Modified stem cell secretome topical eye compositions and uses thereof

Abstract

Disclosed herein are methods for preparing modified secretome compositions, such as stem cell secretome compositions comprising reduced levels of pro-angiogenic factors relative to an unmodified stem cell secretome. Accordingly, disclosed are methods of cultivating cells and modifying the secretome thereof, such as by removing pro-angiogenic factors and including anti-angiogenic factors. Also disclosed herein are compositions comprising the modified secretome and methods of using such compositions, such as in the treatment of ocular diseases.

Description

MODIFIED STEM CELL SECRETOME TOPICAL EYE COMPOSITIONS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 536,835 filed on September 6, 2023, entitled “MODIFIED STEM CELL SECRETOME COMPOSITIONS AND USES THEREOF,” the entire disclosure of which is expressly incorporated by reference herein.REFERENCE TO SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing XML which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML copy, created September 6, 2024, is named “Theratome_st26” and is 19.2KB in size.FIELD OF THE INVENTION

[0003] This disclosure generally relates to methods of modifying the pro-angiogenic capacity of stem cell secretomes, such as by reducing the content of factors that can promote undesirable effects, such as angiogenesis. The disclosure further relates to compositions comprising the modified stem cell secretomes and methods of using the same to treat ocular diseases, such as Keratoconjunctivities Sicca (KCS), commonly known as dry eye disease.BACKGROUND

[0004] The prevalence and persistence of certain ocular conditions, such as Keratoconjunctivities Sicca (KCS), commonly known as dry eye disease, underscore the need for improved therapies. While the efficacy of cell-based therapies, such as platelet rich plasma and stem cell secretome compositions, has been explored, the presence of pro-angiogenic factors therein present limitations to use in certain ocular disease contexts. For example, pro-angiogenicstimulation of new blood vessel formation can be problematic when neovascularization is contraindicated. For context, uncontrolled neovascularization can have serious adverse consequences, including partial vision loss or blindness.

[0005] Cell medium conditioned by stem cells, such as mesenchymal stem cell conditioned medium (MSC-CM), contains various biologically active molecules, including angiogenic factors, growth factors, cytokines, and extracellular vesicles. The repertoire of compounds, peptides, and cell-derived membranous structures secreted by stem cells into the conditioned medium, also referred to as the secretome, has demonstrated therapeutic value across diverse treatment contexts. However, current methods of preparing secretomes, and the resultant compositions, lack selectivity for producing specific secretome compositions having advantageous characteristics in select treatment contexts, such as Keratoconjunctivities Sicca (KCS). Accordingly, there exists a need to provide improved treatment options for ocular diseases, such as secretome compositions having reduced pro-angiogenic factor content, the capacity to inhibit angiogenesis, or a combination thereof. Additionally, there is a need to develop methods for selectively and reliably producing such secretome compositions. Aspects of the invention disclosed herein address these needs.INCORPORATION BY REFERENCE

[0006] Each patent, publication, and non-patent literature cited in the application is hereby incorporated by reference in its entirety as if each was incorporated by reference individually, and as if each is fully set forth herein. However, where such reference is made, and whether to patents, publications, non-patent literature, or other sources of information, it is for the general purpose of providing context for discussing features of the invention. Accordingly, unless specifically stated otherwise, the reference is not to be construed as an admission that the document or underlyinginformation, in any jurisdiction, is prior art, or forms part of the common general knowledge in the art.SUMMARY OF THE INVENTION

[0007] A first aspect of the invention includes methods of preparing a modified secretome composition for topical delivery to an eye of a mammalian subject, which includes producing a mesenchymal stem cell-conditioned medium (MSC-CM) and reducing the angiogenic capacity of the MSC-CM.

[0008] A second aspect of the invention includes compositions containing a modified secretome composition.

[0009] A third aspect of the invention includes methods of treating an ocular condition in a mammal, such as by topically administering a modified mesenchymal stem cell secretome composition to the eye of the mammal.

[0010] A first embodiment is a method of preparing a modified secretome composition for topical delivery to an eye of a mammalian subject including conditioning a medium with mesenchymal stem cells, thereby producing a mesenchymal stem cell-conditioned medium (MSC- CM), and reducing the angiogenic capacity of the MSC-CM, where reducing the angiogenic capacity of the MSC-CM includes removing an angiogenic factor from the MSC-CM, thereby preparing the modified secretome composition.

[0011] A second embodiment is a method of preparing a modified secretome composition where the mesenchymal stem cells are derived from bone marrow, peripheral blood, adipose tissue, placenta, or umbilical cord tissue of a mammal.

[0012] A third embodiment is a method of preparing a modified secretome composition where the mesenchymal stem cells express a cell surface marker profile selected from the groupconsisting of lin- / CD45- / c-kit- / CD90+, lin- / CD45- / c-kit- / CD90+ / Sca-l+, lin- / CD45- / c- kit- / CD90+ / CD34+, and lin- / CD45- / c-kit- / CD90+ / CD34-.

[0013] A fourth embodiment is a method of preparing a modified secretome composition where the medium includes conditioning media.

[0014] A fifth embodiment is a method of preparing a modified secretome composition where the mesenchymal stem cells are cultured under conditions including 1-21% O2.

[0015] A sixth embodiment is a method of preparing a modified secretome composition where the mesenchymal stem cells are cultured under hypoxic conditions for about 24-72 hours.

[0016] A seventh embodiment is a method of preparing a modified secretome composition where the mesenchymal stem cells are cultured in the presence of at least one cytokine, where the cytokine is selected from the group consisting of IL-1, IL-ip, IL-4, IL-6, IL-7, IL-10, IL-12, TNF- a, IL-23 / IL-23, IFN-a, IFNy, CTLA-4, CXCL8, GM-CSF, TNF-a, TNF- 0, TGF-0, and combinations thereof.

[0017] An eighth embodiment is a method of preparing a modified secretome composition where the angiogenic factor is angiogenin (ANG), an endothelial growth factor, a fibroblast growth factor (FGF) protein, a hepatocyte growth factor (HGF) protein, an insulin-like growth factor binding protein, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-0), a vascular endothelial growth factor (VEGF) protein, or a combination thereof.

[0018] A ninth embodiment is a method of preparing a modified secretome composition where the VEGF protein and any one or more of the endothelial growth factor, the fibroblast growth factor (FGF) protein, the hepatocyte growth factor (HGF) protein, the insulin-like growth factor binding protein, platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-0) are removed from the MSC-CM.

[0019] A tenth embodiment is a method of preparing a modified secretome composition where the endothelial growth factor is angiopoietin-1 (Angl) and / or angiopoietin-2 (Ang2).

[0020] An eleventh embodiment is a method of preparing a modified secretome composition where the FGF protein is FGF1 and / or FGF2.

[0021] A twelfth embodiment is a method of preparing a modified secretome composition where the insulin-like growth factor binding protein is insulin-like growth factor binding protein 3 (IGFBP-3), insulin-like growth factor-binding protein 7 (IGFBP-7), an isoform thereof, or a combination thereof.

[0022] A thirteenth embodiment is a method of preparing a modified secretome composition where the VEGF protein is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (P1GF), an isoform thereof, or a combination thereof.

[0023] A fourteenth embodiment is a method of preparing a modified secretome composition where removing the angiogenic factor from the MSC-CM includes subjecting the MSC-CM to antibody-based affinity chromatography, bead collection and removal, buffer exchange, filtration, immunoprecipitation, size exclusion, or a combination thereof.

[0024] A fifteenth embodiment is a method of preparing a modified secretome composition where removing the angiogenic factor from the MSC-CM includes contacting the MSC-CM with a peptide targeting the angiogenic factor, thereby forming an angiogenic factor-peptide complex.

[0025] A sixteenth embodiment is a method of preparing a modified secretome composition where removing the angiogenic factor-peptide complex from the MSC-CM includes subjecting the MSC-CM including the angiogenic factor-peptide complex to antibody-based affinity chromatography, buffer exchange, filtration, immunoprecipitation, size exclusion, or a combination thereof.

[0026] A seventeenth embodiment is a method of preparing a modified secretome composition where the peptide is an antibody, a mimotope, or a nanobody.

[0027] An eighteenth embodiment is a method of preparing a modified secretome composition where the antibody, the mimotope, or the nanobody is present on a solid matrix, where the solid matrix is a bead.

[0028] A nineteenth embodiment is a method of preparing a modified secretome composition where the antibody is bevacizumab, brolucizumab-dbll, or ranibizumab, where the antibody targets VEGF-A, VEGF-B, P1GF, or a combination thereof.

[0029] A twentieth embodiment is a method of preparing a modified secretome composition where removing the angiogenic factor reduces the amount of the angiogenic factor by at least about 25% relative to the amount of the angiogenic factor present in the MSC-CM prior to removal of the angiogenic factor.

[0030] A twenty -first embodiment is a method of preparing a modified secretome composition where removing the angiogenic factor reduces the amount of the angiogenic factor by at least about 50%, 75%, or 100% relative to the amount of the angiogenic factor present in the MSC-CM prior to removal of the angiogenic factor.

[0031] A twenty-second embodiment is a method of preparing a modified secretome composition where reducing the angiogenic capacity of the MSC-CM further includes supplementing the modified secretome composition with an anti -angiogenic factor, where the anti- angiogenic factor is an exogenous cell adhesion molecule, an exogenous antibody, an exogenous pigment epithelium-derived factor (PEDF), a corticosteroid, a decoy receptor, a flavonoid, an immunosuppressant, a kinase inhibitor, or any combination thereof.

[0032] A twenty -third embodiment is a method of preparing a modified secretome composition where the exogenous cell adhesion molecule is intercellular adhesion molecule- 1(ICAM-1) and / or lymphocyte function-associated antigen 1 (LFA-1).

[0033] A twenty-fourth embodiment is a method of preparing a modified secretome composition where the exogenous antibody is AMG780, bevacizumab, brolucizumab-dbll, figitumumab, fresolimumab, ganitumab, MEDI3617, nesvacumab, ramucirumab, ranibizumab, or a combination thereof.

[0034] A twenty-fifth embodiment is a method of preparing a modified secretome composition where the exogenous PEDF protein is Serpin 1 (SEQ ID NO. 1), or a truncated or short derivative thereof.

[0035] A twenty-sixth embodiment is a method of preparing a modified secretome composition where the corticosteroid is dexamethasone, difluprednate, fluoromethoIone, loteprednol, prednisolone, prednisone, or a combination thereof.

[0036] A twenty-seventh embodiment is a method of preparing a modified secretome composition where the decoy receptor is aflibercept and / or conbercept.

[0037] A twenty-eighth embodiment is a method of preparing a modified secretome composition where the flavonoid is kaempferol.

[0038] A twenty-ninth embodiment is a method of preparing a modified secretome composition where the immunosuppressant is abatacept, adalimumab, anakinra, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab, etanercept, infliximab methotrexate, mycophenolate mofetil (MMF), rituximab, sirolimus, tacrolimus, or a combination thereof.

[0039] A thirtieth embodiment is a method of preparing a modified secretome composition where the kinase inhibitor is crenolanib, dovitinib, erdafitinib, infigratinib, imatinib, nintedanib, pazopanib, pegaptanib, sunitinib, trebananib, or a combination thereof.

[0040] A thirty-first embodiment is a method of preparing a modified secretome composition where the method further includes adjusting the pH of the modified secretome composition to a pH of about 4.7 to about 7.5, where adjusting the pH includes formulating the modified secretome composition with a buffer, where the buffer includes boric acid, citric acid, disodium phosphate, monosodium phosphate, sodium citrate, sodium tetraborate, tris(hydroxymethyl)aminomethane hydrochloride, or a combination thereof.

[0041] A thirty-second embodiment is a method of preparing a modified secretome composition where the method further includes formulating the modified secretome composition with a tonicity modifying agent, where the tonicity modifying agent includes dextrose, glycerin, mannitol, potassium chloride, sodium chloride, sorbitol, sucrose, or a combination thereof.

