Bioengineered scaffold for modulation of immune system, and use of the same

Abstract

To provide a composition and a method for modulating an immune system in a subject.SOLUTION: A composition and a method provide means for treating and / or preventing a disease associated with deficiency and / or dysregulation of a T-cell. The composition can include: a porous scaffold, a growth factor which is present at approximately 1ng to approximately 1000ng per scaffold, and an effective amount for inducing formation of a tissue or an organ in the scaffold and mobilizing cells into the scaffold; and a differentiation factor which induces differentiation of mobilized cells into a T-cell precursor cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 780,727, filed December 17, 2018, and U.S. Provisional Application No. 62 / 798,100, filed January 29, 2019. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0002] Government support This invention was made with Government support under grants HL129903, EB015498, EB014703, and EB023287 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention Long-term immunodeficiency in patients undergoing hematopoietic stem cell transplantation (HSCT) remains one of the most significant obstacles in managing life-threatening blood or bone marrow diseases, such as multiple myeloma and leukemia. Prior to transplantation, recipients undergo conditioning cytotoxic radiation and chemotherapy regimens to destroy diseased cells. A side effect of the conditioning process is severe lymphopenia as a result of the destruction of T and B cells of the adaptive immune system. Severe post-transplant immunodeficiency, characterized by a dramatic reduction in T and B cell numbers as well as a reduction in their diversity, can last for 1-2 years. Severe opportunistic infections (approximately 30%), cancer recurrence (greater than 50% for acute myeloid leukemia), and graft-versus-host disease (GVHD) (approximately 40%) associated with immunodeficiency are the most common complications and causes of morbidity and mortality in patients undergoing HSCT. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need for novel compositions and methods that are useful for improving immune system reconstitution after HSCT. There is also a need for compositions and methods that can reduce the risks associated with HSCT and improve patient outcomes. [Means for solving the problem]

[0005] Summary of the Invention Disclosed herein is a novel composition and method for modulating the immune system of a subject.The composition and method disclosed herein provide a means for treating and / or preventing diseases associated with T cell deficiency and / or dysregulation.

[0006] Thus, in one aspect, the present invention provides a composition for modulating the immune system in a subject. The composition includes a porous scaffold, a growth factor present at about 1 ng to about 1000 ng per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and to recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into T cell progenitors. In one embodiment, the growth factor is present at about 0.03 ng / mm2 based on the volume of the scaffold. 3 ~about 350ng / mm 3 It exists in.

[0007] In another aspect, the present invention provides a composition for modulating the immune system in a subject, the composition comprising a porous scaffold and a cellular component comprising about 0.03 ng / mm2 of a cellular component based on the volume of the scaffold. 3 ~about 350ng / mm 3 and a growth factor present in an amount effective to induce formation of a tissue or organ within the scaffold and to recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into T cell progenitor cells.

[0008] In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold comprises a hydrogel. In one embodiment, the scaffold comprises a cryogel. In another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyric acid, poly-epsilon-caprolactone, polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic® polyol, poloxamer, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), and any combination thereof. In yet another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of alginate, alginate derivatives, and combinations thereof. In yet another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and combinations thereof.

[0009] In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold comprises pores having a diameter of about 1 μm to 100 μm. In one embodiment, the scaffold comprises macropores. In another embodiment, the macropores have a diameter of about 50 μm to 80 μm. In yet another embodiment, the scaffold comprises macropores of different sizes.

[0010] In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold is injectable.

[0011] In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold comprises methacrylated alginate (MA-alginate).

[0012] In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold comprises hyaluronic acid or a hyaluronic acid derivative.

[0013] In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold comprises a click hydrogel or a click cryogel. In one embodiment, the scaffold comprises a click alginate, click gelatin, or click hyaluronic acid.

[0014] In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold comprises porogen hydrogel microbeads and bulk hydrogel, where the porogen hydrogel microbeads degrade at least 10% more rapidly than the bulk hydrogel polymer scaffold after administration of the scaffold to a subject. In one embodiment, the porogen hydrogel microbeads comprise oxidized alginate.

[0015] In various embodiments of the above aspect or any other aspect of the invention described herein, the cells are stem cells, hi one embodiment, the stem cells are hematopoietic stem cells.

[0016] In various embodiments of the above aspect or any other aspect of the invention described herein, the cell is a progenitor cell.

[0017] In various embodiments of the above aspects or any other aspects of the invention described herein, the tissue or organ comprises bone tissue or hematopoietic tissue. In one embodiment, the tissue or organ is formed about 7-21 days after administration of the composition to the subject. In another embodiment, the tissue or organ is formed about 14 days after administration of the composition to the subject.

[0018] In various embodiments of the above aspect or any other aspect of the invention described herein, the cell is a stromal cell.

[0019] In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold has a thickness of about 100 μm 3 ~about 10cm 3 In one embodiment, the scaffold is approximately 10 mm in size. 3 ~ approx. 100mm 3 In another embodiment, the scaffold is sized at about 30 mm. 3 The size is.

[0020] In various embodiments of the above aspects or any other aspects of the invention described herein, the growth factor comprises a protein belonging to the transforming growth factor protein beta (TGF-β) superfamily. In one embodiment, the growth factor comprises a protein selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, growth differentiation factor (GDF)-1, GDG-2, GDF-3, GDF-5, GDF-6, GDF-8, GDF-9, GDF-10, GDF-11, GDF-15, anti-Mullerian hormone (AMH), activin, Nodal, TGF-β1, TGF-β2, TGF-β3, TGF-β4, and any combination thereof. In another embodiment, the growth factor comprises BMP-2. In yet another embodiment, the growth factor comprises TGF-β1.

[0021] In various embodiments of the above aspect or any other aspect of the invention described herein, the growth factor is present at about 5 ng to about 500 ng. In one embodiment, the growth factor is present at about 5 ng to about 250 ng. In another embodiment, the growth factor is present at about 5 ng to about 200 ng. In yet another embodiment, the growth factor is present at about 200 ng. In yet another embodiment, the growth factor is present at about 6 ng / mm 3 ~about 10ng / mm 3 In yet another embodiment, the growth factor is present at about 6.5 ng / mm 3 ~ approx. 7.0ng / mm 3 It exists in.

[0022] In various embodiments of the above aspect or any other aspect of the invention described herein, the growth factor retains its bioactivity for at least 12 days after the growth factor is incorporated into the scaffold.

[0023] In various embodiments of the above aspects or any other aspects of the invention described herein, the T cell progenitor cells are capable of differentiating into T cells. In one embodiment, the T cells are CD4 + T cells, CD8 + T cells, regulatory T cells (T reg In another embodiment, the T cells include cells selected from the group consisting of T reg Includes.

[0024] In various embodiments of the above aspect or any other aspect of the invention described herein, the differentiation factor binds to a Notch receptor. In one embodiment, the Notch receptor is selected from the group consisting of Notch-1 receptor, Notch-2 receptor, Notch-3 receptor, Notch-4 receptor, and any combination thereof. In another embodiment, the differentiation factor is selected from the group consisting of Delta-like 1 (DLL-1), Delta-like 2 (DLL-2), Delta-like 3 (DLL-3), Delta-like 3 (DLL-3), Delta-like 4 (DLL-4), Jagged 1, Jagged 2, and any combination thereof.

[0025] In various embodiments of the above aspect or any other aspect of the invention described herein, the differentiation factor is covalently linked to the scaffold. In one embodiment, the differentiation factor is covalently linked to the scaffold using click chemistry. In another embodiment, the differentiation factor is covalently linked to the scaffold using N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocyclooctyne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or disulfide chemistry.

[0026] In various embodiments of the above aspect or any other aspect of the invention described herein, the differentiation factor is present in an amount of about 1 ng to about 1000 μg per scaffold. In one embodiment, the differentiation factor is present in an amount of about 1 μg to about 100 μg per scaffold. In another embodiment, the differentiation factor is present in an amount of 1 μg to about 10 μg per scaffold. In yet another embodiment, the differentiation factor is present at about 6 μg per scaffold.

[0027] In various embodiments of the above aspect or any other aspect of the invention described herein, the molecular factor retains its bioactivity for at least about three months after the differentiation factor is incorporated into the scaffold.

[0028] In various embodiments of the above aspects or any other aspects of the invention described herein, the mobilized cells are transplant cells. In one embodiment, the mobilized cells are autologous. In another embodiment, the mobilized cells are allogeneic. In yet another embodiment, the mobilized cells are xenogeneic.

[0029] In various embodiments of the above aspect or any other aspect of the invention described herein, the mobilized cells are not transplant cells.

[0030] In various embodiments of the above aspects or any other aspects of the invention described herein, the differentiated cells are capable of migrating out of the scaffold, hi one embodiment, the differentiated cells are capable of homing to a tissue in a subject after administration of the composition to the subject.

[0031] In various embodiments of the above aspects or any other aspects of the invention described herein, the composition further comprises a homing factor capable of promoting recruitment of cells to the scaffold. In one embodiment, the homing factor comprises stromal cell-derived factor (SDF-1).

[0032] In one aspect, the present invention provides a method of modulating the immune system in a subject by administering an aforementioned composition of the present invention, thereby modulating the immune system in the subject.

[0033] In another aspect, the present invention provides a method of reducing immune hyper-reactivity in a subject by administering an aforementioned composition of the present invention, thereby reducing immune hyper-reactivity in the subject.

[0034] In yet another aspect, the present invention provides a method of increasing donor chimerism in a subject receiving a transplant by administering an aforementioned composition of the present invention, thereby increasing donor chimerism in the subject.

[0035] In yet another aspect, the present invention provides a method of promoting balanced T cell reconstitution in a subject by administering an aforementioned composition of the present invention, thereby promoting balanced T cell reconstitution in the subject.

[0036] In various embodiments of the above aspect or any other aspect of the invention described herein, the method further comprises administering to the subject hematopoietic stem or progenitor cells. In one embodiment, the composition is administered to the subject simultaneously with or after administration of said hematopoietic stem or progenitor cells to the subject. In another embodiment, the composition is administered to the subject at about 1×10 per kilogram of the subject's body weight. 5 ~About 50×10 6 In yet another embodiment, about 1×10 hematopoietic stem and / or progenitor cells are administered to the subject per kilogram of the subject's body weight. 5 ~Approx. 1×10 6 The hematopoietic stem or progenitor cells are administered to the subject.

[0037] In various embodiments of the above aspect or any other aspect of the invention described herein, the method enhances T cell reconstitution in a subject. In one embodiment, the method enhances T cell neogenesis. In another embodiment, the enhanced T cell neogenesis is characterized by enhanced T cell receptor excision circle (TREC).

[0038] In various embodiments of the above aspects or any other aspects of the invention described herein, the method enhances T cell diversity in the subject. In one embodiment, the T cell diversity is characterized by an enhanced T cell receptor (TCR) repertoire.

[0039] In various embodiments of the above aspect or any other aspect of the invention described herein, the method further comprises: reg ) increase the levels of cells.

[0040] In various embodiments of the above aspect or any other aspect of the invention described herein, the subject is a human with a defective immune system. In one embodiment, the subject has a defective immune system due to immunosenescence. In another embodiment, the subject is over 30 years old, over 40 years old, over 50 years old, over 60 years old, over 70 years old, or over 80 years old. In one embodiment, the subject has a defective immune system due to congenital immune deficiency. In yet another embodiment, the subject has an acquired immune deficiency.

[0041] In another aspect, the invention provides a method of reducing immune hyper-reactivity in a subject, comprising administering to the subject a composition comprising a porous scaffold, a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells to the scaffold, and a differentiation factor to induce differentiation of the recruited cells into T cell progenitors, thereby reducing immune hyper-reactivity in the subject.

[0042] In yet another aspect, the invention provides a method of increasing donor chimerism in a subject receiving a transplant comprising administering to the subject a composition comprising a porous scaffold, a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into T cell progenitors, thereby increasing donor chimerism in the subject.

[0043] In yet another aspect, the invention provides a method for promoting balanced T cell reconstitution in a subject, comprising administering to the subject a composition comprising a porous scaffold, a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into T cell progenitor cells, thereby resulting in balanced T cell reconstitution in the subject.

[0044] In yet another aspect, the invention provides a method of modulating the immune system of a human having an immune system deficiency comprising administering to the human a composition comprising a porous scaffold, a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells to the scaffold, and a differentiation factor to induce differentiation of the recruited cells into T cell progenitor cells, thereby modulating the immune system of the human, wherein the human has an immune system deficiency due to immunosenescence, congenital immune deficiency, or acquired immune deficiency.

[0045] In various embodiments of the above aspects, the scaffold comprises a hydrogel. In one embodiment, the scaffold comprises a cryogel. In another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyric acid, poly-epsilon-caprolactone, polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic® polyol, poloxamer, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), and any combination thereof. In yet another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of alginate, alginate derivatives, and combinations thereof. In yet another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and combinations thereof.

[0046] In various embodiments of the above aspects, the scaffold comprises pores. In one embodiment, the pores have a diameter of about 1 μm to about 100 μm. In another embodiment, the pores have a diameter of about 50 μm to 80 μm. In yet another embodiment, the scaffold comprises pores of different sizes.

[0047] In various embodiments of the above aspects, the scaffold is injectable. In one embodiment, the scaffold comprises methacrylated alginate (MA-alginate). In another embodiment, the scaffold comprises hyaluronic acid or a hyaluronic acid derivative.

[0048] In various embodiments of the above aspects, the scaffold comprises a click hydrogel or a click cryogel. In one embodiment, the scaffold comprises a click alginate, a click gelation, or a click hyaluronic acid.

[0049] In various embodiments of the above aspects, the scaffold comprises porogen hydrogel microbeads and bulk hydrogel, and the porogen hydrogel microbeads degrade at least 10% more rapidly than the bulk hydrogel after administration to a subject. In one embodiment, the porogen hydrogel microbeads comprise oxidized alginate.

[0050] In various embodiments of the above aspects, the cells are stem or progenitor cells. In one embodiment, the cells are selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, recombinant hematopoietic stem cells, recombinant hematopoietic progenitor cells, and any combination thereof. In another embodiment, the cells are selected from the group consisting of hematopoietic bone marrow cells, mobilized peripheral blood cells, recombinant hematopoietic bone marrow cells, recombinant mobilized peripheral blood cells, and any combination thereof.

[0051] In various embodiments of the above aspects, the tissue or organ comprises bone tissue or hematopoietic tissue. In one embodiment, the tissue or organ is formed about 7-21 days after administration of the composition to the subject. In another embodiment, the tissue or organ is formed about 14 days after administration of the composition to the subject. In yet another embodiment, at least two compositions are administered to the subject. In yet another embodiment, the compositions are of similar size.

[0052] In various embodiments of the above aspects, the cells are stromal cells.

[0053] In various embodiments of the above aspects, the scaffold has a thickness of about 100 μm. 3 ~about 10cm 3 In one embodiment, the scaffold is approximately 10 mm in size. 3 ~ approx. 100mm 3 In another embodiment, the scaffold is sized at about 30 mm. 3 The size is.

[0054] In various embodiments of the above aspects, the growth factor comprises a protein belonging to the transforming growth factor protein beta (TGF-β) superfamily. In one embodiment, the growth factor comprises a protein selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, growth differentiation factor (GDF)-1, GDG-2, GDF-3, GDF-5, GDF-6, GDF-8, GDF-9, GDF-10, GDF-11, GDF-15, anti-Mullerian hormone (AMH), activin, Nodal, TGF-β1, TGF-β2, TGF-β3, TGF-β4, and any combination thereof. In another embodiment, the growth factor comprises BMP-2. In yet another embodiment, the growth factor comprises TGF-β1.

[0055] In various embodiments of the above aspects, the growth factor retains its bioactivity for at least 12 days after the growth factor is incorporated into the scaffold.

[0056] In various embodiments of the above aspects, the T cell progenitor cells are capable of differentiating into T cells. In one embodiment, the T cells are CD4 + T cells, CD8 + T cells, regulatory T cells (T reg In another embodiment, the T cells include cells selected from the group consisting of T reg Includes.

[0057] In various embodiments of the above aspect, the differentiation factor binds to a Notch receptor. In one embodiment, the Notch receptor is selected from the group consisting of Notch-1 receptor, Notch-2 receptor, Notch-3 receptor, Notch-4 receptor, and any combination thereof. In another embodiment, the differentiation factor is selected from the group consisting of Delta-like 1 (DLL-1), Delta-like 2 (DLL-2), Delta-like 3 (DLL-3), Delta-like 3 (DLL-3), Delta-like 4 (DLL-4), Jagged 1, Jagged 2, and any combination thereof.

[0058] In various embodiments of the above aspects, the differentiation factor is covalently linked to the scaffold. In one embodiment, the differentiation factor is covalently linked to the scaffold using click chemistry. In another embodiment, the differentiation factor is covalently linked to the scaffold using N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) chemistry, avidin-biotin reaction, azide and dibenzocyclooctyne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or disulfide chemistry.

[0059] In various embodiments of the above aspects, the differentiation factor retains its bioactivity for at least about three months after the differentiation factor is incorporated into the scaffold.

[0060] In various embodiments of the above aspects, the mobilized cells are transplant cells. In one embodiment, the mobilized cells are autologous. In another embodiment, the mobilized cells are allogeneic. In yet another embodiment, the mobilized cells are xenogeneic.

[0061] In various embodiments of the above aspects, the mobilized cells are not transplant cells.

[0062] In various embodiments of the above aspects, the differentiated cells can migrate out of the scaffold, hi one embodiment, the differentiated cells can home to a tissue in a subject after administration of the composition to the subject.

[0063] In various embodiments of the above aspects, the composition further comprises a homing factor capable of promoting recruitment of cells to the scaffold. In one embodiment, the homing factor comprises stromal cell-derived factor (SDF-1).

[0064] In various embodiments of the above aspects, the growth factor is present at about 1 ng to about 1000 μg. In one embodiment, the growth factor is present at about 1 ng to about 1000 ng. In another embodiment, the growth factor is present at about 5 ng to about 500 ng. In yet another embodiment, the growth factor is present at about 5 ng to about 250 ng. In yet another embodiment, the growth factor is present at about 5 ng to about 200 ng. In yet another embodiment, the growth factor is present at about 200 ng.

[0065] In various embodiments of the above aspects, the growth factor is at about 0.03 ng / mm based on the volume of the scaffold. 3 ~about 350ng / mm 3 In one embodiment, the growth factor is present at about 6 ng / mm 3 ~about 10ng / mm 3 In another embodiment, the growth factor is present at about 6.5 ng / mm 3 ~ approx. 7.0ng / mm 3 It exists in.

[0066] In various embodiments of the above aspects, about 5×10 per kilogram of subject body weight 5 ~About 50×10 6 hematopoietic stem and / or progenitor cells are administered to the subject. In one embodiment, about 5×10 hematopoietic stem and / or progenitor cells are administered per kilogram of the subject's body weight. 5A number of hematopoietic stem cells and / or hematopoietic progenitor cells are administered to the subject. In another embodiment, the hematopoietic cells are selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, recombinant hematopoietic stem cells, recombinant hematopoietic progenitor cells, and any combination thereof. In another embodiment, the hematopoietic cells are selected from the group consisting of hematopoietic bone marrow cells, mobilized peripheral blood cells, recombinant hematopoietic bone marrow cells, recombinant mobilized peripheral blood cells, and any combination thereof.

[0067] In various embodiments of the above aspects, the method reduces autoimmunity in the subject.

[0068] In various embodiments of the above aspects, the method prevents or treats an autoimmune disease. In one embodiment, the autoimmune disease is a disease selected from the group consisting of type 1 diabetes, rheumatoid arthritis, psoriasis, arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, and allergies.

[0069] In various embodiments of the above aspects, the method reduces graft-versus-host disease (GVHD). In one embodiment, the GVHD is associated with hematopoietic stem cell transplantation (HSCT) to the subject. In another embodiment, the composition is administered simultaneously with or after hematopoietic stem cell transplantation (HSCT). In yet another embodiment, the GVHD is associated with solid organ transplantation. In yet another embodiment, the composition is administered to the subject prior to transplantation. In yet another embodiment, the GVHD is acute GVHD. In one embodiment, the GVHD is chronic GVHD. In another embodiment, the method reduces GVHD-associated morbidity, GVHD-associated mortality, or reduced long-term survival associated with GVHD.

[0070] In various embodiments of the above aspects, the transplant is a hematopoietic stem cell transplant (HSCT).

[0071] In various embodiments of the above aspects, the increase in donor chimerism comprises T cell chimerism. In one embodiment, the T cells are CD4 + T cells, CD8+ T cells, or T reg Contains cells.

