hours apart) followed two months later by a third single dose.

[0081] Symptom Assessment and Statistics: A new 10-parameter questionnaire was developed to address symptoms commonly associated with hEDS (FIG. 2). Subjects were asked by the physician, either on site in person or by follow up phone call, to rank their symptom severity from 0 (no or least severity) to 10 (most or worst severity).

[0082] Individual score results for each subject were compiled and analyzed using GraphPad Prism 9.5.1 (GraphPad Software). Baseline scores were obtained for all subjects one to three days prior to receiving the first infusion of IP. Scores were also obtained one to three days prior to each subsequent injection. All score results were compared to baseline scores using a paired t- test assuming a Gaussian distribution.

[0083] Results

[0084] Safety: No adverse events were detected in any patient with these dose regimens. One subject (6-H) from the single first dose group failed to return for their fourth and final infusion. For another subject from the back-to-back, double first infusion group (8-1), it was not possible to perform the third and final infusion due to difficulty finding a suitable vein on that day. All other subjects received the intended full allotment of infusions.

[0085] Efficacy: Overall across all subjects, from initial baseline scores to their last score recorded after their final dose, statistically significant improvements were observed for seven of the nine analyzed scoring criteria (FIG. 3). Note that the questionnaire criterion “Other Pain Medication Use” was not included in the analyses because it became clear that this question led to more qualitative rather than quantitative responses regarding the specific types of alternative pain medications used, making it difficult to score on a 0 to 10 scale. These results for the entire group suggest that, over time, the IP can improve patient symptoms across several categories.

[0086] For each subject, plotting the same initial baseline and final assessment scores used to develop FIG. 3 illustrates that there is response heterogeneity among the subjects (FIG. 4). For example, for the “Overall Feelings of Well Being” category, subject 6-H improved by only one point and subject 4-F showed no change, while all others exhibited substantial improvements. Similarly, while most subjects improved in the “Headaches” category, the same two subjects (6- H and 4-F) also showed worsening symptoms. These observations suggest that there may be heterogeneity of responsiveness to the IP across patients.

[0087] It was found that, by taking several patient assessments per week, score improvements could take up to about two weeks to develop in those subjects who responded. To further investigate the possibility of different degrees of responsiveness amongst subjects, the scores from each assessment category were summed to give an overall score for each subject. Thesummed scores obtained after each dose were taken two weeks following each dose and statistical analyses were performed to compare the group’s summed scores to the initial baseline summed scores before the dosing regimen began (FIG. 5). The subjects were analyzed in two groups: the six subjects who received a single initial dose (FIGs. 5A and 5B) and the four who received a back-to-back initial double dosage (FIGs. 5C and 5D). Statistically significant improvement was seen for the initial single dose group (FIGs. 5A and 5B) after just the first dose (p=0.024) and that significant effect was maintained after the group received a back-to- back second dosage approximately four weeks later (p=0.0352). After the fourth and single dose was administered the apparent improvements did not reach statistical significance although a trend was apparent (p=0.0610). This latter result is likely because only four of the initial six subjects provided results at this point due to deviations from the protocol for the other two. It is also apparent from this group (FIG. 5B) that subjects who eventually failed to respond (subject 6-H) or responded modestly (subject 5-J) were apparent after the first single dosage; they did not dramatically improve with additional doses. While subject 2-B improved over the first two dosages from a high score of 35 to a low of 12, partial return of symptoms was apparent after the fourth dose (score = 23) and this same score (23) was also observed 3.5 months after the fourth and final dose.

[0088] For the four subjects who received an initial back-to-back double dose (FIGs. 5C and 5D), a trend towards improved symptoms was apparent after the first two doses (p=0.0929) and after the third dose (p=0.1131), but the results were not statistically significant. Subject 8-1 appeared to be weakly responsive, but it was not possible to confirm this over time as the subject was unable to receive a third dose due to difficulty initiating the IV infusion.

[0089] By analyzing the summed scores for each of the 10 subjects, regardless of the dosing regimen, we conclude that only one subject (6-H) failed to respond favorably to the IP. Three I5-J, 2-B, and 8-1) showed a partial or unsustained response. The remaining six subjects (60%) showed clear rapid and sustained favorable responses.