[0042] A thirty-third embodiment is a method of preparing a modified secretome composition where the human subject has dry eye disease, macular degeneration, diabetic retinopathy, a cataract, cornea transplant, or a combination thereof.

[0043] A thirty-fourth embodiment is a modified secretome composition prepared by the process of any of the preceding embodiments.

[0044] A thirty-fifth embodiment is a modified secretome composition including mesenchymal stem cell-conditioned medium (MSC-CM) including less than 500 pg / ml each of an angiogenic factor.

[0045] A thirty-sixth embodiment is a modified secretome composition where the mesenchymal stem cells are derived from bone marrow, peripheral blood, adipose tissue, placenta, or umbilical cord tissue.

[0046] A thirty-seventh embodiment is a modified secretome composition where the mesenchymal stem cells are derived from adipose tissue.

[0047] A thirty-eighth embodiment is a modified secretome composition where the angiogenic factor is angiogenin (ANG), angiopoietin-1 (Angl), angiopoietin-2 (Ang2), FGF1, FGF2, insulinlike growth factor binding protein 3 (IGFBP-3), insulin-like growth factor-binding protein 7 (IGFBP-7), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-P), VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (P1GF), an isoforms thereof, or a combination thereof.

[0048] A thirty-ninth embodiment is a modified secretome composition where the angiogenic factor is VEGF-A, VEGF-B, VEGF-C, or VEGF-D and a) ANG, Angl, Ang2, or a combination thereof; b) FGF1 and / or FGF2; c) IGFBP-3 and / or IGFBP-7; d) PDGF; or e) TGF-p.

[0049] A fortieth embodiment is a modified secretome composition including an exogenous cell adhesion molecule, an exogenous antibody, an exogenous pigment epithelium-derived factor (PEDF), a corticosteroid, a decoy receptor, a flavonoid, an immunosuppressant, a kinase inhibitor, or any combination thereof.

[0050] A forty-first embodiment is a modified secretome composition where the exogenous cell adhesion molecule is intercellular adhesion molecule-1 (ICAM-1) and / or lymphocyte function-associated antigen 1 (LFA-1).

[0051] A forty-second embodiment is a modified secretome composition where the exogenous antibody is AMG780, bevacizumab, brolucizumab-dbll, figitumumab, fresolimumab, ganitumab,MEDI3617, nesvacumab, ramucirumab, ranibizumab, or a combination thereof.

[0052] A forty-third embodiment is a modified secretome composition where the exogenous PEDF protein is Serpin 1 (SEQ ID NO. 1), or a truncated or short derivative thereof,

[0053] A forty-fourth embodiment is a modified secretome composition where the corticosteroid is dexamethasone, difluprednate, fluoromethoIone, loteprednol, prednisolone, prednisone, or a combination thereof.

[0054] A forty-fifth embodiment is a modified secretome composition where the decoy receptor is aflibercept and / or conbercept.

[0055] A forty-sixth embodiment is a modified secretome composition where the flavonoid is quercetin, kaempferol, myricetin, or a combination thereof.

[0056] A forty-seventh embodiment is a modified secretome composition where the immunosuppressant is abatacept, adalimumab, anakinra, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab, etanercept, infliximab methotrexate, mycophenolate mofetil (MMF), rituximab, sirolimus, tacrolimus, or a combination thereof.

[0057] A forty-eighth embodiment is a modified secretome composition where the kinase inhibitor is crenolanib, dovitinib, erdafitinib, infigratinib, imatinib, nintedanib, pazopanib, pegaptanib, sunitinib, trebananib, or a combination thereof.

[0058] A forty-ninth embodiment is a modified secretome composition where the composition further includes a buffer, where the buffer includes boric acid, citric acid, disodium phosphate, monosodium phosphate, sodium citrate, sodium tetraborate, tris(hydroxymethyl)aminomethane hydrochloride, or a combination thereof.

[0059] A fiftieth embodiment is a modified secretome composition where the pH of the composition is about 4.7 to about 7.5, inclusive.

[0060] A fifty-first embodiment is a modified secretome composition, which includes a tonicity modifying agent, where the tonicity modifying agent includes dextrose, glycerin, mannitol, potassium chloride, sodium chloride, sorbitol, sucrose, or a combination thereof.

[0061] A fifty-second embodiment is a method of treating an ocular condition including topically administering to the eye of a mammal the modified mesenchymal stem cell secretome composition of any of the preceding embodiments.

[0062] A fifty -third embodiment is a method of treatment for an ocular condition in a mammal in need thereof including administering to the mammal a modified mesenchymal stem cell secretome composition, where the composition includes less than 500 pg / ml each of an angiogenic factor.

[0063] A fifty-fourth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the modified secretome composition is derived from MSC-CM.

[0064] A fifty-fifth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the mesenchymal stem cells are derived from bone marrow, peripheral blood, adipose tissue, placenta, or umbilical cord tissue.

[0065] A fifty-sixth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the mesenchymal stem cells are derived from adipose tissue.

[0066] A fifty-seventh embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the angiogenic factor is any one or more of angiogenin (ANG), angiopoietin-1 (Angl), angiopoietin-2 (Ang2), FGF1, FGF2, insulin-like growth factor binding protein 3 (IGFBP-3), insulin-like growth factor-binding protein 7 (IGFBP-7), platelet-derivedgrowth factor (PDGF), transforming growth factor beta (TGF-P), VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (P1GF), and isoforms thereof.

[0067] A fifty-eighth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the angiogenic factor is VEGF-A, VEGF-B, VEGF-C, or VEGF- D and a) ANG, Angl, Ang2, or a combination thereof; b) FGF1 and / or FGF2; c) IGFBP-3 and / or IGFBP-7; d) PDGF; or e) TGF-p.

[0068] A fifty-ninth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the method further includes co-administering to the mammal an exogenous cell adhesion molecule, an exogenous antibody, an exogenous pigment epithelium-derived factor (PEDF), a corticosteroid, a decoy receptor, a flavonoid, an immunosuppressant, a kinase inhibitor, or any combination thereof.

[0069] A sixtieth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the exogenous cell adhesion molecule is intercellular adhesion molecule- 1 (ICAM-1) and / or lymphocyte function-associated antigen 1 (LFA-1).

[0070] A sixty-first embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the exogenous antibody is AMG780, bevacizumab, brolucizumab-dbll, figitumumab, fresolimumab, ganitumab, MEDI3617, nesvacumab, ramucirumab, ranibizumab, or a combination thereof.

[0071] A sixty-second embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the exogenous PEDF protein is Serpin 1 (SEQ ID NO. 1), or a truncated or short derivative thereof.

[0072] A sixty -third embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the corticosteroid is dexamethasone, difluprednate, fluoromethoIone, loteprednol, prednisolone, prednisone, or a combination thereof.

[0073] A sixty-fourth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the decoy receptor is aflibercept and / or conbercept.

[0074] A sixty -fifth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the flavonoid is quercetin, kaempferol, myricetin, or a combination thereof.

[0075] A sixty-sixth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the immunosuppressant is abatacept, adalimumab, anakinra, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab, etanercept, infliximab methotrexate, mycophenolate mofetil (MMF), rituximab, sirolimus, tacrolimus, or a combination thereof.

[0076] A sixty-seventh embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the kinase inhibitor is crenolanib, dovitinib, erdafitinib, infigratinib, imatinib, nintedanib, pazopanib, pegaptanib, sunitinib, trebananib, or a combination thereof.

[0077] A sixty-eighth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the modified secretome composition is administered to the mammal at least once per day.

[0078] A sixty-ninth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the ocular condition is dry eye disease, macular degeneration, diabetic retinopathy, a cataract, cornea transplant, or a combination thereof.

[0079] A seventieth embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the mammal has had at least one bone marrow or stem cell transplant and / or the mammal has graft versus host disease.

[0080] A seventy-first embodiment is a method of treatment for an ocular condition in a mammal in need thereof, where the mammal is a canine, a feline, an equine, a caprine, a lagomorph, a rodent, or a human.DESCRIPTION OF FIGURES

[0081] Figure 1 : Graphical representation of the fraction of original VEGF concentration present in the secretome composition following removal of angiogenic factor VEGF using antibody-coated beads in two separate trials.

[0082] Figure 2: Graphical representation of the fraction of original VEGF concentration present in the secretome composition following removal of angiogenic factor VEGF using VEGF specific siRNA oligos.

[0083] Figure 3: Graphical representation of the endothelial proliferation in modified (VEGF depleted) secretome and unmodified secretome relative to basal media.DETAILED DESCRIPTION

[0084] A 2017 study estimated that >16 million US adults have diagnosed dry eye disease, noting that the prevalence is higher among women than men and increases with age (Farrand et al. Am J Ophthalmol. 2017 Oct; 182:90-98). The causes of dry eye are varied, and the condition can result from diabetes, thyroid problems, and autoimmune disorders. In another example, dry eye disease is a common permanent side effect from stem cell or bone marrow transplants due to graft versus host disease. Treatment for severe cases of KCS include blood-derived eye drops, such as autologous serum (AS), which lubricate the eyes and provide factors that improve the ocularsurface and facilitate wound healing. Studies have shown that platelet-rich plasma (PRP) is also an effective treatment for dry eye disease (Metheetrairut et al., Sci Rep. 2022; 12: 8906). See also Rawat et al., Indian J Ophthalmol. 2022 May;70(5): 1549-1553 and Murtaza et al., Clin Ophthalmol. 2022 Jul 6; 16:2199-2208. Common between the two are epitheliotrophic factors, which are thought to mediate treatment efficacy, including epidermal growth factor (EGF), fibronectin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-betal (TGF-pi). Such factors may also be identified in the secretome of stem cells, the repertoire of small molecules, peptides, and extracellular vesicles secreted by stem cells cultivated or conditioned in cell media. While understood to drive beneficial effects, these factors are also pro- angiogenic. For example, platelet rich plasma extract promotes angiogenesis through the angiopoietinl-Tie2 pathway (Mammoto et al., Mier avascular Research, 2013;89: 15-24.

[0085] Angiogenesis, which is regulated by pro-angiogenic proteins, is the physiological process of new blood vessel formation from pre-existing vessels. Pro-angiogenic factors, i.e., proteins implicated in driving angiogenesis in several biological processes, e.g., embryonic development, wound healing, and tumor growth, include vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs), hepatocyte growth factors (HGF), platelet-derived growth factors (PDGFs), and transforming growth factor beta (TGF-P). Such pro-angiogenic factors can stimulate the proliferation, migration, and tube formation of endothelial cells, which result in the development of blood vessels. See, e.g., Adair & Montani, “Chapter 1 : Overview of Angiogenesis,” San Rafael (CA): Morgan & Claypool Life Sciences; 2010, Raica & Simpean, Pharmaceuticals (Basel). 2010 Mar; 3(3): 572-599, and Tahergorabi & Khazaei, Iran J Basic Med Sci. 2012 Nov-Dec; 15(6): 1110-1126.

[0086] While valuable in the treatment of certain conditions, the promotion of angiogenesis in the eye may result in the development in angiogenic ocular conditions, which represent a major cause of irreversible vision loss. See, e.g., Penn et al., Prog Retin Eye Res. 2008 Jul; 27(4): 331— 371. As the level of relevant blood proteins can vary between patients, it is difficult to regulate the content of AS and PRP treatments. In one example, higher levels of inflammatory cytokines in the blood of certain subjects can cause negative effects and reduce treatment efficacy. In comparison to AS and PRR, stem cell secretome compositions are amenable to standardization. Treatment of ocular conditions with a secretome, e.g., by topical administration of the secretome to the eye of a subject, may benefit from specifically tuning the angiogenic capacity of the secretome, such as by selectively removing pro-angiogenic factors and / or adding anti-angiogenic factors. Among other aspects, now described are methods of selectively modifying the angiogenic capacity of stem cell secretomes, compositions thereof, and methods of using the same, such as in the treatment of ocular conditions and diseases.

