Compositions including decellularized extracellular matrix and a HIF-1 alpha stabilizing compound, and methods of using the same

Abstract

The present invention relates to a hydrogel composition including a decellularized extracellular matrix (ECM), wherein the ECM originates from a tissue lacking thrombospondin- 2 (TSP2), and a HIF-la stabilizing compound. The invention further relates to compositions and methods for promoting tissue regeneration in a subject by administering a hydrogel composition including a decellularized extracellular matrix (ECM), wherein the ECM originates from a tissue lacking thrombospondin-2 (TSP2), and a HIF-la stabilizing compound. The invention further provides methods for preparing a hydrogel composition including a decellularized extracellular matrix (ECM), wherein the ECM originates from a tissue lacking thrombospondin-2 (TSP2), and a HIF-la stabilizing compound.

Description

[0001] Attorney Docket No.: 047162-7519WO 1(02692)

[0002] COMPOSITIONS INCLUDING DECELLULARIZED EXTRACELLULAR MATRIX AND A HIF-1 ALPHA STABILIZING COMPOUND, AND METHODS OF USING THE SAME

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 706,808, filed October 14, 2025, which is incorporated herein byreference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under DK132645 awarded by National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Regenerative material requires use of biocompatible scaffolds, examples of which include synthetic polymers and decellularized extracellular matrix (ECM). ECM is a complex network of materials, such as proteins and polysaccharides, that are secreted locally by cells and remain closely associated with them. This non-cellular network of materials is present within all tissues and organs; it provides not only essential physical scaffolding for the cellular constituents, but also initiates crucial biochemical and biomechanical cues that are required for tissue morphogenesis, differentiation and homeostasis.

[0009] Advantages of decellularized ECM over synthetic materials include: existence of native ECM structure, retention of matrix-bound grow th factors and other bioactive components, and a favorable host response. Nevertheless, decellularized materials are not without limitations. While synthetic materials can be engineered to fit almost any need, decellularized materials can be difficult to customize, because they rely on a natural source (either animal tissues or cells grown in vitro).

[0010] Thrombospondin-2 (TSP2) is an anti-angiogenic, math cellular protein that interacts with ECM proteins and with a variety of cell surface receptors including CD36, CD47. heparin sulfate proteoglycan, low-density lipoprotein receptor-related protein, and ohfE. The phenotype of TSP2 knock-out (TSP2KO) mice is dominated by abnormalities in connective tissue and a platelet aggregation defect that manifests an abnormal bleeding tendency. ECM Attorney Docket No.: 047162-7519WO 1(02692) lacking TSP2 (“TSP2-null” or “TSP2KO’' ECM) derived from cells or tissue has been used to generate tunable hydrogels see, PCT / US2024 / 024208, WO 2019 / 083842, and WO 2017 / 189480).

[0011] Hydrogels are materials composed of polymers swollen with water and can be fabricated with synthetic or natural starting materials. Hydrogels formulated from natural sources are attractive because they should maintain a level of biochemical complexity not achievable with purified polymers.

[0012] There remains a need in the art for decellularized ECM, methods of preparing the decellularized ECM, and its use for regenerative medicine.

[0013] Diabetes affects 1 in 10 people in the United States and the global patient population is projected to exceed 693 million by 2024. With this increased prevalence, diabetes has become one of the leading causes of death and a substantial health concern worldwide. Moreover, diabetic patients are 2-4 times more likely to suffer from peripheral vascular diseases due to poor circulation and associated complications For example, these individuals are prone to the formation of diabetic foot ulcers (DFU), predominantly in the lower extremities due to vascular dysfunction. 15-25% of patients will develop DFU at some point during their lifetime, and the 5-year mortality rate of DFU is 44%. Notwithstanding the severity of the disease, the options of therapeutic agents are limited, problematic, and inefficient. For example, therapies that utilize growth factors are linked with increased cancer risk.

[0014] Thus, more effective, and safe therapies for DFU are needed.

[0015] SUMMARY

[0016] In one aspect, provided herein is a method for promoting tissue regeneration in a subject in need thereof, the method including administering to the subject a hydrogel which includes: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and aHIF-la stabilizing compound.

[0017] In another aspect, provided herein is a hydrogel composition which includes: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-la stabilizing compound.

[0018] In another aspect, provided herein is a method for preparing a hydrogel composition, the method including: providing a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and contacting the decellularized extracellular matrix (ECM) with a HIF-1 a stabilizing compound. Attorney Docket No.: 047162-7519WO 1(02692)

[0019] In another aspect, provided herein is a method of treating, ameliorating, and / or preventing diabetic foot ulcer on a subject in need thereof, the method comprising: applying to the ulcer a hydrogel composition comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-la stabilizing compound.

[0020] Other aspects, features and advantages will be apparent from the description, drawings and claims.

[0021] SUMMARY

[0022] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0023] Method for promoting tissue regeneration

[0024] In some aspects, the present invention is directed to a method for promoting tissue regeneration in a subject in need thereof.

[0025] In some embodiments, the method comprising administering to the subject a hydrogel.

[0026] In some embodiments, the hydrogel comprises a decellularized extracellular matrix (ECM) of a tissue, and a HIF-la stabilizing compound.

[0027] In some embodiments, the tissue lacks functional thrombospondin-2 (TSP2);

[0028] In some embodiments, the tissue is a musculoskeletal tissue.

[0029] In some embodiments, the HIF-la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI).

[0030] In some embodiments, the tissue is a muscle, a cartilage, a connective tissue, a tendon, a ligament, or a bone.

[0031] In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is a 2- oxoglutarate mimetic compound.

[0032] In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, and / or vadadustat.

[0033] In some embodiments, cells of the tissue lacking functional TSP2 comprise a TSP2- null knockout allele.

[0034] In some embodiments, cells of the tissue lacking functional TSP2 comprise uppressed TSP2 gene expression.

[0035] In some embodiments, the method enhances at least one biological response at the Attorney Docket No.: 047162-7519WO 1(02692) treatment site, as compared to a site administered a decellularized ECM originating from a tissue lacking functional TSP2 and lacking a HIF- la stabilizing compound.

[0036] In some embodiments, the method enhances at least one biological response at the treatment site, as compared to a site administered a decellularized ECM originating from a tissue comprising functional TSP2.

[0037] In some embodiments, the method enhances at least one biological response at the treatment site, as compared to an untreated site.

[0038] In some embodiments, the biological response at the treatment site is selected from the group consisting of cellular migration towards the treatment site, cellular invasion of the treatment site, vascular growth and maturation, innervation, angiogenesis, and wound repair.

[0039] In some embodiments, the tissue lacking functional TSP2 is muscle and wherein the subject suffers from at least one condition selected from the group consisting of type 1 diabetes and type 2 diabetes.

[0040] In some embodiments, the subject is a mammal.

[0041] In some embodiments, the tissue originates from a mammal selected from the group consisting of a mouse, a pig, anon-human primate, and a human.

[0042] In some embodiments, the subject is a human.

[0043] In some embodiments, the ECM is formulated with at least one additional therapeutic agent.

[0044] In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of an immunosuppressive agent, an anti-inflammatory agent, an antimetabolite, an antibiotic, an antibody, a growth factor, a cytokine, a gene therapy, an immunomodulator, and any combination thereof.

[0045] Hydrogel composition

[0046] In some aspects, the present invention is directed to a hydrogel composition.

[0047] In some embodiments, the hydrogel composition comprises: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF- la stabilizing compound.

[0048] In some embodiments, the tissue is a musculoskeletal tissue.

[0049] In some embodiments, the HIF-la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI).

[0050] In some embodiments, the tissue is muscle, cartilage, a connective tissue, a tendon, a ligament, or a bone. Attorney Docket No.: 047162-7519WO 1(02692)

[0051] In some embodiments, the HIF-PHI is a 2-oxoglutarate mimetic compound.

[0052] In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, and / or vadadustat.

[0053] In some embodiments, the cells of the tissue lacking functional TSP2 comprise a TSP2-null knockout allele.

[0054] In some embodiments, the cells of the tissue lacking functional TSP2 comprise suppressed TSP2 gene expression.

[0055] In some embodiments, the tissue lacking functional TSP2 originates from a mammal selected from the group consisting of a mouse, a pig, a non-human primate, and a human.

[0056] In some embodiments, the ECM is formulated with at least one additional therapeutic agent.

[0057] In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of an immunosuppressive agent, an anti-inflammatory agent, an antimetabolite, an antibiotic, an antibody, a growth factor, a cytokine, a gene therapy, an immunomodulator, and any combination thereof.

[0058] Method for preparing a hydrogel composition

[0059] In some aspects, the present invention is directed to a method for preparing a hydrogel composition.

[0060] In some embodiments, the method comprises providing a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and contacting the decellularized extracellular matrix (ECM) with a HIF-la stabilizing compound.

[0061] In some embodiments, the method further comprises formulating the ECM as a hydrogel in the presence of the HIF-la stabilizing compound.

[0062] In some embodiments, the tissue is a musculoskeletal tissue.

[0063] In some embodiments, the HIF-la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI).

[0064] In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is a 2- oxoglutarate mimetic compound.

[0065] In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat. desidustat, enarodustat, molidustat, roxadustat, or vadadustat. Attorney Docket No.: 047162-7519WO 1(02692)

[0066] In some embodiments, the cells of the tissue lacking functional TSP2 comprise a TSP2-null knockout allele.

[0067] In some embodiments, the cells of the tissue lacking functional TSP2 comprise suppressed TSP2 gene expression.

[0068] Method of treating, ameliorating, and / or preventing diabetic foot ulcer

[0069] In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing diabetic foot ulcer on a subject in need thereof.

[0070] In some embodiments, the method comprises applying to the ulcer a hydrogel composition comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-la stabilizing compound.

[0071] BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the appended drawings. For illustrating the invention, specific embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0073] FIGS. 1A-1H show that diabetic wounds are more hypoxic than WT and DBDB TSP2KO wounds. Representative images of immunohistochemistry of hydroxy probe in (FIG. 1 A) WT, (FIG. I B) DBDB, and (FIG. 1 C) DBDB TSKP2KO wounds at D7, and (FIGS. 1D- 1F) D10 (Corresponding whole wound area images are shown in FIG. 7). Hypoxia staining area quantified in WT, DBDB, and DBKO whole wound sections at (FIG. 1G) D7 and (FIG. 1H) D10. Scale bar = 50 pm. (n=5, one-way ANOVA, *, p<0.05. **, p< 0.01, ***. p<0.001).

[0074] FIGS. 2A-2F show that the expression level of TSP2 is significantly increased in diabetic wounds but the expression level of HIF-1 alpha is similar between WT, diabetic (DBDB), and diabetic TSP2KO (DBKO) wounds. (FIG. 2A) Western blot of TSP2 and HIF- 1 alpha in protein extracted from WT, DBDB. DBKO w ounds at D7. Quantification of (FIG. 2B) TSP2 and (FIG. 2C) HIF-1 alpha expression in western blot. (FIG. 2D) Immunofluorescence detection of CD31, Vimentin, and HIF-la in D7 WT, DBDB, DBKO wounds. Quantification of (FIG. 2E) CD31+HIF-la+ / CD31+ and (FIG. 2F) Vimentin+HIF- la+ / Vimentin+ by colocalization analysis (n=3, one-way ANOVA, *, p<0.05, **, p< 0.01, ***, pO.001).

[0075] FIGS. 3A-3E show' that diabetic DFs are resistant to hypoxia in terms of TSP2 Attorney Docket No.: 047162-7519WO 1(02692) expression. (FIG. 3A) Western blot of TSP2 in cell lysates from WT and DBDB primary DFs cultured with hypoxia chamber (denoted by hypo) (0.1% O2). and DMOG (2 mM) (denoted by DM) for 24 hours. Quantification of TSP2 expression in (FIG. 3B) WT and (FIG. 3C) DBDB DFs. Detection of mRNA expression level of HIF- la target genes, (FIG. 3D) GLUT1, and PGK-1 in WT, DBDB, and DBKO DFs. (n=3, one-way ANOVA, *, p<0.05, **, p<0.01, ***, pO.OOl).

[0076] FIGS. 4A-4C show that TSP2KO + DMOG hydrogel reduces hypoxia in D7 and D10 diabetic wounds. Representative images (FIG. 4A) and quantifications (FIG. 4B, FIG. 4C) of immunochemistry staining of hydroxy probe in D7 and D10 diabetic wounds that are untreated or treated with KO hydrogel, KO+DMOG hydrogel, or DMOG. (blue star indicates gel area, n=4-7, ANOVA, *. p<0.05, **, p<0.01, ****, p<0.0001).

[0077] FIGS. 5A-5K show that KO + DMOG hydrogel could further improve diabetic wound healing. (FIG. 5A) Representative photos of diabetic wounds that are untreated or treated with KO, KO+DMOG gel, or DMOG. Quantification of wound closure in (FIG. 5B) D7 and (FIG. 5C) D14 wound (%). Representative images of trichrome staining on diabetic wounds that are (FIG. 5D) untreated, or treated with (FIG. 5E) KO, (FIG. 5F) KO+DMOG gel, or (FIG. 5G) DMOG. Quantification of (FIG. 5H) wound gap, (FIG. 51) granulation area in four groups, and (FIG. 5J) remaining gel thickness, and (FIG. 5K) number of closed epithelial gaps in KO and KO+DMOG groups. (Arrow points to the unclosed epithelium, yellow star indicates the gel. n=4-16, ANOVA, *. p<0.05, **. p<0.01, ***. p< 0.001, ****, p<0.0001).

[0078] FIG. 6A-6H shows characterization on DI 4 diabetic wounds that are untreated, or treated by KO, KO+DMOG hydrogel, and DMOG. (FIG. 6A) The representative images of CD31 staining. (FIG. 6B) The quantification of CD31+lumen / HPF in the D14 wounds. The representative images of immunofluorescence staining of (FIG. 6C) Vimentin, (FIG. 6E) Mac3, and (FIG. 6G) a-SMA. The quantification of (FIG. 6D) Vimentin+ Area, (FIG. 6F) Mac3+ area, and (FIG. 6H) a-SMA+lumen / HPF in the D14 wounds. (n=4-l 1, ANOVA, *, p<0.05, **, p<0.01).

[0079] FIG. 7 shows representative images of D7 and D10 whole wound area showing hydroxyprobe detection via immunohistochemistry in WT, DBDB, and DBDB TSKP2KO (DBKO).