[0090] To illustrate the characteristics of responsive and non-responsive subjects further FIG. 6 shows full profiles of subjects of each type and from each dosing protocol two weeks after the initial treatments. The two responders (FIGs. 6A and 6C) indicate that improvements occurred in all criteria, but that there is also interpatient variability in terms of the extent of responses in different criteria. In addition, the non-responders (FIGs. 6B and 6D) were generally non- responsive across all symptom categories.

[0091] In the six responsive subjects, notably, in the parameter “Overall Feelings of Wellbeing”, subjects showed substantial improvement and responses to this query were accompanied with such comments as: “Much improved nightly sleep...”, “Drastic difference in symptoms...”,“Good experience in the clinical trial...”, “I am where I want to be physically...”, “I feel better than I have in years...”, “It feels like I have hours back in my day...”, “...better than I ever thought I could be...”, “...improved outlook on life...” and “...a miracle...”.

[0092] Discussion

[0093] The effect and safety of an IP that comprises HBM-MSC EVs, currently in advanced stages of clinical development, was evaluated on the symptoms reported by a group of patients with hEDS. The IP, a HBM-MSC EV preparation that is manufactured to cGMP standards and subject to strict quality assurance processes confer all the documented regenerative and immunoregulatory properties of intact HBM-MSC, without the manufacturing risks and costs or the liability risks of stem cells (see Example 1). The IP contains over 1,000 bioactive molecular species, including miRNAs, other small RNAs, cytokines, chemokines, and lipids with regenerative potential. Regardless of the dosing scheme, no treatment related adverse events were reported by the subjects or noted by the investigators. By the end of the study, the group exhibited statistically significant improvements in seven of the nine parameter categories that were analyzed (FIG. 3 and FIG. 4). Within one to two weeks of the first infusions, seven out of the 10 subjects self-reported a substantial reduction in symptoms based upon a ten-parameter scale recorded first on a daily, then on a weekly basis (FIG. 5). Of the remaining three one (5-J) exhibited a partial reduction in symptoms with additional infusions. Lastly, there was clear evidence that a minority of the subjects was not responsive to the IP as provided in the dosing regimens studied here (FIG. 6). From these results it’s concluded that the IP, a single therapeutic drug candidate, safely, effectively, and favorably modified multiple symptoms in this group of hEDS patients. The mechanism(s) of action of the IP can include the immunomodulatory capacity of the IP that may contribute to improved regulation of the MCAS that is implicated in hEDS.

[0094] Relatively specific criteria for the diagnosis of hEDS have been proposed. The condition is subsumed under the heading of Hypermobile Spectrum Disorder (HSD) which encompasses a broad group of loose-joint conditions. The first usual clue to hEDS, however, is the presence of painful, hypermobile joints. Not all hypermobile joints are painful. Rheumatology evaluation and genetics consultation are usually in order. In this study, significant reductions in both joint pain and NSAID scores were observed, which indicates a significant reduction in pain symptoms. In addition, while all subjects in this study were advised to continue any currently utilized pain reduction modalities and medications during the study, the responsive subjects voluntarily either reduced or discontinued use of these as the study continued.

[0095] The fact that some subjects were unresponsive or moderately responsive could be due to many reasons; however, this observation is consistent with the heterogenous character of thedisease and the lack of a defined genetic basis for the disease. There may be patients with phenotypical hEDS with differing and varied unknown genotypes - some of whom are classified as “responders” and others as “non-responders” to HBM-MSC EVs in this study. No definitive clinical or laboratory readings was determined based on laboratory histories of the subjects to explain differences between responders and non-responders. Both HLA-B27 positive patients were responders. No response was seen in the one subject with a MTHFR mutation. One subject (5-J), initially thought to be a non-responder, began to respond only after several infusions and ultimately reaching a favorable, albeit incomplete, result. It is unknown why this patient had such an apparently delayed response. Another subject (2-B) initially showed a 50% reduction in total symptom score (FIG. 5B) but appeared to partially regress by the fourth and final dose. It is possible that additional dosing could restore their more favorable status and this proposal is supported by the following observations. In the extensive weekly assessment scores, not all of which were included in the statistical analyses, for those five among the first group of six that did respond to the first dose, symptoms began to return by 10 to 12 weeks post infusion. This prompted a change in the protocol to infuse subjects every two months rather than every four months with the goal of keeping symptoms in check. It was observed that cumulative beneficial effects typically occurred in responders with each of the subsequent infusions. Durability of the symptom reduction in this group of patients beyond the first three or four doses was not addressed in this initial study.