[0087] In some aspects, disclosed herein are methods of modifying a stem cell secretome with the at least the aims of 1) reducing angiogenic capacity and 2) retaining or increasing the anti- angiogenic, anti-inflammatory, and immunosuppressive properties of the secretome. In other aspects, disclosed herein are modified secretome compositions having diminished angiogenic capacity and enhanced anti-angiogenic, anti-inflammatory, or immunosuppressive properties. Various methods to evaluate such properties are available to one of skill in the art.

[0088] The angiogenic capacity of a secretome refers to its ability to stimulate angiogenesis.Various assays are available to assess the angiogenic capacity of a composition, such as a stem cell derived secretome, including, e.g., endothelial cell proliferation assays, endothelial cell migration assays, endothelial cell growth and network formation assays, Boyden chamber assays,phagokinetic track assays, endothelial cell differentiation assays, network formation assays, and corneal angiogenesis assays. See, e.g., Nowak-Sliwinska et al., Angiogenesis. 2018 Aug;21(3):425-532; Merfeld-Clauss et al., Tissue Eng Part A 2010 Sep;16(9):2953-66; Tahergorabi & Khazaei, Iran J Basic Med Sci. 2012 Nov-Dec; 15(6): 1110-1126.

[0089] In some examples, the methods disclosed herein, such as removing an angiogenic factor from a stem cell secretome, promote the relative anti-inflammatory properties of the secretome. Anti-inflammatory properties of a disclosed modified secretome composition may be evaluated by a variety of methods, including assessments of oxidative stress and prevention thereof. In some examples, the disclosed modified secretome compositions reduce inflammation, which may be determined by an increase in anti-inflammatory markers, a decrease in inflammatory markers, e.g., IL-8, or a combination thereof. Exemplary assays which may be used to assess the antiinflammatory activity of a disclosed modified secretome composition include measuring reactive oxygen species (ROS) in cells, such as immune cells, exposed to the modified secretome and determining whether the modified secretome induces production of anti-inflammatory cytokines, such as IL-10 and TGF-P, and reduces production of pro-inflammatory cytokines, e.g., TNF-a and IL-6.

[0090] In additional examples, the methods disclosed herein, such as removing an anti- angiogenic factor from a stem cell secretome, promote the relative immunosuppressant properties of the secretome. Additionally, the immunomodulatory properties of a disclosed modified secretome composition may be assessed, e.g., by observing the effects of the composition on inflammatory cytokine production, such as inflammatory cytokine suppression, macrophage polarization, T-cell proliferation and suppression, regulatory T-cell induction, and migration and homing behavior of immune cells. In preferred examples, the capacity of a disclosed modifiedsecretome composition to stimulate angiogenesis is depleted, whereas the immunomodulatory effects, specifically immunosuppressive effects, are retained or enhanced.

[0091] The term “adipose stromal cells” refers to the “non-adipocyte” fraction of adipose tissue. The cells can be fresh, or in culture. Adipose stromal cells contain pluripotent cells, which have the ability to differentiate into cell types including but not limited to adipocytes, cardiomyocytes, endothelial cells, hematopoietic cells, hepatic cells, chondrocytes, osteoblasts, neuronal cells, and myotubes. “Adipose stem cells” are cells within the adipose stromal fraction which exhibit a stem cell phenotype, such as CD45- / Sca-l+ / c-kit- or CD45- / CD34+ / c-kit-

[0092] The term “secretome” or “secretome composition” refers to a mesenchymal cell- conditioned medium (MSC-CM) composition prepared according to the methods described herein. The term “modified secretome” or “modified secretome composition” refers to a secretome composition which having reduced angiogenicity by depletion of angiogenic factors or pro- angiogenic factors and / or increase in the antiangiognic factors, anti-inflammatory factors, and / or immunosuppressive activity in the composition.

[0093] “Therapeutically effective amount” or “therapeutically effective dose” of a disclosed composition, such as a modified secretome composition, refers to an amount that is effective to achieve a desired therapeutic result, e.g., treating an ocular disease, such as dry eye disease. Therapeutically effective amounts will typically depend upon the EC50 and safety profile of the specific agent being administered.

[0094] Methods of Preparing Modified Secretome Compositions

[0095] In some aspects, provided herein are methods of preparing a modified secretome composition. In some embodiments, disclosed methods of preparing a modified secretome composition comprise culturing mesenchymal stem cells in medium, thereby producing amesenchymal stem cell-conditioned medium (MSC-CM), and removing an angiogenic factor from the conditioned medium, thereby preparing the modified secretome composition. The removal of angiogenic factors is important for the use as a therapeutic for ocular diseases, but not for other therapeutic uses of secretome. The disclosed methods yield a modified secretome composition comprising a reduced amount of an angiogenic factor compared to modification of the mesenchymal stem cell-conditioned medium to remove the angiogenic factor.

[0096] Cell Collection and Cultivation

[0097] In some embodiments, MSCs are collected from a mammalian donor, such as a mammal. In some embodiments, the mammal is a canine, e.g., a dog, a feline, e.g., a cat, an equine, e.g., a horse, a caprine, e.g., a sheep or a goat, a lagomorph, e g., a rabbit, or a rodent, e.g., a guinea pig, hamster, or gerbil. In some embodiments, the mammal is human. In preferred embodiments, the mammal is a female human. In additional preferred embodiments, the human donor is under the age of 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70.

[0098] In some embodiments, the MSC-CM comprises medium conditioned by contact with MSCs derived from embryonic stem cells, fetal stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs). In preferred embodiments, the MSC-CM comprises medium conditioned by contact with MSCs derived from iPSCs.

[0099] In some embodiments, MSC-CM comprises medium conditioned by contact with MSCs derived from bone marrow, peripheral blood, adipose tissue, placenta, umbilical cord tissue, or a combination thereof. In some embodiments, MSC-CM comprises medium conditioned by contact with MSCs derived from umbilical cord tissue, such as the umbilical cord wall or lining, umbilical cord matrix, Wharton’s jelly, or a combination thereof.

[0100] Culture conditions described herein facilitate production of a conditioned medium, e.g., as described in US Patent Publication No. US20160145576A1. In preferred embodiments, the MSC-CM comprises medium conditioned by contact with MSCs derived from adipose tissue. In some embodiments, MSCs are separated from conditioned media (MSC-CM) prior to removal of an angiogenic factor. In other embodiments, MSCs are not separated from conditioned media (MSC-CM) prior to removal of an angiogenic factor.

[0101] In some embodiments, the MSCs are cultured under normoxic conditions. In some embodiments, the MSCs are cultured under hypoxic conditions. In some embodiments, the MSCs are cultured under anoxic conditions. In some embodiments, the MSCs are cultured under a combination of hypoxic and anoxic conditions. “Normoxic conditions" refer to oxygen tensions ranging from approximately 18-21 % O2, inclusive. "Hypoxic conditions" refer to oxygen tensions of greater than 1% O2 and less than 15% O2, less than 10% O2, or less than 5% O2, preferably ranging from approximately 1-5 % O2, inclusive. "Anoxic conditions" refer to oxygen tensions below approximately 1 % O2, preferably below 0.5% O2. Method of managing oxygen conditions in cell culture are known to one of skill in the art. See, e.g., Place et al., Free Radio Biol Med. 2017 Dec;113:311-322 and Wenger et al., Hypoxia (Auckl). 2015; 3: 35-43.

[0102] In some embodiments, the MSCs are cultured under hypoxic conditions selected from culture conditions comprising less than 15% O2, less than 10% O2, less than 5% O2, or about 1% O2. Hypoxia may be induced in the MSC cell culture in a variety of ways, such as, for example, by altering the gas composition the cells are exposed to or by providing one or more chemical inducer of hypoxia to the MSCs in culture.

[0103] In some embodiments, the MSCs are cultured under hypoxic conditions for up to about1 hour, 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, or 24 hours. In someembodiments, the MSCs are cultured under anoxic conditions for up to about 30 minutes, 60 minutes, 90 minutes, or 120 minutes.

[0104] In some embodiments, MSCs are cultured under conditions including inflammatory stimuli. In some embodiments, MSCs are cultured under conditions including inflammatory stimuli prior to collection of the modified secretome. In some embodiments, MSCs are cultured in combination with at least one pro-inflammatory factor. In some embodiments, the pro- inflammatory factor is a cytokine. In some embodiments, the cytokine is an interleukin, an interferon, or a tumor necrosis factor. In some embodiments, MSCs are cultured in combination with IL-1, IL-ip, IL-4, IL-6, IL-7, IL- 10, IL- 12, TNF-a, IL-23 / IL-23, IFN-a, IFNy, CTLA-4, CXCL8, GM-CSF, TNF-a, TNF- p, TGF-P, or a combination thereof. Culturing MSCs in the presence may enhance anti-inflammatory properties and / or immunosuppressant properties of the cells. See, e.g., Xie et al., Stem Cells. 2015 Feb;33(2):468-78.

[0105] In some embodiments, medium may be conditioned with MSCs for a range of times to obtain suitable MSC-CM. In some embodiments, medium is conditioned with MSCs for at least about 20 minutes, at least about 40 minutes, at least about 60 minutes, at least about 80 minutes, at least about 100 minutes, at least about 120 minutes, at least about 140 minutes, at least about 160 minutes, at least about 180 minutes, at least about 200 minutes, at least about 220 minutes, at least about 240 minutes, at least about 260 minutes, at least about 280 minutes, at least about 300 minutes, at least about 320 minutes, at least about 340 minutes, at least about 360 minutes, at least about 380 minutes, at least about 400 minutes, at least about 420 minutes, at least about 440 minutes, at least about 460 minutes, at least about 480 minutes, at least about 500 minutes.

[0106] In some embodiments, medium is conditioned with MSCs for a time period of between about 20 minutes to 96 hours, 20 minutes to 72 hours, 20 minutes to 60 hours, 20 minutes to 48hours, 20 minutes to 36 hours, 20 minutes to 24 hours, 20 minutes to 12 hours or 20 minutes to 6 hours. In some embodiments, medium is conditioned with MSCs for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, medium is conditioned with MSCs in a continuously flowing or circulating system, such as a bioreactor comprising channels.

[0107] In some embodiments, medium is conditioned with MSCs until the MSCs reach about 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100% confluency. 50% to 90% , 60% to 90%, 70% to 90% , or 80% to 90% confluency. In some embodiments, medium is conditioned with MSCs until the MSCs reach about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% confluency.

[0108] In various embodiments, the MSCs used to condition the CM may be “preconditioned” with one or more treatments. By preconditioned, we mean exposed to a treatment, such as, for example, an environmental condition, one or more small molecules and / or proteins.

[0109] In preferred embodiments, disclosed methods are performed with adipose-derived MSCs (Ad-MSCs). Ad-MSCs, also referred to herein as adipose stem cells (ASCs), are present in adult and pediatric adipose tissue. Ad-MSCs can be cultured in a cell culture medium, in vitro. After a period of time in culture, in accordance with disclosed methods, the Ad-MSCs can be separated from the medium, and the medium collected. This medium, conditioned with Ad-MSCs, is referred to as Ad-MSC-conditioned medium (Ad-MSC-CM), and it contains various components secreted by the Ad-MSCs during the period of culture time. The Ad-MSC secretome has been characterized and described in, e.g., Alvarez-Llamas et al., Mol Cell Proteomics. 2007Apr;6(4):589-600, Kapur & Katz, Biochimie. 2013 Dec;95(12):2222-8, Li et al., Stem Cell ResTher. 2015; 6(1): 55, and Pires et al., Stem Cells Dev. 2016 Jul 15;25(14): 1073-83.