[0072] In various embodiments of the above aspects, balanced T cell reconstitution is achieved by maintaining a homeostatic CD4 + :CD8 + Characterized by T cell ratio.

[0073] In various embodiments of the above aspects, the human has a defective immune system due to immunosenescence. In one embodiment, the human is over 30 years old, over 40 years old, over 50 years old, over 60 years old, over 70 years old, or over 80 years old.

[0074] In various embodiments of the above aspects, the human has a compromised immune system due to a congenital immune deficiency.

[0075] In various embodiments of the above aspects, the human has a compromised immune system due to an acquired immune deficiency.

[0076] In various embodiments of the above aspects, the method further comprises controlling T(T reg ) increase the levels of cells.

[0077] In various embodiments of the above aspects, the composition is administered by injection. In one embodiment, the injection is a subcutaneous injection.

[0078] In yet a further aspect of the invention, the invention provides a syringe comprising a needle, a reservoir containing the composition of various embodiments of the above aspect or any other aspect of the invention described herein, and a plunger.

[0079] In yet a further aspect of the invention, the invention provides a kit comprising a composition of the various embodiments of the above aspects or any other aspect of the invention described herein, and instructions for administering the composition.

[0080] In various embodiments of the above aspects, the method further comprises administering to the subject a stem cell or progenitor cell mobilizing agent in an amount effective to induce stem cell or progenitor cell migration from bone marrow to blood. In one embodiment, the stem cell or progenitor cell mobilizing agent is selected from the group consisting of IL-1, IL-2, IL-3, IL-6, GM-CSF, G-CSF, plerixafor, PDGF, TGF-beta, NGF, IGF, growth hormone, erythropoietin, thrombopoietin, or a combination thereof. In another embodiment, the stem cell or progenitor cell mobilizing agent is administered before, simultaneously with, or after administration of the composition. In yet another embodiment, the method further comprises administering to the subject a therapeutically effective amount of electromagnetic radiation.

[0081] Other features and advantages of the present invention will become apparent from the following detailed description and drawings. [Brief description of the drawings]

[0082] Figures 1A-1E, 1J, and 1M show that alginate-PEG-DLL4 based bone marrow cryogels (BMCs) present DLL4 and BMP-2 and preferentially expand common lymphoid precursors (CLPs). Figures 1F-1I, 1K, and 1L show an extensive characterization of BMC bioactivity.

[0083] [Figure 1A] FIG. 1A is a schematic diagram for the fabrication of covalently cross-linked BMCs.

[0084] [Figure 1B] Figure 1B is a representative cross-sectional scanning electron micrograph (SEM) image of BMC. Scale bar, 1 mm.

[0085] [Figure 1C] Figure 1C is a representative SEM of the pore shape and structure in the cross-section of a BMC. Scale bar = 200 μm.

[0086] [Figure 1D]FIG. 1D shows the release kinetics of encapsulated BMP-2 and covalently tethered DLL4 (n=5 per group).

[0087] [Figure 1E] FIG. 1E shows surface plasmon resonance measuring the binding kinetics of DLL4 before and after modification with a methacrylate linker.

[0088] [Figure 1F] FIG. IF shows the bioactivity of pooled released BMP-2, measured using alkaline phosphatase enzyme activity in MC3T3-E1 preosteoblasts, compared to BMP-2 that does not become incorporated into BMCs (native BMP-2) and medium without added BMP-2 (growth medium).

[0089] [Figure 1G] FIG. 1G shows the bioactivity of BMP-2 quantified at different time intervals after release using alkaline phosphatase enzyme activity in MC3T3-E1 preosteoblastic cells.

[0090] [Figure 1H] FIG. 1H shows the in vitro bioactivity of the Notch ligand DLL-4 measured using a colorimetric assay.

[0091] [Figure 1I] Figure 1I shows representative fluorescence microscopy images of Citrine expression in CHO-K1+2xHS4-UAS-H2B-Citrine-2xHS4 cH1+hNECD-Gal4esn c9 Notch-reporter cell line at different time intervals for duplicate BMCs (top row) and blank BMCs (bottom row).

[0092] [Figure 1J] Figure 1J shows in vitro differentiation of isolated mouse and human hematopoietic stem / progenitor cells onto CLPs as a function of the degree of functionalization of methacrylate groups on the polymer backbone (n=5).

[0093] [Figure 1K] Figures 1K and 1L show the fold expansion and viability of mouse (Figure 1K) and human (Figure 1L) hematopoietic cells after 7 days of in vitro culture. [Figure 1L] Figures 1K and 1L show the fold expansion and viability of mouse (Figure 1K) and human (Figure 1L) hematopoietic cells after 7 days of in vitro culture.

[0094] Data in Figures 1F-1I, 1K, and 1L are means ± sd of n = 5 and representative from three independent experiments (*P<0.05, **P<0.01, ***P<0.001, analysis of variance (ANOVA) with Tukey post-hoc test).

[0095] [Figure 1M] FIG. 1M shows the percentage of Lin − common lymphoid and myeloid mouse progenitors quantified in growth medium, blank, single factor BMC and dual factor BMC.

[0096] Images and pore size quantification in Figures 1B and 1C are representative of 10 independent replicates. Data in Figures 1D, 1J and 1M show the mean ± sd of 5 337 experimental replicates and are representative of 3 independent experiments. Separate samples were assayed individually.

[0097] Figures 2B-2K show the in vivo location of BMC and its integration into the host. Figures 2A, 2L, and 3B-3F show the extensive in vivo characterization of BMC.

[0098] [Figure 2A] FIG. 2A shows representative flow cytometry profiles of bone marrow cells before and after lineage depletion for use in transplantation (five independent experiments).

[0099] [Figure 2B]Figure 2B shows the schedule of L-TBI, administration of HSCT and co-injection of BMC. B6 mice were irradiated with 1000 cGy (1 dose) and then transplanted with 5x105 lineage-depleted syngeneic GFP BM cells within 48 hours after L-TBI.

[0100] [Figure 2C] FIG. 2C shows the volume of BMC nodules in vivo as a function of time after delivery of various combinations of BMP-2 and DLL-4 into BMCs.

[0101] [Figure 2D] FIG. 2D shows a confocal microscopy image of donor GFP+ cells (green) identified within BMCs (red).

[0102] [Figure 2E-F] Figures 2E and 2F show representative micro-computed tomography (microCT, scale bar = 1 mm) imaging (Figure 2E) and histological examination (scale bar = 1 mm) (Figure 2F) of dual-functionalized BMCs 3 weeks after injection, with bone shells (green arrows) and hematopoietic tissues (yellow arrows).

[0103] [Figure 2G] FIG. 2G shows images of BMCs (blue) in the subcutaneous tissue at various times after injection.

[0104] [Figure 2H-I] Figures 2H and 2I show histological Verhoeff-Van Gieson stained sections of BMCs at 10, 30, 40, and 90 days after transplantation in which blood vessels were identified (blue arrows) (Figure 2H) and quantification of blood vessel density within these sections (Figure 2I).

[0105] [Figure 2J]Figures 2J and 2K show histological Safranin-O stained sections of BMCs at 10, 20, 40, 60, and 90 days after implantation in which alginate was identified (red thread-like staining) (Figure 2J) and quantification of the accessible area of ​​alginate within these sections (Figure 2K). [Figure 2K] Figures 2J and 2K show histological Safranin-O stained sections of BMCs at 10, 20, 40, 60, and 90 days after implantation in which alginate was identified (red thread-like staining) (Figure 2J) and quantification of the accessible area of ​​alginate within these sections (Figure 2K).

[0106] [Figure 2L] Figure 2L shows images of the edges of BMCs extracted from the subcutaneous tissue at given time intervals after implantation. The collagen (blue-green) and cell (black) boundaries of the BMCs can be identified, and in some sections, alginate (red) can be observed using Safranin-O staining (10x objective magnification).

[0107] Data in Figure 2C represent the mean ± sd of five experimental replicates and are representative of two independent experiments (*P<0.05, **P<0.01, ***P<0.001, analysis of variance (ANOVA) with Tukey post-hoc test). Images in Figures 2D-2G, and 2J are representative of four independent samples. Data in Figures 2I and 2K represent the mean ± sd from eight samples and are representative of two independent experiments (*P<0.05, **P<0.01, ns, not significant, ANOVA with Tukey post-hoc test). Separate samples were assayed individually. Data in Figure 2L are the mean ± sd of n=5 and are representative of two independent experiments.

[0108] Figures 3A, and 3G-3P show enhanced mobilization of donor cells into BMCs and seeding of thymic precursors in vivo.

[0109] [Figure 3A]FIG. 3A shows the total number and type of donor-derived GFP+ cells in BMCs containing the combination of BMP-2 and DLL-4 and blank BMCs.

[0110] [Figure 3B] Figure 3B shows representative flow cytometry profiles of bone marrow and BMCs (dual and BMP-2 only) 28 days after transplantation. Donor GFP, myeloid, HSC, lymphoid prestimulated multipotent precursors (LMPP), CLP and myeloid precursors are identified.

[0111] [Figure 3C] Figure 3C shows host mesenchymal stromal cells in BMC and endogenous bone marrow and representative flow cytometry plots. Sca-1+ precursors are represented as a fraction of CD45- cells. CD44-, CD73-, CD29-, CD105- and CD106-expressing cells are represented as a fraction of Sca-1+ precursors.

[0112] [Figure 3D] FIG. 3D shows quantification of bone alkaline phosphatase (bALP) and Oil-red-O (ORO) in bone and BMC 20 days after subcutaneous injection (n=6-7).

[0113] [Figure 3E] FIG. 3E shows colony forming unit assays using bone marrow cells from transplant-only and double BMC-treated mice at days 10, 35, and 70 post-transplant.

[0114] [Figure 3F] Figure 3F shows the concentrations of the homing factor SDF-1α and the lymphocyte precursor-supporting cytokine IL-7 in the harvested BMCs. Mice post-HSCT treated with BMP-2 BMCs and mice post-HSCT treated with dual BMCs were analyzed and compared to cytokine concentrations in the bone marrow of the same groups.

[0115] Data in Figures 3C-3F are means ± sd of n = 4, n = 7, n = 4, and n = 4, respectively, and are representative of two independent experiments. Data in Figure 3B are from n = 10, and are representative of two independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, analysis of variance (ANOVA) with Tukey post-hoc test).

[0116] [Figure 3G] FIG. 3G shows the absolute number of donor GFP+CLPs and the percentage of Ly6D-CLPs in BMP-2 and dual factor BMCs.

[0117] [Figure 3H] FIG. 3H shows a schematic diagram of the experimental setup for surgical transplantation of BMCs harvested from mice after HSCT into sublethally irradiated mice.

[0118] [Figure 3I] FIG. 3I shows double positive (DP), single positive (SP) CD4+ and SP CD8+ cells quantified in the thymus 20 days after surgical transfer of BMC.

[0119] [Figure 3J] FIG. 3J shows total numbers of early T-lineage precursors (ETPs; CD44+CD25-c-kit+) quantified as a function of lineage-depleted transplanted cell dose compared to BMC treatment with the lowest cell dose at multiple time points post-transplant, and representative FACS plots (five experimental replicates per time point, two independent experiments).

[0120] [Figure 3K-M] Figures 3K-3P show the total numbers of early T-lineage precursors (ETPs; CD44+CD25-c-kit+), DN2 (CD44+CD25-), DN3 (CD44+CD25-), DP, SP4, and SP8 thymocyte subsets compared across different treatment conditions at multiple time points post-transplant. [Figure 3N-O]Figures 3K-3P show the total numbers of early T-lineage precursors (ETPs; CD44+CD25-c-kit+), DN2 (CD44+CD25-), DN3 (CD44+CD25-), DP, SP4, and SP8 thymocyte subsets compared across different treatment conditions at multiple time points post-transplant. [Figure 3P] Figures 3K-3P show the total numbers of early T-lineage precursors (ETPs; CD44+CD25-c-kit+), DN2 (CD44+CD25-), DN3 (CD44+CD25-), DP, SP4, and SP8 thymocyte subsets compared across different treatment conditions at multiple time points post-transplant.

[0121] For Figures 3A, 3G, and 3K-3P, mice were administered 5 × 10 5 Lineage-depleted syngeneic GFP BM cells were transplanted within 48 h after L-TBI 375 (1 × 1000 cGy). In Figures 3H and 3I, the first set of mice contained 5 × 10 5 Lineage-depleted syngeneic GFP BM cells were transplanted within 48 h after L-TBI. Subsequent sets of mice were In Figure 3J, mice received 5 × 10 4 pieces~5×10 5 lineage-depleted GFP cells were transplanted. In Figure 3A, all groups were compared to transplant-only controls (*P<0.05, **P<0.01, ***P<0.001, analysis of variance (ANOVA) with Tukey post-hoc test). Data in Figure 3A represent the mean ± sd of 10 mice per group and are representative of two independent experiments. Data in Figures 3G, and 3I-3P represent the mean ± sd from 5 mice per group at each time point in Figures 3I-3P and are representative of two independent experiments. Comparisons were to the lowest cell dose group in Figure 3J and to the transplant-only group in Figures 3I-3P (*P<0.05, **P<0.01, ***P<0.001, analysis of variance (ANOVA) with Tukey post-hoc test). Separate samples were assayed individually.

[0122] Figures 3Q-3T show an extensive characterization of thymic cellularity and weight after transplantation. B6 mice were irradiated with a 1×1000 cGy L-TBI dose followed by 5×10 5 Lineage-depleted syngeneic GFP BM cells were transplanted within 48 h after L-TBI and treated as indicated in the figure.

[0123] [Figure 3Q] FIG. 3Q shows total thymocytes quantified 32 and 42 days after transplantation.

[0124] [Figure 3R] FIG. 3R shows thymus weights quantified between days 12 and 42 after HSCT.

[0125] [Figure 3S] FIG. 3S shows mTECs, cTECs, fibroblasts and endothelial cells quantified 22 days after HSCT.

[0126] [Figure 3T] Figure 3T shows the total numbers of early T-lineage precursors (ETP; CD44+CD25-c-kit+), DN2 (CD44+CD25-), DN3 (CD44+CD25-), DP, SP4, and SP8 thymocyte subsets compared across different treatment conditions 22 days after HSCT.

[0127] Thymuses from mice after HSCT without BMC (transplant only), mice after HSCT treated with BMP-2 BMC, and mice after HSCT treated with dual BMC were harvested, weighed, and compared to irradiated non-treated mice. All groups in Figures 3Q, 3S, and 3T are compared to transplant-only controls. Ten days after HSCT, BMCs were explanted and surgically placed into subcutaneous pockets in a second set of B6 mice irradiated with 500 cGy of SL-TBI. Values ​​are expressed as absolute numbers. Data in Figures 3Q-3T represent the mean ± sd from five mice per group at each time point and are representative of at least two independent experiments (*P<0.05, **P<0.01, ***P<0.001, analysis of variance (ANOVA) with Tukey post-hoc test).

[0128] Figures 4A, and 4D-4L show the enhanced T cell reconstitution mediated by BMCs. Figures 4B, 4C, 4M, and 4N show the extensive characterization of blood cell analysis following HSCT.

[0129] [Figure 4A] Figure 4A shows total CD3+CD4+ and CD3+CD8+ in peripheral blood of mice after HSCT. B6 mice were irradiated with an L-TBI dose of 1x1000cGy. Mice were then transplanted with 5x105 lineage-depleted syngeneic GFP BM cells within 48 hours after L-TBI and treated as indicated.

[0130] [Figure 4B] FIG. 4B shows a representative FACS gating strategy for measuring immune cell reconstitution after HSCT in C57BL / 6J mice transplanted with GFP+ donor hematopoietic stem and progenitor cells from five independent experiments.

[0131] [Figure 4C]Figure 4C shows B cell and myeloid cell reconstitution in vivo. B6 mice were irradiated with an L-TBI dose of 1x1000cGy and then transplanted with 5x105 lineage-depleted syngeneic GFP BM cells within 48 hours after L-TBI. Peripheral blood from mice after HSCT without BMC (transplant only), treated with BMP-2 BMC, and treated with dual BMC was analyzed and the measured counts were compared to the immune cell concentrations before radiation.

[0132] [Figure 4D] Figures 4D-4F show measurements of recovery of the ratio of CD4+ to CD8+ T cells in blood (Figure 4D), spleen (Figure 4E) and bone marrow (Figure 4F) as a function of time, with non-irradiated treated mice for comparison with the same group as in Figure 4A. In Figures 4A and 4D-4F, mice after HSCT treated without BMC (transplant only), mice after HSCT treated with bolus BMP-2 and DLL-4 injections, mice after HSCT treated with BMC containing BMP-2 (BMP-2 BMC) or mice after HSCT treated with BMC containing BMP-2 and DLL4 (dual BMC) were analyzed. [Figure 4E-F] Figures 4D-4F show measurements of recovery of the ratio of CD4+ to CD8+ T cells in blood (Figure 4D), spleen (Figure 4E) and bone marrow (Figure 4F) as a function of time, with non-irradiated treated mice for comparison with the same group as in Figure 4A. In Figures 4A and 4D-4F, mice after HSCT treated without BMC (transplant only), mice after HSCT treated with bolus BMP-2 and DLL-4 injections, mice after HSCT treated with BMC containing BMP-2 (BMP-2 BMC) or mice after HSCT treated with BMC containing BMP-2 and DLL4 (dual BMC) were analyzed.

[0133] [Figure 4G-H]In Figures 4G-4L, B6 mice were irradiated with 500 cGy SL-TBI and then transplanted with 5 × 105 lineage-depleted bone marrow cells within 48 h after radiation. Total numbers of DP thymocytes (Figure 4G), SP4 thymocytes (Figure 4H), and SP8 thymocytes (Figure 4I), as well as peripheral CD4+ T cells (Figure 4J), CD8+ T cells (Figure 4K), and B cells (Figure 4L) in the spleens of SL-TBI syn-HSCT mice with and without treatment with double BMC 28 days after transplantation. Data in Figures 4A, 4D-4F represent the mean ± s.d. of n=8 mice per group for each time point and are representative of at least three independent experiments. Data in Figures 4G-4L represent the mean ± sd of n = 10 mice and are representative of two independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, analysis of variance (ANOVA) with Tukey post-hoc test). [Figure 4I-J] In Figures 4G-4L, B6 mice were irradiated with 500 cGy SL-TBI and then transplanted with 5 × 105 lineage-depleted bone marrow cells within 48 h after radiation. Total numbers of DP thymocytes (Figure 4G), SP4 thymocytes (Figure 4H), and SP8 thymocytes (Figure 4I), as well as peripheral CD4+ T cells (Figure 4J), CD8+ T cells (Figure 4K), and B cells (Figure 4L) in the spleens of SL-TBI syn-HSCT mice with and without treatment with double BMC 28 days after transplantation. Data in Figures 4A, 4D-4F represent the mean ± s.d. of n=8 mice per group for each time point and are representative of at least three independent experiments. Data in Figures 4G-4L represent the mean ± sd of n = 10 mice and are representative of two independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, analysis of variance (ANOVA) with Tukey post-hoc test). [Figure 4K-L]In Figures 4G-4L, B6 mice were irradiated with 500 cGy SL-TBI and then transplanted with 5 × 105 lineage-depleted bone marrow cells within 48 h after radiation. Total numbers of DP thymocytes (Figure 4G), SP4 thymocytes (Figure 4H), and SP8 thymocytes (Figure 4I), as well as peripheral CD4+ T cells (Figure 4J), CD8+ T cells (Figure 4K), and B cells (Figure 4L) in the spleens of SL-TBI syn-HSCT mice with and without treatment with double BMC 28 days after transplantation. Data in Figures 4A, 4D-4F represent the mean ± s.d. of n=8 mice per group for each time point and are representative of at least three independent experiments. Data in Figures 4G-4L represent the mean ± sd of n = 10 mice and are representative of two independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, analysis of variance (ANOVA) with Tukey post-hoc test).

[0134] [Figure 4M] Figure 4M shows representative FACS plots after HSCT of donor and host chimerism in thymocytes (DP, SP4, SP8) and splenocytes (CD4+, CD8+, B220+) at day 28 post-transplant in BM-treated and transplant-only mice.

[0135] [Figure 4N] FIG. 4N shows representative flow cytometry plots of host chimerism (CD45.2) and donor chimerism (CD45.1) in sublethally irradiated mice 28 days after transplantation.

[0136] In Figures 4M and 4N, B6 mice were irradiated with 500 cGy SL-TBI and then 5 × 10 5Lineage-depleted bone marrow cells were transplanted within 48 hours after radiation. One group was treated with BMC. Data in Figure 4C represent the mean ± sd from five mice per group at each time point. Data in Figures 4C, 4M, and 4N are representative of two independent experiments (*P<0.05, **P<0.01, ***P<0.001, analysis of variance (ANOVA) with Tukey post-hoc test).