[0080] FIG. 8 shows TSP2 expression overlaps with hypoxic region in diabetic wounds. Immunofluorescence-staining of hypoxia and TSP2 in WT and DBDB wounds at D10 (arrows point to signals).

[0081] FIG. 9 shows mRNA expression level of HIF-la in D7 wounds. Attorney Docket No.: 047162-7519WO 1(02692)

[0082] FIG. 10 shows flow cytometry of fibroblast marker Vimentin suggests over 90% fibroblast population in isolated DFs.

[0083] FIGS. 11 A-l 1C show that DMOG can inhibit TSP2 upregulation in fibroblasts caused by hyperglycemia and promote cell migration. (FIG. 11 A) Western blot of TSP2 in media from NIH 3T3s cultured in low / high glucose media with or without DMOG (ImM) at different time points. (FIG. 1 IB) Scratch assay and (FIG. 11C) Quantification of closed area (%) in the two groups in low / high glucose (n=3, Unpaired Student T-test, **, p<0.01).

[0084] FIG. 12 shows that TSP2 is not required for HIF-la stabilization. mRNA expression of GLUT1 and PGK-1 in TSP2KO DFs cultured in control or hypoxia chamber for 24 hrs (n=3, Unpaired Student T-test, *, p<0.05).

[0085] FIG. 13A shows representative images of SEM photos of KO hydrogels (6mg / ml) without and with DMOG incorporated. FIG. 13B shows the cumulative release curve of DMOG from WT and TSP2KO hydrogels (6mg / ml) in PBS.

[0086] FIGS. 14A-14B show in vitro dose-dependent DMOG toxicity in hydrogel. FIG. 14A, DAPI / Phalloidin staining of NIH3T3s cultured on KO hydrogels with different concentrations of DMOG at DI. FIG. 14B, NIH3T3 proliferation rate on hydrogels with different DMOG concentrations at DI, D2, and D3. (N = 3, ANOVA, *, P < 0.05, **, PO.01).

[0087] FIGS. 15A-15C show interactions between NIH3T3 fibroblasts and hydrogels. (FIG. 15 A) DAPI / Phalloidin staining of NIH3T3s cultured on WT, KO, and KO+DMOG hydrogels at DI and D5, and quantification of their proliferation. (FIG. 15B) H&E staining of NIH3T3s invasion in the WT, KO, and KO+DMOG hydrogels. (FIG. 15C) Phase contrast images of NIH3T3s transmigrated to the bottom of trans-well (N = 3).

[0088] FIG. 16 shows KO+DMOG hydrogel promotes in vitro angiogenesis. Representative light microscopic images of HUVECs seeded on WT, KO, and KO+DMOG hydrogel at 24 hours, and quantification of angiogenesis (n = 3, ANOVA, *, p<0.05).

[0089] FIG. 17A shows representative images of western blot on D7 diabetic wounds treated with TSP2KO hydrogel with / without DMOG. FIG. 17B: Densitometric analysis of TSP2 expression, (n = 5, Unpaired Student T-test, *, p< 0.05).

[0090] FIGS. 18A-18E show (FIG. 18A) Representative images of hydroxy probe, Vimentin, CD31, and HIF-la immunofluorescence in D7 diabetic wounds treated by TSP2KO hydrogel with / without DMOG (KO vs KO+DMOG). Asterisk and orange boxes indicate the gel and the area analyzed, respectively. High-magnification images of the latter are shown and numbers indicate percent positive area. Quantification of (FIG. 18B) Vimentin+, (FIG. 18C) Attorney Docket No.: 047162-7519WO 1(02692)

[0091] CD31+, (FIG. 18D) total HIF-la+ area (%), and (FIG. 18E) HIF-la+ area / number of nuclei (a.u.) inside the gel. (n = 3, Unpaired Student T-test, *, p< 0.05).

[0092] FIG. 19 shows representative images of trichrome staining on D7 diabetic wounds that are untreated, or treated with KO, KO+DMOG hydrogels, or DMOG.

[0093] DETAILED DESCRIPTION

[0094] DFU has a ven’ complex pathobiology involving hyperglycemia and hyperinsulinemia that lead to irregular cell signaling including nitric oxide (NO) / eNOS and hypoxia-inducible factor 1 alpha (HIF-la) pathways, resulting in vascular dysfunction. Macro- and microvascular dysfunction causes chronic tissue hypoxia, peripheral neuropathy, abnormal inflammatory responses, and extracellular matrix (ECM) remodeling. Additionally, irregular hypoxia and ECM modulation interfere with cell functions, inhibit neovascularization, and delay repair. Due to the complexity and severity of these abnormalities, effective therapies have not been realized. Emerging strategies are designed to target multiple processes during healing. Therefore, delineating the interactions between critical factors like hyperglycemia, hypoxia, and ECM remodeling can enhance our understanding of DFU and lead to the discovery of new molecular targets and the development of new7therapeutic strategy7.

[0095] Thrombospondin-2 (TSP2), an ECM matricellular protein, is dysregulated in diabetes and associated with the etiology of DFU. TSP2. mainly produced by fibroblasts and smooth muscle cells (SMCs), is present at low7to negligible levels in intact skin. However, TSP2 is induced and expressed at high levels during the ECM remodeling phase of w ound healing and dictates a series of physiological processes. Primarily, TSP2 exerts an anti-angiogenic effect via inhibition of proliferation and induction of apoptosis of endothelial cells (ECs). Furthermore, TSP2 coordinates cell-ECM interactions and ECM assembly by modulating growth factor release and levels of extracellular enzymes like matrix metalloproteinases (MMPs) levels, demonstrated by increased levels of soluble vascular endothelial growth factor (VEGF) and MMPs in wounds and cells of TSP2KO mice. Additionally, numerous studies using animal models have shown that the rate of wound healing is negatively correlated with TSP2 expression. For example, increased TSP2 expression is associated with delayed wound closure. It is also found that the expression level of TSP2 was elevated in wounds of both diabetic patients and a genetic mouse model of type 2 diabetes (DBDB). Strikingly, genetic depletion of TSP2 in diabetic mice improved wound healing with enhanced angiogenesis, suggesting that TSP2 could be a novel target in DFU. How ever, a Attorney Docket No.: 047162-7519WO 1(02692) small molecule targeting TSP2 has not been described, and its complex structure and function do not allow simple screening of candidate drugs. Targeting TSP2-associated processes, on the other hand, could be an alternative approach. TSP2 can be regulated by hyperglycemia- and hypoxia- related pathways in vitro. Specifically, high glucose stimulates TSP2 production in fibroblasts mainly via the hexosamine pathway. Hypoxia, on the contrary, inhibits TSP2 expression in vitro via HIF-la.

[0096] In one aspect, a method for promoting tissue regeneration in a subject in need thereof is described, the method comprising administering to the subject a hydrogel comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-la stabilizing compound. In some embodiments, the tissue is from a mammal. Throughout the application, thrombospondin-2 can be represented with TSP2, THBS2, or Thbs2 and can be used interchangeably. In some embodiments, the HIF-la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI). In some embodiments, the HIF proly l hy droxy lase inhibitor (HIF-PHI) is a 2-oxoglutarate mimetic compound. In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, or vadadustat.

[0097] In another aspect, a hydrogel composition is described, the hydrogel composition comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-la stabilizing compound. In some embodiments, the HIF-l a stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF- PHI). In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is a 2- oxoglutarate mimetic compound. In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, and / or vadadustat.

[0098] In another aspect, a method for preparing a hydrogel composition is described, the method comprising: providing a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and contacting the decellularized extracellular matrix (ECM) with a HIF-la stabilizing compound. In some embodiments, the HIF-la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF- PHI). In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is a 2- oxoglutarate mimetic compound. In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat. desidustat, enarodustat, molidustat, roxadustat, and / or vadadustat. Attorney Docket No.: 047162-7519WO 1(02692)

[0099] In another aspect, a method of treating, ameliorating, and / or preventing diabetic foot ulcer on a subject in need thereof is provided. In certain embodiments, the method comprises: applying to the ulcer a hydrogel composition comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF- la stabilizing compound. In some embodiments, the HIF-1 a stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI). In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is a 2-oxoglutarate mimetic compound. In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, and / or vadadustat.

[0100] Definitions

[0101] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0102] It is also to be understood that the terminology7used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0103] The articles “a” and “an” are used herein to refer to one or to more than one (i.e.. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0104] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0. 1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0105] The term “additional therapeutic agent” refers to a therapeutic agent additional to a HIF-la stabilizing compound in the hydrogel compositions described herein.

[0106] The term “allogeneic” refers to organs, tissues, or cells originating from different individuals of the same species.

[0107] By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a marker or clinical indicator as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10%-100% change in measured levels (e.g, 10, 20, 30, 40, 50, 60, 75, 80, 85, 90, 95, 100%). Attorney Docket No.: 047162-7519WO 1(02692)

[0108] The term “autologous’' with reference to an organ, tissue, or cell denotes that the organ, tissue, or cell originates from the same individual.

[0109] The term “biocompatibility” refers to the properties of materials, such as a medical device or an implant, device being biologically compatible by not eliciting unwanted local or systemic responses from a living system or tissue. In other embodiments, the device does not elicit any significantly and / or measurably deleterious responses from the living system or tissue. A biocompatible device is substantially non-toxic, non-injurious or non-inhibiting or non-inhibitory to cells, tissues, organs, and / or organ systems that would come into contact with the device, scaffold, composition, etc.

[0110] The term “coating” refers to a covering, layer or film, of a substance applied to the surface of a substrate. The coating may be an all-over coating, completely covering the substrate, or it may only cover parts of the substrate.

[0111] As used herein, the term “comminute” and any other word forms or cognates thereof, such as, without limitation, “comminuting”, refers to the process of reducing larger particles into smaller particles, including, without limitation, by grinding, blending, shredding, slicing, milling, or cutting. ECM can be comminuted while in any form, including, but not limited to, hydrated forms, frozen, air-dried, lyophilized, powdered, sheet-form.

[0112] The expression “difference in the level of’ or “differentially present” refers to differences in the quantity and / or the frequency of a marker present in a sample taken from subjects having a disease as compared to a control subject. A marker can be differentially present in terms of quantity, frequency or both. A difference in the level of a polypeptide is present between two samples if the amount of the polypeptide in one sample is statistically significantly different from the amount of the polypeptide in the other sample. Alternatively or additionally, a polypeptide is differentially present between two sets of samples if the frequency of detecting the polypeptide in a diseased subjects’ samples is statistically significantly higher or lower than in the control samples. A marker that is present in one sample, but undetectable in another sample is differentially present.

[0113] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, antitumor activity as determined by any means suitable in the art. Attorney Docket No.: 047162-7519WO 1(02692)

[0114] “Encoding"’ refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (z.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0115] As used herein “endogenous” with reference to a material denotes that the material is from or produced inside an organism, cell, tissue or system.

[0116] As used herein, the term “exogenous” with reference to a material denotes that the material is introduced from or produced outside an organism, cell, tissue or system.

[0117] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0118] The terms “extracellular matrix” or “ECM” refer to proteins that are secreted by cells and assembled in a three dimensional manner to provide structural support for cells. Generally, extracellular matrix comprises proteins such as collagens (e.g. type I, III, IV, and V collagens), vitronectin, fibronectin, laminin, thrombospondin, entactin. and nidogen: and glycosaminoglycans and proteoglycans. However, it is noted that the extracellular matrix can vary in composition, and structural assembly, depending on its anatomic origin. In some instances, ECMs include an isolated basement membrane produced by vascular endothelial cells and a membrane on which the cells rest in vivo. Non limiting examples of ECMs are those originating from a tissue of a mammal, such as muscle (e.g., skeletal muscle, smooth muscle, or cardiac muscle), cartilage, a connective tissue, a tendon, a ligament, or bone. While matrices may differ somewhat in their composition, they are primarily composed of collagens (e.g. type I, III, IV, VI collagens), fibronectin, laminins, and other matricellular proteins. Despite the variation due to anatomic origin, extracellular matrix from any anatomic site could be useful in the present invention. Of particular interest in the present invention, are ECMs that comprise extracellular molecules that form a three-dimensional structure supporting cell and tissue growth. The ECMs of the present invention originate from a tissue lacking functional TSP2.

[0119] The term “HIF-1 a stabilizing compound” as used herein refers to a compound that, Attorney Docket No.: 047162-7519WO 1(02692) when exposed to cells expressing HIF-la, reduces, inhibits, slows or otherwise mitigates degradation of HIF-la under normoxic conditions. In some embodiments, a HIF-l a stabilizing compound can be an HIF prolyl hydroxylase inhibitor (HIF-PHI). In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is a 2-oxoglutarate mimetic compound. In some embodiments, the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, or vadadustat. Salts and tautomers of the above-mentioned HIF-PHI compounds are also contemplated.

[0120] The term “hydrogel” as used herein refers to a gel in which the liquid component comprises water.

[0121] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0122] The term “immune response” as used herein is defined as a host response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0123] As used herein, the terms “immunosuppression” or “immunosuppressive therapy (1ST)” involve an act that reduces the activation or efficacy of the immune system. Deliberately induced immunosuppression is performed to prevent the body from rejecting an organ transplant, treating graft-versus-host disease after a bone marrow transplant, or for the treatment of auto-immune diseases such as rheumatoid arthritis or Crohn’s disease.

[0124] As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may. for example, be affixed to a container which contains the nucleic acid, peptide, and / or composition of the invention or be shipped together with a container Attorney Docket No.: 047162-7519WO 1(02692) which contains the nucleic acid, peptide, and / or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

[0125] As used herein, the phrases “lacks functional TSP2” and “lacking functional TSP2” with reference to a tissue denote that the expression of wild-ty pe TSP2 or any functional variant thereof in the cells of the tissue is eliminated or significantly reduced compared to a wild-type tissue that is otherwise the same type of tissue. In some embodiments, a tissue lacking or that lacks functional TSP2 comprises cells which comprise one or both of (a) a TSP2-null knockout (KO) allele; and (b) suppressed TSP2 gene expression. TSP2 nucleic acid and / or amino acid sequence are disclosed by NCBI ascension numbers (NCBI Gene IDs: 7058 and 21826): NG 022911.2, NM 001381939.1, NM 001381940.1, NM 001381941.1, NM_001381942.1, NM_003247.5, NP_001368868.1, NP_001368869.1, NP_001368870.1, NP_001368871.1, NP_003238.2, NM_011581.3, and NP_035711.2, each of which is herein incorporated by reference in its entirety7. In some embodiments, the TSP2 nucleic acid and / or amino acid sequence comprises a sequence as set forth in NG_022911.2, NM_001381939. 1, NM_001381940.1. NM_001381941.1, NM_001381942.1, NM_003247.5, NP_001368868.1, NP 001368869.1, NP_001368870.1, NP_001368871.1, NP_003238.2, NM_011581.3, or NP_035711.2.