[0096] Conclusion

[0097] This study shows HBM-MSC EV infusion can relieve symptoms of hEDS. These findings support a trial by clinicians of IV infusion of HBM-MSC EVs as a treatment option for those with the diagnosis of hEDS.D. Example 4 - Clinical Study of Bone Marrow Derived Mesenchymal Stem Cell Isolate as a Treatment for Ehlers-Danlos Syndrome (hEDS)

[0098] In this Example, a biologic drug investigational product (IP) comprising a human bone marrow-derived mesenchymal stem cell secretome is used to treat Ehlers-Danlos Syndrome (EDS). The human bone marrow-derived mesenchymal stem cell secretome is prepared according to Example 1.

[0099] Subjects are selected. The human subjects have been diagnosed with EDS. The subjects may have hypermobile EDS. The subject may have vascular EDS. The human subjects may harbor a mutation in a collagen gene. The human subjects may harbor a mutation in a COL3A1 gene. The subjects may have distinctive facial features. The distinctive facial features include but are not limited to a thin nose, a thin upper lip, one or more small earlobes, and prominenteyes. The human subjects may exhibit a range of physical features including but not limited to thin skin, translucent skin, bruised skin, visible blood vessels, weak blood vessels, weak uterine walls, and weak intestinal walls. The human subject exhibit one or more symptoms including but not limited to: hyperflexible joints, joint dislocations, chronic pain, soft and velvety skin, easy bruising, hyper-elastic skin, delayed wound healing, altered gait, atrophic scars, muscle weakness, internal bleeding, sprains, strains, chronic fatigue, arterial rupture, heart valve problems, gastrointestinal issues, flat feet, scoliosis, fragile blood vessels, tendon or ligament ruptures, hypotonia, frequent sprains, chronic pain syndromes, sleep apnea, dysautonomia, visible veins, brittle teeth, frequent headaches, shortness of breath, early osteoarthritis, skin that tears easily, poor proprioception, varicose veins, excessive sweating, increased scarring, uterine rupture or complications in pregnancy, delayed puberty, frequent nosebleeds, hernias, easy tissue damage, restless legs, sleep disturbances, dental crowding, teeth with a translucent appearance, fragile corneas, soft or thin nails, hypermobility of the fingers, corneal rupture or tearing, early onset arthritis, increased skin infections, reduced range of motion in some joints, irregular heart rhythms, and increased risk of blood clots.

[0100] The human bone marrow-derived mesenchymal stem cell secretome is administered to the subjects. The administration is intravenous. The secretome composition is diluted in saline. From 5 to 15 mL of the secretome composition is diluted in from 30 to 95 mL of saline. The administration takes places over 15 to 60 minutes. The secretome composition is administered in from 1 to 5 doses. The doses are administered over the course of from 1 week to 12 weeks.