[0137] Figures 5A, 5B, 5D-5G, 5I, 5J, and 5L-5P show enhanced T cell reconstitution and mitigation of GVHD in NSG-BLT mice and mice following allogeneic HSCT. Figures 5H, 5K, and 5Q show extensive flow cytometric characterization of BMC-generated T cells and culture-generated T cell precursors.

[0138] [Figure 5A-B] Figures 5A, 5B, and 5D show CD3+ T cell reconstitution (Figure 5A) and CD19+ B cell reconstitution (Figure 5B) at CD4+:CD8+ ratio (Figure 5D) in humanized NSG-BLT mice at day 75 with exemplary flow cytometry plots.

[0139] [Figure 5C] Figure 5C shows the extensive characterization of blood cell analysis in NSG-BLT mice. Figure 5C shows pre-B CFU quantified from bone marrow of NSG-BLT mice with or without BMC treatment at two time points after transplantation. Data are mean ± sd of n = 4 and from a single donor in one experiment (*P<0.05, **P<0.01, ***P<0.001, analysis of variance (ANOVA) with Tukey post-hoc test). [Figure 5D] Figures 5A, 5B, and 5D show CD3+ T cell reconstitution (Figure 5A) and CD19+ B cell reconstitution (Figure 5B) at CD4+:CD8+ ratio (Figure 5D) in humanized NSG-BLT mice at day 75 with exemplary flow cytometry plots.

[0140] [Figure 5E]FIG. 5E is an exemplary flow cytometry plot at day 75.

[0141] [Figure 5F] Figures 5F and 5G show survival of NSG-BLT mice (n=10 per group) (Figure 5F) and reconstitution of human regulatory T cells in the thymus and spleen of NSG-BLT mice (Figure 5G). [Figure 5G] Figures 5F and 5G show survival of NSG-BLT mice (n=10 per group) (Figure 5F) and reconstitution of human regulatory T cells in the thymus and spleen of NSG-BLT mice (Figure 5G).

[0142] In Figures 5A, 5B, and 5D-5G, xenogeneic humanized BLT (bone marrow-liver-thymus) mice were generated and used as previously described (Brainard, D. M. et al., Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice. Journal of virology 83, 7305-7321 (2009)). Mice without BMC (NSG-BLT) and with double BMC (NSG-BLT+double BMC) with human donor tissue from the same source were analyzed.

[0143] [Figure 5H] FIG. 5H shows representative flow cytometry profiles of isotypes used to identify FoxP3+ cells among CD4+ cells and Treg cells in the thymus and spleen (three independent experiments).

[0144] [Figure 5I-J]Figures 5I and 5J show the survival (Figure 5I) and reconstitution (Figure 5J) of donor-derived regulatory T cells in the thymus and spleen of allografted Balb / c mice. In Figures 5I and 5J, BALB / cJ recipient mice received 850cGy of L-TBI. Within 48 hours after radiation, mice were transplanted with allogeneic GFP 5x105 lineage-depleted GFP BM cells + 106 GFP splenocytes. One group was treated with double BMC at the same time.

[0145] [Figure 5K] FIG. 5K shows representative FACS profiles of sorted HSCs (Lin-ckit+Sca-1+) and CD44 / CD25-expressing T cell precursors after 14 days of coculture with OP9-DL1 cells (two independent experiments).

[0146] [Figure 5L] Figures 5L-5P show a comparison of T cell reconstitution in mice treated with BMC or OP9-DL1 derived pro-T cells. Balb / cJ recipient mice received 850 Gy of L-TBI and received either 5x106 allogeneic GFP T cell precursors from OP9-DL1 cultures + 103 syngeneic HSCs or double BMC + lineage-depleted allogeneic GFP BM cells. Total numbers of DP thymocytes (Figure 5L) and SP4 thymocytes (Figure 5M) and SP8 thymocytes (Figure 5N) in the thymus and peripheral CD4+ T cells (Figure 5O) and CD8+ T cells (Figure 5P) in the spleen 28 days after transplant in transplanted mice 424. Data in a-d are the mean ± sd of n=10 mice at the start of the 425 study and are representative of three donors. [Fig. 5M-N]Figures 5L-5P show a comparison of T cell reconstitution in mice treated with BMC or OP9-DL1 derived pro-T cells. Balb / cJ recipient mice received 850 Gy of L-TBI and received either 5x106 allogeneic GFP T cell precursors from OP9-DL1 cultures + 103 syngeneic HSCs or double BMC + lineage-depleted allogeneic GFP BM cells. Total numbers of DP thymocytes (Figure 5L) and SP4 thymocytes (Figure 5M) and SP8 thymocytes (Figure 5N) in the thymus and peripheral CD4+ T cells (Figure 5O) and CD8+ T cells (Figure 5P) in the spleen 28 days after transplant in transplanted mice 424. Data in a-d are the mean ± sd of n=10 mice at the start of the 425 study and are representative of three donors. [Figure 5O-P] Figures 5L-5P show a comparison of T cell reconstitution in mice treated with BMC or OP9-DL1 derived pro-T cells. Balb / cJ recipient mice received 850 Gy of L-TBI and received either 5x106 allogeneic GFP T cell precursors from OP9-DL1 cultures + 103 syngeneic HSCs or double BMC + lineage-depleted allogeneic GFP BM cells. Total numbers of DP thymocytes (Figure 5L) and SP4 thymocytes (Figure 5M) and SP8 thymocytes (Figure 5N) in the thymus and peripheral CD4+ T cells (Figure 5O) and CD8+ T cells (Figure 5P) in the spleen 28 days after transplant in transplanted mice 424. Data in a-d are the mean ± sd of n=10 mice at the start of the 425 study and are representative of three donors.

[0147] Data in Figures 5G and 5J are means ± sd of n = 7 mice. Data in Figure 5I are means ± sd of n = 10 mice. Data in Figures 5G, 5I, and 5J are representative of two independent experiments. Data in Figures 5L-5P are means ± sd of n = 10 mice and are representative of two independent experiments. Separate samples were assayed individually (*P<0.05, **P<0.01, ***P<0.001, analysis of variance (ANOVA) with Tukey post-hoc test).

[0148] [Figure 5Q] FIG. 5Q shows representative flow cytometry profiles of ckit and the isotypes used to identify ETPs in the thymus.

[0149] Figures 6A-6H show quantitative analysis of T cell output, immune repertoire and vaccination with regenerated T cells in mice.

[0150] [Figure 6A-B] Figures 6A and 6B show (a) signal joint T-cell receptor excision circle (sjTREC) analysis of isolated thymus (Figure 6A) and spleen (Figure 6B) in mice.

[0151] [Figure 6C] Figure 6C shows the antigen receptor diversity of T cells analyzed by sequenced CDR3 beta chain V and J segments in BMC and transplanted mice. Each bar represents a single clone. The plot presents the depth (bar length) and diversity (bar number) of T cells in the mouse. Samples were pooled from 5 mice for each group and combined data are presented.

[0152] [Figure 6D] FIG. 6D shows a timeline for the analysis of antigen-specific donor T cell responses following vaccination.

[0153] [Figure 6E] 6E and 6F show SIINFEKL-tetramer+ donor CD8+ T cells enumerated in vaccinated mice after syn-HSCT (FIG. 6E) and allo-HSCT (FIG. 6F). [Figure 6F] 6E and 6F show SIINFEKL-tetramer+ donor CD8+ T cells enumerated in vaccinated mice after syn-HSCT (FIG. 6E) and allo-HSCT (FIG. 6F).

[0154] [Figure 6G]Figures 6G and 6H show that splenocytes from 440 OP9-DL1 T cell precursor and double BMC-treated groups were stimulated and stained for surface markers and intracellular cytokines using antibodies specific for CD45.1, CD4, IFN-γ, and TNF-α on days 22 and 42 after HSCT. Cells were gated on CD4+ or CD8+ donor cells and analyzed for IFN-γ- and TNF-α-positive cells. In Figures 6A-6C and 6E, B6 recipients received 1000cGy of L-TBI and 5x105 lineage-depleted syngeneic GFP BM cells. Mice after HSCT without BMC (transplant only), mice after HSCT treated with BMP-2 BMC, and mice after HSCT treated with double BMC were analyzed and compared to irradiated untreated mice that did not receive transplant or vaccine. In Figure 6B, sjTRECs are normalized to 105 CD4+ splenocytes. In Figures 6F-6H, Balb / cJ recipient mice received 850 Gy of L-TBI and received either 5x106 allogeneic GFP T cell precursors from OP9-DL1 cultures + 103 syngeneic HSCs or 5x105 double BMC + lineage-depleted allogeneic GFP BM cells. Data in Figures 6A and 6B are the mean ± sd of n = 10 mice, data in Figures 6E and 6F are the mean ± sd of n = 5 mice, and data in Figures 6G and 6H are the mean ± sd of n = 7 mice. All experiments are representative of two independent experiments (*P<0.05, **P<0.01, ***P<0.001, analysis of variance (ANOVA) with Tukey post-hoc test). [Figure 6H]Figures 6G and 6H show that splenocytes from 440 OP9-DL1 T cell precursor and double BMC-treated groups were stimulated and stained for surface markers and intracellular cytokines using antibodies specific for CD45.1, CD4, IFN-γ, and TNF-α on days 22 and 42 after HSCT. Cells were gated on CD4+ or CD8+ donor cells and analyzed for IFN-γ- and TNF-α-positive cells. In Figures 6A-6C and 6E, B6 recipients received 1000cGy of L-TBI and 5x105 lineage-depleted syngeneic GFP BM cells. Mice after HSCT without BMC (transplant only), mice after HSCT treated with BMP-2 BMC, and mice after HSCT treated with double BMC were analyzed and compared to irradiated untreated mice that did not receive transplant or vaccine. In Figure 6B, sjTRECs are normalized to 105 CD4+ splenocytes. In Figures 6F-6H, Balb / cJ recipient mice received 850 Gy of L-TBI and received either 5x106 allogeneic GFP T cell precursors from OP9-DL1 cultures + 103 syngeneic HSCs or 5x105 double BMC + lineage-depleted allogeneic GFP BM cells. Data in Figures 6A and 6B are the mean ± sd of n = 10 mice, data in Figures 6E and 6F are the mean ± sd of n = 5 mice, and data in Figures 6G and 6H are the mean ± sd of n = 7 mice. All experiments are representative of two independent experiments (*P<0.05, **P<0.01, ***P<0.001, analysis of variance (ANOVA) with Tukey post-hoc test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0155] Detailed Description of the Invention I. Definition In order that the present invention may be more readily understood, certain terms are first defined.

[0156] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. The meaning and spirit of the terms should be clear, however, in the event of potential ambiguity, the definitions provided herein shall take precedence over any dictionary or extrinsic definitions.

[0157] The use of the terms "a," "an," and "the," and similar references in the context of describing the present invention (particularly in the context of the claims which follow), shall be construed to include both the singular and the plural (i.e., one or more), unless otherwise indicated herein or clearly contradicted by context. The terms "including" and "containing" shall be construed as open ended (i.e., meaning "including but not limited to"), unless otherwise indicated. Statements of ranges of values ​​herein are intended to serve as a shorthand method of referring individually to each separate value recited or falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited.

[0158] The term "about" or "approximately" typically means within 5%, or more preferably within 1%, of a given value or range.

[0159] In general, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent to a patient, or to an isolated tissue or cell line derived from a patient, having a disease, a symptom of a disease, or a predisposition to a disease, with the intent to cure, heal, alleviate, relieve, alter, treat, mitigate, improve, or affect a disease, a symptom of a disease, or a predisposition to a disease. Thus, treating can include suppressing, inhibiting, preventing, treating, or a combination thereof. Treating refers to, among other things, prolonging the time to sustained progression, hastening remission, inducing remission, increasing remission, accelerating recovery, increasing the effectiveness of or decreasing resistance to alternative therapies, or a combination thereof. "Suppressing" or "inhibiting" refers to, among other things, delaying the onset of symptoms, preventing recurrence of disease, reducing the number or frequency of recurrent episodes, lengthening the time between symptomatic episodes, reducing the severity of symptoms, reducing the severity of acute episodes, reducing the number of symptoms, reducing the occurrence of disease-related symptoms, reducing the duration of symptoms, alleviating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or combinations thereof. In one embodiment, the symptoms are primary, while in another embodiment, the symptoms are secondary. "Primary" refers to symptoms that are a direct result of a disorder, e.g., diabetes, while secondary refers to symptoms that originate from or are the result of a primary cause. The symptoms can be any manifestation of a disease or condition.

[0160] Thus, as used herein, the term "treatment" or "treating" includes any administration of a composition described herein, and includes (i) preventing a disease from occurring in a subject who may be predisposed to the disease but who is not experiencing or exhibiting pathology or symptoms of the disease, (ii) inhibiting the disease (i.e., halting further development of the pathology and / or symptoms) in a subject who is experiencing or exhibiting pathology or symptoms of the disease, or (iii) alleviating the disease (i.e., reversing the pathology and / or symptoms) in a subject who is experiencing or exhibiting pathology or symptoms of the disease.

[0161] "Treatment," "prevention," or "alleviation" of a disease or disorder refers to delaying or preventing the onset of such disease or disorder, reversing, alleviating, mitigating, inhibiting, slowing, or halting the progression, worsening, or exacerbation of the conditions associated with such disease or disorder, or the progression or severity of the symptoms thereof. In one embodiment, the symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0162] The effectiveness of treatment is determined in association with any known method for diagnosing disorder.The alleviation of one or more symptoms of disorder indicates that the composition provides clinical benefit.Any of the above-mentioned therapeutic methods can be applied to any suitable subject, including, for example, mammals, for example, dogs, cats, cows, horses, rabbits, monkeys, and most preferably humans.

[0163] As used herein, the term "subject" includes any subject that may benefit from administration of the hydrogel or implantable drug delivery device of the present invention. The term "subject" includes animals, e.g., vertebrates, amphibians, fish, mammals, non-human animals, including humans and primates, e.g., chimpanzees, monkeys, etc. In one embodiment of the present invention, the subject is a human.

[0164] The term "subject" also includes production livestock, such as cattle, sheep, goats, horses, pigs, donkeys, camels, buffalo, rabbits, chickens, turkeys, ducks, geese, and bees, as well as household pets, such as dogs, cats, domesticated birds, and ornamental fish, and also so-called test animals, such as hamsters, guinea pigs, rats, and mice.

[0165] II. Compositions for modulating the immune system The present invention features compositions and methods for modulating the immune system of a subject. The compositions of the invention include a porous scaffold, a growth factor present in an amount effective to recruit cells to the scaffold and / or induce the formation of a tissue or organ, and a differentiation factor that induces differentiation of the recruited cells into T cell progenitors.

[0166] The compositions and methods of the present invention provide advantages over the prior art. For example, the compositions and methods of the present invention enhance thymic T cell precursor seeding, T cell neogenesis, and T cell receptor repertoire expansion. In addition, the compositions and methods of the present invention promote donor CD4+ regulatory T cell generation and improved survival after allogeneic HSCT. Compared with adoptive transfer of T cell precursors, the compositions and methods of the present invention increase donor chimerism, T cell generation, and induce stronger antigen-specific T cell response to stimulation. The compositions of the present invention may represent an easy-to-administer ready-made approach to T cell regeneration and alleviation of GVHD in HSCT.

[0167] In certain embodiments, the compositions and methods of the present invention also reduce toxicity resulting from the use of significantly reduced levels of growth factor, e.g., 1 ng to about 1000 μg of growth factor, or 1 ng to about 1000 ng of growth factor, compared to those taught in the art. In addition, the compositions and methods of the present invention exhibit surprisingly enhanced activity, thereby allowing for comparable efficacy when transplanting reduced numbers of cells.

[0168] Scaffold The composition of the present invention includes a scaffold, for example, a polymer scaffold. The scaffold may include one or more biomaterials. Preferably, the biomaterial is a biocompatible material that is non-toxic and / or non-immunogenic. As used herein, "biocompatible material" refers to any material that, when implanted in or adjacent to a subject's biological tissue, does not induce significant immune or adverse tissue reaction over time, e.g., toxic reaction or significant irritation.

[0169] The scaffold may include a biomaterial that is non-biodegradable or biodegradable. In certain embodiments, the biomaterial may be a non-biodegradable material. Exemplary non-biodegradable materials include, but are not limited to, metals, plastic polymers, or silk polymers. In certain embodiments, the polymer scaffold includes a biodegradable material. Biodegradable materials can be degraded by physical or chemical action, such as levels of hydration, heat, oxidation, or ion exchange, or cellular action by nearby or present cells, such as assimilation of enzymes, peptides, or other compounds. In certain embodiments, the polymer scaffold may include both non-degradable and degradable materials.

[0170] In some embodiments, the scaffold composition may degrade at a predetermined rate based on physical parameters selected from the group consisting of temperature, pH, hydration state, and porosity, crosslink density, type, and chemistry, or susceptibility to degradation of the backbone linkages. Alternatively, the scaffold composition degrades at a predetermined rate based on chemical polymer ratios. For example, a high molecular weight polymer composed solely of lactide will degrade over a period of years, e.g., 1-2 years, while a low molecular weight polymer composed of a 50:50 mixture of lactide and glycolide will degrade within a few weeks, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, or 10 weeks. Calcium cross-linked gels composed of high molecular weight, high guluronic acid alginates will degrade in vivo over a period of months (1, 2, 4, 6, 8, 10, or 12 months) to years (1, 2, or 5 years), whereas gels composed of low molecular weight alginates and / or partially oxidized alginates will degrade within a period of weeks.

[0171] In certain embodiments, one or more compounds or proteins disclosed herein (e.g., growth factors, differentiation factors, and homing factors) are linked or bound to the scaffold composition by covalent or non-covalent bonds. In various embodiments, one or more compounds or proteins disclosed herein are incorporated into or present in the structure or pores of the scaffold composition.

[0172] In some embodiments, the scaffold comprises a biomaterial that has been modified, e.g., oxidized or reduced. The degree of modification, e.g., oxidation, can vary from about 1% to about 100%. As used herein, the degree of modification refers to the molar percentage of sites on the biomaterial that are modified with functional groups. For example, the degree of modification may be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51% , 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Values ​​and ranges intermediate to the recited values ​​are intended to be part of the invention. Exemplary modified biomaterials, eg, hydrogels, include, but are not limited to, reduced alginate, oxidized alginate, MA-alginate (methacrylated alginate), or MA-gelatin.

[0173] Exemplary biomaterials suitable for use as scaffolds in the present invention include glycosaminoglycans, silk, fibrin, MATRIGEL®, poly-ethylene glycol (PEG), polyhydroxyethyl methacrylate, polyacrylamide, poly(N-vinylpyrrolidone), (PGA), polylactic-co-glycolic acid (PLGA), poly-e-carpolactone (PCL), polyethylene oxide, polypropylene fumarate (PPF), polyacrylic acid (PAA), polyhydrochloride (PHA), polyisocyanate ... Examples of suitable biomaterials include butyric acid, hydrolyzed polyacrylonitrile, polymethacrylic acid, polyethyleneamine, esters of alginic acid, pectinic acid, and alginates, fully or partially oxidized alginates, hyaluronic acid, carboxymethylcellulose, heparin, heparin sulfate, chitosan, carboxymethyl chitosan, chitin, pullulan, gellan, xanthan, collagen, gelatin, carboxymethyl starch, carboxymethyl dextran, chondroitin sulfate, cationic guar, cationic starch, and combinations thereof. In certain embodiments, the biomaterial is selected from the group consisting of alginates, fully or partially oxidized alginates, and combinations thereof.

[0174] The scaffold of the present invention may include an exterior surface. Alternatively or additionally, the scaffold may include an interior surface. The exterior or interior surface of the scaffold of the present invention may be solid or porous. The pore size of the scaffold may be less than about 10 nm, between about 100 nm and 20 μm, or greater than about 20 μm, e.g., up to 1000 μm in diameter. For example, the pores may be nanoporous, microporous, or macroporous. For example, the diameter of the nanopores is less than about 10 nm, the diameter of the micropores is in the range of about 100 nm to 20 μm, and the diameter of the macropores is greater than about 20 μm, e.g., greater than about 50 μm, e.g., greater than about 100 μm, e.g., greater than about 400 μm, e.g., greater than 600 μm, or greater than 800 μm. In some embodiments, the diameter of the pores is about 50 μm to about 80 μm.