[0126] A TSP2 knock out allele or suppressed TSP2 gene expression can be obtained via a genetic engineering technique comprising a nuclease. Exemplary nucleases include, but are not limited to, a clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease, a transcription activator-like effector nuclease (TALEN), and a zinc- finger nuclease. A TSP2KO allele may comprise a non-functional TSP2 variant and / or may comprise a mutation or variant which eliminates expression of functional TSP2. Alternatively, or in addition, cells of the tissue lacking functional TSP2 may comprise an inhibitory RNA molecule which suppresses TSP2 gene expression. Examples of such inhibitory RNA molecules include, but are not limited to, an RNA interference (RNAi) RNA, a short hairpin RNA (shRNA). a small interfering RNA (siRNA), a trans-acting siRNA (tasiRNA), a micro RNA (miRNA), an antisense RNA (asRNA), a long noncoding RNA (IncRNA), a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a guide RNA (gRNA), a single guide RNA (sgRNA), a double-stranded RNA (dsRNA), a ribozy me, and any combination thereof.

[0127] By "marker" is meant any protein or polynucleotide having an alteration in level or activity that is associated with a disease or disorder. Attorney Docket No.: 047162-7519WO 1(02692)

[0128] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids therein.

[0129] The term “model organism” refers to a non-human species that is easy to maintain and breed in a laboratory setting and has particular experimental advantages. Model organisms as used herein provide an in vivo model to research the effects of a human disease or condition and / or biological activities associated with a disease or condition, such as thrombosis.

[0130] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0131] “Monitoring” refers to recording changes in a continuously varying parameter (e.g. monitoring progression of a disease).

[0132] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0133] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0134] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0135] The language “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the Attorney Docket No.: 047162-7519WO 1(02692) formulation, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable earners include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, ■‘pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity7of the compound, and are physiologically acceptable to the subject. Supplementary7active compounds may also be incorporated into the compositions.

[0136] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any' means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library7or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0137] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more Attorney Docket No.: 047162-7519WO 1(02692) amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0138] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0139] The terms “purified”, “biologically pure” or “isolated” as used herein mean having been increased in purity, wherein “purity” is a relative term, and not to be necessarily construed as absolute purity. For example, the purity of a substance, for example, but not limited to a nucleic acid, can be at least about 50%, can be greater than 60%, 70%, 80%, 90%, 95%, or can be 100%. The terms “purified”, “biologically pure” or “isolated” refer to material that is free to vary ing degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically7determined using analytical chemistry7techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially Attorney Docket No.: 047162-7519WO 1(02692) purified form, or can exist in a non-native environment such as, for example, a host cell.

[0140] As used herein, '’sample'’ or “biological sample’7refers to anything, which may contain an analyte (e.g, polypeptide, polynucleotide, or fragment thereof) for which an analyte assay is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. In certain embodiments, a biological sample is a salivary7sample. Such a sample may include diverse cells, proteins, and genetic material. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s). Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like.

[0141] By the term “specifically binds,’' as used herein with respect to an antigen binding molecule is meant an antigen binding molecule which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antigen binding molecule that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity7does not itself alter the classification of an antigen binding molecule as specific. In another example, an antigen binding molecule that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antigen binding molecule as specific. In some instances, the terms “specific binding’' or “specifically binding,’" can be used in reference to the interaction of an antigen binding molecule, an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g, an antigenic determinant or epitope) on the chemical species; for example, an antigen binding molecule or an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antigen binding molecule is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antigen binding molecule, will reduce the amount of labeled A bound to the antigen binding molecule.

[0142] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g, mammals). A “subject” or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, non-human primates, livestock, and pets, such as simian, ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is human.

[0143] A “target site” or “target sequence” refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under Attorney Docket No.: 047162-7519WO 1(02692) conditions sufficient for binding to occur.

[0144] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0145] As used herein, the term “tissue” refers to a mammalian tissue, such as connective tissue, muscle tissue, nervous tissue, and epithelial tissue. Examples of connective tissue include bone, cartilage, blood, and fat. Examples of muscle tissue include skeletal muscle, smooth muscle, and cardiac muscle. Nervous tissue is found in the nervous system, including the brain and spinal cord. Epithelial tissue is found in the skin, the lining of the mouth and nose, and the lining of the digestive system. In certain embodiments, the tissue of the present invention is not skin. In some embodiments, the tissue is muscle (e.g., skeletal muscle). In some embodiments, the tissue is bone. In some embodiments, the tissue is cartilage.

[0146] As used herein, the term “transplantation” refers to the process of taking a cell, tissue, or organ, called a “transplant” or “graft” from one individual and placing it or them into a (usually) different individual. The individual who provides the transplant is called the “donor” and the individual who received the transplant is called the “host” (or “recipient”). An organ, or graft, transplanted between two genetically different individuals of the same species is called an “allograft”. A graft transplanted between individuals of different species is called a “xenograft”.

[0147] As used herein, “transplant rejection” refers to a functional and structural deterioration of the organ due to an active immune response expressed by the recipient, and independent of non-immunologic causes of organ dysfunction.

[0148] As used herein, the term “tolerance” is a state of immune unresponsiveness specific to a particular antigen or set of antigens induced by previous exposure to that antigen or set. Tolerance is generally accepted to be an active process and. in essence, a learning experience for T cells. Tolerance, as used herein, refers to the inhibition of a graft recipient’s ability to mount an immune response which would otherwise occur, e.g., in response to the introduction of a non-self MHC antigen into the recipient. Tolerance can involve humoral, cellular, or both humoral and cellular responses.

[0149] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely- eliminated.

[0150] The term “xenogeneic” with reference to an organ, tissue, or cell denotes that the Attorney Docket No.: 047162-7519WO 1(02692) organ, tissue, or cell originates from an individual that is of a different species than the recipient.

[0151] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3. 4, 5, 5.3. and 6. This applies regardless of the breadth of the range.

[0152] The following abbreviations are used herein: ECM, extracellular matrix; HA, hyaluronic acid; H&E, hematoxylin and eosin; HUVECs, Human umbilical vein endothelial cell; KO, knock-out; SEM, scanning electron microscope; TSP2, thrombospondin-2; WT, wild-type.

[0153] Description

[0154] The present invention relates to compositions and methods for promoting tissue regeneration in a subject in need thereof. In certain aspects, the subject is administered a hydrogel composition including a decellularized ECM of a tissue, wherein the tissue lacks functional TSP2, and a HIF-la stabilizing compound. In some embodiments, the tissue is from a mammal. In certain embodiments, the mammal is a human.

[0155] In certain aspects, the invention provides a hydrogel composition including decellularized ECM of a tissue, wherein the tissue lacks TSP2, and a HIF-la stabilizing compound. In some embodiments, the tissue is from a mammal. In certain embodiments, the mammal is a human.

[0156] In some embodiments, the ECM is formulated as a hydrogel, such as a tunable hydrogel. In certain embodiments, the decellularized ECM is chemoattractant to at least one cell type selected from the group consisting of endothelial cell, mesenchymal cell, myocyte, fibroblast, and osteoblast. In some embodiments, the ECM and compositions of the invention are useful for the methods recited elsewhere herein.

[0157] Methods of the invention Attorney Docket No.: 047162-7519WO 1(02692)

[0158] Method of promoting tissue regeneration

[0159] In some aspects, the invention provides a method for promoting tissue regeneration in a subject in need thereof, the method comprising administering to the subject a hydrogel composition including a decellularized ECM of a tissue, wherein the tissue lacks functional TSP2, and a HIF-la stabilizing compound. In some embodiments, the tissue is from a mammal.

[0160] In some embodiments, the tissue is a musculoskeletal tissue. The tissue can be a muscle, a cartilage, a connective tissue, a tendon, a ligament, or a bone.

[0161] In some embodiments, the HIF-la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI). The HIF prolyl hydroxylase inhibitor (HIF-PHI) can be a 2-oxoglutarate mimetic compound. The HIF prolyl hydroxylase inhibitor (HIF-PHI) can be dimethyloxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, and / or vadadustat.

[0162] In some embodiments, cells of the tissue lacking functional TSP2 can comprise:

[0163] (a) a TSP2-null knockout allele; and / or

[0164] (b) suppressed TSP2 gene expression.

[0165] In some embodiments, the TSP2 null knockout allele or suppressed TSP2 gene expression is obtained via a genetic engineering technique comprising a nuclease selected from the group consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease, a transcription activator-like effector nuclease (TALEN). and a zinc-finger nuclease.

[0166] In some embodiments, cells of the tissue lacking functional TSP2 comprise an inhibitory RNA molecule which suppresses TSP2 gene expression.

[0167] In some embodiments, the inhibitory RNA molecule is selected from the group consisting of: an RNA interference (RNAi) RNA, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a trans-acting siRNA (tasiRNA), a micro RNA (miRNA), an antisense RNA (asRNA), a long noncoding RNA (IncRNA), a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a guide RNA (gRNA), a single guide RNA (sgRNA), a double-stranded RNA (dsRNA), a ribozyme, and any combination thereof.

[0168] In some embodiments, the method enhances at least one biological response at the treatment site, as compared to (1) a site administered a hydrogel composition comprising a decellularized ECM originating from a tissue lacking functional TSP2 and lacking a HIF-la stabilizing compound, (2) a site administered a hydrogel composition comprising a decellularized ECM originating from a tissue comprising functional TSP2, with or without a Attorney Docket No.: 047162-7519WO 1(02692)

[0169] HIF-la stabilizing compound, or (3) an untreated site. The biological response at the treatment site can be selected from the group consisting of cellular migration towards the treatment site, cellular invasion of the treatment site, vascular growth and maturation, innervation, angiogenesis, and wound repair.

[0170] In certain embodiments, the tissue lacking functional TSP2 is muscle and the subject suffers from at least one condition selected from the group consisting of a muscle injury, a myopathy, a genetic myopathy, an inflammatory myopathy, an endocrine myopathy, a neuromuscular disorder, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, multiple sclerosis, a muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Limb Girdle muscular dystrophy, spinal muscular atrophy, Guillain-Barre Syndrome, Chronic Inflammatory Demyelinating Polyneuropathy, Multifocal Motor Neuropathy, Myasthenia Gravis, Pompe’s Disease, Drop Head Syndrome (floppy head syndrome), multisystemic smooth muscle dysfunction syndrome, peripheral vascular disease, congenital heart disease, a cardiomyopathy, coronary artery disease, heart attack, heart valve disease, hypertension, hernia, type 1 diabetes, type 2 diabetes, alcohol use, and tobacco use.

[0171] In some embodiments, the tissue lacking functional TSP2 is muscle and the subject suffers from at least one condition selected from the group consisting of type 1 diabetes and type 2 diabetes.

[0172] In some embodiments, the tissue lacking functional TSP2 is bone and the subject suffers from at least one condition selected from the group consisting of a bone injury, a bone disorder, osteoporosis, osteopetrosis, osteonecrosis, osteogenesis imperfecta, osteoarthritis, rheumatoid arthritis, ty pe 1 diabetes, type 2 diabetes, lupus, celiac disease, hyperthyroidism, infection of bone or joint, Paget's Disease of Bone, fibrous dysplasia, tobacco use, and weight loss surgery’.

[0173] In some embodiments, the tissue lacking functional TSP2 is cartilage and wherein the subject suffers from at least one condition selected from the group consisting of osteoarthritis, rheumatoid arthritis, avascular necrosis, costochondritis, a fracture, lupus, Maffucci syndrome, osteoporosis, achondroplasia, conclusion, and herniation.

[0174] In some embodiments, the subject is a mammal. In some embodiments, the tissue originates from a mammal selected from the group consisting of a mouse, a pig, a non-human primate, and a human. In some embodiments, the subject is a human.

[0175] The tissue lacking functional TSP2 can be autologous, allogeneic, or xenogeneic relative to the subject. In some embodiments, the tissue lacking functional TSP2 is not skin.

[0176] In some embodiments, the ECM is formulated with at least one additional therapeutic Attorney Docket No.: 047162-7519WO 1(02692) agent. The at least one additional therapeutic agent can be selected from the group consisting of an immunosuppressive agent, an anti-inflammatory agent, an antimetabolite, an antibiotic, an antibody, a growth factor, a cytokine, a gene therapy, an immunomodulator, and any combination thereof.

[0177] Promoting tissue regeneration in a subject includes, but is not necessarily limited to, promoting cellular migration, cellular invasion, vascular growth and maturation, and / or wound repair in the site of ECM administration. In some embodiments, promoting tissue regeneration comprises accelerating cellular migration, enhancing cellular invasion, enhancing vascular grow th and maturation of a location to be treated, and enhancing wound repair in a subject in need thereof. In certain embodiments, cellular migration, cellular invasion, vascular growth and maturation, and / or wound repair are enhanced or accelerated at the site of ECM administration to the subject as compared to a location administered an ECM originating from a wild-type tissue, or an untreated location.

[0178] In certain embodiments, the tissue lacking functional TSP2 is selected from the group consisting of connective tissue, muscle tissue, nervous tissue, and epithelial tissue. Examples of connective tissue include bone, cartilage, blood, and fat. Examples of muscle tissue include skeletal muscle, smooth muscle, and cardiac muscle. Nervous tissue is found in the nervous system, including the brain and spinal cord. Epithelial tissue is found in the skin, the lining of the mouth and nose, and the lining of the digestive system. In certain embodiments, the tissue lacking functional TSP2 is not skin. In some embodiments, the tissue lacking functional TSP2 is muscle (e.g., skeletal muscle, smooth muscle, or cardiac muscle). In some embodiments, the tissue lacking functional TSP2 is skeletal muscle. In some embodiments, the tissue lacking functional TSP2 is bone.

[0179] In certain embodiments, the tissue lacking functional TSP2 is formulated as a hydrogel. In some embodiments, the ECM is dried and rehydrated in an aqueous solution to form the hydrogel. In some embodiments, the ECM is treated to form a pre-gel as described herein and the pre-gel is further treated to form the hydrogel as described herein. The pre-gel or the hydrogel can be contacted with a HIF-la stabilizing compound prior to administering the hydrogel to the subject.