[0110] Reducing the Angiogenic Capacity of a Stem Cell Derived Secretome

[0111] In some aspects, provided herein are methods of reducing the angiogenic capacity of a stem cell-derived secretome. In some embodiments, reducing the angiogenic capacity of a stem cell-derived secretome increases the relative anti-inflammatory properties and / or the relative immunosuppressant properties of the stem cell-derived secretome. In some embodiments, disclosed methods comprise removing an angiogenic factor, such as a pro-angiogenic factor, from a stem cell-derived secretome, such as mesenchymal stem cell conditioned medium (MSC-CM). In some embodiments, the stem cell-derived secretome comprises medium conditioned by embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs), hematopoietic stem cells (HSCs), or a combination thereof. In some embodiments the stem cell derived secretome comprises medium conditioned by stem cells derived from bone marrow, peripheral blood, adipose tissue, placenta, or umbilical cord tissue. In preferred embodiments, the secretome comprises medium conditioned by mesenchymal stem cells derived from adipose tissue.

[0112] In some embodiments, disclosed methods comprise removing at least one angiogenic factor from the secretome, such as a pro-angiogenic factor, thereby providing a modified stem cell secretome. In some embodiments, the removed angiogenic factor is any one or more of angiogenin (ANG), an endothelial growth factor, a fibroblast growth factor (FGF) protein, a hepatocyte growth factor (HGF) protein, an insulin-like growth factor binding protein, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-P), a vascular endothelial growth factor (VEGF)protein, and isoforms thereof. Additional details regarding protein structure, function, and isoforms are available to one of skill in the art, e.g., by accessing UniProtKB (www.uniprot.org).

[0113] In some embodiments, disclosed methods comprise removing angiogenin from a cell derived secretome. Angiogenin induces vascularization of normal and malignant tissues (Dickson et al., Biochemistry. 2009 May 12;48(18):3804-6). An exemplary protein sequence of human angiogenin is represented by SEQ ID NO:2.

[0114] In some embodiments, disclosed methods comprise removing an endothelial growth factor from a cell derived secretome. In some embodiments, the endothelial growth factor is an angiopoietin, such as angiopoietin-1 (Angl), angiopoietin-2 (Ang2), or angiopoietin-4 (Ang4). In some embodiments, the endothelial growth factor is VEGF. Along with VEGF and FGF2 (bFGF), angiopoietins are involved in the regulation of chemotactic endothelial cell migration during angiogenesis. See, e.g., Lamalice et al., Circulation Research, 2007; 100(6): 782-794 and Akwii et al., Cells. 2019 May; 8(5): 471. Exemplary protein sequences of human Angl, Ang2, and Ang4 are represented by SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively.

[0115] In some embodiments, disclosed methods comprise removing an FGF protein from a cell derived secretome, wherein the FGF protein is FGF1, FGF2, which is also known as basic FGF (bFGF), or a combination thereof. In preferred embodiments, the FGF protein is bFGF or an isoform thereof. FGF proteins include FGF1, FGF2, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, and isoforms thereof. The mammalian Fibroblast Growth Factor (FGF) family includes at least eighteen secreted proteins, which interact with four signaling tyrosine kinase FGF receptors (FGFRs). FGF proteins and their functions are described in, e.g., Omitz & Itoh, Wiley Interdiscip Rev Dev Biol. 2015 May - Jun;4(3):215-66, Goetz & Mohammadi, Nat Rev Mol Cell Biol. 2013 Mar; 14(3): 166-180, andBeenken & Mohammadi, Nat Rev Drug Discov. 2009 Mar; 8(3): 235-253. Exemplary canonical protein sequences of human FGF1 and bFGF are represented by SEQ ID NO:6 and SEQ ID NO:7, respectively, and isoforms thereof are available to one of skill in the art, e.g., by accessing UniprotKB.

[0116] In some embodiments, disclosed methods comprise removing an insulin growth factor (IGF) from a stem cell derived secretome, wherein the IGF is IGF1, IGF2, an isoform thereof, or a combination thereof. An exemplary protein sequences of human IGF1 is represented by SEQ ID NO:8.

[0117] In some embodiments, disclosed methods comprise removing an insulin-like growth factor binding protein (IGFBP) from a stem cell derived secretome, wherein the IGFBP is IGFBP- 1, IGFBP-3, IGFBP-7, an isoform thereof, or a combination thereof. Insulin-like growth factor binding proteins include insulin growth factor binding protein 1 (IGFBP- 1), insulin growth factor binding protein 2 (IGFBP-2), insulin-like growth factor binding protein 3 (IGFBP-3), insulin growth factor binding protein 4 (IGFBP-4), insulin growth factor binding protein 5 (IGFBP-5), insulin growth factor binding protein 6 (IGFBP-6), insulin-like growth factor-binding protein 7 (IGFBP-7), and isoforms thereof. The structure and function of insulin-like growth factor-binding proteins, such as their involvement in angiogenesis, are described in, e.g., Slater et al., Cytokine Growth Factor Rev. 2019 Apr;46:28-35. An exemplary protein sequence of human IGFBP2 is represented by SEQ ID NO: 9.

[0118] In some embodiments, disclosed methods comprise removing platelet derived growth factor (PDGF) from a stem cell derived secretome. PDGF isoforms include PDGF-AA (PDGFA), -BB (PDGFB), -CC (PDGFC), and -DD (PDGFD), and -AB (a PDGFA andPDGFB heterodimer). PDGF and isoforms thereof are described in, e.g., Fredriksson et al., Cytokine & Growth FactorReviews. 2004;! 5(4): 197-204. Exemplary protein sequences of human PDGFA and PDGFB are represented by SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

[0119] In some embodiments, disclosed methods comprise removing transforming growth factor P (TGF- ) or an isoform thereof from a stem cell derived secretome. TGF-P isoforms include TGF- i, TGF- 2, and TGF- 3. In some embodiments, disclosed methods comprise removing latent transforming growth factor binding protein 2 (LTBP-2), transforming growth factor-induced protein IG-H3, an isoform thereof from a stem cell derived secretome. An exemplary protein sequence of human TGFpi proprotein is represented by SEQ ID NO: 12.

[0120] In some embodiments, disclosed methods comprise removing a VEGF protein from a stem cell derived secretome, wherein the VEGF protein is VEGF -A, VEGF-B, VEGF-C, VEGF- D, placental growth factor (P1GF), an isoform thereof, or a combination thereof. The structure and function of VEGF proteins, such as the pro-angiogenic activity, are described in, e.g., Park et al., BMB Rep. 2018 Feb;51(2):73-78 and Melincovici et al., Rom J Morphol Embryol. 2018;59(2):455- 467. Exemplary protein sequences of VEGF A and VEGFB are represented by SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0121] In some embodiments, disclosed methods comprise removing a VEGF protein and angiogenin (ANG) from a stem cell derived secretome. In some embodiments, disclosed methods comprise removing a VEGF protein and Angl and / or Ang2 from a stem cell derived secretome. In some embodiments, disclosed methods comprise removing a VEGF protein and FGF1 and / or FGF2 from a stem cell derived secretome. In some embodiments, disclosed methods comprise removing a VEGF protein and PDGF from a stem cell derived secretome. In some embodiments, disclosed methods comprise removing a VEGF protein and TGF-P from a stem cell derivedsecretome. In some embodiments, the VEGF protein is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (P1GF), or an isoform thereof.

[0122] In some embodiments, removing an angiogenic factor, such as a pro-angiogenic factor, from an MSC-CM comprises use of antibody-based affinity chromatography, countercurrent tangential chromatography, protein concentration, protein precipitation, buffer exchange methods, size exclusion methods, such as ultrafiltration, immunoprecipitation, or a combination thereof. Ultrafiltration alone is described in US10758571B1 to achieve removal of VEGF. However, this approach is non-selective, and inevitably various other proteins above or below the size or weight cutoff threshold are excluded from the secretome composition. In one contrasting example, immunoprecipitation provides a higher degree of selectivity, as it relies on use of antibodies that specifically target an angiogenic factor. Accordingly, to ensure a high degree of selectivity, exclusion methods based on size or weight may be performed in conjunction with methods that facilitate greater specificity of the factors being removed, e.g., immunoprecipitation.

[0123] In some embodiments, removing angiogenic factors from the MSC-CM comprises use of an antibody, aptamer, mimotope, peptide, or small molecule targeting the angiogenic factor. Exemplary peptides targeting VEGF or interfering with VEGF signaling are represented by SEQ ID NO: 15 and SEQ ID NO: 16. See, e.g., Shoari et al., ResPharm Sci. 2021 Dec; 16(6): 559-574; Binetruy-Tournaire et al., EMBOJ. 2000; 19(7): 1525-1533; Zhang et al., Signal Transduction and Targeted Therapy, 2017; 2: 17010. The antibody, mimotope, peptide, or small molecule may be loaded or immobilized onto a surface, such as a solid surface, e.g., a bead. See, e g., Sakr et al., J Control Release. 2016 Feb 28;224: 199-207. In some embodiments, the solid surface is a microplate, a membrane, a chip, a slide, a bead, a magnetic bead, a nanoparticle, or a microfluidic device.

[0124] In some embodiments, disclosed methods comprise removing angiogenic factors via antibody-based affinity chromatography. Affinity removal of selected factors involves: i) preparing the sample to load on the affinity column; ii) loading and incubation of the sample with the antibody to promote binding of the antibody to the column, depleting the selected factor(s); iii) washing away the desired non-bound preparation from the column; and possibly iv) eluting (recovering) the bound antibodies from the immobilized matrix. See, e.g., Arora et al., Methods. 2017 Mar 1;116:84-94. Alternatively, the antibody, or other binding ligand, such as an aptamer, which can selectively bind to the factor to be depleted can be pre-attached to the immobilized matrix, and then the sample can be exposed to the immobilized ligand, and eluted after depletion of the selected factor.

[0125] In some embodiments, disclosed methods comprise removing angiogenic factors via immunoprecipitation. In a general immunoprecipitation process, an antibody targeting the angiogenic factor is added to a cell-derived secretome, thereby facilitating formation of an angiogenic factor-peptide complex. The complex may then be precipitated using, e g., bead collection and removal by centrifugation or magnetic beads, and eluted proteins may be analyzed by Western blot, ELISA, or mass spectrometry. See, e.g., DeCaprio & Kohl, Cold Spring Harb Protoc 2017.

[0126] In some embodiments, removing an angiogenic factor from MSC-CM comprises contacting the MSC-CM with an antibody targeting the angiogenic factor. In some embodiments, the antibody is an anti-VEGF antibody. In some embodiments, the anti-VEGF antibody is aflibercept, bevacizumab, brolucizumab-dbll, or ranibizumab. The preceding in any combination may also be used to remove VEGF from a secretome, thereby preparing a modified secretome as disclosed herein.

[0127] In some embodiments, disclosed methods comprise inhibiting gene expression or silencing gene expression of an angiogenic factor in at least one stem cell, e.g., a mesenchymal stem cell. In some embodiments, disclosed methods comprise inhibiting gene expression or silencing gene expression of angiogenin (ANG), an endothelial growth factor, a fibroblast growth factor (FGF) protein, a hepatocyte growth factor (HGF) protein, an insulin-like growth factor binding protein, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF- ), a vascular endothelial growth factor (VEGF) protein, or an isoform thereof, in the at least one stem cell.

[0128] In some embodiments, inhibiting gene expression of an angiogenic factor in a stem cell comprises transfecting the stem cell, such as a mesenchymal stem cell, with an antisense oligonucleotide or an siRNA. In some embodiments, an expression vector, such as a viral vector transiently expresses the antisense oligonucleotide or the siRNA in the stem cell. In some embodiments, an expression vector, such as a viral vector, encoding the antisense oligonucleotide or the siRNA is stably integrated into the genome of the stem cell.

[0129] In some embodiments, inhibiting gene expression of an angiogenic factor in a stem cell comprises gene editing via delivery of at least one RNA-guided endonuclease system to the cell. In some embodiments, the at least one RNA-guided endonuclease system is a CRISPR system comprising a CRISPR nuclease and a guide RNA. In some embodiments, the CRISPR nuclease is Cas9, Cpfl, a homolog thereof, a modified version thereof, a codon-optimized version thereof, or any combination thereof.