[0175] In some embodiments, the scaffolds of the invention are organized in various geometries (e.g., disks, beads, pellets), niches, planar layers (e.g., thin sheets). For example, disks with diameters of about 0.1 to 200 millimeters, e.g., 5, 10, 20, 40, or 50 millimeters, may be implanted subcutaneously. The disks may have thicknesses of 0.1 to 10 millimeters, e.g., 1, 2, or 5 millimeters. The disks are easily compressed or lyophilized for administration to a patient. An exemplary disk for subcutaneous administration has the following dimensions: 8 millimeters diameter and 1 millimeter thickness.

[0176] In some embodiments, the scaffold may include multiple components and / or compartments. In certain embodiments, the multi-compartment device is assembled in vivo by applying successive layers of similarly or differently doped gel or other scaffold material to the target site. For example, the device is formed by using a needle to sequentially inject the next inner layer into the center of the previously injected material, thereby forming a concentric spheroid. In certain embodiments, non-concentric compartments are formed by injecting material into different locations of the previously injected layer. A multi-headed injection device extrudes the compartments in parallel and simultaneously. The layers are made of similar or different biomaterials that are differently doped with pharmaceutical compositions. Alternatively, the compartments are self-assembled based on their hydrophilic / hydrophobic characteristics or secondary interactions within each compartment. In certain embodiments, a multi-component scaffold is optionally constructed into multiple concentric layers, each of which is characterized by different physical properties, e.g., percentage of polymer, percentage of crosslinks of the polymer, chemical composition of the hydrogel, pore size, porosity and pore architecture, stiffness, toughness, ductility, viscoelasticity, growth factors, differentiation factors, and / or homing factors incorporated therein, and / or any other compositions incorporated therein.

[0177] Hydrogel and cryogel scaffolds In certain embodiments, the scaffold of the present invention comprises one or more hydrogels. Hydrogels are polymer gels that contain a network of crosslinked polymer chains. Hydrogels are compositions that contain polymer chains that are usually hydrophilic. The network structure of hydrogels allows them to absorb significant amounts of water. Some hydrogels are highly stretchable and elastic, while others are viscoelastic. Hydrogels can also be found as colloidal gels in which water is the dispersion medium. In certain embodiments, hydrogels are highly absorbent (can contain more than 99% water (volume / volume)) natural polymers due to their significant water content, or synthetic polymers that have a degree of flexibility very similar to natural tissue. In certain embodiments, hydrogels can have the property that upon application of an appropriate shear stress, the deformable hydrogel can be dramatically and reversibly compressed (up to 95% of its volume), resulting in an injectable scaffold with preformed macropores. Hydrogels have been used in therapeutic applications, for example, as vehicles for the in vivo delivery of therapeutic agents, such as small molecules, cells, and biologics. Hydrogels are generally produced from polysaccharides, such as alginate. Polysaccharides may be chemically engineered to modulate their properties and those of the resulting hydrogel.

[0178] The hydrogels of the present invention may be porous or non-porous. Preferably, the compositions of the present invention are formed from porous hydrogels. For example, the hydrogels may be nanoporous, with pores having a diameter of less than about 10 nm, microporous, with pores having a diameter of preferably about 100 nm to 20 μm, or macroporous, with pores having a diameter of more than about 20 μm, more preferably more than about 100 μm, and even more preferably more than about 400 μm. In certain embodiments, the hydrogels are macroporous, with pores having a diameter of about 50 to 80 μm. In certain embodiments, the hydrogels are macroporous, with aligned pores having a diameter of about 400 to 500 μm. Methods for preparing porous hydrogel products are known in the art. (See, for example, U.S. Patent No. 6,511,650, which is incorporated herein by reference).

[0179] Hydrogels can be constructed from a number of different rigid, semi-rigid, flexible, gel-like self-assembling liquid crystal or fluid compositions, such as peptide polymers, polysaccharides, synthetic polymers, hydrogel materials, ceramics (e.g., calcium phosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans, metals, and metal alloys. The compositions are assembled into hydrogels using methods known in the art, such as injection molding, freeze-drying of preformed structures, printing, self-assembly, phase inversion, solvent casting, melt processing, gas foaming, fiber formation / processing, particle leaching, or combinations thereof. The assembled device is then implanted or administered into the body of the individual to be treated.

[0180] Compositions including hydrogels can be assembled in vivo in several ways. Hydrogels are made from gelling materials that are introduced into the body in their non-gelled form and gel in situ. Exemplary methods of delivering the components of the composition to the site where assembly occurs include injection with a needle or other extrusion tool, spraying, painting, or deposition methods at the tissue site, such as delivery using an application device inserted by a cannula. In some embodiments, non-gelled or unformed hydrogel material is mixed with at least one pharmaceutical composition before or during introduction into the body. The resulting in vivo / in situ assembled device, e.g., hydrogel, contains a mixture of at least one pharmaceutical composition.

[0181] In situ assembly of hydrogels can occur as a result of spontaneous association of polymers or by synergistically or chemically catalyzed polymerization. Synergistic or chemical catalysis can be initiated by some intrinsic factor or condition at or near the assembly site, such as body temperature, ions, or pH, or by an external factor or condition provided to the assembly site by an operator, such as photon, heat, electricity, sound, or other irradiation induced there after the non-gelling material is introduced. Energy is induced into the hydrogel material by a radiation beam, or by a heat or light conductor, such as a wire or fiber optic cable or an ultrasonic transducer. Alternatively, shear-thinning materials, such as amphiphiles, are used that recrosslink after the shear stress applied to them is released, for example by passing through a needle.

[0182] In some embodiments, the hydrogel may be assembled ex vivo. In some embodiments, the hydrogel is injectable. For example, the hydrogel is made outside the body as a macroporous scaffold. When injected into the body, the pores collapse and the gel becomes very small, allowing it to fit through a needle. See, for example, International Publication No. WO2012 / 149358 and Bencherif et al., 2012, Proc. Natl. Acad. Sci. USA 109.48:19590-5, the contents of which are incorporated herein by reference).

[0183] Hydrogels suitable for assembly of hydrogel devices both in vivo and ex vivo are well known in the art and are described, for example, in Lee et al., 2001, Chem. Rev. 7:1869-1879. Amphiphilic peptide approaches for assembly by self-assembly are described, for example, in Hartgerink et al., 2002, Proc. Natl. Acad. Sci. USA 99:5133-5138. Reversible gels after shear thinning Methods for purification are exemplified in Lee et al., 2003, Adv. Mat. 15:1828-1832.

[0184] In certain embodiments, exemplary hydrogels are composed of materials compatible with the encapsulation of materials, including polymers, nanoparticles, polypeptides, and cells. Exemplary hydrogels are made from alginate, polyethylene glycol (PEG), PEG-acrylate, agarose, hyaluronic acid, or synthetic proteins, such as collagen or engineered proteins (i.e., self-assembly peptide-based hydrogels). For example, commercially available hydrogels include BD™ PuraMatrix™. BD™ PuraMatrix™ peptide hydrogels are synthetic matrices used to create defined three-dimensional (3D) microenvironments for cell culture.

[0185] In some embodiments, hydrogels are biocompatible polymer matrices that are fully or partially biodegradable. Examples of materials that can form hydrogels include alginates and alginate derivatives, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA) polymers, gelatin, collagen, agarose, hyaluronic acid, hyaluronic acid derivatives, natural and synthetic polysaccharides, polyamino acids, such as polypeptides, particularly poly(lysine), polyesters, such as polyhydroxybutyric acid and poly-epsilon.-caprolactone, polyanhydrides, polyphosphazines, poly(vinyl alcohol), poly(alkylene oxides), particularly poly(ethylene oxide), poly(allylamine) (PAM), poly(acrylates), modified styrene polymers, such as poly(4-aminomethylstyrene), Pluronic® polyols, polyoxamers, poly(uronic acid), poly(vinylpyrrolidone), and copolymers of the above, including graft copolymers. Synthetic and naturally occurring polymers, including but not limited to collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels, can also be used. The term "derivative" as used herein refers to a compound derived from a similar compound by chemical reaction. For example, oxidized alginate, which is derived from alginate by oxidation reaction, is a derivative of alginate.

[0186] The implantable composition may have virtually any regular or irregular shape, including, but not limited to, spheroids, cubes, polyhedrons, cylinders, rods, discs, or other geometric shapes. Thus, in some embodiments, the implant is of a cylindrical shape with a diameter of about 0.5 to about 10 mm and a length of about 0.5 to about 10 cm. Preferably, the diameter is about 1 to about 5 mm, and the length is about 1 to about 5 cm.

[0187] In some embodiments, the compositions of the present invention are in spherical form. When the composition is in spherical form, its diameter may range from about 0.5 to about 50 mm in some embodiments. In some embodiments, the diameter of the spherical implant is about 5 to about 30 mm. In an exemplary embodiment, the diameter is about 10 to about 25 mm.

[0188] In certain embodiments, the scaffold comprises a click hydrogel and / or a click cryogel. A click hydrogel or cryogel is a gel in which crosslinking between hydrogel or cryogel polymers is facilitated by a click reaction between the polymers. Each polymer may contain one of the additional functional groups useful in click reactions. Given the high level of specificity of functional group pairs in click reactions, active compounds can be added to the preformed device prior to or simultaneously with the formation of the hydrogel device by click chemistry. Non-limiting examples of click reactions that can be used to form click hydrogels include copper I-catalyzed azide-alkyne cycloaddition, strain-promoted assize-alkyne cycloaddition, thiol-ene photocoupling, Diels-Alder reaction, inverse electron demand Diels-Alder reaction, tetrazole-alkene photoclick reaction, oxime reaction, thiol-Michael addition, and aldehyde-hydrazide coupling. Non-limiting embodiments of click hydrogels are described in Jiang et al., 2014, Biomaterials, 35:4969-4985, in their entirety. The contents are incorporated herein by reference.

[0189] In various embodiments, click alginates are utilized (see, e.g., PCT International Patent Application Publication No. WO 2015 / 154078, published October 8, 2015, which is incorporated by reference in its entirety).

[0190] In certain embodiments, a hydrogel (e.g., cryogel) system can deliver one or more agents (e.g., growth factors, e.g., BMP-2, and / or differentiation factors, e.g., DLL-4) while creating space for cells (e.g., stem cells, e.g., hematopoietic stem cells (HSC) infiltration and trafficking). In some embodiments, a hydrogel system according to the present invention delivers BMP-2, which acts as a hematopoietic stem cell (HSC) and / or hematopoietic progenitor cell enhancing / mobilizing factor, and DLL-4 as a differentiation factor that promotes T cell lineage specification of hematopoietic stem cells and / or hematopoietic progenitor cells.

[0191] In some embodiments, cryogel compositions, such as those formed from MA-alginate, may serve as delivery platforms by creating local niches, such as specific niches for enhancing T lineage specification. In some embodiments, cryogels create local niches that can control the encounter of cells, such as mobilized stem or progenitor cells, and various exemplary agents of the present invention, such as growth and / or differentiation factors. In certain embodiments, cells and exemplary agents of the present invention are localized in a small volume, and cell and agent contact can be quantitatively controlled in space and time.

[0192] In certain embodiments, hydrogels (e.g., cryogels) can be engineered to spatially and temporally coordinate the delivery of both growth and differentiation factors, potentially enhancing overall immune modulation functions by regulating the differentiation and / or specification of recruited cells, e.g., hematopoietic stem or progenitor cells. In certain embodiments, cells and growth / differentiation factors are localized to a small volume, and spatial and temporal factor delivery can be quantitatively controlled. Growth / differentiation factors are released locally, and little systemic action is expected, in contrast to systemically delivered agents, e.g., growth factors.

[0193] Examples of polymer compositions from which cryogels or hydrogels are made are described throughout this disclosure, including alginate, hyaluronic acid, gelatin, heparin, dextran, carob gum, PEG, PEG derivatives including PEG-co-PGA and PEG-peptide conjugates. These techniques can be applied to any biocompatible polymer, such as collagen, chitosan, carboxymethylcellulose, pullulan, polyvinyl alcohol (PVA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-isopropylacrylamide) (PNIPAAm), or poly(acrylic acid) (PAAc). For example, in certain embodiments, the composition comprises an alginate-based hydrogel / cryogel. In another example, the scaffold comprises a gelatin-based hydrogel / cryogel.

[0194] Cryogels are a class of materials with highly porous interconnected structures produced using cryotropic gelation (or cryogelation) techniques. Cryogels also have a highly porous structure. Typically, active compounds are added to the cryogel device after freezing the pore / wall structure of the cryogel. Cryogels are characterized by high porosity, e.g., at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% pores in thin pore walls characterized by a high density of polymer crosslinks. As used herein, the term "porosity" refers to the percentage of pore volume relative to the volume of the scaffold. Values ​​and ranges intermediate to the recited values ​​are intended to be part of the invention. The walls of cryogels are typically dense and highly crosslinked, allowing them to be compressed through a needle into a subject without permanent deformation or substantial structural damage.

[0195] In various embodiments, the pore walls comprise at least about 10, 15, 20, 25, 30, 35, or 40% (weight / volume) polymer. Values ​​and ranges intermediate to the recited values ​​are intended to be part of the invention. In other embodiments, the pore walls comprise about 10-40% polymer. In some embodiments, a polymer concentration (before cryogelation) of about 0.5-4% (weight / volume) is used, with the concentration increasing substantially once cryogelation is complete. Non-limiting aspects of cryogel gelation and the increase in polymer concentration after cryogelation are discussed in Beduer et al., 2015 Advanced Healthcare Materials 4.2: 301-312, the entire contents of which are incorporated herein by reference.

[0196] In certain embodiments, cryogelation is a technique in which the polymerization-crosslinking reaction is carried out in a pseudo-frozen reaction solution. Non-limiting examples of cryogelation techniques are described in U.S. Patent Application Publication No. 20140227327, published August 14, 2014, the entire contents of which are incorporated herein by reference. The advantage of cryogels compared to traditional macroporous hydrogels obtained by phase separation is their high reversible deformability. Cryogels are extremely soft, but can deform and reform their shape. In certain embodiments, cryogels can be very robust and can withstand high levels of deformation, e.g., stretching and twisting, and can also be squeezed under mechanical force to expel their solvent content. The improved deformation properties of alginate cryogels are due to the high crosslink density of the non-frozen liquid channels of the reaction system.

[0197] In the cryogelation process, during the freezing of the macromonomer (e.g., methacrylated alginate) solution, the macromonomer and initiator system (e.g., APS / TEMED) are expelled from the ice concentrate in the channels between the ice crystals, so that the reaction occurs only in these non-frozen liquid channels. After polymerization and after the ice melts, a porous material is produced whose microstructure is a negative replica of the ice formed. The ice crystals act as porogens. The desired pore size is achieved, in part, by altering the temperature of the cryogelation process. For example, the cryogelation process is typically performed by rapidly freezing the solution at -20°C. Reducing the temperature, for example, to -80°C, will result in more ice crystals and smaller pores. In some embodiments, cryogels are produced by low-temperature polymerization of at least methacrylated (MA)-alginate and MA-PEG. In some embodiments, the cryogel is produced by low temperature polymerization of at least MA-alginate, growth factors, differentiation factors, and MA-PEG.

[0198] In some embodiments, the present invention also features gelatin scaffolds, e.g., gelatin hydrogels, e.g., gelatin cryogels, which are cell-responsive platforms for biomaterial-based therapeutics. Gelatin is a mixture of polypeptides derived from collagen by partial hydrolysis. These gelatin scaffolds have distinct advantages over other types of scaffolds and hydrogels / cryogels. For example, the gelatin scaffolds of the present invention support cell attachment, proliferation, and survival, and are degraded by cells, e.g., by the action of enzymes, e.g., matrix metalloproteinases (MMPs) (e.g., recombinant matrix metalloproteinases-2 and -9).

[0199] In certain embodiments, pre-fabricated gelatin cryogels, when injected subcutaneously into a subject (e.g., a mammal, e.g., a human, dog, cat, pig, or horse), rapidly assume their approximate original shape ("shape memory") and elicit little or no adverse host immune response (e.g., immune rejection) following injection.

[0200] In some embodiments, the hydrogel (e.g., cryogel) comprises a modified polymer, e.g., a site of the polymer molecule is modified with methacrylic acid groups (methacrylate (MA)) or acrylic acid groups (acrylate). An exemplary modified hydrogel / cryogel is MA-alginate (methacrylated alginate) or MA-gelatin. For MA-alginate or MA-gelatin, 50% corresponds to the degree of methacrylation of the alginate or gelatin. This means that every other repeat unit contains a methacrylated group. The degree of methacrylation can vary from about 1% to about 100%. Preferably, the degree of methacrylation varies from about 1% to about 90%.

[0201] In certain embodiments, the polymer may also be modified with acrylated groups instead of methacrylated groups. The product will then be referred to as an acrylated polymer. The degree of methacrylation (or acrylate) can vary for most polymers. However, some polymers (e.g., PEG) maintain their water-soluble properties even with 100% chemical modification. After crosslinking, the polymer usually reaches nearly complete methacrylate group conversion, exhibiting a crosslinking efficiency of approximately 100%. As used herein, the term "crosslinking efficiency" refers to the percentage of macromonomers that are linked by covalent bonds. For example, the polymers in hydrogels are 50-100% crosslinked (covalently). The degree of crosslinking correlates with the durability of the hydrogel. Therefore, a high level of crosslinking (90-100%) in the modified polymer is desirable.

[0202] For example, highly crosslinked hydrogel / cryogel polymer compositions are characterized by at least about 50% polymer crosslinking (e.g., about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, with intermediate values ​​and ranges of the recited values ​​being contemplated as part of the present invention). The high level of crosslinking provides mechanical robustness to the structure. Preferably, the percentage of crosslinking is less than about 100%. The compositions are formed using free radical polymerization and cryogelation processes. For example, cryogels are formed by low temperature polymerization of methacrylated gelatin, methacrylated alginate, or methacrylated hyaluronic acid. In some embodiments, the cryogel comprises a concentration of methacrylated gelatin macromonomer or methacrylated alginate macromonomer of about 1.5% (weight / volume) or less (e.g., about 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less, values ​​and ranges intermediate the recited values ​​are intended to be part of the invention). In some embodiments, the methacrylated gelatin or alginate macromonomer concentration is about 1% (weight / volume).

[0203] In certain embodiments, the cryogel comprises at least about 75% (volume / volume) pores, e.g., about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (volume / volume) pores or more. Values ​​and ranges intermediate to the recited values ​​are intended to be part of the invention. In some embodiments, the pores are interconnected. Interconnectivity is important for the function of hydrogels and / or cryogels, since without interconnectivity, water would become trapped within the gel. Pore interconnectivity allows the passage of water (and other compositions, e.g., cells and compounds) into and out of the structure. In certain embodiments, in a fully hydrated state, a hydrogel (e.g., a cryogel) comprises at least about 90% water (volume of water / volume of scaffold) (e.g., about 90-99%, at least about 92%, 95%, 97%, 99%, or more). For example, at least about 90% (e.g., at least about 92%, 95%, 97%, 99%, or more) of the volume of the cryogel is made up of liquid (e.g., water) contained in the pores. Values ​​and ranges intermediate to the recited values ​​are intended to be part of the invention. In certain embodiments, up to about 50%, 60%, 70% of the water is absent in a compressed or dehydrated hydrogel, e.g., a cryogel comprises less than about 25% water (e.g., about 20%, 15%, 10%, 5%, or less).

[0204] In certain embodiments, the cryogels of the present invention contain pores large enough for cells to pass through. For example, the cryogels contain pores with diameters of about 20-500 μm, e.g., about 20-30 μm, about 30-150 μm, about 50-500 μm, about 50-450 μm, about 100-400 μm, about 200-500 μm. In some embodiments, the hydrated pore size is about 1-500 μm (e.g., about 10-400 μm, about 20-300 μm, about 50-250 μm). In certain embodiments, the cryogels contain pores with diameters of about 50-80 μm.

[0205] In some embodiments, the injectable hydrogel or cryogel is further functionalized by the addition of a functional group selected from the group consisting of amino, vinyl, aldehyde, thiol, silane, carboxyl, azide, or alkyne. Alternatively, or in addition, the cryogel is further functionalized by the addition of an additional crosslinker (e.g., a multi-armed polymer, a salt, an aldehyde, etc.). The solvent may be water-soluble, and in particular may be acidic or alkaline. The water-soluble solvent may include a water-miscible solvent (e.g., methanol, ethanol, DMF, DMSO, acetone, dioxane, etc.).

[0206] For cryogels, low-temperature crosslinking can occur in the mold, and the cryogels (which can be injected) can be degradable. The pore size can be controlled by the choice of the main solvent used, the incorporation of porogens, the freezing temperature and rate applied, the crosslinking conditions (e.g., polymer concentration), and also by the type and molecular weight of the polymer used. The shape of the cryogel can be determined by the mold and therefore can take any shape desired by the manufacturer, for example, various sizes and shapes (disks, cylinders, squares, strings, etc.) are prepared by low-temperature polymerization.