[0180] The method for promoting tissue regeneration in a subject in need thereof is performed at one or more treatment site(s) of the subject. That is, in certain embodiments, the hydrogel is administered to one or more treatment site(s) of the subject. In some embodiments, the treatment site(s) may be any site in need of tissue regeneration, such as, but not limited to, a wound or injury. In other embodiments, the hydrogel is administered by any Attorney Docket No.: 047162-7519WO 1(02692) method known in the art, e.g., subcutaneous, intramuscular, intraosseous, or topical administration.

[0181] In certain embodiments, tissue lacking functional TSP2 is obtained by a method described herein. For example, cells of the tissue lacking functional TSP2 comprise one or both of (a) a TSP2 knockout allele, and (b) suppressed TSP2 gene expression.

[0182] Methods of preparing tissue lacking functional TSP2, and of preparing ECM from such tissues, are described in, for example, WO 2017 / 189480, WO 2019 / 083842, and PCT / US2024 / 24208.

[0183] In various aspects, the subject of the method is a mammal, such as, but not limited to, a human, a non-human primate, a pig, a sheep, or a mouse. In certain embodiments, the subject is a human. In certain embodiments, the tissue lacking functional TSP2 originates from a mammal, such as, but not limited to, a mouse, a pig, a non-human primate, and a human. In other embodiments, the tissue lacking functional TSP2 is autologous, allogeneic, or xenogeneic relative to the subject. In the case of allogeneic or xenogeneic tissue, the tissue may be genetically engineered to be more immunologically compatible with the subject.

[0184] In certain embodiments, the hydrogel composition including decellularized ECM is formulated with at least one additional therapeutic agent beyond the HIF-la stabilizing compound. In some embodiments, the method for promoting tissue regeneration further comprises administering a therapeutic agent, or a pharmaceutical composition comprising a therapeutic agent, to the subject. Suitable therapeutic agents to be formulated with the decellularized ECM or administered separately from the decellularized ECM are known to those in the art and include, but are not limited to, an immunosuppressive agent, an antiinflammatory agent, an antimetabolite, an antibiotic, an antibody, a growth factor, a cytokine, a gene therapy, an immunomodulator, and any combination thereof.

[0185] In still further embodiments, the tissue (e.g., bone, cartilage, muscle) that lacks functional TSP2 can be a source of material that provides for a decellularized ECM prepared therefrom that lacks the functional TSP2. In other embodiments, hydrogels, scaffolds, and other materials lacking the functional TSP2 can be prepared from the decellularized ECM lacking the functional TSP2.

[0186] In some embodiments, the decellularized ECM lacking the functional TSP2, and the hydrogels, scaffolds, and other material prepared therefrom that also lack the functional TSP2 and include a HIF-la stabilizing compound, are capable of promoting re-endothelialization.

[0187] Methods of preparing decellularized ECM Attorney Docket No.: 047162-7519WO 1(02692)

[0188] In some embodiments, the ECM is obtained from a mammalian tissue lacking functional TSP2. In certain embodiments, the tissue is obtained from a mammal using methods known to those skilled in the art, such as cutting, excision, and / or shaving. In some embodiments, tissue can be derived from aggregates of cells, an organ, portions of an organ, or combinations of organs. In some instances, the tissue is isolated from a mammal, for example and without limitation, human, non-human primate, monkey, pig, cattle, sheep, and mouse. In some embodiments, the tissue is isolated from any tissue of a subject (e.g.. mammal), for example and without limitation, muscle (e.g., skeletal muscle, connective, myocardium), bone, urinary bladder, liver, central nerv ous system (CNS), adipose tissue, small intestine, large intestine, colon, esophagus, pancreas, vascular tissue (e.g., artery, vein) and heart.

[0189] The ECM is the natural substrate on which cells migrate, proliferate, and differentiate. These components are linked in such a way that the resulting structure is tri-dimensional scaffolding in vivo. Thus, in some embodiments, the decellularized ECM of the invention provides scaffolding, support and strength to cells grown on and / or therein, allowing those cells to differentiate and mediate physiologic responses. ECMs from different anatomic sites may vary in their ability to support and allow for proper differentiation of cells not from that respective anatomic site. In some embodiments, without wishing to be bound by any theory, the decellularized ECM is of tissues derived from the same species as the recipient or from species known to in the art to have compatibility with the recipient. In some embodiments, if the recipient is a human, the decellularized ECM originates from a tissue of an animal known to have compatibilities with humans such as, but not limited to, a primate or a pig. In some embodiments, the animal has been genetically engineered, such as a genetically engineered pig or primate, to be more compatible with the human recipient compared to the wild-type tissue.

[0190] Tissue lacking functional TSP2

[0191] Generation of a tissue lacking functional TSP2 can be accomplished in several ways. In some aspects, the absence of a functional TSP2 in the tissue may be achieved by a full or partial knock-out of the TSP2 gene in the tissue. Methods of gene knock-out are well known in the art. In some embodiments, knock-out can be accomplished through a variety7of well- established molecular techniques. In some embodiments, individual stem cells are genetically- transfected with the DNA construct for the goal of creating a transgenic organism (e.g., mammal) that has the altered gene. Embry onic stem cells are genetically transformed and Attorney Docket No.: 047162-7519WO 1(02692) inserted into early embryos. The resulting transgenic animals with the genetic alteration in their germline cells then pass the knock-out to future generations. For instance, a knock-out mouse refers to a mouse in which a gene or genes have been mutated such that the activity of the gene has been reduced or eliminated. In some embodiments, the TSP2 gene is knocked out in a genetically engineered organism such as a mouse or a pig. In other aspects, the TSP2 gene is knocked down in a tissue using molecular techniques known in the art such as, but not limited to, RNA interference (RNAi), small hairpin RNA (shRNA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs). Knocked-down expression of TSP2 is useful for generation of tissue lacking functional TSP2 in organisms where knock-out of TSP2 is not usually possible or desired, e.g., in humans. In some embodiments, the tissue lacking functional TSP2 comprises a TSP2 knockout genotype. In other embodiments, expression of TSP2 in the tissue has been knocked down. In certain embodiments, expression of TSP2 is diminished when compared with wild type expression, and / or is eliminated altogether. In some aspects, the characteristics of an ECM produced from a TSP2 knockdown are optimized and similar to those of an ECM produced from a TSP2 knock-out.

[0192] In some embodiments, cells of the tissue lacking functional TSP2 comprise one or both of the following: (a) a TSP2-null knockout allele; and (b) suppressed TSP2 gene expression. In certain embodiments, the TSP2 null knockout allele or suppressed TSP2 gene expression is obtained via a genetic engineering technique comprising a nuclease selected from the group consisting of a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CAS) nuclease, a transcription activator-like effector nuclease (TALEN), and a zinc-finger nuclease. In some embodiments, cells of the tissue lacking functional TSP2 comprise an inhibitory RNA molecule which suppresses TSP2 gene expression. Inhibitory RNA molecules are well-known in the art and include, but are not limited to, an RNA interference (RNAi) RNA, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a trans-acting siRNA (tasiRNA), a micro RNA (miRNA), an antisense RNA (asRNA), a long noncoding RNA (IncRNA), a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a guide RNA (gRNA), a single guide RNA (sgRNA), a double-stranded RNA (dsRNA), a ribozyme, and any combination thereof.

[0193] In some embodiments, the tissue is a muscle, a cartilage, a connective tissue, a tendon, a ligament, or a bone. In some embodiments, the tissue lacking functional TSP2 is muscle, such as skeletal muscle, smooth muscle, or cardiac muscle. In some embodiments, the tissue lacking functional TSP2 is bone. In some embodiments, the tissue lacking functional TSP2 is cartilage. In certain embodiments, the tissue lacking functional TSP2 is Attorney Docket No.: 047162-7519WO 1(02692) not skin.

[0194] Decellularizing tissue

[0195] The tissue of this invention can be decellularized by methods known in the art. In one aspect, decellularization of the tissue is performed to prevent a pro-inflammatory response in the subject. As such, in one aspect, a decellularized ECM refers to ECM material that is decellularized to the extent that a pro-inflammatory response, and thus growth of fibrotic tissue, is not elicited to any substantial degree in favor of constructive remodeling.

[0196] In certain embodiments, decellularization of the tissue comprises treating the tissue with a decellularization solution comprising one or more of tiypsin-EDTA, TRIS, triton, sodium deoxy cholate (SDC), and the like. In certain embodiments, the decellularization solution does not comprise sodium dodecyl sulfate (SDS).

[0197] In certain embodiments, the decellularized ECM as described herein retains activity of at least a portion of its structural and non-structural biomolecules, including, but not limited to, collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and / or growth factors. In certain embodiments, the activity of the biomolecules within the ECM can be removed chemically or mechanically, for example, by cross-linking and / or by dialyzing the ECM. In one aspect, the decellularized ECM composition of this invention is cross-linked by addition of a chemical cross-linking agent. In other aspects, the ECM materials described herein essentially have not been cross-linked and / or dialyzed. Thus, in one aspect, the ECM material is not cross-linked and / or dialyzed in anything but a trivial manner which does not substantially affect the gelation and functional characteristics of the ECM material in its uses described herein.

[0198] In certain embodiments, the tissue lacking functional TSP2 is muscle. In some embodiments, decellularizing the muscle tissue comprises treating the muscle at room temperature (RT) sequentially with (i) an aqueous trypsin-EDTA solution for about 5-7 hours; (ii) an aqueous H2O2 solution for about 15 minutes; (iii) an aqueous Triton X-100 / EDTA / Tris solution for about 5-7 hours; (iv) a fresh aqueous Triton X-100 / EDTA / Tris solution for about 5-15 hours; and (v) an aqueous Triton X-100 / sodium deoxycholate (SDC) solution for about 5-7 hours. Exemplary concentrations for the decellularization solutions include: (i) 0.25% trypsin-EDTA solution; (ii) 3% H2O2 solution; (iii) 1% Triton X-100 I 0.26% EDTA / 0.69% Tris; (iv) 1% Triton X-100 / 0.26% EDTA / 0.69% Tris; and (v) 1% Triton X-100 / 0.2% SDC.

[0199] In certain embodiments, the tissue lacking functional TSP2 is cartilage. Generally, Attorney Docket No.: 047162-7519WO 1(02692) prior to decellularization, the sample has skin and fat remnants removed. The sample is then sterilized with ethanol and oxidized with 3% hydrogen peroxide. Decellularization comprises mixing the sample with 1% Triton solution / EDTA / Tris for about 36 hours. After, the sample is washed with ethanol / peracetic acid / ddH2O. Finally, the sample is incubated in FBS free culture media with 1% antibiotics overnight and then lyophilized.

[0200] In certain embodiments, the tissue lacking functional TSP2 is bone. Generally, the bone is sectioned to produce fragments. The bone is then demineralized to generate demineralized bone matrix (DBM). Bone demineralization can be achieved using any method known in the art. For example, in some embodiments, the bone is demineralized under agitation, e.g., at about 300 rpm, in an aqueous acid solution at about RT for about 24 hours (i.e., about 20-28 hours). In some embodiments, the aqueous acid solution comprises about 0.5 N HC1 (approximately 25 ml / g bone). For embodiments where the tissue is bone, decellularizing the tissue comprises decellularizing the DBM. In some embodiments, decellularizing the DBM comprises incubating the DBM with agitation with an aqueous trypsin-EDTA solution at approximately 37 °C for about 24 hours (i.e., about 20-28 hours). Exemplary concentrations for the trypsin-EDTA solution include 0.05% trypsin and 0.02% EDTA.

[0201] Formulation of the ECM

[0202] In certain embodiments, the decell ularized ECM of this invention is formulated as sheet of material such as but not limited to STRATTICE™ or ALLODERM™ regenerative tissue matrix (Allergan, Dublin, Ireland).

[0203] In certain embodiments, the decellularized ECM is formulated as a hydrogel. In some embodiments, the tissue is muscle and the ECM is dried, such as by lyophilization, and then rehydrated to generate the hydrogel. Suitable solutions for rehydrating the ECM include, but are not limited to, water, saline, phosphate-buffered saline. In some embodiments, the solution for rehydrating the ECM includes the HIF-la stabilizing compound. In some embodiments, the hydrogel is contacted with the HIF-la stabilizing compound (e.g., as a solution containing the the HIF-la stabilizing compound) after the hydrogel has been fully rehydrated.

[0204] In some embodiments, the tissue is bone, and the decellularized ECM is formed into a pre-gel by removing residual cellular materials and digesting with pepsin for about 96 hours (e.g., for about 84 to about 108 hours) at RT. Next, the pepsin digestion is stopped and the digested ECM pre-gel is incubated for about 1 hour at higher temperature, such as at Attorney Docket No.: 047162-7519WO 1(02692) approximately 37 °C, whereby the pre-gel forms into a hydrogel. In some embodiments, the pre-gel is contacted with the HIF- la stabilizing compound, before and / or during incubation at higher temperature.

[0205] In some instances, such as when the tissue is bone, the hydrogel is produced from a reverse gel (i.e., a pre-gel), which forms a hydrogel upon an increase in temperature. As the temperature rises above a certain temperature in a reverse gel, a hydrogel is formed. The general concept of reverse gelation of polymers and, e.g., its relation to lower critical solution temperature (LCST) are broadly known in the chemical arts. The ECM compositions described herein are prepared, for example, from decellularized or devitalized, intact ECM as described elsewhere herein. An ECM gel is prepared by digestion of the ECM material with an acid protease, neutralization of the material to form a pre-gel, and then raising the temperature of the pre-gel above a gelation temperature, for example the LCST of the pre-gel, to cause the pre-gel to become a gel. As used herein, the term “gel” includes hydrogels. The transition temperature for acid-protease-digested from solution to gel is ty pically within the range of from about 10 °C to 40 °C and any increments or ranges therebetween, for example from about 20 °C to 35 °C. For example, the pre-gel can be warmed to about 37 °C to form a hydrogel.

[0206] In some instances, such as when the tissue is cartilage, the decellularlized ECM is formed into a hydrogel by weighing lyophilized tissues and then the tissues undergo Cryomill™ or cryogenic grinding prior to digestion.

[0207] Tissue for preparation of ECM, ECM-derived pre-gel solutions, and gels as described herein may be harvested in any useful manner. Decellularized or devitalized ECM can be dried, either lyophilized (freeze-dried) or air dried. The ECM composition is optionally comminuted at some point, for example prior to acid protease digestion in preparation of an ECM gel, for example prior to or after drying. In certain embodiments, the decellularized TSP2-null ECM of the invention is comminuted. The comminuted ECM can also be further processed into a powdered form by methods, for example and without limitation, such as grinding or milling in a frozen or freeze-dried state.