[0130] Supplemental Anti-Angiogenic Factors

[0131] In some aspects, provided are methods which not only reduce the angiogenic capacity of a stem cell derived secretome, such as mesenchymal stem cell conditioned medium (MSC-CM),but also retain or enhance the anti angiogenic capacity, the anti-inflammatory activity, the immunosuppressive activity, or a combination thereof, of the secretome. In some embodiments, disclosed methods comprise supplementing the modified secretome composition with an anti- angiogenic factor, wherein the anti -angiogenic factor or the anti-inflammatory factor is an exogenous cell adhesion molecule, an exogenous antibody, an exogenous pigment epithelium- derived factor (PEDF), a corticosteroid, a decoy receptor, a flavonoid, an immunosuppressant, a kinase inhibitor, or any combination thereof.

[0132] In some embodiments, disclosed methods comprise adding an exogenous cell adhesion molecule to a secretome modified by reduction or removal of an angiogenic factor, such as a modified secretome as described herein. In some embodiments, disclosed methods comprise adding intercellular adhesion molecule- 1 (ICAM-1), lymphocyte function-associated antigen 1 (LFA-1), or a combination thereof to the modified secretome.

[0133] In some embodiments, disclosed methods comprise adding an exogenous antibody to a secretome modified by reduction or removal of an angiogenic factor, such as a modified secretome as described herein. In some embodiments, disclosed methods comprise adding AMG780, bevacizumab, brolucizumab-dbll, figitumumab, fresolimumab, ganitumab, MEDI3617, nesvacumab, ramucirumab, ranibizumab, or a combination thereof to the modified secretome.

[0134] In some embodiments, disclosed methods comprise adding an exogenous pigment epithelium-derived factor (PEDF) protein to a secretome modified by reduction or removal of an angiogenic factor, such as a modified secretome as described herein. In some embodiments, disclosed methods comprise adding full-length PEDF, truncated PEDF, short PEDF, or a combination thereof to the modified secretome. An exemplary short PEDF peptide is represented by SEQ ID NO: 17. In some embodiments, disclosed methods comprise adding SerpinFl (SEQ IDNO:1) to the modified secretome. Such peptides may be added in an amount effective to improve corneal repair and stimulate growth expansion of limbal epithelial stem cells and meibomian gland stem cells. While enhancing anti-inflammatory signalling, PEDF proteins may further reduce angiogenic activity.

[0135] In some embodiments, disclosed methods comprise adding a corticosteroid to a secretome modified by reduction or removal of an angiogenic factor, such as a modified secretome as described herein. In some embodiments, disclosed methods comprise adding dexamethasone, difluprednate, fluoromethoIone, loteprednol, prednisolone, or prednisone, or a combination thereof to the modified secretome.

[0136] In some embodiments, disclosed methods comprise adding a decoy receptor to a secretome modified by reduction or removal of an angiogenic factor, such as a modified secretome as described herein. In some embodiments, the disclosed methods comprise adding aflibercept, conbercept, or a combination thereof to the modified secretome.

[0137] In some embodiments, disclosed methods comprise adding a flavonoid to a secretome modified by reduction or removal of an angiogenic factor, such as a modified secretome as described herein. In some embodiments, disclosed methods comprise adding quercetin, kaempferol, myricetin, or a combination thereof to the modified secretome. Kaempferol, e.g., has been shown to inhibit expression of VEGF and PGF (Xu et al., Braz. J. Med. Biol. Res. 2017;50 (3).

[0138] In some embodiments, disclosed methods comprise adding an immunosuppressant to a secretome modified by reduction or removal of an angiogenic factor, such as a modified secretome as described herein. In some embodiments, disclosed methods comprise adding abatacept, adalimumab, anakinra, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab,etanercept, infliximab methotrexate, mycophenolate mofetil (MMF), rituximab, sirolimus, tacrolimus, or a combination thereof to the modified secretome.

[0139] In some embodiments, disclosed methods comprise adding a kinase inhibitor to a secretome modified by reduction or removal of an angiogenic factor, such as a modified secretome as described herein. In some embodiments, disclosed methods comprise adding crenolanib, dovitinib, erdafitinib, infigratinib, imatinib, nintedanib, pazopanib, pegaptanib, sunitinib, trebananib, or a combination thereof to the modified secretome.

[0140] In some embodiments, disclosed methods comprise adjusting the pH of the modified secretome composition. In some embodiments, the pH of the modified secretome is adjusted to about 4.7 to about 7.5. In some embodiments, the pH of the MSC secretome composition is adjusted to about 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, or 7.5.

[0141] In some embodiments, the pH of the modified secretome composition is adjusted by formulating the modified secretome composition with a buffer system, wherein the buffer comprises boric acid, citric acid, disodium phosphate, monosodium phosphate, sodium citrate, sodium tetraborate, tri s(hydroxymethyl)aminom ethane hydrochloride, or a combination thereof.

[0142] In some embodiments, disclosed methods further comprise formulating modified secretome compositions with a tonicity modifying agent. In some embodiments, the tonicity modifying agent comprises dextrose, glycerin, mannitol, potassium chloride, sodium chloride, sorbitol, sucrose, or a combination thereof.

[0143] Modified Secretome Compositions

[0144] In some aspects, provided are modified secretome compositions. In some embodiments, disclosed compositions comprise a reduced amount of an angiogenic factors compared to an unmodified secretome composition.

[0145] In some embodiments, disclosed compositions comprise a reduced amount of an angiogenic factor relative to an unmodified secretome composition, i.e., a secretome composition that has not been modified by removal of a pro-angiogenic factor as described herein. In some embodiments, the angiogenic factor is any one or more of angiogenin (ANG), angiopoietin-1 (Angl), angiopoietin-2 (Ang2), FGF1, FGF2, insulin-like growth factor binding protein 3 (IGFBP- 3), insulin-like growth factor-binding protein 7 (IGFBP-7), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-P), VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (P1GF), and isoforms thereof.

[0146] In some embodiments, the reduced amount of any one angiogenic factor is at least about 5% less, at least about 10% less, at least about 15% less, at least about 20% less, at least about 25% less, at least about 30% less, at least about 35% less, at least about 40% less, at least about45% less, at least about 50% less, at least about 55% less, at least about 60% less, at least about65% less, at least about 70% less, at least about 75% less, at least about 80% less, at least about85% less, at least about 90% less, at least about 95% less, at least about 100% less, at least about105% less, at least about 110% less, at least about 115% less, at least about 120% less, at least about 125% less, at least about 130% less, at least about 135% less, at least about 140% less, at least about 145% less, at least about 150% less, at least about 155% less, at least about 160% less, at least about 165% less, at least about 170% less, at least about 175% less, at least about 180% less, at least about 185% less, at least about 190% less, at least about 195% less, at least about200% less, at least about 205% less, at least about 210% less, at least about 215% less, at leastabout 220% less, at least about 225% less, at least about 230% less, at least about 235% less, at least about 240% less, at least about 245% less, at least about 250% less, at least about 255% less, at least about 260% less, at least about 265% less, at least about 270% less, at least about 275% less, at least about 280% less, at least about 285% less, at least about 290% less, at least about 295% less, at least about 300% less, at least about 305% less, at least about 310% less, at least about 315% less, at least about 320% less, at least about 325% less, at least about 330% less, at least about 335% less, at least about 340% less, at least about 345% less, at least about 350% less, at least about 355% less, at least about 360% less, at least about 365% less, at least about 370% less, at least about 375% less, at least about 380% less, at least about 385% less, at least about 390% less, at least about 395% less, at least about 400% less, at least about 405% less, at least about 410% less, at least about 415% less, at least about 420% less, at least about 425% less, at least about 430% less, at least about 435% less, at least about 440% less, at least about 445% less, at least about 450% less, at least about 455% less, at least about 460% less, at least about 465% less, at least about 470% less, at least about 475% less, at least about 480% less, at least about 485% less, at least about 490% less, at least about 495% less, or at least about 500% less than the amount of the same angiogenic factor prior to modification of the mesenchymal stem cell- conditioned medium, i.e., removal of the angiogenic factor.

[0147] In some embodiments, disclosed compositions do not comprise detectable levels of any one or more of angiogenin (ANG), an endothelial growth factor, e.g., An l and Ang2, a fibroblast growth factor (FGF) protein, e.g., FGF1 and FGF2, an insulin-like growth factor binding protein, e.g., IGFBP-3 and IGFBP-7, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-P), a vascular endothelial growth factor (VEGF) protein, and isoforms thereof. A detectable level, such as the lower limit of quantitation (LLOQ), varies across analytical techniquesand is the lowest amount of an analyte in a sample that can be quantitatively determined with suitable precision and accuracy.

[0148] In some embodiments, disclosed compositions are substantially free from any one or more of angiogenin (ANG), an endothelial growth factor, e.g., Angl and Ang2, a fibroblast growth factor (FGF) protein, e.g., FGF1 and FGF2, a hepatocyte growth factor (HGF) protein, e.g. NK1 and NK2, an insulin-like growth factor binding protein, e.g., IGFBP-3 and IGFBP-7, platelet- derived growth factor (PDGF), transforming growth factor beta (TGF-P), a vascular endothelial growth factor (VEGF) protein, and isoforms thereof.

[0149] In some embodiments, a disclosed composition comprises less than about 50 pg / ml, 100 pg / ml, 150 pg / ml, 200 pg / ml, 250 pg / ml, 300 pg / ml, 350 pg / ml, 400 pg / ml, 450 pg / ml, 500 pg / ml, 550 pg / ml, 600 pg / ml, 650 pg / ml, 700 pg / ml, 750 pg / ml, 800 pg / ml, 850 pg / ml, 900 pg / ml, 950 pg / ml, or 1000 pg / ml, inclusive, each of any one or more of angiogenin (ANG), an endothelial growth factor, a fibroblast growth factor (FGF) protein, a hepatocyte growth factor (HGF) protein, an insulin-like growth factor binding protein, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-P), a vascular endothelial growth factor (VEGF) protein, or a combination thereof.

[0150] In some embodiments, the modified MSC secretome composition comprises less than about 50 pg / ml, 100 pg / ml, 150 pg / ml, 200 pg / ml, 250 pg / ml, 300 pg / ml, 350 pg / ml, 400 pg / ml, 450 pg / ml, 500 pg / ml, 550 pg / ml, 600 pg / ml, 650 pg / ml, 700 pg / ml, 750 pg / ml, 800 pg / ml, 850 pg / ml, 900 pg / ml, 950 pg / ml, or 1000 pg / ml, inclusive, each of any one or more of VEGF- A, VEGF-B, VEGF-C, VEGF-D, P1GF, ANG, Angl, Ang2, Ang4, FGF1, bFGF, IGFBP-1, IGFBP- 3, IGFBP-7, PDGF, PDGF A, PDGFB, PDGFC, PDGFD, and PDGF -AB, TGF-P, TGF-P 1, TGF- P2, and TGF-P3.

[0151] In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml VEGF-A. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml VEGF-B. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml VEGF-C. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml VEGF-D. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml P1GF.

[0152] In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml angiogenin.

[0153] In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml Angl. In some embodiments, the modified MSC secretome comprises up to and including about 0 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml Ang2. In some embodiments, the modified MSC secretome comprises up to and including about 0 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml Ang4.

[0154] In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml FGF1. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml bFGF (FGF2).

[0155] In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml IGF-1. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml IGF-2.

[0156] In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml IGFBP-1. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml IGFBP-3. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml IGFBP-7.

[0157] In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml PDGF. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml PDGFA. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml,50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml PDGFB. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml PDGFC. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml PDGFD. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml PDGF-AB.