[0207] Injectable cryogels can be prepared on the micrometer to centimeter scale. Exemplary volumes are several hundred μm 3 (For example, about 100 to 500 μm 3 ) ~ approx. 10cm 3 In certain embodiments, the exemplary scaffold composition has a thickness of about 100 μm. 3 ~100mm 3 In various embodiments, the scaffold is approximately 10 mm in size. 3 ~ approx. 100mm 3 In one particular embodiment, the scaffold is about 30 mm 3 The size is.

[0208] In some embodiments, the cryogel is hydrated, loaded with a compound, and loaded into a syringe or other delivery device. For example, the syringe is pre-filled and stored refrigerated until use. In another example, the cryogel is dehydrated, e.g., lyophilized, and the gel is loaded with a compound (e.g., growth or differentiation factors) as needed, and stored dry or refrigerated. Prior to administration, the cryogel-loaded syringe or device may be contacted with a solution containing the compound to be delivered. For example, the barrel of a syringe pre-loaded with a cryogel is filled with a physiologically compatible solution, e.g., phosphate buffered saline (PBS). Alternatively, the cryogel may be administered to the desired anatomical site, followed by administration, optionally with other components, e.g., a physiologically compatible solution containing growth and / or differentiation factors, or one or more compounds disclosed herein. The cryogel is then rehydrated and regains its shape integrity in situ. In certain embodiments, the volume of PBS or other physiological solution administered after placement of the cryogel is generally about 10 times the volume of the cryogel itself.

[0209] Cryogels also have the advantage that when compressed, the cryogel composition maintains structural integrity and shape memory properties. For example, cryogels are injectable through hollow needles. For example, a cryogel returns to its approximately original geometry after passing through a needle (e.g., a 16 gauge (G) needle having an inner diameter of 1.65 mm). Other exemplary needle sizes are 16 gauge, 18 gauge, 20 gauge, 22 gauge, 24 gauge, 26 gauge, 28 gauge, 30 gauge, 32 gauge, or 34 gauge needles. Injectable cryogels are designed to pass through hollow structures, e.g., ultra-fine needles, e.g., 18-30G needles. In certain embodiments, a cryogel returns to its approximately original geometry after passing through a needle in a short period of time, e.g., less than about 10 seconds, less than about 5 seconds, less than about 2 seconds, or less than about 1 second.

[0210] The cryogel can be injected into a subject using any suitable injection device. For example, the cryogel can be injected through a needle using a syringe. The syringe can include a plunger, a needle, and a reservoir containing the composition of the present invention. The injectable cryogel can also be injected into a subject using a catheter, a cannula, or a stent.

[0211] Injectable cryogels can be molded into the desired shape in the form of rods, squares, disks, spheres, cubes, fibers, foams. In some cases, the cryogels are in the shape of disks, cylinders, squares, rectangles, or strings. For example, the cryogel composition can be in the shape of about 100 μm. 3 ~10cm 3 Size, for example, 10mm 3 ~100mm 3 For example, the cryogel composition is about 1 mm in diameter to about 50 mm in diameter (e.g., about 5 mm). Optionally, the cryogel has a thickness of about 0.2 mm to about 50 mm (e.g., about 2 mm).

[0212] Three exemplary cryogel material systems are described below.

[0213] a) Methacrylated Gelatin Cryogels (CryoGeIMA) - Exemplary cryogels utilizing methacrylated gelatin and results are described in detail in U.S. Patent Application Publication No. 2014-0227327, published Aug. 14, 2014, the entire contents of which are incorporated herein by reference.

[0214] b) Methacrylated Alginate Cryogels (CryoMA Alginate) - Exemplary cryogels and results utilizing methacrylated alginate are described in U.S. Patent Application Publication No. 2014-0227327, published Aug. 14, 2014, the entire contents of which are incorporated herein by reference.

[0215] c) Click Alginate Cryogel with Laponite Nanoplatelets (CryoClick) - The substrate is click alginate (PCT International Patent Application Publication No. WO 2015 / 154078, published October 8, 2015, incorporated herein by reference in its entirety). In some examples, the substrate contains Laponite, a commercially available silicate clay used in many consumer products such as cosmetics. Laponite has a large surface area and high negative charge density, which allows it to adsorb to positively charged moieties on various proteins and other biologically active molecules by electrostatic interactions, thereby allowing drug loading. When placed in an environment with a low concentration of drug, the adsorbed drug is released from Laponite in a sustained manner. This system allows for the release of a more flexible array of different agents, e.g., growth factors, compared to the substrate alone.

[0216] Various embodiments of the present subject matter include a delivery vehicle that includes a pore-forming scaffold composition. For example, pores (e.g., macropores) are formed in situ in the hydrogel after the hydrogel is injected into a subject. The pores formed in situ by the degradation of the sacrificial porogen hydrogel in the surrounding hydrogel (bulk hydrogel) facilitate cell recruitment and transport, as well as the release of any composition or agent of the present invention, such as growth factors, e.g., BMP-2, differentiation factors, or homing factors, or any combination thereof. In some embodiments, the sacrificial porogen hydrogel, the bulk hydrogel, or both the sacrificial porogen hydrogel and the bulk hydrogel can include any composition or agent of the present invention, such as growth factors, differentiation factors, and / or homing factors, or any combination thereof.

[0217] In various embodiments, the pore-forming composition becomes macroporous over time when present in the body of a recipient animal, e.g., a mammalian subject. For example, the pore-forming composition can include a sacrificial porogen hydrogel and a bulk hydrogel, where the sacrificial porogen hydrogel is degraded at least about 10% faster (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% faster) than the bulk hydrogel. Values ​​and ranges intermediate to the recited values ​​are intended to be part of the invention. The sacrificial porogen hydrogel can be degraded to leave macropores in their place. In certain embodiments, the macropores are interconnected open macropores. In some embodiments, the sacrificial porogen hydrogel may be degraded more rapidly than bulk hydrogels because (i) it is highly soluble in water (including a low solubility index), (ii) it is crosslinked to protease-mediated degradation motifs as described in U.S. Patent Application Publication No. 2005-0119762, published June 2, 2005, which is incorporated by reference in its entirety, (iii) it comprises short polymers that degrade more rapidly than those of longer bulk hydrogel polymers, (iv) it has been modified (e.g., by oxidation) to be more hydrolytically degradable than bulk hydrogels, and / or (v) it is more susceptible to enzymatic degradability compared to bulk hydrogels, thereby degrading more rapidly than bulk hydrogels.

[0218] In various embodiments, the scaffold is loaded (e.g., impregnated) with one or more active compounds after polymerization. In certain embodiments, the device or scaffold polymer forming material is mixed with one or more active compounds before polymerization. In some embodiments, the device or scaffold polymer forming material is mixed with one or more active compounds before polymerization and then further loaded with the same or one or more additional active compounds after polymerization.

[0219] In some embodiments, the pore size or total pore volume of the composition or scaffold is selected to affect the release of the compound from the device or scaffold. Exemplary porosity (e.g., nanoporous, microporous, and macroporous scaffolds and devices) and total pore volume (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more of the volume of the scaffold) are described herein. Values ​​and ranges intermediate the listed values ​​are intended to be part of the invention. Increasing the pore size and total pore volume increases the amount of compound that can be delivered to a tissue, for example, bone marrow or nearby. In some embodiments, the pore size or total pore volume is selected to increase the rate at which the active ingredient leaves the composition or scaffold. In various embodiments, the active ingredient can be incorporated into a hydrogel or cryogel scaffold material to achieve continuous release of the active ingredient from the scaffold or device over an extended period of time compared to, for example, the active ingredient that can diffuse out of the pore spaces.

[0220] Porosity affects the recruitment of cells to the devices and scaffolds and the release of materials from the devices and scaffolds. The pores can be, for example, nanoporous, microporous, or macroporous. For example, nanopores have a diameter of less than about 10 nm. Micropores have a diameter ranging from about 100 nm to about 20 μm. Macropores have a diameter greater than about 20 μm (e.g., greater than about 100 μm or greater than about 400 μm). Exemplary macropore sizes include diameters of about 50 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, and about 600 μm. Values ​​and ranges intermediate to the recited values ​​are intended to be part of the invention. The macropores are of a size that allows eukaryotic cells to move in and out of the composition. In one example, the macroporous composition has pores with a diameter of about 400 μm to about 500 μm. The preferred pore size depends on the application. In certain embodiments, the pores have a diameter of about 50 μm to about 80 μm.

[0221] In various embodiments, the composition is manufactured in one step, where one layer or compartment is made and impregnated or coated with one or more compounds. Exemplary bioactive compositions include polypeptides or polynucleotides. In certain embodiments, the composition is manufactured in two or more (three, four, five, six, .... ten or more) steps, where one layer or compartment is made and impregnated or coated with one or more compounds, followed by the construction of a second, third, fourth or more layers, which are then impregnated or coated with one or more compounds in turn. In some embodiments, each layer or compartment is identical to the others, or is differentiated from each other by the number or mixture of bioactive compositions, as well as by different chemical, physical, and biological properties. The polymer can be formulated for a particular application by controlling the molecular weight, rate of degradation, and method of scaffold formation. Coupling reactions can be used to covalently attach bioactive agents, such as differentiation factors, to the polymer backbone.

[0222] In some embodiments, one or more compounds are added to the scaffold composition using known methods, including surface absorption, physical binding, e.g., using phase change to entrap substances in the scaffold material. For example, growth factors are mixed with the scaffold composition while in an aqueous or liquid phase, and after a change in environmental conditions (e.g., pH, temperature, ion concentration), the liquid gels or solidifies, thereby entrapping the bioactive substance. In some embodiments, covalent coupling, e.g., using alkylating or acylating agents, is used to provide stable long-term presentation of compounds on the scaffold in a defined configuration. Exemplary reagents for covalent coupling of such substances are provided in the table below. [Table 1]

[0223] Alginate scaffold In certain embodiments, the compositions of the present invention include alginate hydrogels. Alginates are versatile polysaccharide-based polymers that can be formulated for specific applications by controlling the molecular weight, rate of degradation, and method of scaffold formation. Alginate polymers are composed of two different monomer units, (1-4)-linked β-D-mannuronic acid (M unit) monomers and α L-guluronic acid (G unit) monomers, which can vary in even and continuous distribution along the polymer chain. Alginate polymers can be cleaved with divalent cations (e.g., Ca +2 , Mg +2 , B.A. +2 ), which forms a stable hydrogel when exposed to these molecules. See Martinsen A., et al., 1989, Biotech. & Bioeng., 33: 79-89). For example, calcium cross-linked alginate hydrogels are useful for dental applications, wound coverings, chondrocyte transplants, and as matrices for other cell types. Without wishing to be bound by theory, it is believed that G units are preferentially cross-linked using calcium cross-links, while click reaction-based cross-links are more indiscriminate toward G or M units (i.e., both G and M units can be cross-linked by click chemistry). Alginate scaffolds and methods for making them are known in the art. See, for example, International Patent Application Publication No. WO2017 / 075055 A1, published May 4, 2017, the entire contents of which are incorporated herein by reference.

[0224] Alginate polymers useful in the context of the present invention may have an average molecular weight of about 20 kDa to about 500 kDa, for example, about 20 kDa to about 40 kDa, about 30 kDa to about 70 kDa, about 50 kDa to about 150 kDa, about 130 kDa to about 300 kDa, about 230 kDa to about 400 kDa, about 300 kDa to about 450 kDa, or about 320 kDa to about 500 kDa. In one example, an alginate polymer useful in the present invention may have an average molecular weight of about 32 kDa. In another example, an alginate polymer useful in the present invention may have an average molecular weight of about 265 kDa. In some embodiments, the alginate polymer has a molecular weight of less than about 1000 kDa, e.g., less than about 900 kDa, less than about 800 kDa, less than about 700 kDa, less than about 600 kDa, less than about 500 kDa, less than about 400 kDa, less than about 300 kDa, less than about 200 kDa, less than about 100 kDa, less than about 50 kDa, less than about 40 kDa, less than about 30 kDa, or less than about 25 kDa. In some embodiments, the alginate polymer has a molecular weight of about 1000 kDa, for example, about 900 kDa, about 800 kDa, about 700 kDa, about 600 kDa, about 500 kDa, about 400 kDa, about 300 kDa, about 200 kDa, about 100 kDa, about 50 kDa, about 40 kDa, about 30 kDa, or about 25 kDa. In one embodiment, the molecular weight of the alginate polymer is about 20 kDa.

[0225] Coupling reactions can be used to covalently attach bioactive agents, such as atoms, chemical groups, nucleosides, nucleotides, nucleic acid bases, sugars, nucleic acids, amino acids, peptides, polypeptides, proteins, or protein complexes, to the polymer backbone.

[0226] The term "alginate", which is used interchangeably with the term "alginate polymer", includes unmodified or modified alginates. Modified alginates include oxidized alginates (e.g., containing one or more algoxalate monomer units) and / or reduced alginates (e.g., containing one or more argoxinol monomer units). The oxidized alginates include, but are not limited to, alginates containing one or more aldehyde groups or alginates containing one or more carboxylate groups. In other embodiments, the oxidized alginates include highly oxidized alginates, for example, containing one or more alkoxalate units. The oxidized alginates may also contain a relatively small number of aldehyde groups (e.g., less than 15% on a molar basis, e.g., 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% aldehyde groups or oxidation). Values ​​and ranges intermediate to the recited values ​​are intended to be part of the invention. The term "alginate" or "alginate polymer" may also include alginates, such as unmodified alginates, oxidized or reduced alginates, or methacrylated or acrylated alginates. Alginates may also refer to any number of alginic acid derivatives, such as calcium, sodium, or potassium salts, or propylene glycol alginates. See, for example, International Publication No. WO1998012228A1, which is incorporated herein by reference.

[0227] Hyaluronic acid In certain embodiments, the composition of the present invention comprises hyaluronic acid hydrogel.Hyaluronic acid (HA, conjugate base hyaluronate) is an anionic non-sulfurized glycosaminoglycan that is widely distributed throughout connective tissue, epithelial tissue, and nervous tissue.As one of the major components of extracellular matrix, hyaluronic acid contributes significantly to cell proliferation and migration.Natural hyaluronic acid is an important component of articular cartilage, muscle connective tissue, and skin.

[0228] Hyaluronic acid is a disaccharide polymer composed of alternating β-(1→4) and β-(1→3) glycosidic bonds between D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid can be 25,000 disaccharide repeats long. Polymers of hyaluronic acid can range in size from 5,000 to 20,000,000 Da. Hyaluronic acid can also contain silicon.

[0229] Hyaluronic acid is energetically stable due in part to the stereochemistry of its constituent disaccharides: the bulky groups on each sugar molecule are in sterically favorable positions, while the smaller hydrogens are in less favorable axial positions.

[0230] Hyaluronic acid can be degraded by a family of enzymes called hyaluronidases, which are present in many mammals, including humans. Hyaluronic acid can also be degraded by non-enzymatic reactions. These include acid and alkaline hydrolysis, ultrasonic destruction, thermal decomposition, and degradation by oxidizing agents.

[0231] Due to its high biocompatibility and its common presence in the extracellular matrix of tissues, hyaluronic acid is used to form hydrogels, e.g., cryogels, as biomaterial scaffolds in tissue engineering research. Hyaluronic acid hydrogels are formed through crosslinking. Hyaluronic acid can form hydrogels, e.g., cryogels, into the desired shape for delivering therapeutic molecules to the host. Hyaluronic acid can be crosslinked by binding thiols, methacrylates, hexadecylamides, and tyramines for use in the present compositions. Hyaluronic acid can also be crosslinked directly with formaldehyde or divinylsulfone.

[0232] The term "hyaluronic acid" includes unmodified hyaluronic acid or modified hyaluronic acid. Modified hyaluronic acid includes, but is not limited to, oxidized hyaluronic acid and / or reduced hyaluronic acid. The term "hyaluronic acid" or "hyaluronic acid polymer" can also include hyaluronic acid, such as unmodified hyaluronic acid, oxidized hyaluronic acid or reduced hyaluronic acid, or methacrylated hyaluronic acid or acrylated hyaluronic acid. Hyaluronic acid can also refer to any number of hyaluronic acid derivatives.

[0233] Porous and Pore-Forming Scaffolds The scaffolds of the present invention may be non-porous or porous. In certain embodiments, the scaffolds of the present invention are porous. The porosity of the scaffold composition affects the migration of cells through the device. The pores may be nanoporous, microporous, or macroporous. For example, the diameter of a nanopore is less than about 10 nm. A micropore has a diameter ranging from about 100 nm to about 20 μm. A macropore has a diameter greater than about 20 μm (e.g., greater than about 100 μm or greater than about 400 μm). Exemplary macropore sizes include diameters of about 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, and 600 μm. Intermediate values ​​and ranges of the recited values ​​are intended to be part of the invention. The macropores are of a size that allows eukaryotic cells to migrate in and out of the composition. In certain embodiments, the macroporous composition has pores with a diameter of 400 μm to about 500 μm. The size of the pores can be adjusted for different purposes. For example, for cell recruitment and cell release, the pore diameter can be greater than 50 μm. In certain embodiments, the macroporous composition has pores with a diameter of about 50 μm to about 80 μm.

[0234] In some embodiments, the scaffold comprises pores prior to administration to a subject. In some embodiments, the scaffold comprises a pore-forming scaffold composition. Pore-forming scaffolds and methods for making pore-forming scaffolds are known in the art. See, for example, U.S. Patent Publication No. US2014 / 0079752A1, the contents of which are incorporated herein by reference. In certain embodiments, the pore-forming scaffold is not initially porous, but becomes macroporous over time while present in the body of a recipient animal, e.g., a mammalian subject. In certain embodiments, the pore-forming scaffold is a hydrogel scaffold. The pores can be formed at different times, for example, about 12 hours after administration, i.e., after being present in the body of a subject, or 1, 3, 5, 7, or 10 days or more after administration.

[0235] In certain embodiments, the pore-forming scaffold comprises a first hydrogel and a second hydrogel, where the first hydrogel is degraded at least about 10% faster than the second hydrogel (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 2 times faster, or at least about 5 times faster). Values ​​and ranges intermediate to the recited values ​​are intended to be part of the invention. In certain embodiments, the first hydrogel comprises a porogen that degrades leaving pores in place. For example, the first hydrogel is a porogen, and the pores obtained in situ after degradation are within 25% of the size of the initial porogen, e.g., within 20%, within 15%, or within 10% of the size of the initial porogen. Preferably, the resulting pores are within 5% of the size of the initial porogen. Values ​​and ranges intermediate to the recited values ​​are intended to be part of the invention. The first hydrogel may degrade faster than the second hydrogel due to differences in their physical, chemical, and / or biological properties.In certain embodiments, the first hydrogel degrades faster than the second hydrogel because it is more soluble in water (comprises a low solubility index).In certain embodiments, the first hydrogel degrades faster because it is crosslinked to the protease-mediated degradation motif described in US Patent Publication US2005 / 0119762A1, the contents of which are incorporated herein by reference.

[0236] In certain embodiments, the molecular mass of the polymer used to form the first hydrogel composition (porogen) is approximately 50 kilodaltons (kDa) and the molecular mass of the polymer used to form the second hydrogel composition (bulk) is approximately 250 kDa. Shorter polymers (e.g., those of the porogen) are degraded more rapidly compared to longer polymers (e.g., those of the bulk composition). In certain embodiments, the composition is modified to be more hydrolytically degradable based on the presence of sugar groups (e.g., approximately 3-10% sugars in the alginate composition). In certain embodiments, the porogen hydrogel is chemically modified, e.g., oxidized, to be more susceptible to degradation. In some embodiments, the porogen hydrogel is more enzymatically degradable compared to the bulk hydrogel. The composite (first and second hydrogel) composition is permeable, e.g., to bodily fluids containing enzymes that degrade the porogen hydrogel upon exposure to the composition. In some embodiments, the second hydrogel is crosslinked around the first hydrogel, i.e., the porogen (first hydrogel) is completely physically entrapped in the bulk (second) hydrogel.

[0237] The click reagents disclosed herein can be provided in a bulk hydrogel or a porogen hydrogel. In an exemplary embodiment, the click reagent, e.g., a polymer or nanoparticle, is provided in a bulk hydrogel.

[0238] In certain embodiments, hydrogel microbeads ("porogens") are formed. The porogens are encapsulated in a "bulk" hydrogel that is either non-degradable or degraded at a slower rate compared to the porogens. The composite material immediately after hydrogel formation or injection into the desired site in vivo is devoid of pores. Porogen degradation subsequently results in the formation of pores in situ. The size and distribution of the pores are controlled during porogen formation and mixing with the polymer that forms the bulk hydrogel.