[0208] To prepare solubilized ECM tissue, the ECM is digested with an acid protease in an acidic solution to form a digest solution. As used herein, the term “acid protease” refers to an enzyme that cleaves peptide bonds, wherein the enzyme has increased activity of cleaving peptide bonds in an acidic pH. For example and without limitation, acid proteases include pepsin and trypsin and mixtures thereof.

[0209] As an example, the digest solution of ECM is kept at a constant stir for a certain Attorney Docket No.: 047162-7519WO 1(02692) amount of time at room temperature. In one aspect, the pH is maintained at less than pH 4.0 or at pH 2.0 ± 0.3 during acid protease digestion of the decellularized tissue as described herein. The ECM digest can be used immediately or can be stored at -20 °C or frozen at, for example and without limitation, -20 °C or -80 °C. In certain aspects, the ECM digest is snap frozen in liquid nitrogen. To form a “pre-gel” solution, the pH of the digest solution is raised to a pH between 6.8 and 7.8. The pH can be raised by adding one or more of a base or an isotonic buffered solution, for example and without limitation, NaOH or PBS at pH 7.4. In some aspect, the pre-gel solution is freeze dried and stored at -20 °C or -80 °C until needed. The method optionally does not include a dialysis step prior to gelation, yielding a more complete ECM-like matrix that typically gels at 37 °C more slowly than comparable collagen or dialyzed ECM preparations. The gel therefore retains more of the qualities of native ECM due to retention of many native soluble factors, such as, without limitation, cytokines. Without intending to be limited to any particular theory, these factors contribute to chemoattraction of cells and proper rearrangement of tissue at the site of wound or injury, rather than a fibrotic response that leads to unwanted scarring. In other embodiments, the ECM is dialyzed prior to gelation to remove certain soluble components.

[0210] As used herein, the term “isotonic buffered solution” refers to a solution that is buffered to a pH between 6.8 and 7.8, e.g., pH 7.4, and that has a balanced concentration of salts to promote an isotonic environment. As used herein, the term “base” refers to any compound or a solution of a compound with a pH greater than 7. For example and without limitation, the base is an alkaline hydroxide or an aqueous solution of an alkaline hydroxide. In certain embodiments, the base is NaOH, or NaOH in PBS. This “pre-gel” solution can, at that point be incubated at a suitably warm temperature, for example and without limitation, at about 37 °C to gel.

[0211] In the method of preparing an ECM gel, the ECM may be partially or completely digested with the acid protease, such as pepsin. The digested ECM is then neutralized to a pH of 6.8-7.8, e.g, 7.2-7.6, or 7.4 and the neutralized and digested ECM material is gelled by incubation at a temperature at which the material gels, e.g., at a temperature above 20, 25, 30, or 35 °C, such as at 37°. The degree of digestion can be determined by comparison on a gel, or by ascertaining the degree of degradation of hyaluronic acid, for example by Western blot (anti-hyaluronic acid antibodies are commercially available from multiple sources) or chromatographic methods, as are broadly known. For example in a partial digestion, hyaluronic acid is digested less than 50%, 40%, 30%, 25%, 20% or 10%.

[0212] Compositions and methods of the present invention are useful for treatment of Attorney Docket No.: 047162-7519WO 1(02692) mammals, and particularly humans. In certain embodiments, the mammal is immune compromised, suffers from an autoimmune disease, has or will have transplant, or suffers from a condition with high risk for wounds. In certain embodiments, the mammal suffers from at least one condition selected from the group consisting of: diabetes, hernia, mastectomy, peripheral vascular disease, and neuropathy. In certain embodiments, the mammal is in need for regenerative medicine to replace or repair a tissue or organ that has been damaged by a disease, a trauma or a congenital issue (such as, but not limited to, empty nose syndrome). In other embodiments, the compositions and methods of the present invention are useful for aesthetic purposes.

[0213] Combination Therapies

[0214] The hydrogel compositions described herein are also useful when combined with at least one additional compound (e.g., beyond the HIF-la stabilizing compound). The additional compound may comprise commercially available compounds known to promote wound healing, or to treat, prevent, or reduce the symptoms associated with graft transplants or implantation of a device into a subject.

[0215] In one aspect, the present invention contemplates that the hydrogel compositions of the invention may be used in combination with a therapeutic agent such as an immunosuppressive agent. Non-limiting examples of immunosuppressive agents known in the art are cyclosporine, azathioprine, everolimus and glucocorticoids, mycophenolic acid, fingolimod. Anti metabolites (such as, but not limited to, methotrexate, fluorouracil), antibiotics (such as, but not limited to, dactinomycin, mitomycin C, bleomycin), and antibodies (such as, but not limited to, Atgam, Muromonab-CD3, basiliximab, daclizumab).

[0216] In another aspect, the present invention provides the hydrogel compositions of the invention as a delivery vehicle for one or more active pharmaceutical agents or drugs. In certain embodiments, the hydrogel compositions of the invention further comprises at least one active pharmaceutical agent selected from the group consisting of a Rael inhibitor, a NFKB inhibitor, a p38 MAPK inhibitor, a RhoA inhibitor, a growth factor (including, but not limited to, VEGF, PDGF and BMP-2), Fasudil, Ripasudil, an antibiotic, immune modulators (including, but not limited to, IL-4, IL-33 and IL- 10), an anti -infl ammatory (including, but not limited to, glucocorticoids and NSAIDs), a cytokine and oligonucleotides (including, but not limited to, siRNA, shRNA, plasmid DNA, and / or virus for gene therapy). In yet other embodiments, the at least one active pharmaceutical agent is an anti-inflammatory drug that influences the response of macrophages, such as, but not limited to BAY-1 1. In yet other Attorney Docket No.: 047162-7519WO 1(02692) embodiments, the at least one pharmaceutical agent is selected from the group consisting of CAS 1177865-17-6, SB 202190 SB203580, RKI-1447, and Y-27632.

[0217] Pharmaceutical Compositions and Formulations

[0218] The invention also includes the use of a pharmaceutical composition combined with the hydrogel compositions as described herein for use in the methods of the invention. In some embodiments, the method for promoting tissue regeneration further comprises administering an additional therapeutic agent (i.e., additional to the HIF-la stabilizing compound), or a pharmaceutical composition comprising an additional therapeutic agent, to the subject. Methods for promoting tissue regeneration further comprises administering a therapeutic agent, or a pharmaceutical composition comprising a therapeutic agent, to the subject. Suitable therapeutic agents to be formulated with the hydrogel compositions or administered separately from the hydrogel compositions are known to those in the art and include, but are not limited to, an immunosuppressive agent, an anti-inflammatory agent, an antimetabolite, an antibiotic, an antibody, a growth factor, a cytokine, a gene therapy, an immunomodulator, and any combination thereof.

[0219] Such a pharmaceutical composition is in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The various components of the pharmaceutical composition may be present in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0220] In an embodiment, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In another embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng / kg / day and 500 mg / kg / day.

[0221] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary’, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0222] Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of Attorney Docket No.: 047162-7519WO 1(02692) administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically- based formulations. The route(s) of administration is readily apparent to the skilled artisan and depends upon any number of factors including the type and severity of the disease or wound being treated, the type and age of the veterinary7or human patient being treated, and the like.

[0223] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory7methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory7ingredients, and then, if necessary7or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0224] As used herein, a “unit dose’’ is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as. for example, one-half or one- third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0225] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it is understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary- pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0226] In certain embodiments, the compositions are formulated using one or more pharmaceutically acceptable excipients or carriers. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable earners are described in Attorney Docket No.: 047162-7519WO 1(02692)

[0227] Remington’s Pharmaceutical Sciences, 1991, Mack Publication Co., New Jersey.

[0228] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or poly alcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0229] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0230] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzy l alcohol and 0.05% to 0.5% sorbic acid.

[0231] The composition preferably includes an antioxidant and a chelating agent which inhibits the degradation of the compound. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. Preferably, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Particularly preferred chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about Attorney Docket No.: 047162-7519WO 1(02692)

[0232] 0.01% to 0.20% and more preferably in the range of 0.02% to 0. 10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition which may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0233] Kit

[0234] In one aspect of the invention, a commercial kit is provided comprising the hydrogel compositions described herein. A kit comprises suitable packaging material and the hydrogel compositions, or key components required for a user to prepare the hydrogel compositions using the components supplied in the kit. In certain embodiments, the kit comprises a decellularized ECM formulated as a sheet of material (e.g. STRATTICETMor ALLODERM™) or a digested decellularized ECM solution in a vessel, which may be the packaging, or which may be contained within packaging; and a HIF- la stabilizing compound, optionally in a separate vessel. The HIF-la stabilizing compound may be supplied as a solid or in solution.

[0235] In certain embodiments, if the sheet of material or digest solution is neutralized, it may be frozen, cooled; e.g., kept at near-freezing temperatures, such as, without limitation, below about 4 °C or kept at room temperature, e.g., 20-25 °C. In another embodiment, the kit comprises a first vessel containing an acidic solution comprising a pre-neutralization digest as described elsewhere herein, and a second vessel comprising a neutralizing solution comprising a base and / or buffer(s) to bring the acidic solution of the first vessel to physiological ionic strength and pH. to form a neutralized digest. In a further embodiment, the first vessel contains a terminally sterilized, lyophilized, pre-neutralization digest that can be hydrated using water or a suitable aqueous solution that optionally neutralizes the acid. In some embodiments, the neutralizing solution can optionally include the HIF-la stabilizing compound. In this embodiment, a second vessel is optionally provided comprising a neutralization solution as described above that is capable of both hydrating the lyophilized product and neutralizing it, or optionally a third vessel comprising water or any other suitable solution useful in hydrating the lyophilized product prior to neutralization with the neutralization solution. This kit also optionally comprises a mixing needle and / or a cold-pack. The vessel may be a vial, syringe, tube or any other container suitable for storage and transfer in commercial distribution routes of the kit. Attorney Docket No.: 047162-7519WO 1(02692)

[0236] The kit can optionally include instructions for the user, instructing how to store, handle, prepare, and use the hydrogel compositions.

[0237] Administration / Dosing

[0238] The regimen of administration may affect what constitutes an effective amount. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0239] The compositions described herein find use as, without limitation, a composition to promote tissue regeneration, e.g., wound healing (for example, of a diabetic foot ulcer) an injectable graft (e.g., xenogeneic, allogeneic or autologous) for tissues, for example, bone or soft tissues, in need of repair or augmentation most typically to correct a wound, a trauma or disease-induced tissue defects. The compositions also may be used as a filler for implant constructs comprising, for example, a molded construct formed into a desired shape for use in cosmetic or trauma-treating surgical procedures.

[0240] Administration of the compositions of the present invention to a subject (being a patient), preferably a mammal, more preferably a human, may be carried out using know n procedures, at dosages and for periods of time effective to treat the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination wi th the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0241] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the Attorney Docket No.: 047162-7519WO 1(02692) unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of wound in a patient.

[0242] Routes of Administration

[0243] One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route.

[0244] Routes of administration of any of the compositions of the invention include parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral. vaginal (e.g.. trans- and perivaginally), (intra)nasal, and (trans )rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intraosseous, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, sheets of material (e.g. STRATTICE ™ or ALLODERM™) or hydrogels that could be sutured into wounds or used as supporting meshes / slings, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

[0245] In certain embodiments, the composition of the invention is applied to a bandage or dressing, which is then applied to the wound or treatment site of a subject. For example. In certain embodiments, a dressing is soaked in a liquid solution or liquid suspension comprising the hydrogel composition. In other embodiments, the hydrogel composition is incorporated into a pharmaceutical formulation including topical ointments, creams, aerosol sprays, and the like.

[0246] In certain embodiments, the administration of the hydrogel composition is at least one selected from the group consisting of subcutaneous, intramuscular, intraosseous, and topical. In other embodiments, the composition of the invention is injected, seeded or surgically- implanted to the region to be treated. Attorney Docket No.: 047162-7519WO 1(02692)

[0247] EXPERIMENTAL EXAMPLES

[0248] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0249] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0250] Materials And Methods

[0251] Animals

[0252] C57bl / 6J. db / db (B6.BKS(D)-Leprdb / J). and db / db TSP2KO mice aged 12 to 14 weeks were used. C57bl / 6J and db / + mice were purchased from Jackson Laboratory. The generation of db / db (DBDB) and db / db TSP2KO (DBKO) animals was described previously18. All animal study procedures were approved by the Yale Institutional Animal Care and Use Committee (IACUC). All mice were kept in a 12-hour light / dark environment and fed ordinary feed. All authors complied with ARRIVE guidelines.

[0253] In Vivo Wound Healing

[0254] For hypoxia detection experiments, twelve-week-old wild-type C57BL / 6J, diabetic db / db mice (B6.BKS(D)-Leprdb / J, The Jackson Laboratory), and db / db TSP2KO were used for wound creation as described previously (Kunkemoeller B, Bancroft T, Xing H, et al. Elevated thrombospondin 2 contributes to delayed wound healing in diabetes. Diabetes. 2019;68(10):2016-2023. doi:10.2337 / dbl8-1001; Morris AH, Lee H, Xing H, Stamer DK, Tan M, Kyriakides TR. Tunable Hydrogels Derived from Genetically Engineered Extracellular Matrix Accelerate Diabetic Wound Healing. ACSAppl Mater Interfaces. 2018;10(49):41892-41901. doi: 10.1021 / acsami.8b08920). Two full-thickness wounds were created on the dorsal region using a 6mm biopsy (Acu-Punch) after hair removal. Buprenorphine (0.05-0. 1 mg / kg) and Rimadyl (5.0 mg / kg) were administered prior to and after surgery. For hydrogel evaluation experiments, wounds in db / db mice were either untreated or filled with 40 pL of pre-gel solution which gelled in situ, or 40 pL 1.4 mg / ml DMOG solution, and then covered with Tegaderm (3M) secured in place by sutures. The Attorney Docket No.: 047162-7519WO 1(02692) animals were monitored for 14 days and photos were taken every other day for wound evaluation.

[0255] Wounds were excised with surrounding tissue for analysis following euthanasia at days 7, 10, and 14 post-wounding. Half of the wounds were snap frozen using liquid nitrogen and stored in -80°C for later protein and RNA extraction. The other half were fixed in 10% neutral -buffered formalin (NBF) (Sigma- Aldrich) and embedded in paraffin. Five- micrometer sections were used for H&E and Masson's Trichrome staining and analyzed by immunohistochemistry or immunofluorescence.