[0158] In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml TGF-p. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml TGF-pi. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml TGF-P2. In some embodiments, the modified MSC secretome comprises up to and including about 1 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, or 100 pg / ml TGF-P3.

[0159] Methods of detecting, identifying, and quantifying secretome components, such as proteins, are known to one of skill in the art. For example, HPLC, ELISA, and multiplex methods may generally be used to detect the presence and the quantity of various secretome proteins, e.g., VEGF. See, e.g., Amirthalingam et al., IntJMol. 2019;2:5, Kong et al., Stem Cell Res Ther. 2021Jan 8;12(1):47, Kuljanin et al., Cell Rep. 2018;25(9):2524-2536. e2524, and Wangler et al., StemCell Res Ther. 2021 ; 12(1): 1-17.

[0160] In some embodiments, disclosed compositions comprise extracellular vesicles (EVs), including but not limited to exosomes, concentrated in the MSC-CM. EVs contain cargos of factors that may be unstable in the extracellular milieu, such as microRNAs. In a particularly preferred embodiment, the composition provided herein comprises exosomes separated from MSC-CM. In an embodiment, it is contemplated that a composition suitable for use in the disclosed method comprises exosomes separated from MSC-CM, such as, for example, Ad-MSC-CM. An MSC composition comprising exosomes may be beneficial, at least because, relative to an MSC composition comprising all the contents of and MSC-CM, its composition may be easier to define, standardize, assay for toxicity, and / or store for a time period (e.g., improved shelf life).

[0161] Preferably, the extracellular vesicle concentration is from 40 million particles per ml to 1.3 billion particles per ml, from 40 million particles per ml to 1.0 billion particles per ml, . 40 million particles per ml to 750 million particles per ml, 40 million particles per ml to 500 million particles per ml, 40 million particles per ml to 250 million particles per ml, 40 million particles per ml to 100 million particles per ml. This preferred EV concentration encompasses both prior to and post-VEGF depletions. Most preferably, the EV concentration is around 700 million per ml.

[0162] In some embodiments, disclosed compositions comprise an anti -angiogenic factor, wherein the anti-angiogenic factor is an exogenous cell adhesion molecule, an exogenous antibody, an exogenous pigment epithelium-derived factor (PEDF), a corticosteroid, a decoy receptor, a flavonoid, an immunosuppressant, a kinase inhibitor, or any combination thereof. In some embodiments, the MSC secretome comprises at least about 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml, inclusive, of the anti-angiogenic factor, such as a single anti-angiogenic factor.

[0163] In some embodiments, disclosed compositions comprise an exogenous cell adhesion molecule. In some embodiments, the MSC secretome comprises at least about 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml, inclusive, of the cell adhesion molecule, such as a single cell adhesion molecule. In some embodiments, disclosed compositions comprise adding intercellular adhesion molecule- 1 (ICAM- 1), lymphocyte function-associated antigen 1 (LFA-1), or a combination thereof.

[0164] In some embodiments, disclosed compositions comprise an exogenous antibody. In some embodiments, the MSC secretome comprises at least about 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml, inclusive, of the exogenous antibody, such as a single exogenous antibody. In some embodiments, disclosed compositions comprise adding AMG780, bevacizumab, brolucizumab-dbll, figitumumab, fresolimumab, ganitumab, MEDI3617, nesvacumab, ramucirumab, ranibizumab, or a combination thereof.

[0165] In some embodiments, disclosed compositions comprise an exogenous pigment epithelium-derived factor (PEDF) protein. In some embodiments, the MSC secretome comprises at least about 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml, inclusive, of the PEDF protein, such as single PEDF protein. In some embodiments, disclosed compositions comprise full-length PEDF, truncated PEDF, short PEDF, or a combination thereof. In some embodiments, disclosed compositions comprise SerpinFl (SEQ ID NO: !).

[0166] In some embodiments, disclosed compositions comprise a corticosteroid. In some embodiments, the MSC secretome comprises at least about 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml, inclusive, of thecorticosteroid, such as a single corticosteroid. In some embodiments, disclosed compositions comprise dexamethasone, difluprednate, fluoromethoIone, loteprednol, prednisolone, or prednisone, or a combination thereof to the modified secretome.

[0167] In some embodiments, disclosed compositions comprise a decoy receptor. In some embodiments, the MSC secretome comprises at least about 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml, of the decoy receptor, such as a single decoy receptor. In some embodiments, the disclosed compositions comprise aflibercept, conbercept, or a combination thereof.

[0168] In some embodiments, disclosed compositions comprise a flavonoid. In some embodiments, the MSC secretome comprises at least about 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml, inclusive, of the flavonoid, such as a single flavonoid. In some embodiments, disclosed compositions comprise quercetin, kaempferol, myricetin, or a combination thereof.

[0169] In some embodiments, disclosed compositions comprise an immunosuppressant. In some embodiments, the MSC secretome comprises at least about 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml, inclusive, of the immunosuppressant, such as a single immunosuppressant. In some embodiments, disclosed compositions comprise abatacept, adalimumab, anakinra, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab, etanercept, infliximab methotrexate, mycophenolate mofetil (MMF), rituximab, sirolimus, tacrolimus, or a combination thereof.

[0170] In some embodiments, disclosed compositions comprise a kinase inhibitor. In some embodiments, the MSC secretome comprises at least about 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml, inclusive, of the kinaseinhibitor, such as a single kinase inhibitor. In some embodiments, disclosed compositions comprise crenolanib, dovitinib, erdafitinib, infigratinib, imatinib, nintedanib, pazopanib, pegaptanib, sunitinib, trebananib, or a combination thereof.

[0171] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5. In some embodiments, the MSC secretome composition has a pH of about 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, or 7.5. In some embodiments, the MSC secretome is formulated in a buffer system selected from the group consisting of di / mono sodium phosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0172] In some embodiments, the MSC secretome composition further comprises a tonicity modifying agent. In some embodiments, the tonicity modifying agent is selected from the group consisting of NaCl, KC1, mannitol, dextrose, sucrose, trehalose, sorbitol, and glycerin.

[0173] In some embodiments, the MSC secretome further comprises mono / di- sodium phosphate, mannitol, and trehalose, wherein the composition has a pH of about pH 7.4.

[0174] In some embodiments, the MSC secretome further comprises divalent cations. In some embodiments, the divalent cations are selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0175] In some embodiments, the MSC secretome further comprises di-sodium phosphate / citric acid, mannitol, and trehalose, wherein the composition has a pH of about pH 6.4.

[0176] In some embodiments, disclosed compositions comprise a pharmaceutically acceptable excipient. In some embodiments, disclosed compositions comprise a lubricant. In some embodiments, disclosed compositions comprise saline.

[0177] Methods of Treating Ocular Conditions and Diseases

[0178] In some aspects, provided are methods of treating a subject suffering from an ocular condition by administering the disclosed compositions to the subject. In some embodiments, the ocular disease is an ocular neovascular disease. In some embodiments, disclosed compositions are administered to the subject via topical application to the eye of the subject. In some embodiments, the subject is a mammal.

[0179] In some embodiments, the mammal is a canine, e.g., a dog, a feline, e.g., a cat, an equine, e.g., a horse, a caprine, e.g., a sheep or a goat, a lagomorph, e.g., a rabbit, or a rodent, e.g., a guinea pig, hamster, or gerbil. In preferred embodiments, the mammal is a human.

[0180] In some embodiments, the ocular condition is dry eye disease, dry eye disease associated with graft versus host disease, macular degeneration, e.g., wet age-related macular degeneration, diabetic retinopathy, a cataract, cornea transplant, retinopathy of prematurity, retinal vein occlusion, corneal neovascularization, or a combination thereof.

[0181] Pathological angiogenesis is a key feature of numerous ocular neovascular diseases, which include wet age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, and corneal neovascularization. See, e.g., Chen et al., MedComm-Future Medicine, 2023;2:e33. Ocular angiogenesis, which may occur in the retina, choroid, and cornea, can lead to severe visual impairment. Methods of diagnosing such conditions, e.g., diagnostic imaging tools, are available to one of skill in the art. See, e.g., Dreyfuss et al., J Ophthalmol. 2015; 2015: 892043.

[0182] In some embodiments, the ocular disease is dry eye disease, which is also known as dry eye syndrome. The two main subtypes of dry eye disease include evaporative dry eye disease and aqueous deficient dry eye. Several conditions can contribute to dry eye disease, including graft versus host disease (see, e.g., Munir & Aylward, Optom Vis Sci. 2017 May;94(5):545-555),Sjorgen’s syndrome, rheumatoid arthritis, lupus, thyroid disorders, such as hypothyroidism and hyperthyroidism, diabetes, allergies, blepharitis, certain medication, e.g., antihistamines, decongestants, antidepressants, and hormonal therapies, and environmental factors, e.g., humidity levels, screen time, or exposure to air flow, such as air conditioning or wind, or smoke. Other conditions that contribute to dry eye syndrome are known or within the understanding of one of skill in the art.

[0183] Dry eye disease may be recognized or diagnosed using a variety of methods available to one of skill in the art. For example, dry eye disease may be diagnosed according to patient history and patient symptoms, evaluation of the ocular surface or eyelid, e.g., to determine the presence of signs of inflammation or damage to the ocular surface or eyelid, or tear film assessment, e.g., Schirmer’s test, tear breakup time, or tear osmolarity.

[0184] In some embodiments, disclosed compositions are administered to the subject, such as by topical application to an eye or the eyes of the subject, at least once per day, at least twice per day, or at least three times per day. In some embodiments, disclosed compositions are administered to the subject once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times per day.

[0185] Co-administration

[0186] In some embodiments, disclosed methods of treatment further comprise coadministering a disclosed composition and an exogenous agent, such as an exogenous cell adhesion molecule, a corticosteroid, an immunosuppressant, or any combination thereof, to a subject suffering from an ocular disease, such as dry eye disease. In some embodiments, the exogenous agent co-administered with a disclosed composition is administered via topicalapplication, such as topically to an eye or the eyes of a subject, orally, or parenterally, e.g., subcutaneously, intravenously, or intrathecally.

[0187] In some embodiments, disclosed methods comprise co-administering a disclosed composition and an exogenous cell adhesion molecule to a subject in need thereof. In some embodiments, the exogenous cell adhesion molecule is intercellular adhesion molecule- 1 (ICAM- 1) or lymphocyte function-associated antigen 1 (LFA-1). In some embodiments, the exogenous cell adhesion molecule is intercellular adhesion molecule- 1 (ICAM-1) and lymphocyte function- associated antigen 1 (LFA-1).

[0188] In some embodiments, disclosed methods comprise co-administering a disclosed composition and a corticosteroid to a subject in need thereof. In some embodiments, the corticosteroid is dexamethasone, difluprednate, fluorometholone, loteprednol, prednisolone, or prednisone, or any combination thereof.

[0189] In some embodiments, disclosed methods comprise co-administering a disclosed composition and an immunosuppressant to a subject in need thereof. In some embodiments, the immunosuppressant is abatacept, adalimumab, anakinra, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab, etanercept, infliximab methotrexate, mycophenolate mofetil (MMF), rituximab, sirolimus, tacrolimus, or any combination thereof.

[0190] In some embodiments, disclosed methods of treatment further comprise coadministering a disclosed composition and an exogenous antibody. In some embodiments, the exogenous antibody targets an angiogenic factor. In some embodiments, the antibody targets an angiogenic factor or its corresponding receptor, e.g., an anti-VEGF antibody or an anti-VEGFR antibody. In some embodiments, the exogenous antibody targets the angiogenic factor or the corresponding receptor of vascular endothelial growth factor (VEGF), angiogenin (ANG),angiopoietin-1 (Angl), angiopoietin-2 (Ang2), basic fibroblast growth factor (bFGF), a hepatocyte growth factor (HGF) protein, an insulin-like growth factor, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-P), or any combination thereof.