[0101] Subjects are evaluated for symptoms of EDS before, during, and after the administration. Subjects are evaluated for one or more outcomes including but not limited to improvements in joint stability, reduction in pain and inflammation, enhanced skin elasticity, fewer instances of joint dislocations or subluxations, improved wound healing, reduced bruising, increased mobility and strength, better cardiovascular function, improved quality of life, reduced frequency of gastrointestinal issues, decreased fatigue, improved muscle tone, fewer soft tissue injuries, reduction in scarring, improved sleep quality, enhanced mental health and mood, fewer headaches or migraines, increased endurance, and overall better management of symptoms related to connective tissue fragility. A significant improvement in one or more outcomes in observed in the majority of subjects who were administered the secretome composition.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of treating Ehlers-Danlos Syndrome (EDS) or a symptom thereof in a subj ect, the method comprising administering to the subject a composition comprising one or more extracellular vesicles (EVs), wherein the composition comprises NUP85, DAPP1, PDGF R alpha, SLITRK5, FAP, Artemin, DPPII, cIAP-1, Pentraxin 3, Visfatin, Neprilysin, Albumin, Galectin-1, UNC5H3, IL-20 R beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midkine, Calreticulin, Osteoactivin, Legumain, TAZ, Cathepsin L, RBP4, Serpin A4, JAM- A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin 3, SIRP alpha, and Syndecan-4, GSTM1, NUP85, MeprinA, IL-1 F10, TGFbl, Ephrin-A4, Aminopeptidase LRAP, GDF-9, PDGF R alpha, PAPP -A, Arylsulfatase A, LAIR2, ULBP-4, TFPI, SOX2, SLITRK5, Spinesin, ENPP-2, CD97, CTACK, Integrin alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin DI, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Persephin, TRAIL Rl, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, P0N1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Folli statin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mcl-1, DPPIV, SREC- II, Norrin, JAM-C, Bcl-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gpl30, C-myc, LILRB4, BMP-2,, MIA, CD34, IFNab R2, Glypican 2, MSP R, DSCAM, Matriptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nidogen-1, CD2, Kallikrein 1, TSLP R, TROY, VCAM-1, Siglec-11, S100A1, PARI, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6„ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 alpha, 0X40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, Semaphorin 4D, ROBO2, PDX-1, APRIL, Neurturin, Kremen-2, EMMPRIN, Activin RIB, Neuroligin 2, Epiregulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF Rl, DSPG3, SorCSl, Matrilin-2, sFRP-3, EphB3, NCK1, Prolactin,Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Pappalysin-2, Syndecan-3, Jagged 1, AKR1C4, Olfactomedin-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA125, HAO-1, PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, R0R1, CMG-2, 4-1BB Ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Legumain, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 beta, TAFA1, Podocalyxin, Rai A, CRELD2, GRAP2, SP-D, BID, GFR alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1 R3, SALM4, GBA3, R0B04, OSCAR, VEGF, IGSF3, Biglycan, Neudesin, ILT4, uPAR, Axl, WIF-1, IL-7 R alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Cochlin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17 RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, Prolactin R, FGF-20, CD28, Nogo- A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, ADAM23, NOV, Galectin-2, Neurexin 3 beta, TLR3, Sirtuin 2, Numb, IL-28 R alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA I3, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-Selectin, Hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-lra, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin- 4, SCF, Serpin A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, 0X40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, R0R2, EphB4, Glypican 1, LAP(TGFbl), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, Nestin, TPO, ErbB3, Kirrel3, FLRT1, Galectin-3, CXCL16, JAM-B, DR6,Nogo Receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R alpha 1, AMIG02, HCC-1, TFPI-2, ULBP-2, Desm oglein 2, Aggrecan, Syntaxin 4,VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurogranin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-Spondin 2, CK19, Cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospondin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, R0B03, RANTES, Endocan, Granulysin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin 3, ARSB, LIF R alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-lb, NKp46, MCP-3, IL-32 alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Syntaxin 6, GRO, Bcl-w, Lipocalin-2, PDGF-AA, IL-2 Ra, Angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP -2, PU.1, ED AR, ADAMTS13, Kynureninase, PTH1R, IFN-gamma Rl, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein 11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP- 10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecan-4, IL-2 Rg, MICA, Dopa Decarboxylase, NPDC-1, MCP-4, EG- VEGF, Glycoprotein V, Semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, Endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, Galectin-8, CA4, Cystatin E M, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, Kallikrein 7, or IL-13, or a combination of two or more thereof.

2. A method of treating Ehlers-Danlos Syndrome (EDS) or a symptom thereof in a subject, the method comprising administering to the subject a composition comprising one or more extracellular vesicles (EVs), wherein the composition comprises TIMP-1 (tissue inhibitor of metalloproteinases 1), TIMP-2 (tissue inhibitor of metalloproteinases 2), CD63 antigen, Ferritin, IGFBP-4 (Insulin-like growth factor binding protein-4), MIF (Macrophage migration inhibitory factor), LAMB1 (Laminin Subunit Beta 1), FBN1 (Fibrillin 1), HSPG2 (Heparin Sulphate Proteoglycan 2), BGN (Biglycan), or FN1 (Fibronectin 1), or a combination of two or more thereof.