[0256] Hydroxyprobe Assay

[0257] Hypoxyprobe™-! (Hypoxyprobe, Inc.) solution was prepared according to manufacturer's protocol. Briefly, solid pimonidazole HC1 was dissolved in 0.9% saline water to make a 50mg / ml stock solution and sterile filtered. This pimonidazole solution was injected into mice intraperitoneally at a dose of 60mg / kg body weight. After one hour, mice were euthanized by CO2 and the wounds were processed as previously described.

[0258] Tissue Immunohistochemistry (IHC) and Immunofluorescence (IF)

[0259] Paraffin-embedded tissue slides were processed according to standard protocols. For antigen retrieval, slides were boiled for 30 min in citrate buffer (10 mM Citric Acid, 0.05% Tween 20 pH=6.0). Prior to IHC staining, slides were treated with 3% H2O2 / 0. 1% sodium azide in methanol for 30 min to quench endogenous peroxidase activity and with 1% BSA in PBS for 30 min to block non-specific binding. Incubation with the primary antibody was performed at 4°C overnight and incubation with the secondary antibody, either biotin- conjugated or fluorescence-tagged, was conducted for 1 hr at RT. Immune reactions were visualized with the Vector ABC Elite and DAB peroxidase (HRP) substrate kits (Vector Laboratories). Nuclei were then counter-stained with either methyl green or DAPI.

[0260] Dermal Fibroblasts Isolation and Cell Culture

[0261] Dermal fibroblasts (DFs) were isolated from mouse dorsal skin as described previously (Xing H, Huang Y, Kunkemoeller BH, et al. Dysregulation of TSP2-Racl- WAVE2 axis in diabetic cells leads to cytoskeletal disorganization, increased cell stiffness, and dysfunction. Sci Rep. 2022;12(l):22474. doi:10.1038 / s41598-022-26337-l). Briefly, mouse dorsal skins were shaved, excised, antibiotic-treated, and incubated in 25 pg / ml Try psin (Sigma) overnight at 4°C. After separation from the epidermis and adipose tissue, the dermis was digested using collagenase IV (Worthington Biochemical) to extract primary fibroblasts. Primary fibroblasts were then cultured in 25mM glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco) with 10% (v / v) fetal bovine serum (FBS, Peak Serum Inc) Attorney Docket No.: 047162-7519WO 1(02692) and 1% (v / v) penicillin-streptomycin (P / S, Gibco). Flow cytometry with Vimentin antibody was used to evaluate fibroblast purity. Cells between passage 1 and 2 were used for experiments.

[0262] NIH3T3 cells (ATCC) were maintained in 25mM glucose DMEM with 10% FBS and 1% P / S and switched to either 5mM or 30mM glucose DMEM per experimental design. Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Yale Vascular Biology & Therapeutics Program Tissue Culture Core and maintained in Ml 99 with 20% FBS and supplemented with 0.1% Endothelial Cell Growth Supplement (ECGS). To starve cells overnight for migration assays, media with 0.1% FBS and 1% P / S was used. All cells were maintained at 37°C with 5% CO2.

[0263] Hypoxia Chamber and DMOG Treatment

[0264] NIH3T3s or primary dermal fibroblasts were seeded in a 6-well plate and used for experiments once they reached 80% confluency. Cells were exposed either to hypoxia (0.5% oxygen) in a ProxOxC nitrogen-induced hypoxia sy stem (BioSpherix, Red Field, NY) or normoxia (18% oxygen) in a normal cell incubator for 24 hours. For drug treatment, DMOG (Cayman Chemical Company, 71210) was added to complete media at a concentration of 1 mM or 2 mM for NIH3T3s and primary dermal fibroblasts, respectively. During the 24-hour time course, cells demonstrated no visible sign of apoptosis. At 24 hours, media, cell lysates, and total mRNA were collected for further analysis.

[0265] RNA Isolation and qRT-PCR

[0266] RNA was isolated from cells or tissue using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Reverse transcription was completed with the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time PCR (qRT-PCR) was performed with the iTaq Universal SYBR Green One-Step Kit (Bio-Rad) on a CFX96 Touch Real-Time PCR machine (Bio-Rad). Primers were ordered from the Keck Oligonucleotide Facility (Yale University) from sequences found in PrimerBank (Harvard University) or published literature. Data were normalized to GAPDH or pl-act in expression.

[0267] Western Blot

[0268] Cell or tissue lysates were extracted using RIPA buffer (pH = 7.4. Boston BioProducts, Inc.) supplemented with cOmplete EDTA-free protease inhibitor cocktail (Roche). Protein concentration was determined using BCA protein assay kit according to supplier’s instructions (Thermofisher). Standard western blot was then performed using a mini-PROTEAN TGX Stain- Free Gel (10%, Bio-Rad) and the Licor Odyssy CLx system was used to visualize blots. Densitometry analysis was performed using ImageJ Gels plugin. Attorney Docket No.: 047162-7519WO 1(02692)

[0269] Skin Decellularization and Hydrogel Preparation

[0270] Murine skin was isolated and decellularized according to established protocols (Morris AH, Stamer DK, Kunkemoeller B, Chang J, Xing H, Kyriakides TR. Decellularized materials derived from TSP2-KO mice promote enhanced neovascularization and integration in diabetic wounds. Biomaterials. 2018;169:61-71. doi: 10.1016 / J.BIOMATERIALS.2018.03.049). Briefly, after removing hair and fat, the isolated skin was incubated in 0.25% Trypsin-EDTA (ThermoFisher) for 6 hours at room temperature to be accomplish decellularization. After washing with double distilled water (ddH2O) and ethanol, the skin was incubated in 3% H2O2 and 1% Triton X-100 in 0.26%EDTA / 0.69%Tris sequentially to remove remaining nuclei. As sterile decellularized skin was desired, the skin was further incubated in 0.1% peracetic acid in 4% ethanol for 2 hours before extensive washing in ddH2O. The decellularized skin ECM was then incubated in DMEM overnight and lyophilized.

[0271] To prepare hydrogel, dry WT or TSP2KO mouse skin ECM was weighted and digested in 1 mg / mL pepsin (Sigma) in 0.01 N HC1 (J.T. Baker) for 72 h with a concentration of 10 mg / ml. The solubilized ECM was then neutralized and buffered with sodium hydroxide (1 / 10 digest volume) and 10x phosphate-buffered saline (PBS) (1 / 9 digest volume). Next, the buffered ECM was diluted to the desired concentration using PBS to form a pre-gel solution and stored on ice until use. To make TSP2KO+DMOG hydrogels, 50 mg / ml DMOG stock solution was added to the pre-gel solution and vortexed. Gelation then induced by incubating the pre-gel solution at 37°C for 1 hr.

[0272] Scratch Assay

[0273] Cell scratch assay was used to evaluate the migration ability and conducted based on the protocol described previously (Kunkemoeller B. Kyriakides TR. Redox Signaling in Diabetic Wound Healing Regulates Extracellular Matrix Deposition. Antioxid Redox Signal. 2017;27(12):823-838. doi: 10.1089 / ars.2017.7263). Briefly, NIH3T3 fibroblasts were seeded in a 6-well plate and allowed to grow to 100 % confluence in 5mM glucose DMEM. A scratch was created in each well with a 200 qL pipet tip. The cells were then washed with PBS and incubated in 5 mM or 30 mM glucose DMEM with or without DMOG. The plates were then monitored and photographed using a Zeiss Axio Vert. Al inverted light microscope for the following 24 hours. The area of the scratch at each time point was measured with ImageJ, and relative wound closure (%) calculated for each well. All experiments were performed at least three times in triplicate.

[0274] DMOG Release Attorney Docket No.: 047162-7519WO 1(02692)

[0275] The DMOG cumulative release curve from the hydrogel was determined and plotted using ultraviolet- visible spectroscopy. 40 pL WT or TSP2KO skin hydrogel with 1.4mg / ml DMOG was plated on the 96-well-plate and placed in 37°C incubator for Ihr to induce gelation. Then 200 pL PBS was added on the top of the gel to start the drug release. At 1, 2, 3, 6, 24, 48, and 72 hours, 100 pL of the PBS was taken out from the top solution and replenished. The absorbance of DMOG in the PBS extracted was read by Nanodrop (Thermo Scientific) at a wavelength of 218 nm (determined by scanning the whole spectrum of DMOG in PBS). Then the concentration of DMOG was converted from the absorbance based on the standard curve determined before.

[0276] Scanning Electron Microscopy (SEM)

[0277] To prepare SEM samples, hydrogels were fixed in 2.5% paraformaldehyde in 0.1 cacodylate buffer (Electron Microscopy Sciences), dehydrated using an ethanol gradient, incubated in hexamethyldisilazane (Electron Microscopy Sciences), and air-dried overnight. Samples were then coated with 8-nm thick gold using a sputtering tool (Cressington 208) and imaged under a Hitachi SU-70 scanning electron microscope.

[0278] Cell-Gel Interactions

[0279] Cell proliferation on hydrogels was examined using Cell Counting Kit-8 (CCK-8, ab228554, Abeam) to test the cytotoxicity' and biocompatibility' of the hydrogels. Specifically, a total number of 3x 103NIH 3T3 or HUVECs were seeded on the surface of 2 mg / ml WT, KO, and KO+DMOG hydrogel per well in a 96-well plate. Cell proliferation rate was determined by replacing the media with the media containing 10% CCK-8 solution and reading the O.D. using plate-reader every day in the following five days. Media was changed every other day. Cells were also fixed by 4% paraformaldehyde (PF A, J.T. Baker), and stained with DAPI (Invitrogen) and rhodamine-phalloidin (Invitrogen) for morphological analysis.

[0280] In vitro angiogenesis assay was conducted using HUVECs. Briefly, 100 pL 2mg / ml WT, KO, and KO+DMOG hydrogels were plated on a 48-well plate, and a total number of lOOxlO3HUVECs were seeded on top of hydrogels. HUVECs activities were monitored for 48 hours and images were taken using light microscope at 1, 6, 24, and 48 hours. Cells were also fixed by 4% PF A, stained by DAPI and Phalloidin, and imaged.

[0281] Cell migration and invasion abilities in hydrogels were investigated using trans-well assay. Briefly, 50 or 100 pL 2mg / ml WT, KO, and KO+DMOG hydrogels were plated on the top chamber of a Transwell (Coming) for transmigration and invasion experiments, respectively. After incubating the gel under 37 °C for Ih, a total number of 50x 103HUVECs Attorney Docket No.: 047162-7519WO 1(02692) or NIH3T3 cells pre-starved overnight were seeded onto the top chamber with 100 pL serum- free media. 700 pL normal cell culture media with 10% FBS was added in the lower chamber. After 6 and 24 hours, media was removed, and the unattached cells were washed using PBS. For cell invasion experiments, samples were then fixed with z-fix overnight, embedded in paraffin, and sectioned. Sections were then stained with H&E, and the distance of cell invasion in the gel was then examined. For transmigration, samples were fixed using methanol. After carefully removing the gel on the top chamber, the bottom of the trans-well was stained by a rapid kit and visualized using light microscope.

[0282] Reagents

[0283] The following primary antibodies were used for flow cytometry, IHC or IF: affinity purified rabbit antipimonidazole antibody (1: 100, Hypoxyprobe, Inc., PAb2627AP), antimouse CD107b (1:200, BD Biosciences, 550292), anti-CD31 (1:50, AF3628, R&D Systems), anti-aSMA (1 :50, AF3628, R&D Systems), anti-Vimentin (1:500, AB5733, EMD Millipore), anti- HIF-la (1: 100 Novus Biologicals, NB100-134), and anti-TSP2 (1:250, GenScript).

[0284] The following primary antibodies were used for western blot: anti-TSP2 (1:250, GenScript), anti- HIF-la (1 : 1000. Cell Signaling. 141795). anti-GAPDH (1 : 1000, Cell Signaling, 5174S), and anti-|3-actin (1 :1000, Abeam, ab8226).

[0285] The following secondary' antibodies were used for immunohistochemistry: Biotin anti-rabbit IgG (1 :250, Vector, BA-1000), and Biotin anti-rat IgG (1:250, Novex. A-18731). The following secondary antibodies were used for immunofluorescence staining or flow cytometry: anti-rabbit IgG Alexa Fluor 488 (1 : 1000, Invitrogen, al 1008), anti-chicken IgG Alexa Fluor 488 (1: 1000, Abeam, abl50169), anti-rabbit IgG Alexa Fluor 555 (1 : 1000, Abeam, abl50078), anti-rat IgG Alexa Fluor 594 (1: 1000, Life Technologies, a21471), antigoat IgG Alexa Fluor 647 (1:500, Invitrogen, a21447), and anti -goat IgG Alexa Fluor 488 (1: 1000, Invitrogen, a21467). The following secondary antibodies were used for western blots in the Licor Odyssy DLx system: anti -mouse IgG Alexa Fluor 680 (1 : 1000, Invitrogen, a!0038), and anti-rabbit IgG Alexa Fluor 800 (1: 1000, Invitrogen, a32735).

[0286] Image Analysis and Statistical Analyses

[0287] Images were analyzed with ImageJ or Metamorph software (Molecular Devices). Colocalization between HIF-la and CD31 or Vimentin was analyzed via Coloc2 in ImageJ. For wound histological analysis, the entire wound area was quantified. For co-staining of hypoxia and CD31 or vimentin, only area around gel was quantified. Error bars represent standard deviation (SD) unless stated otherwise. An unpaired student's t-test was performed when comparing two groups. For comparisons betw een three or four groups, one-way Attorney Docket No.: 047162-7519WO 1(02692) analysis of variance (ANOVA) tests with Tukey’s post hoc tests and multiple comparisons was utilized. All statistical analyses were performed using GraphPad Prism 9. QPCR and western blot quantifications were standardized based on the control that was set as 1. By default, figures are labeled to denote only groups that achieve significance in terms of differences.

[0288] Example 1: Diabetic wounds are more hypoxic than WT and depletion of TSP2 could ameliorate excess hypoxia.

[0289] To investigate the interactions between hypoxia and TSP2 in diabetic wounds, we first determined hypoxic regions in Day 7 (D7) and Day 10 (D10) WT, DBDB, and DBKO wounds, which correspond to the peak TSP2 expression. Hydroxyprobe (pimonidazole hydrochloride) can bind to proteins in hypoxic cells, and the generated adducts can be visualized by immunohistochemistry to detect hypoxia. Quantification of IHC-positive areas in wounds showed that there were more hypoxic cells in DBDB than WT wounds at D7 and D10 (FIGs. 1A-1H & FIG. 7). In contrast, DBKO wounds showed significantly less positive area compared to DBDB, indicating an association between TSP2 depletion and decreased hypoxia. Moreover, the detection of hypoxia and TSP2 on serial sections revealed overlapping and non-overlapping patterns in DBDB and WT wounds, respectively (FIG. 8).