[0191] In some embodiments, the exogenous antibody is aflibercept, AMG780, bevacizumab, brolucizumab-dbll, conbercept, crenolanib, dovitinib, erdafitinib, figitumumab, fresolimumab, ganitumab, infigratinib, imatinib, MEDI3617, nesvacumab, nintedanib, pazopanib, pegaptanib, ramucirumab, ranibizumab, sunitinib, trebananib, or a combination thereof.

[0192] In some embodiments, disclosed methods comprise co-administering a disclosed composition and axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, ramucirumab, regorafenib, sorafenib, sunitinib, thalidomide vandetanib, ziv- aflibercept, or a combination thereof to a subject in need thereof.

[0193] Treatment Efficacy

[0194] Various methodologies are available to one of skill in the art to determine treatment efficacy of the disclosed compositions and methods. See, e g., methods of assessing the treatment, such as the improvement, of ocular diseases in US20210346313A1. For example, in the case of dry eye, any of Schirmer's (SH) test scores, tear breakup times (TBUT), corneal fluorescein staining scores (Oxford scale, 0-4), and ocular surface disease indexes may be used to evaluate the effects of disclosed compositions and methods.

[0195] In some examples, treatment in accordance with the compositions and methods described herein may be assessed by evaluating improvement of ocular surface damage, such as with use of fluorescein staining of the (a) total corneal region, (b) central corneal region, (c) nasal corneal region, (d) inferior corneal region, (e) temporal comeal region and (f) superior corneal region.

[0196] In other examples, treatment in accordance with the compositions and methods described herein may be assessed by the self-reported Visual Analog Scale (VAS). In the case of dry eye, symptoms include (a) “severity of dryness” (corresponding to question 1 “dryness” of the 10-item VAS questionnaire), (b) “frequency of dryness”, (c) “awareness of dryness”, (d) “burning / stinging”, (e) “itching”, (f) “sticky feeling”, (g) “blurred vision”, (h) “foreign body sensation”, (i) “sensitivity to light”, (j) “pain.”

[0197] In additional examples, treatment in accordance with the compositions and methods described herein may be assessed using the Total Ocular Surface Disease Index (OSDI). For example, improvement of individual symptoms of dryness can be determined by evaluating various factors, including (a) “sensitivity to light”, (b) “eyes feeling gritty”, (c) “painful or sore eyes”, (d) “blurred vision”, (e) “poor vision”, (f) “reading problems”, (g) “problems with driving at night”, (h) “problems with working with a computer or bank machine (ATM)”, (i) “problems with watching TV”, (j) “uncomfortable under windy conditions”, (k)“uncomfortable in areas with low humidity” and (1) “uncomfortable in areas that are air conditioned.”EXAMPLES

[0198] Example 1: Preparation of an Exemplary Modified Secretome Composition of Mesenchymal Stem Cells (MSCs)

[0199] Mammalian mesenchymal stem cells (MSCs) are used in the preparation of a modified secretome composition having reduced angiogenic capacity and retained or enhanced activity relating to at least one of anti-inflammatory effects and immunosuppressant effects. In some examples, the MSCs are derived from bone marrow, peripheral blood, adipose tissue, placenta, or umbilical cord tissue of the mammal. In some examples, the mammal is a human. Human MSCs are characterized by the surface marker profile of lin- / CD45- / c-kit- / CD90+. Additional cell surfacemarker profiles for use in the preparation of a modified secretome as described herein include lin- / CD45- / c-kit- / CD90+ / Sca-l+ and lin- / CD45- / c-kit- / CD90+ / CD34. Stem cells appropriate for use may display the CD34+ positive marker at the time of isolation but lose this marker during culturing. Therefore, the full marker profile for one stem cell type that may be used according to the present application is lin- / CD45- / c-kit- / CD90+ / CD34, where CD34 is CD34+ or CD34-.

[0200] MSCs were cultured under conditions to produce a stem cell conditioned medium, such as described in US Patent Publication No. 20160145576A1. The pro-angiogenic factor, VEGFwas removed from medium conditioned by mammalian MSCs, i.e., the “secretome,” using VEGF antibody-coated beads.

[0201] A total of 10 mg / mL of VEGF antibody-coated beads were prepared using the anti hVEGF antibody (R &D Systems MAB9947) and the Novex® Life Technologies Dynabead Antibody Coupling Kit). 1 mg of these beads was incubated with 1 mL of secretome for 18 hours at room temperature. Following incubation, the VEGF and HGF concentrations in the secretome were analyzed using the R&D Systems Human VEGF DuoSet® ELISA (Catalog DY-293B) for VEGF. This pro-angiogenic factor depletion process was conducted in two separate trials. As shown in Figure 1, in both trials there is a 80-90% removal of VEGF using the anti-VEGF antibody coated beads resulting in less than 20% of VEGF remaining in the secretome composition.

[0202] Separately, MSCs were cultured under conditions to produce a stem cell conditioned medium, such as described in US Patent Publication No. 20160145576A1. The pro-angiogenic factor, VEGF was removed from medium conditioned by mammalian MSCs, i.e., the “secretome,” using VEGF specific siRNA. The VEGF removal using si RNA was performed as follows: First, oligo reconstitution was prepared: The scrambled siRNA and three VEGF specific siRNA oligos from Origene® were independently reconstituted in RNAse free buffer to give a finalconcentration of 2 nmole. The VEGF specific oligos were pooled to a final concentration of 20 or 60 or 180 pmoles in 0.15ml antibiotic free, phenol red free DMEM. In reparation for Transfection, to 0.15ml antibiotic free, phenol red free DMEM, 0.09ml ofLipofectamine RNAiMAXwas added.

[0203] The two aforementioned preparations were combined and allowed to incubate at room temperature for 5 minutes. Four transfection mixes were prepared1) Scrambled siRNA at 180 pmoles2) VEGF siRNA mix 20 pmoles3) VEGF siRNA mix 60 pmoles4) VEGF siRNA mix 180 pmoles.

[0204] Each mix was distributed dropwise to three pre-labeled wells of a 6-well plate with 50,000 ASC (passage 9) per well. 24h following transfection, the media was changed to 2.0ml antibiotic free, phenol red free DMEM per well. 72h following the media switch, the secretomes from three wells of a given siRNA treatment were pooled and centrifuged at low speed to remove any cellular debris. The supernatant was concentrated to 0.26ml final volume, the Amicon filter was rinsed with 0.14ml and combined to obtain 0.4ml final volume.

[0205] Each of such samples were examined by VEGF specific ELISA to determine the concentration of VEGF. As shown in Figure 2, the concentration of VEGF in the secrotome was below the level of detection after exposure to VEGF specific siRNA.

[0206] Reduction and / or removal of at least one angiogenic factor yields a “modified secretome” composition as described herein.

[0207]

[0208] Example 2: Preparation of an Exemplary Modified Secretome Composition of Adipose-Derived Mesenchymal Stem Cells (Ad-MSCs)

[0209] Adipose-derived mesenchymal stem cells (Ad-MSCs) are used in the preparation of a modified secretome composition having reduced angiogenic capacity and retained or enhancedactivity relating to anti-inflammatory effects and / or immunosuppressant effects. Cells are obtained from frozen stocks of previously collected human lipoaspirate and / or abdominal subcutaneous fat samples from at least three healthy adult donors aged 21-45. Prior to use, adipose-derived mesenchymal stem cells (Ad-MSCs) are routinely analyzed with fluorescent activated cell sorting (FACS) and appropriate differentiation potential to confirm cellular characteristics, as harmonized by multiple groups, and accepted by the International Society for Cellular Therapy.

[0210] Ad-MSC cell stocks are frozen at passages 2 and 3 and phenotypically characterized based on MSC cell surface markers, including, e.g., CD10, CD13, CD29, CD73, CD44. From each donor, Ad-MSC conditioned medium is generated by incubating 10 ml of serum- free medium, or preferably xeno-free medium, over an Ad-MSC monolayer (75 cm2, at 4xl04cells / cm2or equivalent) for at least about 24 to 72 hours, 48 to 72 hours, 24 to 96 hours, 48 to 96 hours or longer and is concentrated by centrifugation through Amicon Ultra Centrifugal Filter units with membranes selective for >3 kDa (EMD Millipore).

[0211] Frozen ASCs are thawed and plated in T flasks at 5000 cells / cm2in sterile filtered (0.22 pm) growth media (0.217 ml / cm2), which includes 90% (v / v)DMEM / Ham's F-12, 10% (v / v) FBS, rhEGF 1 ng / ml, rhFGF-basic 1 ng / ml, 1 pg / ml SoluCortef, and 1 pg / ml ascorbic acid. The cells are fed every 3 days and passaged at <80% confluence then replated at 5000 cells / cm2in growth media. When ASCs reach approximately 70-80 % confluence at desired batch size, e.g., approximately 2L, the ASCs are washed with 3 vol (0.217 ml / cm2) sterile PBS and fed with conditioning media (HyClone’s DMEM with High Glucose, with L-Glutamine, without Phenol Red, Sodium Pyruvate).

[0212] Conditioning media is harvested at 60 h after initial contact with ASCs, then centrifuged in sterile polypropylene tubes at 400 x g for 10 min. The supernatant is filtered througha 0.45 pm PES filter and may be stored overnight at 2-8°C. For downstream processing of conditioned media, small and large tangential flow filtration (TFF) columns are prepared by cleaning with 0.1 N NaOH in sterile water. The TFF column is then washed with sterile PBS until the pH of permeate is pH 7.4-7.6. Ports are capped with sterile luer caps and stored at 2-8°C for up to 2 days.

[0213] Conditioned media is concentrated using TFF to the desired endpoint while the concentrate reservoir rests on cold packs. The process may also be performed in a refrigerated cabinet. Conditioned media is concentrated using the large column (e.g., 1600 cm2) prior to the small column (e.g., 115 cm2), when the volume is reduced to < 500 ml, to achieve highest recovery. A sterile syringe is used to flush each column at the time of changing the column and at end of each run to maximize recovery. Once the final concentrate is retrieved, the conditioned medium may be subject to buffer exchange with sterile PBS.

[0214] The final product is filtered through 0.22 pm sterile syringe filter. Protein concentration is determined using a Bradford assay, and conditioned medium may be diluted with sterile PBS to a desired protein concentration. Conditioned medium may be stored in sterile polypropylene cryovials at < -70°C.

[0215] MSCs may also be exposed to a pro-inflammatory stimulus in culture, such as a cytokine, e.g., an interleukin, an interferon, a tumor necrosis factor, or a combination thereof. Pro- angiogenic factors, such as any one or more of VEGF, PDGF, bFGF, angiopoietins, and TGF-0, are removed from the Ad-MSC conditioned medium, i.e., the “secretome.” Such angiogenic factors may be removed with use of an antibody-mediated method. For example, the secretome may be treated with an anti-VEGF antibody, such as an anti-VEGF antibody loaded orimmobilized on a surface, e.g., a bead. Reduction and / or removal of at least one angiogenic factor yields a “modified secretome” composition as described herein.

[0216] The modified secretome composition may be further modified by adding at least one compound or protein that reduces angiogenic activity, such as an anti-angiogenic factor. The modified secretome composition may be additionally modified by adding at least one compound or protein that enhances anti-angiogenic activity, anti-inflammatory activity, or immunosuppressant activity. Such properties, i.e., anti-angiogenic, anti-inflammatory, and immunosuppressant effects may be evaluated as described in Example 1. An exemplary compound, which reduces angiogenic activity, for addition to the modified secretome composition is kaempferol. See, e.g., Xu et al., Braz J Med Biol Res. 2017 Mar 2;50(3):e5396. Exemplary proteins, which reduce angiogenic activity, for addition to the modified secretome composition include pigment epithelium-derived factor (PEDF) and PEDF-derived short peptide (PDSP).