[0239] In some embodiments, the polymers utilized in the pore-forming scaffold are naturally occurring or synthetically produced. In one example, both the porogen and bulk hydrogels are formed from alginate.

[0240] In certain embodiments, alginate polymers suitable for porogen formation have a molecular weight of 5,000 to 500,000 daltons. The polymers are optionally further modified to promote rapid degradation (e.g., by oxidation with sodium periodate (Bouhadir et al., 2001, Biotech. Prog. 17:945-950, incorporated herein by reference). In certain embodiments, the polymers are immersed in a divalent cation bath (e.g., Ca) through a nebulizer with a coaxial airflow. 2+ or Ba 2+ ) to form hydrogel microbeads. Higher air velocities result in smaller porogen diameters.

[0241] In some embodiments, the porogen hydrogel microbeads contain oxidized alginate. For example, the porogen hydrogel may contain about 1-50% (weight / volume) oxidized alginate. In an exemplary embodiment, the porogen hydrogel may contain about 1-10% oxidized alginate. In one embodiment, the porogen hydrogel contains about 7.5% oxidized alginate.

[0242] In certain embodiments, the concentration of divalent ions used to form the porogen can vary from about 5 to about 500 mM, and the concentration of polymer is from about 1% to about 5% by weight / volume. However, any method that produces a porogen that is significantly smaller than the bulk phase is suitable. The porogen chemistry can be further manipulated to produce porogens that interact with or inhibit interactions with host proteins and / or cells.

[0243] Alginate polymers suitable for forming bulk hydrogels have a molecular weight of about 5,000 to about 500,000 Da. The polymer may be further modified to accelerate degradation (e.g., by oxidation with sodium periodate), so long as the bulk hydrogel degrades more slowly than the porogen. The polymer may also be modified to present biological cues to control the cell response (e.g., integrin-binding adhesion peptides, e.g., RGD). Either the porogen or the bulk hydrogel may also encapsulate bioactive factors, e.g., oligonucleotides, growth factors, or drugs, to further control the cell response. The concentration of the divalent ions used to form the bulk hydrogel may vary from about 5 to about 500 mM, and the concentration of the polymer is about 1% to about 5% by weight / volume. The elastic modulus of the bulk polymer is adjusted for that purpose, e.g., to recruit stem or progenitor cells.

[0244] Methods related to producing the hydrogels described herein include: Bouhadir et al., 1999, Polymer, 40: 3575-84, incorporated herein by reference in its entirety, describe the oxidation of alginate with sodium periodate and characterize the reaction; Bouhadir et al., 2001, Biotechnol. Prog., 17: 945-50, incorporated herein by reference in its entirety, describe the oxidation of high molecular weight alginates to form alginate dialdehydes (alginate dialdehydes are formed when a certain percentage, e.g., 5%, of the sugars in the alginate are oxidized to form aldehydes) and high molecular weight (M w (M ) alginates) and their application to make hydrogels rapidly degrade. Kong et al., 2002, Polymer, 43: 6239-46, incorporated herein by reference in its entirety, describe the weight average molecular weight (M ) of guluronic acid (GA)-rich alginates. wdescribe the use of gamma irradiation to reduce the mannuronic acid, MA block of alginate, without substantially reducing the GA content (e.g., gamma irradiation selectively attacks the mannuronic acid, MA block of alginate). Alginate is composed of GA and MA blocks, and it is the GA block that provides the stiffness (elastic modulus) to alginate. Kong et al., 2002, Polymer, 43: 6239-46 (incorporated herein by reference in its entirety) describe the use of high M w GA-rich alginate and irradiated low M w The binary combination of high GA alginate with calcium crosslinking formed a rigid hydrogel, which was comparable to the high M w Alsberg et al., 2003, J Dent Res, 82(11): 903-8 (incorporated herein by reference in its entirety) showed that irradiated low M w describe the degradation profile of hydrogels made from GA-rich alginates with applications in bone tissue engineering. Kong et al., 2004, Adv. Mater, 16(21): 1917-21 (incorporated herein by reference in its entirety) describe the control of hydrogel degradation profile by combining gamma irradiation procedures and oxidation reactions, with applications in cartilage engineering.

[0245] Techniques for controlling the degradation of hydrogen biomaterials are well known in the art. For example, Lutolf MP et al., 2003, Nat Biotechnol., 21: 513-8 (incorporated herein in its entirety by reference) describe poly(ethylene glycol)-based materials engineered to degrade by mammalian enzymes (MMPs). Bryant SJ et al., 2007, Biomaterials, 28(19): 2978-86 (U.S. Patent No. 7,192,693 B2, incorporated herein in its entirety by reference) describe a method for producing hydrogels with macro-scale pores. A pore template (e.g., poly-methyl methacrylate beads) is encapsulated within the bulk hydrogel, and then acetone and methanol are used to extract the porogens while leaving the bulk hydrogel intact. Silva et al., 2008, Proc. Natl. Acad. Sci USA, 105(38): 14347-52 (US Publication No. 2008 / 0044900, incorporated herein by reference in its entirety) describes the development of endothelial progenitor cells from alginate sponges. The sponges are made by forming an alginate hydrogel and then freeze-drying it (ice crystals form pores). Ali et al., 2009, Nat Mater (incorporated herein by reference in its entirety) describes the use of porous scaffolds to recruit dendritic cells and program them to elicit anti-tumor responses. Huebsch et al., 2010, Nat Mater, 9: 518-26 (incorporated herein by reference in its entirety) describes the use of hydrogel elastic modulus to control the differentiation of encapsulated stromal stem cells.

[0246] In some embodiments, the scaffold composition comprises interconnected open macropores. Alternatively or additionally, the scaffold composition comprises a pore-forming scaffold composition. In certain embodiments, the pore-forming scaffold composition may comprise a sacrificial porogen hydrogel and a bulk hydrogel, where the pore-forming scaffold composition is devoid of macropores. For example, the sacrificial porogen hydrogel may degrade at least 10% more rapidly than the bulk hydrogel after administration of said pore-forming scaffold to a subject, leaving macropores in their place. In some embodiments, the sacrificial porogen hydrogel is in the form of a porogen that degrades to form said macropores. For example, the macropores may comprise pores having diameters of, for example, about 10 to 400 μm.

[0247] Growth factors The composition of the present invention may include a growth factor. The term "growth factor" as used herein refers to an agent that can stimulate cell growth, proliferation, healing, and / or cell differentiation. In certain embodiments, the growth factor is a polypeptide. Growth factor polypeptides typically act as signaling molecules. In certain embodiments, the growth factor polypeptide is a cytokine.

[0248] In certain embodiments, the growth factor can mobilize cells into the scaffold after administering the composition to the subject. The mobilized cells can be autologous. For example, the mobilized cells can be stromal cells derived from the subject. In certain embodiments, the autologous cells can be stem cells (e.g., umbilical cord stem cells) of the subject. The mobilized cells can also be syngeneic, allogeneic, or xenogeneic. As used herein, the term "syngeneic" refers to cells that are genetically identical or sufficiently identical to allow transplantation and immunologically compatible. For example, syngeneic cells can include transplanted cells obtained from identical twins. As used herein, the term "allogeneic" refers to cells that are not genetically similar but are derived from individuals of the same species. As used herein, the term "xenogeneic" refers to cells that are derived from different species and therefore genetically different.

[0249] For example, the mobilized cells can be donor cells in transplantation. In certain embodiments, the transplantation is hematopoietic stem cell transplantation (HSCT). As used herein, HSCT refers to the transplantation of multipotent hematopoietic stem cells or hematopoietic progenitor cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. HSCT can be autologous (the patient's own stem or progenitor cells are used), allogeneic (stem or progenitor cells derived from donor), syngeneic (derived from identical twins), or xenogeneic (derived from different species).

[0250] The growth factors of the present invention can induce the formation of tissues or organs within or around the administered composition. In certain embodiments, the tissue or organ is bone tissue or hematopoietic tissue. Tissue formation can be restricted to the scaffold of the composition.

[0251] Methods of incorporating polypeptides (e.g., growth factor polypeptides) are known in the art. See U.S. Patent Nos. 8,728,456, 8,067,237, and 10,045,947, U.S. Patent Publication No. US20140079752, International Patent Publication No. WO2017 / 136837, which are incorporated by reference in their entireties. The release of growth factor polypeptides can be controlled. Methods of controlled release of polypeptides (e.g., growth factor polypeptides) are known in the art. See U.S. Patent Nos. 8,728,456, 8,067,237, and 10,045,946, which are incorporated by reference in their entireties. In certain embodiments, growth factors (e.g., BMP-2) can be released for an extended period of time, e.g., 7 to 30 days or longer. The controlled release of growth factors can affect the timing of tissue or organ formation within the scaffold, hi certain instances, the release of growth factors is controlled to target the formation of functionally active bone nodules or tissues within 1-2 weeks following subcutaneous injection of the compositions of the invention.

[0252] In certain embodiments, the growth factor retains its bioactivity for an extended period of time. The term "bioactivity" as used herein refers to the beneficial or detrimental effects of an agent, such as a growth factor. The bioactivity of a growth factor can be measured by any suitable means. For example, the bioactivity of BMP-2 can be measured by its ability to induce the formation of bone nodules or tissues and / or recruit cells to the scaffold. In certain examples, the growth factor retains its bioactivity for at least 10 days, 12 days, 14 days, 20 days, or 30 days after the growth factor is incorporated into the scaffold.

[0253] Exemplary growth factors include, but are not limited to, bone morphogenetic proteins (BMPs), epidermal growth factor (EGF), transforming growth factor beta (TGF-β), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), and interleukins.

[0254] In some embodiments, the growth factor comprises a protein belonging to the transforming growth factor beta (TGF-β) superfamily. As used herein, the TGF-β superfamily is a large group of structurally related cell regulatory proteins. The TGF-β superfamily includes four major subfamilies: the TGF-β subfamily, the bone morphogenetic proteins and growth differentiation factors, the activation and inhibin subfamily, and a group that includes various diverse members. Proteins from the TGF-β superfamily are active as homo- or heterodimers in which two chains are linked by a single disulfide bond. TGF-β superfamily proteins interact with a family of conserved cell surface serine / threonine-specific protein kinase receptors and generate intracellular signals using a conserved family of proteins called SMADs. TGF-β superfamily proteins play a key role in the control of fundamental biological processes, such as growth, development, tissue homeostasis, and the immune system.

[0255] Exemplary TGF-β superfamily proteins include, but are not limited to, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, MSTN, NODAL, NRTN, PSPN, TGF-β1, TGF-β2, TGF-β3 and TGF-β4. In certain embodiments, the growth factor is BMP2.

[0256] In certain embodiments, growth factor comprises bone morphogenetic protein (BMP).As used herein, BMP is a protein that belongs to a group of growth factors also known as cytokines and metabologens.BMP induces bone and cartilage formation, constitutes a group of important morphogenetic signals, and can integrate tissue architecture throughout the body.Absence or defect of BMP signaling can be an important factor in disease or disorder.

[0257] In certain embodiments, BMP is selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, and any combination thereof.In certain embodiments, BMP is BMP-2.BMP-2 plays an important role in the development of bone and cartilage.BMP-2 can strongly induce osteoblast differentiation in various cell types.

[0258] In certain embodiments, the growth factor comprises a TGF-β subfamily protein. As used herein, TGF-β subfamily protein or TGF-β is a multifunctional cytokine that includes four different isoforms (TGF-β1, TGF-β2, TGF-β3, and TGF-β4). Activated TGF-β forms a complex with other factors to form a serine / threonine kinase complex that binds to the TGF-β receptor, which is composed of type 1 and type 2 receptor subunits. After binding of TGF-β, type 2 receptor kinase phosphorylates and activates type 1 receptor kinase, which activates a signal transduction cascade. This leads to the activation of different downstream substrates and regulatory proteins, inducing the transcription of different target genes that function in the differentiation, chemotaxis, proliferation, and activation of multiple immune cells.

[0259] In certain embodiments, the growth factor comprises TGF-β1. TGF-β1 promotes the proliferation of induced Tregs (iTregs) with regulatory functions and Tregs that secrete proinflammatory cytokines from CD4+ T cells. h TGF-β1 alone is involved in the expression of Foxp3 and Treg differentiation from activated helper T cells.

[0260] Growth factors (e.g., BMP-2 or TGF-β1) can be isolated from endogenous sources or can be synthesized in vivo or in vitro. Endogenous growth factor polypeptides can be isolated from healthy human tissues. Synthetic growth factor polypeptides are synthesized in vivo following transfection or transformation of a template DNA into a host organism or cell, e.g., a mammalian or human cell line. Alternatively, synthetic growth factor polypeptides are synthesized in vitro by cell-free translation or other art-recognized methods, Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), which is incorporated herein by reference. is incorporated into.

[0261] In certain embodiments, the growth factor (e.g., BMP-2 or TGF-β1) polypeptide may be recombinant. In some embodiments, the growth factor polypeptide is a humanized derivative of a mammalian growth factor polypeptide. Exemplary mammalian species from which the growth factor polypeptide is derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In some embodiments, the growth factor is a recombinant human protein. In some embodiments, the growth factor is a recombinant murine (mouse) protein. In some embodiments, the growth factor is a humanized derivative of a recombinant mouse protein.

[0262] In certain embodiments, the growth factor polypeptides may be modified to increase protein stability in vivo. In certain embodiments, the growth factor polypeptides may be engineered to be more or less immunogenic. The terms "immunogenic" and "immunogenicity" refer to the ability of a particular substance, e.g. For example, it refers to the ability of a protein, antigen, or epitope to elicit an immune response in the human or other animal body.

[0263] In certain embodiments, growth factors may be present from about 0.001 nmol to about 1000 nmol per scaffold, or from about 0.001 to about 100 nmol per scaffold, or from about 0.001 nmol to about 1 nmol per scaffold.

[0264] In some embodiments, the growth factor may be present at about 1 ng to 1000 micrograms per scaffold. For example, the growth factor may be present in an amount of about 1 μg to about 1000 μg, about 1 μg to about 500 μg, about 1 μg to about 200 μg, about 1 μg to about 100 μg, about 1 μg to about 50 μg, or about 1 μg to 10 μg.

[0265] In certain embodiments, the compositions of the present invention contain nanogram amounts of growth factors (e.g., about 1 ng to about 1000 ng of BMP-2). For example, the growth factors can be present in amounts of about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, about 10 ng to about 200 ng, about 25 ng to about 200 ng, about 50 ng to 200 ng, about 100 ng to 200 ng, and about 200 ng. The nanogram amounts of growth factors are also released in a controlled manner. The nanogram amounts of growth factors and / or controlled release may contribute to reduced toxicity of the compositions and methods of the present invention compared to other delivery systems that use high doses of growth factors and have suboptimal release kinetics.

[0266] In various embodiments, the amount of growth factor present in the scaffold may vary depending on the size of the scaffold. For example, the growth factor may be present in a concentration of about 0.03 ng / mm 3 (ratio of amount of growth factor by weight to volume of scaffold) ~ approx. 350ng / mm 3 , for example, about 0.1 ng / mm 3 ~about 300ng / mm 3 , about 0.5ng / mm 3 ~about 250ng / mm 3 , about 1ng / mm3 ~about 200ng / mm 3 , about 2ng / mm 3 ~about 150ng / mm 3 , about 3ng / mm 3 ~ approx. 100ng / mm 3 , about 4ng / mm 3 ~about 50ng / mm 3 , about 5ng / mm 3 ~25ng / mm 3 , about 6ng / mm 3 ~about 10ng / mm 3 , or about 6.5 ng / mm 3 ~ approx. 7.0ng / mm 3 It can exist in.

[0267] In some embodiments, the amount of growth factor is about 300 ng / mm 3 ~Approx. 350μg / mm 3 , for example, about 400 ng / mm 3 ~Approx. 300μg / mm 3 , about 500ng / mm 3 ~about 200μg / mm 3 , about 1μg / mm 3 ~Approx. 100μg / mm 3 , about 5μg / mm 3 ~about 50μg / mm 3 , about 10μg / mm 3 ~about 25μg / mm 3 It can exist in.

[0268] differentiation factor The composition of the present invention may include a differentiation factor. As used herein, a differentiation factor is an agent that can induce differentiation of cells, for example, recruited cells. In certain embodiments, the differentiation factor is a polypeptide. As used herein, "differentiation", "cell differentiation", "cell differentiation", or other similar terms refer to the process by which cells change from one cell type to another. In certain embodiments, cells change to more specialized types, for example, from stem cells or progenitor cells to T cell progenitor cells. Differentiation occurs multiple times during the development of a multicellular organism, changing from a simple zygote to a complex system of tissues and cell types. Differentiation continues into the adult stage, as adult stem cells divide during tissue repair and normal cell turnover to create fully differentiated daughter cells. Differentiation can change the size, shape, membrane potential, metabolic activity, and responsiveness of cells to signals. These changes can result from highly regulated modifications in gene expression.

[0269] In dividing cells, there are multiple levels of cell differentiation potential, which is the ability of a cell to differentiate into other cell types. Higher differentiation potential indicates that multiple cell types can be derived. Cells that can differentiate into all cell types, including placental tissue, are known as totipotent. Cells that can differentiate into all cell types of an adult organism are known as pluripotent. In mammals, such as humans, pluripotent cells can include embryonic stem cells and adult pluripotent cells. Induced pluripotent stem (iPS) cells can be generated from fibroblasts by inducing the expression of certain transcription factors, such as Oct4, Sox2, c-Myc, and KIF4. Multipotent cells are those that can differentiate into multiple different but closely related cell types. Oligopotent cells are more limited than multipotent, but still can differentiate into several closely related cell types. Finally, unipotent cells can only differentiate into one cell type, but are capable of self-renewal.

[0270] In certain embodiments, the differentiation factor of the present invention induces differentiation of stem cells or progenitor cells into T cell precursor cells. As used herein, the term "T cell precursor cells" refers to a precursor cell that can eventually differentiate into a T lymphocyte (T cell). As used herein, the term "lymphocyte" refers to one of the subtypes of white blood cells in the immune system of a vertebrate (e.g., human). Lymphocytes include natural killer cells, T cells, and B cells. Lymphocytes originate from lymphoid common precursors before differentiating into distinct lymphocyte types during hematopoiesis, the process by which stem cells differentiate into several types of blood cells in the bone marrow.

[0271] In some embodiments, the T cell progenitor comprises a common lymphoid progenitor. The term "common lymphoid progenitor" as used herein refers to the earliest lymphoid progenitor cell from which lymphocytes, including T lineage cells, B lineage cells, and natural killer (NK) cells, arise. In various embodiments, the T cell progenitor comprises a T cell competent common lymphoid progenitor. The term "T cell competent common lymphoid progenitor" as used herein refers to a common lymphoid progenitor cell that differentiates into a T lineage progenitor. A T cell competent common lymphoid progenitor cell is usually characterized by the absence of the biomarker Ly6D. The compositions of the present invention can create an ectopic niche that mimics key features of bone marrow and induces differentiation of stem or progenitor cells into T cell progenitors.

[0272] In certain embodiments, lymphocytes include T cells. In some embodiments, the T cells are naive T cells. As used herein, naive T cells are T cells differentiated in bone marrow. Naive T cells include CD4 + T cells, CD8 + T cells, and regulatory T cells (T reg ) may be included.

[0273] In certain embodiments, the differentiation factor induces the differentiation of the mobilized cells into T cell precursor cells. In certain embodiments, the differentiation factor induces the differentiation of the mobilized cells into T cell precursor cells through the Notch signaling pathway. The Notch signaling pathway is a highly conserved cell signaling system present in many multicellular organisms. Mammals have four different Notch receptors, designated Notch1, Notch2, Notch3, and Notch4. Notch signaling plays an important role in T cell lineage differentiation from common lymphoid progenitor cells. In certain embodiments, the differentiation factor binds to one or more Notch receptors and activates the Notch signaling pathway. In certain embodiments, the differentiation factor is selected from the group consisting of Delta-like 1 (DLL-1), Delta-like 2 (DLL-2), Delta-like 3 (DLL-3), Delta-like 3 (DLL-3), Delta-like 4 (DLL-4), Jagged 1, Jagged 2, and any combination thereof. In certain embodiments, binding of a differentiation factor to one or more Notch receptors activates the Notch signaling pathway and induces T cell lineage differentiation.

[0274] In certain embodiments, the differentiation factor is Delta-like 4 (DLL-4).DLL-4 is a protein that is a homologue of the Drosophila Delta protein.The Delta protein family includes Notch ligands that are characterized by DSL domain, EGF repeats, and transmembrane domain.