3. The composition of claim 2, wherein the composition further comprises beta-IG-H3 (Transforming growth factor-beta-induced protein ig-H3), CA2 (Carbonic anhydrase 2), Cathepsin B, CD81 antigen, CD9 antigen, CD44 antigen, CD99 antigen, CD 109 antigen, CNTF (Ciliary neurotrophic factor), Decorin, DcR3 (Tumor necrosis factor receptor superfamily member 6B), Dkk-3 (Dickkopf-related protein 3), Endoglycan (podocalyxin- like protein 2), Furin, GM-CSF Ra (Granulocyte-macrophage colony-stimulating factor receptor subunit alpha), GPR115 (Adhesion G protein-coupled receptor F4), GP73 (Golgi membrane protein 1), HS3ST4 (Heparan sulfate glucosamine 3-O-sulfotransferase 4), IGF-2(Insulin-like growth factor-2), IGFBP-2 (Insulin-like growth factor binding protein-2), IGFBP-3 (Insulin-like binding protein-3), IGFBP-6 (Insulin-like growth factor binding protein-6), IL18BP (Interleukin- 18 Binding Protein), IL-1 R6 (Interleukin 1 Receptor 6), ILFBP-4 (Insulin-like growth factor binding protein-4), LAMP2 (Lysosome-associated membrane glycoprotein 2), Lumican, OPN (Osteopontin), PAI-1 (Plasminogen activator inhibitor- 1 or SERPINE 1), PCK1 (Phosphoenol pyruvate carboxykinase, cytosolic), PF4 (Platelet Factor 4), Periostin, PRDX4 (Peroxidredoxin-4), RGM-C (Hemojuvelin), Semaphorin 6C, Serpin B6, Serpin Fl (Pigment epithelium-derived factor), Sortilin, TGM4 (Protein-glutamine gamma-glutamyltransferase 4), Thrombomodulin, TSP-1 (Thrombospondin 1), TSP-2 (Thrombospondin 2), CAD11 (Cadherin 11), JAM3 (Junctional Adhesion Molecule 3), VIM (Vimentin), FBLN5 (Fibulin 5), FBN2 (Fibrillin 2), Hyaluronic Acid, C0L1A1 (Collagen Type 1 Alpha 1), C0L1A2 (Collagen Type 1 Alpha 2), or Transferrin, or a combination of two or more thereof.

4. The composition of claim 2 or claim 3, wherein the composition further comprises one or more of COL2Al, C0L3A1, C0L5A1, COL5A2, C0L6A1, C0L11A1, or C0L14A1, or a combination of two or more thereof.

5. A method of treating Ehlers-Danlos Syndrome (EDS) or a symptom thereof in a subject, the method comprising administering to the subject a composition comprising one or more extracellular vesicles (EVs), wherein the composition comprises hsa-miR-21-5p, miR-24-3p, hsa-miR-222-3p, hsa-miR-27b-3p, or let-7, or a combination of two or more thereof.

6. The method of claim 5, wherein the composition further comprises hsa-miR-125b-5p, hsa- miR-132-3p, hsa-miR-145-5p, hsa-miR-191-5p, hsa-miR-199a-3p, hsa-miR-221-3p, hsa- miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-miR-27a-3p, hsa-miR-29a-3p, hsa-miR- 29c-3p, hsa-miR-31-5p, hsa-miR-320a, hsa-miR-34a-5p, hsa-miR-423-3p, hsa-miR-424-5p, or hsa-miR-940, or a combination of two or more thereof.

7. A method of treating Ehlers-Danlos Syndrome (EDS) or a symptom thereof in a subject, the method comprising administering to the subject a composition comprising one or more extracellular vesicles (EVs), wherein at least 80% of the one or more EVs are CD63+, CD9-, CD81-.