[0217] MSCs secrete a plurality of paracrine factors, including factors which mediate angiogenesis and anti-inflammatory capacity. A modified secretome composition as described herein has reduced levels of angiogenic factors relative to an unmodified secretome composition, i.e., a secretome composition prepared using comparable methods without the additional step of removing angiogenic factors.

[0218] Example 3: Reduced Angiogenic Capacity of Modified Secretome

[0219] Reduced angiogenic capacity may be evaluated using an endothelial cell proliferation assay, anti-inflammatory activity can be determined by assessment of oxidative stress and inflammatory markers, and immunosuppressive effects can be assessed by evaluating inflammatory cytokine production, e.g., the suppression thereof, such as in comparison to an“unmodified” secretome, i.e., a secretome that has not been modified by removing an angiogenicfactor. Herein we evaluated the angiogenic activity of modified secretome, having reduced concentration of VEGF, using an endothelial cell proliferation assay. In the assay, CBF18 Cord blood endothelial cells from passages 7-9 were plated in a 48-well plate with EGM-2 medium(Lonza) supplemented with 10% defined FBS. After an overnight incubation at 37°C with 5% CO2, some wells were stained with Hoechst stain, and the starting cell count was done. The medium was then replaced with either basal EGM-2 with 5% FBS (baseline media), untreated secretome, or secretome treated with anti-VEGF antibody-coated beads. Following a 3-day incubation, the cells were fixed with 1% paraformaldehyde and stained with DAPI. A fluorescence microscope was used to count the cells and analyze them with ImageJ. As shown in Figure 3, endothelial proliferation was decreased in modified secretome as compared to unmodified secretome.

[0220] Example 4: Exemplary Use of a Modified Secretome Composition

[0221] Disclosed modified secretome compositions, including the exemplary compositions encompassed by Example 1, are used in the treatment of ocular diseases. Such use includes administration of a composition as described herein to a mammalian subject suffering from an ocular disease, preferably a human.

[0222] In one example, disclosed modified secretome compositions are used to treat dry eye, occurs when tears cannot properly lubricate the eyes. Dry eye may be caused by a variety of reasons, including but not limited to illness, e.g., graft versus host disease, rheumatoid arthritis, lupus, Graves' disease, diabetes, scleroderma, and Sjogren's syndrome, hormonal changes, and use of certain medications, e.g., tranquilizers, antihistamines, certain heart medications, diuretics, birth control pills and ulcer medications. In some cases, treatment with a disclosed modified secretome composition may involve co-administration with an additional therapeutic. For example, apharmaceutically acceptable product containing cyclosporine or another immunosuppressant, LFA-1, ICAM-1, a lubricant, or a saline solution may additionally be administered to a subject in conjunction with disclosed modified secretome compositions.

[0223] A disclosed modified secretome is more effective in the treatment of dry eye in comparison to an unmodified secretome composition, i.e., a secretome composition prepared by comparable methods but which retains angiogenic factors. Determining treatment efficacy and / or superiority is within the grasp of one of skill in the art and may include, e.g., analysis of Schirmer's (SH) test scores, tear breakup times (TBUT), corneal fluorescein staining scores (Oxford scale, 0- 4), and ocular surface disease indexes, or any combination of the preceding. See, e.g., Yang et al., Int J Environ Res Public Health. 2021 Mar 1 ; 18(5):2383.

[0224] Evaluation of Secretome for Treatment of Dry Eye Syndrome in Canines

[0225] The standard treatment for dry eye syndrome is a topical lacrimostimulant, such as cyclosporine (used at 0.2-2.0% for canines, which is a significantly higher concentration that used for human dosing), combined with or in addition to tear substitutes. In general, this high- concentration regimen is effective in restoring normal to near-normal ocular surface health for many individuals. Cyclosporine is usually administered twice daily, while artificial tears are often administered four times a day or even more often. Tear substitutes are generally considered safe compounds that can be administered without any harm to the ocular surface. They function in the wetting of the ocular surface and may have mechanical properties that enhance their staying power (or half-life). No modification of the ocular surface structure is typically expected following their use.

[0226] Tear substitutes often contain a compound to increase their viscosity, such as methylcellulose, hydroxypropyl methylcellulose (hypromellose), or hyaluronic acid (HA). It hasbeen shown that HA may lead to an increase in tear stability, thereby reducing some symptoms of the dry eye. Although artificial tears have been used for several years, a formulation that extends the contact time of the viscous compound with the ocular surface is desired, as it may allow for less frequent application, reducing cost and improving compliance.

[0227] In an open label, negatively controlled field study, canines will be randomly assigned based on the order of presentation of eligible animals for enrollment into the treatment group (receiving unmodified secretome). A minimum of 10 animals will be enrolled in the study.

[0228] The objective will be to evaluate the effectiveness of a topically applied adipose- derived secretome (Thera-101) applied three times daily for the treatment of immune-mediated KCS in client-owned dogs. This will be accomplished by standard methods for quantifying the aqueous tear production:1. Evaluation, relative to baseline, of clinical signs associated with KCS, measured prior to and periodically throughout the treatment period.2. Evaluation, relative to baseline, of Feming Test results, measured prior to and periodically throughout the treatment period.3. Evaluation, relative to baseline, of the Schirmer Tear Test (STT) results, measured prior to and periodically throughout the treatment period.4. Evaluation, relative to baseline, of the Tear Film Breakup Time (TFBUT) results, measured prior to and periodically throughout the treatment period.5. Evaluation, relative to baseline, of Rose Bengal Staining measured prior to and periodically throughout the treatment period.

[0229] In the Schirmer’s tear test (STT). A fdter paper is inserted in the conjunctival fornix and the length of wetting of the paper is measured after 1 minute. In dogs, values less than15mm / min are suspicious if combined with clinical signs of KCS, and values less than 1 Omm / min are considered to be diagnostic for the condition.

[0230] Tear film breakup time (TFBUT) is another widely used method of measuring the tear film stability and supporting a presumptive diagnosis of a qualitative tear abnormality. The test evaluates the ability of the corneal surface to retain a homogenous tear film. TFBUT is performed by instilling one drop of 1% or 2% fluorescein stain onto the eye, then manually holding the eyelids open. The time is recorded from the last blink to the appearance of the first dry spot, which appears as a dark area in the yellow-green fluorescein film. The normal reference values reported for dogs should be 20 seconds or longer. In animals affected with qualitative dry eye, the tear breakup usually occurs in less than 5 seconds.

[0231] Rose Bengal is an external ophthalmic stain used in the diagnosis of POTF disorders. Rose Bengal has been demonstrated to stain corneal and conjunctival epithelial cells that are not adequately covered by an intact mucin layer, thereby demonstrating the poor stability of the POTF. (4,7) The tear ferning test is a quick, simple and inexpensive test, which indirectly reflects the ocular surface osmolarity and electrolyte concentration in tears, and has shown a very high sensitivity and specificity for the diagnosis for dry eye in humans and in dogs. The tear sample is collected with a glass capillary tube applied to a glass slide then allowed to dry by evaporation for 10 minutes. The evaporation of the tears results in crystallization and arborization patterns (known as “ferning patterns”).EQUIVALENTS AND SCOPE

[0232] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of thepresent invention is not intended to be limited to the above, but rather is as set forth in the appended claims.

[0233] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0234] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses and descriptive terms, from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0235] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention is / are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity, those embodiments have not been specificallyset forth in haec verba herein. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps.

[0236] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranged can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0237] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of the ordinary skill in the art, which will 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, per the practice in the art. 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, the term can mean within an order of magnitude, for example 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.

[0238] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the method of the invention can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.Sequence Table

Claims

CLAIMSClaim 1. A topical eye composition for treatment of ocular disease in a subject, wherein the topical eye composition comprises a mesenchymal stem cell -conditioned (MSC-CM) medium.Claim 2. The topical eye composition of claim 1, wherein the topical eye composition comprises from 400 million particles per ml to 1.3 billion extracellular vesicle particles per ml.Claim3. The topical eye composition of claim 1 or 2 wherein the MSC-CM is a modified MSC- CM having reduced angiogenic capacity due to a reduced concentration of at least one angiogenic factor in the modified MSC-CM.Claim 4. The topical eye composition of claim 3, wherein the angiogenic factor is angiogenin (ANG), an endothelial growth factor, a fibroblast growth factor (FGF) protein, a hepatocyte growth factor (HGF) protein, an insulin-like growth factor binding protein, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-0), a vascular endothelial growth factor (VEGF) protein, or a combination thereof.Claim 5. The composition of any one of claims 3-4, wherein the concentration of VEGF protein and any one or more of the endothelial growth factor, the fibroblast growth factor (FGF) protein, the hepatocyte growth factor (HGF) protein, the insulin-like growth factor binding protein, platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-P) are reduced in the modified MSC-CM.Claim 6. The composition of claim 4wherein the VEGF protein is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PIGF), an isoform thereof, or a combination thereof.Claim 7. The composition of claim 3 wherein the concentration of the angiogenic factor in the modified MSC-CM is less than 20% of the concentration of the angiogenic factor in an unmodified MSC-CM.Claim 8. The composition of claim 7 wherein the modified MSC-CM has reduced angiogenic activity.Claim 9. The composition of claim 6 wherein the concentration of VEGF in the modified MSC- CM is less than 20% of the concentration of VEGF in an unmodified MSC-CM.Claim 10. A method of treating an ocular condition comprising topically administering to the eye of a mammal the topical eye composition of any one of claims 1-9.Claim 11. The method of claim 10 wherein the mammal is a canine.Claim 12. The method of claim 10 wherein the mammal is a human.Claim 13. A method of preparing a topical eye composition for treatment of ocular disease in a subject comprising conditioning a medium with mesenchymal stem cells, thereby producing a mesenchymal stem cell-conditioned medium (MSC-CM), and reducing the angiogenic capacityof the MSC-CM, wherein reducing the angiogenic capacity of the MSC-CM comprises removing an angiogenic factor from the MSC-CM, thereby preparing a modified MSC-CM composition.Claim 14. The method of claim 13 wherein the angiogenic factor removed is angiogenin (ANG), an endothelial growth factor, a fibroblast growth factor (FGF) protein, a hepatocyte growth factor (HGF) protein, an insulin-like growth factor binding protein, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-0), a vascular endothelial growth factor (VEGF) protein, or a combination thereof.Claim 15. The method of claim 13 wherein the angiogenic factor removed is VEGF protein and any one or more of angiogenic factors selected from endothelial growth factor, the fibroblast growth factor (FGF) protein, the hepatocyte growth factor (HGF) protein, the insulin-like growth factor binding protein, platelet-derived growth factor (PDGF) protein, and transforming growth factor beta (TGF-P).Claim 16. The method of any one of claims 13-15, wherein the VEGF protein is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (P1GF), an isoform thereof, or a combination thereof.Claim 17. The method of claim 13, wherein removing the angiogenic factor from the MSC-CM comprises subjecting the MSC-CM to antibody -based affinity chromatography, bead collection and removal, buffer exchange, filtration, immunoprecipitation, size exclusion, or a combination thereof.Claim 18. The method of claim 17, wherein the antibody is bevacizumab, brolucizumab-dbll, or ranibizumab, wherein the antibody targets VEGF-A, VEGF-B, P1GF, or a combination thereof.Claim 19. The method of claim 13, wherein removing the angiogenic factor from the MSC-CM comprising subjecting the MSC-CM to siRNA specific to the gene corresponding to the angiogenic factor or editing the gene corresponding to the angiogenic factor.Claim 20. The method of any one of claims 13-19, wherein removing the angiogenic factor reduces the amount of the angiogenic factor by 80-90% relative to the amount of the angiogenic factor present in the MSC-CM prior to removal of the angiogenic factor.Claim 21. The method of claim 13, wherein the extracellular vesicles are concentrated in the MSC-CM after the removal of the angiogenic factor.Claim 22. The method of claim 21 wherein the concentration of extracellular vesicles is from 40million particles per ml to 1.3 billion particles per ml.

Images