[0275] In certain embodiments, differentiation factor polypeptides are isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous differentiation factor polypeptides can be isolated from healthy human tissue. Synthetic differentiation factor polypeptides are synthesized in vivo after transfection or transformation of template DNA into a host organism or cell, such as a mammalian or cultured human cell line. Alternatively, synthetic differentiation factor polypeptides are synthesized in vitro by cell-free translation or other methods recognized in the art, Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), which is incorporated herein by reference).

[0276] In certain embodiments, the differentiation factor polypeptide may be recombinant. In some embodiments, the differentiation factor polypeptide is a humanized derivative of a mammalian differentiation factor polypeptide. Exemplary mammalian species from which the differentiation factor polypeptide is derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In some embodiments, the differentiation factor is a recombinant human protein. In some embodiments, the differentiation factor is a recombinant murine (mouse) protein. In some embodiments, the differentiation factor is a humanized derivative of a recombinant mouse protein.

[0277] In certain embodiments, differentiation factor polypeptides may be modified to achieve a desired activity, for example to increase protein stability in vivo, hi certain embodiments, differentiation factor polypeptides may be engineered to be more or less immunogenic.

[0278] In certain embodiments, differentiation factors (e.g., DLL-4) may be covalently linked to the scaffold of the present invention. For example, rather than being released from the scaffold material, the differentiation factors may be covalently bound to the polymer backbone and retained within the composition formed after implantation of the composition into a subject. By covalently binding or coupling differentiation factors to the scaffold material, such differentiation factors will be retained within the scaffold formed after administration of the composition to a subject, and thus will be available to promote differentiation of stem cells or progenitor cells, as contemplated herein. In certain embodiments, differentiation factors are conjugated to the scaffold material using N-hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry. Any method known in the art for covalently binding or coupling differentiation factors may be used, including but not limited to. See "Bioconjugate Techniques Bioconjugate Techniques (Third Addition)", Greg T. Hermanson, Academic Press, 2013 In some embodiments, the differentiation factor may be covalently linked to the scaffold using click chemistry. Methods for covalently binding or coupling the differentiation factor include, but are not limited to, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or disulfide bonds.

[0279] In certain embodiments, the differentiation factor (e.g., DLL-4) of the present invention is linked to a tether (e.g., PEG, PEG 2k) and a methacrylate group (MA). In certain embodiments, the differentiation factor further comprises methacrylated DLL-4-PEG. 2k It is.

[0280] In certain embodiments, the covalent linkage retains the differentiation factor within the scaffold to provide differentiation signals to cells recruited to the scaffold. For example, less than 1% of the total differentiation factor is detected outside the scaffold. The bioactivity of the differentiation factor can be retained for an extended period of time, for example, at least 3 months, after incorporation into the scaffold. The bioactivity of the differentiation factor can be measured by any suitable method, for example, by colorimetric assay for DLL-4.

[0281] In certain embodiments, the differentiation factor may be present at about 0.01 nmol to 1000 nmol, about 0.1 nmol to 100 nmol, or about 1 nmol to 10 nmol per scaffold.

[0282] In some embodiments, the differentiation factor may be present at about 1 ng to about 1000 micrograms per scaffold. For example, the differentiation factor may be present at about 10 ng to about 500 μg, about 50 ng to about 250 μg, about 100 ng to about 200 μg, about 1 μg to about 100 μg, about 1 μg to about 50 μg, about 1 μg to about 25 μg, about 1 μg to about 10 μg, about 2 μg to about 10 μg, or about 6 μg.

[0283] In various embodiments, the amount of differentiation factor present in the scaffold can vary depending on the size of the scaffold. For example, the differentiation factor can be present in a concentration of about 0.03 ng / mm 3 (Ratio of the amount of differentiation factor in weight units to the volume of the scaffold) ~ approx. 350 μg / mm 3 , for example, about 0.1 ng / mm 3 ~Approx. 300μg / mm 3 , about 1ng / mm 3 ~about 250μg / mm 3 , about 10ng / mm 3 ~about 200μg / mm 3 , about 0.1μg / mm3 ~Approx. 100μg / mm 3 , about 0.1μg / mm 3 ~about 50μg / mm 3 , or about 0.1 μg / mm 3 ~about 20μg / mm 3 , about 0.1μg / mm 3 ~about 10μg / mm 3 , about 0.1μg / mm 3 ~about 5μg / mm 3 , about 0.1μg / mm 3 ~Approx. 1μg / mm 3 , about 0.1μg / mm 3 ~0.5μg / mm 3 , or about 0.2 μg / mm 3 It can exist in.

[0284] In certain embodiments, DLL-4 may be present at about 6 μg per scaffold.

[0285] Homing Factor In certain embodiments, the composition of the present invention may further comprise a homing factor.As used herein, the term "homing factor" refers to an agent that can induce the directed migration of cells, such as stem cells or progenitor cells.In certain embodiments, the homing factor of the present invention is a signaling protein that can induce directed chemotaxis in nearby responsive cells.In various embodiments, the homing factor is a cytokine and / or a chemokine.

[0286] In certain embodiments, including such homing factors in the compositions of the invention promotes homing of cells (e.g., transplanted stem and / or progenitor cells) to the scaffold composition administered to a subject. In certain aspects, such homing factors promote infiltration of cells (e.g., transplanted stem or progenitor cells) into the scaffold composition administered to a subject. In some embodiments, the homing factor comprises stromal cell-derived factor (SDF-1). In certain embodiments, the homing factor is encapsulated in the material. In certain embodiments, the homing factor is released from the material over an extended period of time (e.g., about 7-30 days or longer, about 17-18 days).

[0287] In certain embodiments, the homing factor retains its bioactivity for a long period of time.The bioactivity of growth factor can be measured by any suitable means.In certain examples, the homing factor retains its bioactivity for at least 10 days, 12 days, 14 days, 20 days, or 30 days after the homing factor is incorporated into the scaffold.

[0288] In some embodiments, the homing factor may be present at about 0.01 nmol to 1000 nmol, about 0.1 nmol to 100 nmol, or about 1 nmol to 10 nmol per scaffold.

[0289] In some embodiments, the homing factor may be present at about 1 ng to about 1000 micrograms per scaffold. For example, the homing factor may be present at about 10 ng to about 500 μg, about 50 ng to about 250 μg, about 100 ng to about 200 μg, about 1 μg to about 100 μg, about 1 μg to about 50 μg, about 1 μg to about 25 μg, about 1 μg to about 10 μg, about 2 μg to about 10 μg, or about 6 μg.

[0290] In various embodiments, the amount of differentiation factor present in the scaffold can vary depending on the size of the scaffold. For example, the differentiation factor can be present in a concentration of about 0.03 ng / mm 3(Ratio of the amount of differentiation factor in weight units to the volume of the scaffold) ~ approx. 350 μg / mm 3 , for example, about 0.1 ng / mm 3 ~Approx. 300μg / mm 3 , about 1ng / mm 3 ~about 250μg / mm 3 , about 10ng / mm 3 ~about 200μg / mm 3 , about 0.1μg / mm 3 ~Approx. 100μg / mm 3 , about 0.1μg / mm 3 ~about 50μg / mm 3 , or about 0.1 μg / mm 3 ~about 20μg / mm 3 , about 0.1μg / mm 3 ~about 10μg / mm 3 , about 0.1μg / mm 3 ~about 5μg / mm 3 , about 0.1μg / mm 3 ~Approx. 1μg / mm 3 , about 0.1μg / mm 3 ~0.5μg / mm 3 , or about 0.2 μg / mm 3 It can exist in.

[0291] Exemplary Scaffold Compositions The present invention provides a scaffold composition for modulating the immune system in a subject. The composition of the present invention includes a porous scaffold, a growth factor present in an amount effective to induce tissue or organ formation in the scaffold and recruit cells to the scaffold, and a differentiation factor that induces the differentiation of the recruited cells into T cell progenitors.

[0292] In one aspect, the present invention provides a composition for modulating the immune system in a subject, comprising a porous scaffold, a growth factor present in an amount of about 1 ng to about 1000 μg per scaffold and effective to induce formation of a tissue or organ within the scaffold and to recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into lymphocytic T cell progenitors. For example, the growth factor can be present in an amount of about 1 ng to about 500 μg, about 1 ng to about 200 μg, about 1 ng to about 100 μg, about 1 ng to about 50 μg, or about 1 ng to about 10 μg.

[0293] In another aspect, the present invention provides a composition for modulating the immune system in a subject, comprising a porous scaffold, a growth factor present at about 1 ng to about 1000 ng per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and to recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into lymphocytic T cell progenitors. In more specific embodiments, the growth factor is present at about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, or about 200 ng.

[0294] In yet a further aspect, the present invention provides a composition for modulating the immune system in a subject, comprising a porous scaffold and a cellular component comprising about 0.03 ng / mm2 of a cellular component based on the volume of the scaffold. 3 ~about 350ng / mm 3 The present invention is directed to compositions comprising a growth factor, present in an amount effective to induce tissue or organ formation within the scaffold and to recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into T cell progenitor cells. In various embodiments, the growth factor is present in an amount of about 0.1 ng / mm 3 ~about 300ng / mm 3 , about 0.5ng / mm 3 ~about 250ng / mm 3 , about 1ng / mm 3 ~about 200ng / mm 3 , about 2ng / mm 3~about 150ng / mm 3 , about 3ng / mm 3 ~ approx. 100ng / mm 3 , about 4ng / mm 3 ~about 50ng / mm 3 , about 5ng / mm 3 ~25ng / mm 3 , about 6ng / mm 3 ~about 10ng / mm 3 , or about 6.5 ng / mm 3 ~ approx. 7.0ng / mm 3 It can exist in.

[0295] The composition can be designed to release growth factors in a controlled manner. Reduced amounts of growth factors and / or controlled release provide advantages over the prior art, such as reduced toxicity associated with the use of high levels of growth factors, such as BMP-2, and suboptimal release kinetics.

[0296] In various embodiments, the growth factor is a bone morphogenetic protein, such as BMP-2, BMP-4, BMP-7, BMP-12, BMP-14, or any combination thereof. In some embodiments, the growth factor is BMP-2. In certain embodiments, the growth factor is a TGF-β, such as TGF-β1, TGF-β2, TGF-β3, TGF-β4, or any combination thereof. In certain embodiments, the growth factor comprises TGF-β1.

[0297] The porous scaffold of the composition according to the present invention can be any biocompatible and biodegradable scaffold. In certain embodiments, the porous scaffold comprises a hydrogel or cryogel. In various embodiments, the hydrogel or cryogel comprises an alginate or alginate derivative, a gelling or gelatin derivative, or a hyaluronic acid or hyaluronic acid derivative.

[0298] In certain embodiments, the differentiation factor of the present invention comprises a polypeptide that binds to a Notch receptor. In various embodiments, the differentiation factor is selected from the group consisting of Delta-like 1 (DLL-1), Delta-like 2 (DLL-2), Delta-like 3 (DLL-3), Delta-like 3 (DLL-3), Delta-like 4 (DLL-4), Jagged 1, Jagged 2, and any combination thereof. In some embodiments, the differentiation factor comprises DLL-4. In certain embodiments, the differentiation factor is covalently linked to the porous scaffold.

[0299] In a particular embodiment, the composition of the invention comprises an injectable cryogel comprising alginate or an alginate derivative, e.g., methacrylated alginate, or hyaluronic acid or a hyaluronic acid derivative, a growth factor that is a bone morphogenetic protein, e.g., BMP-2, and a differentiation factor that is a Delta-like family protein, e.g., DLL-4. The growth factor is administered at about 200 ng per scaffold, or at about 6.5 ng / mm 3 ~7.0ng / mm 3 It can exist in.

[0300] III. Methods of Modulating the Immune System The present invention features a method of modulating the immune system of a subject. In certain embodiments of the present invention, the method of modulating the immune system of a subject comprises administering to the subject one or more compositions of the present invention. The composition comprises a porous scaffold, a growth factor present in an amount effective to induce formation of a tissue or organ within the scaffold and to recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into T cell progenitors. In certain embodiments, the composition comprises a porous scaffold, a growth factor present in an amount between about 1 ng and about 1000 ng per scaffold and in an amount effective to induce formation of a tissue or organ within the scaffold and to recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into lymphocytes.

[0301] In certain embodiments, the method further comprises administering to the subject hematopoietic stem cells or hematopoietic progenitor cells.

[0302] In certain embodiments, the cell is a stem cell or progenitor cell. As used herein, the term "stem cell" refers to a biological cell that can differentiate into other types of cells and divide to produce more stem cells of the same type. Stem cells include embryonic stem cells isolated from the inner cell mass of blast cells and adult stem cells found in various tissues. In adult organisms, stem cells and progenitor cells function as the body's repair system and replenish adult tissues. In certain embodiments, the stem cell is an embryonic stem cell, fetal stem cell, amniotic fluid stem cell, umbilical cord stem cell, adult stem cell, or induced pluripotent stem cell. In certain embodiments, the stem cell is a hematopoietic stem cell. Hematopoietic stem cells are stem cells that give rise to other blood cells, including blood cells of both myeloid and lymphoid lineages.

[0303] As used herein, the term "progenitor cell" refers to a biological cell that can differentiate into a specific type of cell. Progenitor cells are generally in a more differentiated state than stem cells. Typically, progenitor cells can only divide a limited number of times.

[0304] In certain embodiments, the progenitor cell is a blast cell, such as thymocyte, lymphoblast, myelocyte, or myeloid progenitor cell.In certain embodiments, the progenitor cell is a cell that can differentiate into T cell progenitor cell.In certain embodiments, the lymphocyte includes T cell, such as naive T cell.

[0305] In certain embodiments, the cells that are mobilized are hematopoietic bone marrow cells or mobilized peripheral blood cells.

[0306] In certain embodiments, the cell may be a recombinant cell. The term "recombinant cell" as used herein refers to a cell into which a genetic modification has been introduced. The genetic modification may be at the chromosomal level or extrachromosomal. "Chromosomal level genetic modification" refers to a genetic modification in the genome of a cell, such as an insertion, deletion, and / or substitution in a chromosome of a cell. An extrachromosomal genetic modification refers to a genetic modification that is not located in the genome of a cell. For example, a plasmid containing a protein-coding gene may be introduced into a cell. The plasmid may replicate and be transmitted from a parent cell to a progeny cell.

[0307] In various embodiments, gene modification introduces a gene into a cell. The introduced gene can complement the function of a defective gene in a cell. For example, the cell can contain a mutant defective gene. Gene modification can introduce a wild-type functional gene into a cell to restore the function of the gene. In some embodiments, gene modification can increase or decrease the expression of a specific gene. For example, gene modification can introduce a small interfering RNA (siRNA) specific to a gene to inhibit the expression of the gene.

[0308] Methods for genetically modifying cells are generally known in the art, for example, those described in Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), which is incorporated herein by reference.

[0309] In certain embodiments, genetic modification can be introduced by gene editing, also known as genome editing. Gene editing is a group of techniques that give the skilled artisan the ability to change the DNA of an organism. These techniques allow genetic material to be added, removed, or modified at specific locations in the genome. Gene editing techniques include, but are not limited to, meganuclease system, zinc finger nuclease (ZFN) system, transcription activator-like effector nuclease (TALEN) system, and CRISPR-Cas system. The CRISPR-Cas system, which is an abbreviation for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein system, especially CRISP-Cas9, is faster, cheaper, more accurate, and more efficient than other existing genome editing methods.

[0310] In certain embodiments, the present invention features a method of modulating the immune system of a subject after the subject has undergone a transplant. For example, the subject may undergo a hematopoietic stem cell transplant. In certain embodiments, the composition of the present invention is administered to the subject at the same time as or after the hematopoietic stem cell transplant.

[0311] In certain embodiments, at least two compositions are administered to a subject. The compositions may be of similar size.

[0312] In certain embodiments, the methods of the invention modulate the immune response of a human over the age of 30. For example, the human may be over 40, over 50, over 60, over 70, or over 80 years of age.

[0313] In some embodiments, one or more compositions of the present invention (e.g., bone marrow cryogel) may be administered in conjunction with stem cell mobilization techniques. Stem cell mobilization is a process that uses certain cell mobilizing agents to induce stem cell migration from the bone marrow into the blood, as described, for example, in Hopman and DiPersio, Advances in Stem Cell Mobilization, Blood Rev., 2014, 28(1): 31-40, the contents of which are incorporated herein by reference. Such techniques may also be used to mobilize progenitor cells.

[0314] Thus, in certain embodiments, a stem cell and / or progenitor cell mobilizing agent is administered to a subject in an amount effective to induce stem cell / progenitor cell migration from bone marrow into blood.The released stem cell and / or progenitor cell is then mobilized into the composition of the present invention (e.g., bone marrow cryogel) to differentiate into T cell progenitor cells.The stem cell and / or progenitor cell mobilizing agent can be administered before, simultaneously, or after the administration of the composition (e.g., bone marrow cryogel).

[0315] In various embodiments, the compositions of the present invention (e.g., bone marrow cryogel) can be administered to a subject in combination with stem cell and / or progenitor cell mobilization agents. In certain embodiments, the subject is an elderly human, for example, the human may be over 30 years old, over 40 years old, over 50 years old, over 60 years old, over 70 years old, or over 80 years old. The stem cell and / or progenitor cell mobilization agents can mobilize the subject's own stem cells and / or progenitor cells from the bone marrow, so that these cells can home to the compositions of the present invention, thereby enhancing the generation of T cells in the subject without other conditioning or stem cell transplantation.

[0316] In certain embodiments, the compositions of the present invention (e.g., bone marrow cryogel) can be administered to a subject in conjunction with stem cell and / or progenitor cell mobilization techniques and stem cell transplantation. Transplantation can be autologous, allogeneic, or xenogeneic.

[0317] In some embodiments, a therapeutically effective amount of one or more cell mobilization agents that can stimulate the mobilization, production, and / or function of one or more cell types to the peripheral bloodstream are administered. The agents can be provided through any desired route of administration, including oral, rectal, intravenous, intramuscular, subcutaneous, or aerosol. Some non-limiting embodiments of agents that can stimulate the mobilization, production, and / or function of a cell type to the peripheral bloodstream include IL-1, IL-2, IL-3, IL-6, GM-CSF, G-CSF, plerixafor, PDGF, TGF-beta, NGF, IGF, growth hormone, erythropoietin, thrombopoietin, and the like. In addition to naturally occurring growth factors, growth factor analogs and growth factor derivatives, such as fusion proteins, can be used as well. In some embodiments, the method includes administration of a therapeutically effective amount of G-CSF and a therapeutically effective amount of electromagnetic radiation. In some embodiments, the method includes administering a combination of a therapeutically effective amount of plerixafor and a therapeutically effective amount of electromagnetic radiation. In some embodiments, a therapeutically effective amount of electromagnetic radiation is combined with another agent, which in some embodiments may be a hematopoietic stem cell mobilizing agent. In some embodiments, a therapeutically effective amount of electromagnetic radiation is combined with a combination of two or more of G-CSF, GM-CSF, plerixafor, IL-1, IL-2, IL-3, IL-6, PDGF, TGF-beta, NGF, IGF, growth hormone, erythropoietin, thrombopoietin, or another agent.

[0318] Cell mobilization and differentiation into T cell precursors In one aspect, the invention features a method for recruiting cells to a scaffold and inducing differentiation of the recruited cells into T cell progenitors. Specifically, the method includes administering to a subject one or more compositions of the invention comprising a porous scaffold, a growth factor present in an amount effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into lymphocytes. In certain embodiments, the subject is administered a composition comprising a porous scaffold and a growth factor present in an amount effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold, from about 1 ng to about 1000 ng, e.g., from about 5 ng to about 500 ng, from about 5 ng to about 250 ng, from about 5 ng to about 200 ng, or from about 200 ng per scaffold, and a differentiation factor that induces differentiation of the recruited cells into lymphocytes. In another specific embodiment, a subject is administered a composition comprising a porous scaffold and a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes, present in an amount of about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold. In yet another embodiment, a subject is administered a composition comprising a porous scaffold and a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes, present in an amount of about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold. 3 (ratio of amount of growth factor by weight to volume of scaffold) ~ approx. 350ng / mm 3 , for example, about 0.1 ng / mm 3 ~about 300ng / mm 3 , about 0.5ng / mm 3 ~about 250ng / mm 3 , about 1ng / mm 3 ~about 200ng / mm 3 , about 2ng / mm 3 ~about 150ng / mm 3 , about 3ng / mm 3 ~ approx. 100ng / mm 3 , about 4ng / mm 3 ~about 50ng / mm 3 , or about 5 ng / mm3 ~25ng / mm 3 and the like, and present in an amount effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold, is administered a composition comprising a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes.