8. The method of any one of claims 1-7, wherein the EDS is hypermobile EDS.

9. The method of any one of claims 1-8, wherein the EDS is vascular EDS.

10. The method of any one of claims 1-9, wherein the subject is (i) positive or negative for human leukocyte antigen B27 (HLA-B27), (ii) has, or does not have, a mutation in COL 1 Al and COL3A1 collagen genes, (iii) has, or does not have, a mutation in the MTHFR gene, (iv) is positive or negative for ANA, or (v) a combination of two or more of (i) to (iv).

11. The method of any one of claims 1-10, wherein the symptoms comprise sleeplessness, headaches, brain fog, diminished concentration, fatigue, joint pain, overall pain, joint looseness, joint dislocations, gastrointestinal function, gastrointestinal mobility, CBD or THC use, NSAID use, use of pain medication, or overall feeling of well-being, or a combination of two or more thereof.

12. A method of treating Mast Cell Activation Syndrome (MCAS) or a symptom thereof in a subject, the method comprising administering to the subject a composition comprising a mesenchymal stem cell (MSC) secretome.

13. A method of treating Hypermobile Spectrum Disorder (HSD) or a symptom thereof in a subject, the method comprising administering to the subject a composition comprising a mesenchymal stem cell (MSC) secretome.

14. A method of reducing pain in a subject, the method comprising administering to the subject a composition comprising a mesenchymal stem cell (MSC) secretome.

15. A method of reducing sleeplessness, headaches, brain fog, concentration, fatigue, joint pain, overall pain, joint looseness, joint dislocations, gastrointestinal function, gastrointestinal mobility, CBD or THC use, NSAID use, use of pain medication, or overall feeling of wellbeing in a subject, the method comprising administering to the subject a composition comprising a mesenchymal stem cell (MSC) secretome.

16. The method of any one of claims 1-15, wherein the subject has a collagen synthesis disorder.

17. The method of any one of claims 1-16, wherein the subject has hypermobile joints.

18. The method of any one of claims 1-17, wherein the subject has painful joints.

19. The method of any one of claims 1-18, wherein the administering comprises administering15 mL of the composition.

20. The method of claim 19, wherein the composition further comprises 30 mL of saline.

21. The method of any one of claims 1-20, wherein four doses of the composition are administered.

22. The method of any one of claims 1-20, wherein three doses of the composition are administered.

23. The method of any one of claims 1-20, wherein two doses of the composition are administered.

24. The method of any one of claims 21-23, wherein the second dose is administered 3 to 4 months after administration of the first dose.

25. The method of any one of claims 21-23, wherein the second dose is administered 24 hours after administration of the first dose.

26. The method of claim 21 or 22, wherein the second dose of the composition is administered 3 to 4 months after the administration of the first dose, wherein the third dose is administered 24 hours after the administration of the second dose, and wherein the fourth dose is administered 2 months after the administration of the third dose.

27. The method of claim 21 or 22, wherein the second dose of the composition is administered 24 hours after the administration of the first dose, and wherein the third dose is administered 2 months after the administration of the second dose.

28. The method of any one of claims 1-27, wherein the administering comprises intravenous administration.

29. The method of claim 28, wherein the intravenous administration is carried out over the course of 30, 35, 40, 45, 50, 55, or 60 minutes.

30. The method of any one of claims 1-29, wherein the composition is prepared by a process comprising: (a) culturing bone marrow mesenchymal stem cells (BM-MSCs) under the following conditions to produce an MSC conditioned media: (i) oxygen tension below 5%; and (ii) culture media having a pH below 7; (b) harvesting the MSC conditioned media; and (c) formulating the MSC conditioned media to produce the composition.

31. The method of any one of claims 1-29, the method comprising preparing the composition prior to the administering, wherein the preparing the composition occurs by a process comprising: (a) culturing bone marrow mesenchymal stem cells (BM-MSCs) under the following conditions to produce an MSC conditioned media: (i) oxygen tension below 5%; and (ii) culture media having a pH below 7; (b) harvesting the MSC conditioned media; and (c) formulating the MSC conditioned media to produce the composition.