[0290] Western blot analysis indicated the absence and dramatic elevation of TSP2 in tissue lysates of D7 DBKO and DBDB wounds, respectively (FIGs. 2A and 2B). Next, we investigated the expression level of HIF-l a by western blot in the D7 lysates from the three genotypes. We observed variability in HIF-la levels but densitometric analysis showed comparable expression among the three groups (FIGs. 2A and 2C). Moreover, qPCR analysis revealed a similar pattern of comparable expression (FIG. 9). Furthermore, we explored the HIF-la expression in wound endothelial cells and fibroblasts via co-staining HIF-la with cell markers, CD31 and Vimentin. Colocalization analysis showed decreased HIF-la+ cells in total Vimentin+ cells in DBDB and DBKO wounds compared to WT. In contrast, the ratio of CD31+HIF-la+ / CD31+ was similar between groups (FIGs. 2D, 2E, and 2F).

[0291] Taken together, these findings demonstrate spatially overlapping chronic hypoxia and excess TSP2 in diabetic wounds and indicate an abnormal correlation betw een them in diabetes. Additionally, we detected low HIF-la expression and decreased Vimentin+ / HIF- la+ cells despite increased hypoxia in diabetic wounds, suggesting impaired HIF-la stabilization. Without wishing to be limited by theory, this may be due to hyperglycemia and elevated fatty acid level. Though the causes of impaired HIF-la stabilization are not yet Attorney Docket No.: 047162-7519WO 1(02692) determined, the condition allows for increased TSP2 deposition despite widespread hypoxia in diabetic wounds. More importantly, analysis on DBKO wounds suggested that TSP2 deficiency could prevent severe hypoxia, underscoring the significance of interactions between TSP2 and hypoxia in diabetic wound healing.

[0292] Example 2: Lack of hypoxia-induced repression of TSP2 in diabetic dermal fibroblasts.

[0293] Fibroblasts play a critical role in wound ECM regeneration and contraction and are the major cell type producing TSP2. To investigate how the hypoxia response is altered in diabetic conditions in relation to TSP2 expression, we isolated fibroblasts from WT, DBDB, and DBKO mice and compared TSP2 production as well as HIF-la signaling in response to hypoxia in vitro. Flow cytometric analysis of fibroblast marker Vimentin in isolated DBDB DFs indicated over 90% purity (FIG. 10). Western blot and corresponding densitometric analysis revealed that WT DFs decreased TSP2 production when cultured in a hypoxia chamber or treated with DMOG (FIGS. 3A and 3B). However, DBDB DFs retained high TSP2 expression level when cultured in a hypoxia chamber compared to control conditions. In contrast, DMOG treatment inhibited TSP2 production in DBDB DFs (FIGS. 3A and 3C). We then utilized NIH 3T3 fibroblasts to further probe the functional significance of DMOG. Specifically, we cultured NIH3T3 cells in normal or high glucose and observed reduced TSP2 expression with DMOG (FIG. 11 A). Media with high glucose mimics the diabetic environment and slows cell migration as observed in diabetes. However, DMOG treatment promoted cell migration in high glucose in a scratch assay, indicated by the higher percentage of closed area when compared to no treatment (FIGS. 1 IB-11C).

[0294] HIF-la plays a critical role in hypoxia-regulated pathways and an impairment in its stabilization in diabetes is known. Therefore, we postulated that HIF-la stabilization in diabetic DFs is impeded in response to hypoxia, leading to abnormal TSP2 regulation. To test this hypothesis, we placed WT, DBDB, and DBKO DFs in hypoxia chamber or treated them with DMOG and measured the expression of HIF-la responsive genes, GLUT1 and PGK1. In WT DFs, the mRNA levels of GLUT1 and PGK-1 increased in both hypoxia chamber and DMOG treatment groups, suggesting an activation and stabilization of HIF-la in both conditions. In contrast, DBDB DFs displayed no change in GLUT1 and only a slightly increased mRNA level of PGK-1 in hypoxia. In the same cells, DMOG treatment elevated the expression level of both genes compared to untreated control group, suggesting that DMOG treatment was more efficient than hypoxia. Similarly, DBKO DFs also showed increases in expression of both genes only in the DMOG treatment group. In addition, we have also Attorney Docket No.: 047162-7519WO 1(02692) examined the effect of TSP2 depletion on HIF-1 a activation by placing TSP2KO DFs in hypoxia chamber and determined the mRNA expression level of GLUT1 and PGK-1. Like WT, both genes were upregulated in TSP2 KO DFs when compared to control (FIG. 12). This observation suggests that TSP2 is not required for HIF-la activation.

[0295] Taken together, the above observations verified the insufficient activation of HIF-la in diabetes, which led to increases in TSP2 expression in diabetic DFs. Additionally, TSP2 depletion had no impact on HIF-la activation, suggesting that the reduced hypoxia observed in DBKO wounds was not due to regulation of HIF-la. More importantly, the effect of DMOG in terms of stabilizing HIF-la and repressing TSP2 expression suggested a potential therapeutic benefit in diabetic wounds.

[0296] Example 3: TSP2KO+DMOG ECM hydrogel targets hypoxia and TSP2 in diabetic wounds.

[0297] TSP2KO mouse skin-derived hydrogel demonstrated improved healing in diabetic wound model without interfering with the expression levels of TSP2 and HIF-la in the tissue microenvironment. Despite improvements, wound closure was not complete, which could be due to the persistence of elevated TSP2 levels. Therefore, we hypothesized that stabilizing HIF-la and inhibiting endogenous TSP2 production could further increase treatment efficiency. Since DMOG had a positive effect on HIF-la stabilization and TSP2 repression and promoted fibroblast migration in high glucose conditions (FIGS. 3A-3E and FIGS. 11A- 1 1C), we postulated that it could be incorporated into the hydrogel system as a therapeutic.

[0298] DMOG was introduced into a neutralized pre-gel solution to make a TSP2KO (KO) + DMOG hydrogel. Scanning electron microscopic images revealed no changes in the hydrogel structure with the addition of the drug (FIG. 13 A). Additionally, gels with DMOG were immersed in PBS at 37 °C and cumulative drug release reached 80% within 6 hours (FIG. 13B).

[0299] The drug toxicity and dosing in hydrogel was then investigated in NIH3T3 cells using a CCK8 proliferation assay. Within the range of 0 - 1400 pg / ml DMOG in hydrogel, fibroblast proliferation was not impaired (FIGS. 14A-14B). Moreover, the invasion and transmigration ofNIH3T3 cells using a trans-well assay showed no difference between KO and KO+DMOG groups (FIGS. 15A-C). Similarly, the inclusion of DMOG in the KO hydrogel did not impact HUVEC proliferation and migration. Strikingly, the in vitro angiogenesis assay showed HUVECs cultured on the KO+DMOG gel formed the most cords (FIG. 16), suggesting a beneficial effect of KO+DMOG gel in angiogenesis. Attorney Docket No.: 047162-7519WO 1(02692)

[0300] To assess the therapeutic potential of KO+DMOG hydrogel we utilized a diabetic mouse wound model. Briefly, two 6-mm full-thickness wounds were made on the back of diabetic mice, and one received KO hydrogel treatment while the other received KO+DMOG hydrogel. Untreated diabetic wounds or treated with DMOG one-time topical application were also prepared. Wound progression was monitored over a 14-day period. Despite the rapid release, inclusion of DMOG exerted an inhibitory effect on TSP2 production in diabetic wounds. Specifically, TSP2 western blot analysis of D7 wounds showed reduced levels in wounds treated with KO+DMOG hydrogel (FIGS. 17A-17B). Hydroxy probe immunochemistry of D7 DBDB wounds showed less hypoxic cells in the KO, KO+DMOG hydrogel, and DMOG treatment groups compared to untreated wounds (FIG. 4). Furthermore, immunofluorescence detection of Vimentin and CD31 in areas adjacent to gels revealed a similar composition of fibroblasts and endothelial cells between KO and KO+DMOG treatment groups despite of the variance of hypoxia (5.604% in the former and 2.269% in the latter, FIGS 18A, 18B, 18C). Total HIF-la positive area was also similar between the two groups but a higher HIF-la / cell ratio was observed inside the gel with DMOG (FIGS. 18A, 18D, 18E). Additionally, only wounds treated by KO+DMOG hydrogel continued to have reduced hypoxia at D10 (FIG. 4). The above observations point out that KO+DMOG hydrogel successfully targeted TSP2 and hypoxia in diabetic wounds.

[0301] Example 4: TSP2KO+DMOG hydrogel further improves diabetic wound healing.

[0302] To evaluate wound progression with KO, KO+DMOG hydrogel, and DMOG treatment, both gross images of the wounds and IHC images of tissue sections were analyzed. Quantification of the wound area from images taken at D7 and D14 revealed improved closure in KO+DMOG hydrogel group at both time points when compared to the untreated, KO hydrogel, and DMOG treatment group (FIGS. 5A-5C). Furthermore, trichrome staining of D7 and D14 wounds showed more complete wound closure of the KO+DMOG group (FIG. 16 and FIGS. 5D-5K). None of the wounds in the untreated and DMOG treatment groups showed complete re-epithelialization at D14. In contrast, KO hydrogel treatment promoted re-epithelialization at D14, and inclusion of DMOG increased the number of closed wounds by two-fold (FIG. 5K). Furthermore, KO+DMOG hydrogel treated wounds had the smallest wound gaps than the other three groups (FIG. 5H). However, analysis of granulation tissue area and remaining gel thickness revealed no differences between the KO and KO+DMOG group (FIGS. 5I-5J). Taken together, the above observations indicated that KO+DMOG hydrogel did not affect ECM deposition and gel degradation but enhanced Attorney Docket No.: 047162-7519WO 1(02692) epithelization and wound closure in DBDB wounds.

[0303] Example 5: TSP2KO+DMOG hydrogel promotes neovascularization in diabetic wounds.

[0304] To further probe changes associated with KO+DMOG hydrogel treatment, we examined wounds using IHC and IF. Specifically, endothelial cells and smooth muscle cells were detected by CD31 and a-SMA, respectively, to visualize the formation of vessels. Fibroblasts and macrophages, which play major roles in ECM remodeling and inflammation, were detected using Vimentin and Mac3, respectively. Image analysis showed that there was an increased number of CD31+ lumens in the KO+DMOG group compared to untreated. KO gel. and DMOG treatment groups. Moreover, both KO and KO+DMOG groups had more a- SMA+ lumens compared to untreated and DMOG treated wounds though the number were similar between the two groups (FIGS. 6A-6D). These results suggested that KO+DMOG hydrogel mainly promoted neovascularization, which is consistent with the in vitro angiogenesis data (FIGS. 13A-13B). By contrast, quantification of Vimentin+ and Mac3+ area did not show differences between the four groups though there was a trend of increased Vimentin+ signals and decreased Mac3+ signals in the KO and KO+DMOG hydrogel groups compared to untreated group. These observations indicate that the addition of DMOG in the TSP2KO hydrogel did not exert any toxicity on fibroblasts or interfere with inflammatory cells. Taken together, the in vitro and in vivo observations highlighted the beneficial effect of KO+DMOG hydrogel especially on vascularization. This was likely due, in part, to the reduced TSP2 and decreased hypoxia in D7 and D10 diabetic wounds.

[0305] Remarks

[0306] In the present study, we investigated the relationship between hypoxia and TSP2 using hydroxyprobe to visualize hypoxic regions in WT, DBDB, and DBKO mouse wounds. In WT wounds, TSP2 and hypoxia are distinct from each other. However, an overlapping spatial pattern between hypoxia and TSP2 deposition was observed in DBDB wounds, suggesting that the hyperglycemia overrides the effect of hypoxia on TSP2 production. Diabetes-associated upregulation of TSP2 is observed in human skin and wounds. These tissues have been shown to be hypoxic, but it is unclear if there is a spatial relationship w ith TSP2. This is due to the need to administer chemical reagents, such as hydroxyprobe (used in our study) or EF5 (2-nitroimidazole) that require metabolic conversion in viable tissue. Thus, the approach of co-staining TSP2 and hypoxia used in mice is not feasible because it Attorney Docket No.: 047162-7519WO 1(02692) necessitates administration of reagents into patients. Similarly, physical approaches to detect hypoxia using electronic needles or positron emission tomographic (PET) are complex and difficult to perform and cannot be combined with detection of TSP2. Further molecular investigation revealed similar HIF-la levels between WT, DBDB, and DBKO wounds even though the lack of oxygen is more severe in DBDB.

[0307] To further explore HIF-la cell-specific distribution, double immunofluorescence was performed and revealed no differences in CD31+ cells among the three groups. In contrast, HIF-l + fibroblasts detected by vimentin were reduced in DBDB and DBKO wounds. Overall, fibroblasts exhibited greater percentage of HIF-la + cells when compared to CD31+. Analysis of fetal sheep D8 skin wounds showed that HIF-la + cells are predominantly fibroblasts. However, the cell-specific distribution of HIF-la has not been rigorously examined in diabetic conditions. Impaired HIF-la stabilization was observed in our in vitro experiments using DBDB dermal fibroblasts. Consistent with these observations, we observed that the hypoxia-induced TSP2 repression was absent in diabetic fibroblasts and wounds.

[0308] In normal hypoxic conditions, HIF-la is stabilized due to inhibited hydroxylation and following degradation by VHL, which leads to the fast turnover of HIF-la. Once stabilized, HIF-la has the chance to translocate into the nucleus and form a dimer with HIF-1 , along with other co-activators like p300. This complex then binds to the hypoxia response element in the DNA and regulate transcription of targeted genes. However, in diabetes, PHD- mediated HIF-la degradation is promoted due to increased levels of glucose and fatty acid. In addition, HIF-la transactivation is inhibited by hyperglycemia, impairing HIF-la activation and function. These effects are exerted via the elevated intracellular methylglyoxal (MGO) accumulation induced by hyperglycemia. Moreover, increased reactive oxygen species (ROS) are also reported to modify HIF-la via the MGO pathway. The abnormal level of fatty acids in the type 2 diabetes and insulin-resistant models is also linked to inhibited HIF-la stabilization in cardiomyocytes via decreased succinate level.

[0309] Despite the unclear mechanisms of impaired hypoxia response in diabetic wound healing, efforts have been made to develop better wound care strategies targeting oxygen availability, including hyperbaric oxygen therapies and genetic interventions. Pharmacological agents inhibiting prolyl hydroxylase domain (PHD) activities and stabilizing HIF-la have garnered attention due to their ease of administration and promising results in promoting wound healing. A recent study identified that PDK4, a metabolic enzyme, could Attorney Docket No.: 047162-7519WO 1(02692) inhibit PHD2 activity' and stabilize HIF-la via increasing the level of succinate. Local administration of PDK.4 facilitated fibroblasts functions and diabetic wound healing. Other than macromolecules such as proteins, the small molecules DMOG or DFX have been investigated in multiple disease conditions. Repetitive Injections of DMOG or DFX into diabetic wounds were shown to improve closure, increased oxygen supply, and enhanced vascularization.

[0310] Previous work using TSP2K.0 mouse skin-derived ECM hydrogel to treat diabetic wounds accelerated healing with improved ECM remodeling and cell functions (Morris AH, Lee H, Xing H, Stamer DK, Tan M, Kyriakides TR. Tunable Hydrogels Derived from Genetically Engineered Extracellular Matrix Accelerate Diabetic Wound Healing. ACSAppl Mater Interfaces . 2018;10(49):41892 -41901. doi: 10.1021 / acsami.8b08920). Taking advantage of the TSP2KO ECM hydrogel, we incorporated DMOG into the soft material and delivered the complex as a whole therapy for diabetic wound healing. In vivo experiments showed that it could further improve w ound healing and alleviate hypoxic conditions mainly via increased neovascularization. The successful delivery of the small molecule drug using a hydrogel platform holds promise for improved therapeutic efficacy by inclusion of additional functional agents. Moreover, hydrogel itself is an attractive therapeutic strategy' due to its biocompatibility', versatility', and tunability'. In certain embodiments, the KO+DMOG hydrogel developed here demonstrates a synergistic effect and a greater efficacy in treating diabetic wound healing where hypoxia and ECM regulation are dysfunctional.

[0311] In summary', this study first determined the different levels of hypoxia in WT, DBDB, and DBDB TSP2KO mouse wounds and their spatial relationship w'ith TSP2. Unlike WT tissue repair where TSP2 expression is inhibited by hypoxia, its upregulation in diabetes is independent of hypoxia due to insufficient HIF-la stabilization. As a result, TSP2 deposition and hypoxic regions overlap in diabetic wounds. In vitro experiments using primary fibroblasts confirmed the role of HIF-la in the regulation of hypoxia-induced TSP2 repression and suggested that pharmacological stabilization of HIF-la using DMOG can repress TSP2. A therapeutic strategy was developed using DMOG and TSP2KO mouse skin hydrogel. Improved healing efficiency was verified using an in vivo diabetic wound model that show'ed increased neovascularization and reduced hypoxia following treatment with KO+DMOG gel.

[0312] Enumerated Embodiments

[0313] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance. Attorney Docket No.: 047162-7519WO 1(02692)

[0314] Embodiment 1 provides a method for promoting tissue regeneration in a subject in need thereof, the method comprising administering to the subject a hydrogel comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-la stabilizing compound.

[0315] Embodiment 2 provides the method of Embodiment 1, wherein the tissue is a musculoskeletal tissue.

[0316] Embodiment 3 provides the method of Embodiment 1, wherein the HIF- la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI).

[0317] Embodiment 4 provides the method of any one of Embodiments 1-3, wherein the tissue is a muscle, a cartilage, a connective tissue, a tendon, a ligament, or a bone.

[0318] Embodiment 5 provides the method of Embodiment 3, wherein the HIF prolyl hydroxylase inhibitor (HIF-PHI) is a 2-oxoglutarate mimetic compound.

[0319] Embodiment 6 provides the method of Embodiment 3, wherein the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyl oxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, and / or vadadustat.

[0320] Embodiment 7 provides the method of any one of Embodiments 1 -6, wherein cells of the tissue lacking functional TSP2 comprise: (a) a TSP2-null knockout allele; and / or (b) suppressed TSP2 gene expression.

[0321] Embodiment 8 provides the method of any one of Embodiments 1-7, wherein the method enhances at least one biological response at the treatment site, as compared to (1) a site administered a decellularized ECM originating from a tissue lacking functional TSP2 and lacking a HIF-la stabilizing compound, (2) a site administered a decellularized ECM originating from a tissue comprising functional TSP2, or (3) an untreated site.

[0322] Embodiment 9 provides the method of Embodiment 8, wherein the biological response at the treatment site is selected from the group consisting of cellular migration towards the treatment site, cellular invasion of the treatment site, vascular growth and maturation, innervation, angiogenesis, and wound repair.

[0323] Embodiment 10 provides the method of any one of Embodiments 1-9, wherein the tissue lacking functional TSP2 is muscle and wherein the subject suffers from at least one condition selected from the group consisting of type 1 diabetes and type 2 diabetes.

[0324] Embodiment 11 provides the method of any one of Embodiments 1-10, wherein the subject is a mammal.

[0325] Embodiment 12 provides the method of any one of Embodiments 1-11. wherein the tissue originates from a mammal selected from the group consisting of a mouse, a pig, a non- Attorney Docket No.: 047162-7519WO 1(02692) human primate, and a human.

[0326] Embodiment 13 provides the method of any one of Embodiments 1-12. wherein the subject is a human.

[0327] Embodiment 14 provides the method of any one of Embodiments 1-13, wherein the ECM is formulated with at least one additional therapeutic agent.

[0328] Embodiment 15 provides the method of Embodiment 14, wherein the at least one additional therapeutic agent is selected from the group consisting of an immunosuppressive agent, an anti-inflammatory agent, an antimetabolite, an antibiotic, an antibody, a growth factor, a cytokine, a gene therapy, an immunomodulator, and any combination thereof.

[0329] Embodiment 16 provides a hydrogel composition comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-la stabilizing compound.

[0330] Embodiment 17 provides the hydrogel composition of Embodiment 16, wherein the tissue is a musculoskeletal tissue.

[0331] Embodiment 18 provides the method of any one of Embodiments 16-17, wherein the HIF-la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI).

[0332] Embodiment 19 provides the hydrogel composition of any one of Embodiments 16- 18, wherein the tissue is muscle, cartilage, a connective tissue, a tendon, a ligament, or a bone.

[0333] Embodiment 20 provides the hydrogel composition of Embodiment 18. wherein the HIF-PHI is a 2-oxoglutarate mimetic compound.

[0334] Embodiment 21 provides the hydrogel composition of Embodiment 18, wherein the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, and / or vadadustat.

[0335] Embodiment 22 provides the hydrogel composition of any one of Embodiments 16-

[0336] 21, wherein cells of the tissue lacking functional TSP2 comprise: (a) a TSP2-null knockout allele; and / or (b) suppressed TSP2 gene expression.

[0337] Embodiment 23 provides the hydrogel composition of any one of Embodiments 16-

[0338] 22, wherein the tissue lacking functional TSP2 originates from a mammal selected from the group consisting of a mouse, a pig, a non-human primate, and a human.

[0339] Embodiment 24 provides the hydrogel composition of any one of Embodiments 16-

[0340] 23, wherein the ECM is formulated with at least one additional therapeutic agent.

[0341] Embodiment 25 provides the hydrogel composition of Embodiment 24, wherein the at least one additional therapeutic agent is selected from the group consisting of an Attorney Docket No.: 047162-7519WO 1(02692) immunosuppressive agent, an anti-inflammatory agent, an antimetabolite, an antibiotic, an antibody, a growth factor, a cytokine, a gene therapy, an immunomodulator, and any combination thereof.

[0342] Embodiment 26 provides a method for preparing a hydrogel composition, the method comprising: providing a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and contacting the decellularized extracellular matrix (ECM) with a HIF-l stabilizing compound.

[0343] Embodiment 27 provides the method of Embodiment 26, wherein the method further comprises formulating the ECM as a hydrogel in the presence of the HIF-la stabilizing compound.

[0344] Embodiment 28 provides the method of any one of Embodiments 26-27, wherein the tissue is a musculoskeletal tissue.

[0345] Embodiment 29 provides the method of any one of Embodiments 26-28, wherein the HIF-la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI).

[0346] Embodiment 30 provides the method of Embodiment 29, wherein the HIF prolyl hydroxylase inhibitor (HIF-PHI) is a 2-oxoglutarate mimetic compound.

[0347] Embodiment 31 provides the method of Embodiment 29, wherein the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyl oxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, or vadadustat.

[0348] Embodiment 32 provides the method of any one of Embodiments 26-31 , wherein cells of the tissue lacking functional TSP2 comprise: (a) a TSP2-null knockout allele; and / or (b) suppressed TSP2 gene expression.

[0349] Embodiment 33 provides a method of treating, ameliorating, and / or preventing diabetic foot ulcer on a subject in need thereof, the method comprising: applying to the ulcer a hydrogel composition comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-la stabilizing compound.

[0350] Other Embodiments

[0351] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

Attomey Docket No.: 047162-7519WO 1(02692)CLAIMSWhat is claimed is:

1. A method for promoting tissue regeneration in a subject in need thereof, the method comprising administering to the subject a hydrogel comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-la stabilizing compound.

2. The method of claim 1, wherein the tissue is a musculoskeletal tissue.

3. The method of claim 1, wherein the HIF-la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI).

4. The method of any one of claims 1-3, wherein the tissue is a muscle, a cartilage, a connective tissue, a tendon, a ligament, or a bone.

5. The method of claim 3, wherein the HIF prolyl hydroxylase inhibitor (HIF-PHI) is a 2-oxoglutarate mimetic compound.

6. The method of claim 3, wherein the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, and / or vadadustat.

7. The method of any one of claims 1-6, wherein cells of the tissue lacking functional TSP2 comprise:(a) a TSP2-null knockout allele; and / or(b) suppressed TSP2 gene expression.

8. The method of any one of claims 1-7, wherein the method enhances at least one biological response at the treatment site, as compared to (1) a site administered a decellularized ECM originating from a tissue lacking functional TSP2 and lacking a HIF-la stabilizing compound, (2) a site administered a decellularized ECM originating from a tissue comprising functional TSP2, or (3) an untreated site.Attorney Docket No.: 047162-7519WO 1(02692)9. The method of claim 8, wherein the biological response at the treatment site is selected from the group consisting of cellular migration towards the treatment site, cellular invasion of the treatment site, vascular grow th and maturation, innervation, angiogenesis, and wound repair.

10. The method of any one of claims 1-9, wherein the tissue lacking functional TSP2 is muscle and wherein the subject suffers from at least one condition selected from the group consisting of type 1 diabetes and type 2 diabetes.

11. The method of any one of claims 1-10, wherein the subject is a mammal.

12. The method of any one of claims 1-11, wherein the tissue originates from a mammal selected from the group consisting of a mouse, a pig, a non-human primate, and a human.

13. The method of any one of claims 1-12, wherein the subject is a human.

14. The method of any one of claims 1-13, wherein the ECM is formulated with at least one additional therapeutic agent.

15. The method of claim 14, wherein the at least one additional therapeutic agent is selected from the group consisting of an immunosuppressive agent, an antiinflammatory agent, an antimetabolite, an antibiotic, an antibody, a growth factor, a cytokine, a gene therapy, an immunomodulator, and any combination thereof.

16. A hydrogel composition comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-1 a stabilizing compound.

17. The hydrogel composition of claim 16, wherein the tissue is a musculoskeletal tissue.

18. The method of any one of claims 16-17, wherein the HIF-la stabilizing compound isAttomey Docket No.: 047162-7519WO 1(02692) a HIF prolyl hydroxylase inhibitor (HIF-PHI).

19. The hydrogel composition of any one of claims 16-18, wherein the tissue is muscle, cartilage, a connective tissue, a tendon, a ligament, or a bone.

20. The hydrogel composition of claim 18, wherein the HIF-PHI is a 2-oxoglutarate mimetic compound.

21. The hydrogel composition of claim 18, wherein the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG). daprodustat, desidustat, enarodustat, molidustat. roxadustat. and / or vadadustat.

22. The hydrogel composition of any one of claims 16-21, wherein cells of the tissue lacking functional TSP2 comprise:(a) a TSP2-null knockout allele; and / or(b) suppressed TSP2 gene expression.

23. The hydrogel composition of any one of claims 16-22, wherein the tissue lacking functional TSP2 originates from a mammal selected from the group consisting of a mouse, a pig. a non-human primate, and a human.

24. The hydrogel composition of any one of claims 16-23, wherein the ECM is formulated with at least one additional therapeutic agent.

25. The hydrogel composition of claim 24, wherein the at least one additional therapeutic agent is selected from the group consisting of an immunosuppressive agent, an antiinflammatory agent, an antimetabolite, an antibiotic, an antibody, a grow th factor, a cytokine, a gene therapy, an immunomodulator, and any combination thereof.

26. A method for preparing a hydrogel composition, the method comprising: providing a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and contacting the decellularized extracellular matrix (ECM) with a HIF- la stabilizing compound.Attomey Docket No.: 047162-7519WO 1(02692)27. The method of claim 26, wherein the method further comprises formulating the ECM as a hydrogel in the presence of the HIF-la stabilizing compound.

28. The method of any one of claims 26-27, wherein the tissue is a musculoskeletal tissue.

29. The method of any one of claims 26-28, wherein the HIF-la stabilizing compound is a HIF prolyl hydroxylase inhibitor (HIF-PHI).

30. The method of claim 29, wherein the HIF prolyl hydroxylase inhibitor (HIF-PHI) is a 2-oxoglutarate mimetic compound.

31. The method of claim 29, wherein the HIF prolyl hydroxylase inhibitor (HIF-PHI) is dimethyloxalylglycine (DMOG), daprodustat, desidustat, enarodustat, molidustat, roxadustat, or vadadustat.

32. The method of any one of claims 26-31, wherein cells of the tissue lacking functional TSP2 comprise:(a) a TSP2-null knockout allele; and / or(b) suppressed TSP2 gene expression.

33. A method of treating, ameliorating, and / or preventing diabetic foot ulcer on a subject in need thereof, the method comprising: applying to the ulcer a hydrogel composition comprising: a decellularized extracellular matrix (ECM) of a tissue, wherein the tissue lacks functional thrombospondin-2 (TSP2); and a HIF-la stabilizing compound.

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