32. The method of claim 30 or claim 31, wherein the culture media is serum-free.

33. The method of any one of claims 30-32, wherein the culture media has a glucose concentration below 4.5 g / L.

34. The method of any one of claims 30-33, wherein formulating the MSC conditioned media comprises exchanging the conditioned media for a pharmaceutically acceptable formulation.

35. The method of claim 34, wherein the pharmaceutically acceptable formulation comprises saline.

36. The method of any one of claims 1-35, wherein the composition comprises at least 6xlO10to 8xl010extracellular vesicles per mL and is administered at a dose of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml.

37. The method of claim 36, wherein the dose is administered in combination with normal saline at a final volume of 100 mL.

38. The method of any one of claims 1-37, wherein the composition comprises saline (0.9% sodium chloride).

39. The method of claim 38, wherein the saline is present in the composition at about 80% to about 95% saline.

40. The method of any one of claims 1-39, wherein the composition comprises sodium chloride, sodium lactate, potassium chloride, and / or calcium chloride.

41. The method of any one of claims 1-40, wherein the molecular weight of any non-excipient component of the composition is greater than about 2 kDa (kilodaltons), 8 kDa, 10 kDa, 50kDa, or lOOkDa.

42. The method of any one of claims 1-41, wherein the composition comprises a oligosaccharide.

43. The method of claim 42, wherein the oligosaccharide is present in the composition at from about 0.2 M to about 0.6 M.

44. The method of any one of claims 1-43, wherein any non-excipient component of the composition has a size of less than about 0.2 microns.

45. The method of any one of claims 1-44, wherein the composition is sterile per USP <71>.

46. The method of any one of claims 1-45, wherein the composition is endotoxin free per USP <85>.

47. The method of any one of claims 1-46, wherein the composition is negative for mycoplasma DNA.

48. The method of any one of claims 1-47, wherein the composition is cell-free.

49. The method of any one of claims 1-48, wherein the composition is stored between -80 °C and -60 °C.

50. The method of claim 49, wherein the composition is administered within 6 hours of thaw when maintained at ambient temperature.

51. The method of any one of claims 1-50, wherein the composition is freeze-dried (lyophilized) to a powder cake and stored at or below room temperature.

52. The method of any one of claims 1-51, wherein the composition is reconstituted with water prior to administration.

53. The method of any one of claims 1-52, wherein the composition is present in a glass vial.

54. The method of any one of claims 1-53, wherein the composition is formulated for intravenous administration.

55. The method of any one of claims 1-54, wherein the composition has a pH of about 6 to about 7.5.

56. The method of any one of claims 30-55, wherein the BM-MSCs are negative for CD14, CD31, CD34, and CD45.

57. The method of any one of claims 30-56, wherein the BM-MSCs are positive for CD73, CD 105, CD 166, and CD90.

58. The method of any one of claims 30-57, wherein the BM-MSCs are capable of undergoing trilineage differentiation in vitro toward adipocyte, osteoblast, and chondrocyte phenotypes.

59. The method of any one of claims 30-58, wherein the BM-MSCs are obtained from an iliac crest aspiration of a single donor.

60. The method of any one of claims 1-59, wherein a concentration of the one or more EVs in the composition is measured by nanoparticle tracking analysis (NTA).

61. The method of claim 60, wherein the NTA comprises light scatter and fluorescence evaluation.

62. The method of claim 60 or 61, wherein the concentration of the one or more EVs is about 10 billion to about 250 billion EVs per mL of the composition.

63. The method of any one of claims 60-62, wherein the concentration of the one or more EVs is at about 1 billion to about 40 billion EVs per ml of the composition.

64. The method of any one of claims 1-63, wherein the one or more EVs have an average diameter of about 30 nm to about 170 nm.

65. The method of any one of claims 1-64, wherein a total protein concentration of the composition is about 10 to about 40 ug per ml of the composition.

66. The method of any one of claims 1-65, wherein a total protein concentration of the composition is about 1.5 to about 6 pg per ml of the composition.

67. The method of claim 65 or 66, wherein the total protein concentration is measured by ELISA.

68. The method of any one of claims 4-67, wherein the composition comprises TIMP-1 (tissue inhibitor of metalloproteinases 1), TIMP-2 (tissue inhibitor of metalloproteinases 2), CD63 antigen, Ferritin, IGFBP-4 (Insulin-like growth factor binding protein-4), MIF (Macrophagemigration inhibitory factor), LAMB1 (Laminin Subunit Beta 1), FBN1 (Fibrillin 1), HSPG2 (Heparin Sulphate Proteoglycan 2), BGN (Biglycan), or FN1 (Fibronectin 1), or a combination of two or more thereof.

69. The method of any one of claims 4-68, wherein the composition further comprises beta-IG- H3 (Transforming growth factor-beta-induced protein ig-H3), CA2 (Carbonic anhydrase 2), Cathepsin B, CD81 antigen, CD9 antigen, CD44 antigen, CD99 antigen, CD 109 antigen, CNTF (Ciliary neurotrophic factor), Decorin, DcR3 (Tumor necrosis factor receptor superfamily member 6B), Dkk-3 (Dickkopf-related protein 3), Endoglycan (podocalyxin- like protein 2), Furin, GM-CSF Ra (Granulocyte-macrophage colony-stimulating factor receptor subunit alpha), GPR115 (Adhesion G protein-coupled receptor F4), GP73 (Golgi membrane protein 1), HS3ST4 (Heparan sulfate glucosamine 3-O-sulfotransferase 4), IGF-2 (Insulin-like growth factor-2), IGFBP-2 (Insulin-like growth factor binding protein-2), IGFBP-3 (Insulin-like binding protein-3), IGFBP-6 (Insulin-like growth factor binding protein-6), IL18BP (Interleukin- 18 Binding Protein), IL-1 R6 (Interleukin 1 Receptor 6), ILFBP-4 (Insulin-like growth factor binding protein-4), LAMP2 (Lysosome-associated membrane glycoprotein 2), Lumican, OPN (Osteopontin), PAI-1 (Plasminogen activator inhibitor- 1 or SERPINE 1), PCK1 (Phosphoenol pyruvate carboxykinase, cytosolic), PF4 (Platelet Factor 4), Periostin, PRDX4 (Peroxidredoxin-4), RGM-C (Hemojuvelin), Semaphorin 6C, Serpin B6, Serpin Fl (Pigment epithelium-derived factor), Sortilin, TGM4 (Protein-glutamine gamma-glutamyltransferase 4), Thrombomodulin, TSP-1 (Thrombospondin 1), TSP-2 (Thrombospondin 2), CAD11 (Cadherin 11), JAM3 (Junctional Adhesion Molecule 3), VIM (Vimentin), FBLN5 (Fibulin 5), FBN2 (Fibrillin 2), Hyaluronic Acid, COL1A1 (Collagen Type 1 Alpha 1), COL1A2 (Collagen Type 1 Alpha 2), or Transferrin, or a combination of two or more thereof.

70. The method of any one of claims 1 or 5-69, wherein the composition further comprises one or more of COL2A1, COL3A1, COL5A1, COL5A2, COL6A1, COL11A1, or COL14A1, or a combination of two or more thereof.

71. The method of any one of claims 1-4 or 7-70, wherein the composition comprises hsa-miR- 21-5p, miR-24-3p, hsa-miR-222-3p, hsa-miR-27b-3p, or let-7, or a combination of two or more thereof.

72. The method of any one of claims 1-4 or 7-71, wherein the composition further comprises hsa-miR-125b-5p, hsa-miR-132-3p, hsa-miR-145-5p, hsa-miR-191-5p, hsa-miR-199a-3p, hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-miR-27a-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-31-5p, hsa-miR-320a, hsa-miR-34a-5p, hsa-miR- 423-3p, hsa-miR-424-5p, or hsa-miR-940, or a combination of two or more thereof.

73. The method of any one of claims 1-6 or 8-72, wherein at least 80% of the one or more EVs are CD63+, CD9-, CD81-.

74. Use of a composition comprising a mesenchymal stem cell (MSC) secretome in treating Ehlers-Danlos Syndrome (EDS).

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