[0319] In certain embodiments, the growth factors and / or homing factors function to recruit cells to the scaffold of the invention. The differentiation factors, and optionally the growth factors, of the composition subsequently induce the differentiation of the cells into T cell progenitors.

[0320] In some embodiments, the method further comprises administering to the subject hematopoietic stem or progenitor cells.

[0321] In certain embodiments, growth factors (e.g., BMP-2) and / or homing factors (e.g., SDF-1) mobilize stem or progenitor cells that can differentiate into T cell progenitors. Differentiation factors (e.g., DLL-4) induce differentiation of stem or progenitor cells into T cell progenitors. In certain embodiments, lymphocytes include naive T cells. In certain embodiments, lymphocytes include T cells, e.g., CD4 + T cells, CD8 + T cells, and / or regulatory T cells (T reg ) are included.

[0322] In certain embodiments, the cells to be mobilized are transplant cells. The transplant cells (e.g., hematopoietic stem or progenitor cells) can be autologous. For example, the transplant cells can be the subject's own umbilical cord stem cells or stem cells obtained from the subject before treatment, e.g., radiation treatment. The transplant cells can be syngeneic, e.g., hematopoietic stem or progenitor cells obtained from the subject's identical twin. The transplant cells can also be allogeneic, e.g., hematopoietic stem or progenitor cells obtained from a donor of the same species. The transplant cells can also be xenogeneic, e.g., hematopoietic stem or progenitor cells obtained from a different species.

[0323] In certain embodiments, the cell can be a bone marrow stromal cell derived from a subject. Stromal cell refers to the connective tissue cell of an organ, for example, bone marrow. Stromal cell supports the function of the parenchymal cells of the organ (for example, bone marrow). Bone marrow stromal cell produces DLL-4, which provides an important functional environment for generating T cell-competent lymphoid common progenitor cells. Growth factors (for example, BMP-2) can also promote bone lineage differentiation of stromal cells.

[0324] In certain embodiments, the cells that are mobilized may be cells that are not stem or progenitor cells.

[0325] Reducing immune hyperresponsiveness In one aspect, the invention provides a method of reducing immune hyperresponsiveness in a subject by administering to the subject one or more compositions of the invention comprising a porous scaffold and a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells to the scaffold (e.g., about 1 ng to about 1000 ng, about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, or about 200 ng per scaffold), and a differentiation factor to induce differentiation of the recruited cells into T cell progenitors.

[0326] In another aspect, the invention provides a method of reducing immune hyperresponsiveness in a subject by administering to the subject one or more compositions of the invention comprising a porous scaffold and about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, and present in an amount effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold, and a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes.

[0327] In another aspect, the present invention provides a method of reducing immune hyperresponsiveness in a subject, comprising administering to the subject a porous scaffold and about 0.03 ng / mm3 (ratio of amount of growth factor by weight to volume of scaffold) ~ approx. 350ng / mm 3 , for example, about 0.1 ng / mm 3 ~about 300ng / mm 3 , about 0.5ng / mm 3 ~about 250ng / mm 3 , about 1ng / mm 3 ~about 200ng / mm 3 , about 2ng / mm 3 ~about 150ng / mm 3 , about 3ng / mm 3 ~ approx. 100ng / mm 3 , about 4ng / mm 3 ~about 50ng / mm 3 , or about 5 ng / mm 3 ~25ng / mm 3 and the like, and present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells to the scaffold, and a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes.

[0328] In some embodiments, the method further comprises administering to the subject hematopoietic stem or progenitor cells.

[0329] The immune system is a tightly controlled network that can maintain immune homeostasis balance under normal physiological conditions.Normally, when challenged with foreign antigen, a specific appropriate response is initiated to restore homeostasis.However, under certain circumstances, this balance is not maintained, and immune response is either under-reactive or over-reactive.When immune response is over-reactive, this can lead to conditions such as autoimmune disease, allergy, and / or GVHD.

[0330] In certain embodiments, the present invention relates to a method for detecting and determining whether regulatory T cells (T reg The present invention features a method for increasing levels of T cells, also known as suppressor T cells. regT cells are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent and / or treat autoimmune diseases. reg The cells are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. reg The cells express the biomarkers CD4, FOXP3, and CD25. Without wishing to be bound by any theory, T reg Increasing the levels of the cells is believed to result in a reduction in immune hyper-reactivity.

[0331] autoimmune disease In certain aspects, the present invention provides methods of preventing and / or treating autoimmune disease by administering to a subject one or more compositions of the present invention comprising a porous scaffold, a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells to the scaffold (e.g., about 1 ng to about 1000 ng, about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, or about 200 ng per scaffold), and a differentiation factor to induce differentiation of the recruited cells into T cell progenitors. As used herein, the term "autoimmune disease" refers to a disease, disorder, or illness in which the subject's immune system attacks and / or damages the subject's own tissues. Exemplary autoimmune diseases include, but are not limited to, type 1 diabetes, rheumatoid arthritis, psoriasis, arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and celiac disease.

[0332] In certain embodiments, the subject has T reg A subject may be administered the composition of the present invention and hematopoietic stem or progenitor cells. The hematopoietic stem or progenitor cells may be obtained from the subject. The composition of the present invention is used to inhibit the T cell proliferation and proliferation of hematopoietic stem cells. regIt may contain growth factors (eg, TGF-β family proteins) to induce differentiation into cells.

[0333] allergy In certain embodiments, the invention features a method of preventing and / or treating allergy by administering to a subject one or more compositions of the invention comprising a porous scaffold and a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells to the scaffold (e.g., about 1 ng to about 1000 ng, about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, or about 200 ng per scaffold), and a differentiation factor to induce differentiation of the recruited cells into T cell progenitors.

[0334] Allergies, also known as allergic diseases, are a number of conditions caused by hypersensitivity of the immune system to typically harmless substances in the environment. Without wishing to be bound by any theory, it is believed that the T reg Increasing the levels of the cells can prevent and / or treat allergies.

[0335] GVHD In another aspect, the present invention provides a method of alleviating symptoms associated with graft-versus-host disease (GVHD) by administering to a subject one or more compositions of the present invention comprising a porous scaffold, a growth factor present in an effective amount (e.g., about 1 ng to about 1000 ng, about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, or about 200 ng per scaffold) to induce tissue or organ formation within the scaffold and to recruit cells to the scaffold, and a differentiation factor to induce differentiation of the recruited cells into T cell precursors. GVHD is a medical condition following receipt of transplanted tissue from a genetically different donor. GVHD is commonly associated with stem cell transplantation, e.g., hematopoietic stem cell transplantation. GVHD also occurs in other forms of transplanted tissue, e.g., solid organ transplantation.

[0336] In certain embodiments, the GVHD is associated with hematopoietic stem cell transplantation. In certain embodiments, the GVHD is associated with solid organ transplantation.

[0337] In certain embodiments, the present invention provides a method for the preparation of a reg In one particular embodiment, the method includes increasing the level of T reg The cells differentiate from transplanted hematopoietic stem cells (e.g., donor hematopoietic stem cells). Without wishing to be bound by any theory, donor T cells in hematopoietic stem cell transplantation reg Cell augmentation is performed using donor T reg Given that hematopoietic stem cell transplantation plays an important role in suppressing GVHD, it is likely that hematopoietic stem cell transplantation can contribute to the alleviation of GVHD symptoms in subjects undergoing hematopoietic stem cell transplantation. In certain embodiments, the present invention provides a method for the treatment of hematopoietic stem cell transplantation using autologous hematopoietic stem cell transplantation in a subject. reg The present invention features a method for increasing the levels of a cell.

[0338] Enhancement of donor chimerism in transplant recipients In another aspect, the present invention provides a method of enhancing donor chimerism in a subject receiving a transplant by administering to the subject one or more compositions of the present invention comprising a porous scaffold, a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and to recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into T cell progenitors. In certain embodiments, the subject is administered a composition comprising a porous scaffold, and a growth factor and a differentiation factor that induces differentiation of the recruited cells into T cell progenitors, at about 1 ng to about 1000 ng per scaffold, e.g., about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, or about 200 ng, and present in an amount effective to induce tissue or organ formation within the scaffold and to recruit cells to the scaffold. In another specific embodiment, a subject is administered a composition comprising a porous scaffold and a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes, present in an amount of about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold. In yet another embodiment, a subject is administered a composition comprising a porous scaffold and a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes, present in an amount of about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold. 3 (ratio of amount of growth factor by weight to volume of scaffold) ~ approx. 350ng / mm 3 , for example, about 0.1 ng / mm 3 ~about 300ng / mm 3 , about 0.5ng / mm 3 ~about 250ng / mm 3 , about 1ng / mm 3 ~about 200ng / mm 3 , about 2ng / mm 3 ~about 150ng / mm 3 , about 3ng / mm 3 ~ approx. 100ng / mm 3 , about 4ng / mm 3 ~about 50ng / mm 3 , or about 5 ng / mm 3~25ng / mm 3 and the like, and present in an amount effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold, is administered a composition comprising a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes.

[0339] In some embodiments, the method further comprises administering to the subject hematopoietic stem or progenitor cells.

[0340] Donor chimerism usually occurs when a subject undergoes hematopoietic stem cell transplantation. The term "chimerism" as used herein refers to the presence of lympho-hematopoietic cells of non-host origin. Total or complete chimerism generally refers to the complete replacement of host with donor lympho-hematopoiesis. Mixed chimerism indicates the presence of both donor and recipient cells in a given cell compartment, e.g., lymphocytes. Low levels of donor chimerism are often associated with a defect in T cell generation in allogeneic hematopoietic stem cell or progenitor cell transplantation, which may render patients susceptible to infectious agents and contribute to GVHD.

[0341] Balanced T cell reconstitution In another aspect, the present invention provides a method of providing a balanced T cell reconstitution in a subject by administering to the subject one or more compositions of the present invention comprising a porous scaffold, a growth factor present in an amount effective to induce formation of a tissue or organ within the scaffold and to recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into T cell progenitors. In certain embodiments, the subject is administered a composition comprising a porous scaffold, and a growth factor and a differentiation factor that induce differentiation of the recruited cells into T cell progenitors, at about 1 ng to about 1000 ng per scaffold, e.g., about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, or about 200 ng, and present in an amount effective to induce formation of a tissue or organ within the scaffold and to recruit cells to the scaffold. In another specific embodiment, a subject is administered a composition comprising a porous scaffold and a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes, present in an amount of about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold. In yet another embodiment, a subject is administered a composition comprising a porous scaffold and a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes, present in an amount of about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold. 3 (ratio of amount of growth factor by weight to volume of scaffold) ~ approx. 350ng / mm 3 , for example, about 0.1 ng / mm 3 ~about 300ng / mm 3 , about 0.5ng / mm 3 ~about 250ng / mm 3 , about 1ng / mm 3 ~about 200ng / mm 3 , about 2ng / mm 3 ~about 150ng / mm 3 , about 3ng / mm 3 ~ approx. 100ng / mm 3 , about 4ng / mm 3 ~about 50ng / mm 3 , or about 5 ng / mm 3~25ng / mm 3 and the like, and present in an amount effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold, is administered a composition comprising a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes.

[0342] In some embodiments, the subject is further administered hematopoietic stem or progenitor cells. The compositions of the invention may be administered simultaneously with or after administration of the hematopoietic stem or progenitor cells.

[0343] The term "balanced T cell reconstitution" as used herein refers to CD4 + :CD8 + This refers to the reconstitution of T cells characterized by the ratio of CD4 + Cellular reconstitution is usually slow in HSCT recipients. + It can accelerate T cell reconstitution and result in balanced T cell reconstitution.

[0344] Hematopoietic stem cell transplantation (HSCT) is a curative treatment for several disorders, but allogeneic HSCT is limited by T cell deficiencies and dysregulation. In subjects undergoing allogeneic HSCT, CD4 + T cell recovery is slow, resulting in a reversal of the normal CD4 / CD8 ratio, which is about 0.9 to about 2.5 in peripheral blood, although the ratio may vary in other tissues or organs.

[0345] In certain embodiments, the methods of the present invention involve the use of CD4 + :CD8 + The CD4 ratio stabilizes in the normal range in subjects undergoing HSCT alone. + The T cell compartment is not fully reconstituted. In certain embodiments, balanced T cell reconstitution is achieved by reconstituting homeostatic CD4 + :CD8 +The ratio is characterized by being in the normal range within 30 days following hematopoietic stem cell transplantation and administration of the composition of the invention.

[0346] In certain embodiments according to the invention, a subject, e.g., a human, receives about 1×10 6 cells / kilogram of the subject's body weight in a hematopoietic stem cell transplant. 5 ~About 50×10 6 In certain embodiments, a subject receives about 1 x 10 hematopoietic stem or progenitor cells per kilogram of the subject's body weight. 5 Each recipient receives individual hematopoietic stem cells.

[0347] The methods of the present invention provide similar or better healing and / or therapeutic effects compared to subjects receiving hematopoietic stem cell or T cell precursor infusion alone (i.e., not treated with the compositions of the present invention). For example, treatment with the compositions of the present invention can result in a higher number of T cell precursors and functional T cells in the thymus and periphery, even when used at lower doses compared to, for example, T cell precursor infusion alone. In some embodiments, similar or better healing and / or therapeutic effects can be achieved when less than ten percent (10%) of hematopoietic stem cells or progenitors used in HSCT alone are administered to a subject in combination with the compositions of the present invention.

[0348] In various embodiments, balanced T cell reconstitution is also characterized by enhanced T cell neogenesis. The term "neogenesis" as used herein refers to the generation of new cells. In various embodiments, enhanced T cell neogenesis is characterized by enhanced T cell receptor excision circles (TRECs). In certain embodiments, T cell neogenesis using the compositions and methods of the present invention achieves a baseline or normal number of TRECs. The term "baseline number of TRECs" as used herein refers to the number of TRECs in a subject before the subject undergoes any treatment that impairs the subject's immune system. The term "normal number of TRECs" as used herein refers to the number of TRECs in an individual without a defective immune system. The normal number of TRECs can be within a certain range. In certain embodiments, TRECs can be assessed in humans in a quantitative and non-invasive manner by estimating TRECs in peripheral blood cells.

[0349] Antigen-specific T cell response In another aspect, the present invention provides a method of inducing an antigen-specific T cell response in a subject by administering to the subject one or more compositions of the present invention comprising a porous scaffold, a growth factor present in an amount effective to induce formation of a tissue or organ within the scaffold and to recruit cells to the scaffold, and a differentiation factor that induces differentiation of the recruited cells into T cell precursors, and administering to the subject a vaccine comprising an antigen, thereby inducing an antigen-specific T cell response in the subject. In certain embodiments, the subject is administered a composition comprising a porous scaffold, and a growth factor present in an amount of about 1 ng to about 1000 ng per scaffold, e.g., about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, or about 200 ng, and a differentiation factor that induces differentiation of the recruited cells into T cell precursors, and present in an amount effective to induce formation of a tissue or organ within the scaffold and to recruit cells to the scaffold. In another specific embodiment, a subject is administered a composition comprising a porous scaffold and a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes, present in an amount of about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold. In yet another embodiment, a subject is administered a composition comprising a porous scaffold and a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes, present in an amount of about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold. 3 (ratio of amount of growth factor by weight to volume of scaffold) ~ approx. 350ng / mm 3 , for example, about 0.1 ng / mm 3 ~about 300ng / mm 3 , about 0.5ng / mm 3 ~about 250ng / mm 3 , about 1ng / mm 3 ~about 200ng / mm 3 , about 2ng / mm 3 ~about 150ng / mm 3 , about 3ng / mm 3 ~ approx. 100ng / mm 3 , about 4ng / mm 3 ~about 50ng / mm3 , or about 5 ng / mm 3 ~25ng / mm 3 and the like, and present in an amount effective to induce formation of a tissue or organ within the scaffold and recruit cells to the scaffold, is administered a composition comprising a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes.

[0350] In some embodiments, the method further comprises administering to the subject hematopoietic stem or progenitor cells.

[0351] In certain embodiments, the subject has a defective immune system. As used herein, the term "defective immune system" refers to a state in which the immune system's ability to fight infectious diseases and cancer is defective or completely absent. Many cases of immune system defects are acquired ("secondary") due to foreign factors that affect the subject's immune system. Examples of these foreign factors include HIV infection, age, and environmental factors, such as nutrition. In certain embodiments, immunosuppression by some drugs, such as steroids, can be either a side effect or the intended purpose of treatment. Examples of such use include (i) in organ transplant surgery as an anti-rejection measure, and (ii) in patients suffering from an over-reactive immune system, such as in autoimmune diseases. In certain embodiments, some therapies for cancer, such as radiation therapy and / or chemotherapy, cause a defect in the immune system. A state in which a subject has a defective immune system may be referred to as "immunodeficiency."

[0352] In certain embodiments, the subject undergoes hematopoietic stem cell transplantation.In certain embodiments, the subject has blood or bone marrow cancer, such as myeloma or leukemia.The subject may undergo conditioning cytotoxic radiation and / or chemotherapy regimen to destroy tumor cells, which results in severe lymphopenia due to the destruction of T cells and B cells of adaptive immune system.

[0353] In certain embodiments, the method of inducing an antigen-specific response comprises administering to a subject a vaccine comprising a composition of the invention and an antigen.

[0354] Modulation of the immune system in immune-compromised subjects In one aspect, the invention provides a method of modulating the immune system in an immune-deficient subject by administering to the subject one or more compositions of the invention comprising a porous scaffold and a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells to the scaffold (e.g., about 1 ng to about 1000 ng, about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, or about 200 ng per scaffold), and a differentiation factor to induce differentiation of the recruited cells into T cell progenitors.

[0355] In another aspect, the invention provides a method of modulating the immune system in an immune-compromised subject by administering to the subject one or more compositions comprising a porous scaffold and about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, and present in an amount effective to induce formation of a tissue or organ within the scaffold and to recruit cells to the scaffold, a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes.

[0356] In yet another aspect, the present invention provides a method of modulating the immune system in an immune-compromised subject, comprising administering to the subject a porous scaffold and about 0.03 ng / mm 3 (ratio of amount of growth factor by weight to volume of scaffold) ~ approx. 350ng / mm 3 , for example, about 0.1 ng / mm 3 ~about 300ng / mm 3 , about 0.5ng / mm 3 ~about 250ng / mm 3 , about 1ng / mm 3 ~about 200ng / mm3 , about 2ng / mm 3 ~about 150ng / mm 3 , about 3ng / mm 3 ~ approx. 100ng / mm 3 , about 4ng / mm 3 ~about 50ng / mm 3 , or about 5 ng / mm 3 ~25ng / mm 3 and the like, and present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells to the scaffold, comprising a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes.

[0357] In some embodiments, the subject is further administered hematopoietic stem or progenitor cells.

[0358] In certain embodiments, the subject is further administered a stem cell and / or progenitor cell mobilizing agent so that the released stem cells and / or progenitor cells are mobilized into the composition of the present invention (e.g., bone marrow cryogel). The stem cell / progenitor cell mobilizing agent can be administered before, simultaneously with, or after administration of the composition of the present invention (e.g., bone marrow cryogel).

[0359] In some embodiments, the immune system defect is caused by immunosenescence. The term "immunosenescence" as used herein refers to the deterioration of the immune system brought about by the natural progression of age. It involves both the host's ability to respond to infection and the development of long-term immune memory, especially by vaccination. Immunosenescence is a multifactorial condition that leads to a number of pathologically significant health problems in the elderly population. Immunosenescence is associated with a reduction in the self-renewal capacity of hematopoietic stem cells. Subjects with immunosenescence may show a reduced CD4+ / CD8+ ratio, a reduced antigen recognition repertoire of T cell receptor (TCR) diversity, and / or impaired proliferation in response to antigen stimulation. Immunosenescence can occur at a young age, for example, at age 30 in humans.

[0360] In some embodiments, the subject has a defective immune system due to a congenital immune deficiency. As used herein, "congenital immune deficiency," also known as "primary immune deficiency," refers to a deficiency, absence, or defect in one or more of the major components of the immune system. These disorders are genetically determined and typically manifest in infancy or childhood as frequent, chronic, or opportunistic infections. Classification is based on the major components of the immune system that are defective, absent, or defective. Diagnosis is confirmed with tests such as differential WBC counts, absolute lymphocyte counts, quantitative immunoglobulin (Ig) measurements, and antibody titers. Treatment usually consists of prophylactic antibiotics to manage and prevent infection. Prognosis in primary immune deficiency disorders is variable and depends on the specific disorder.

[0361] Exemplary congenital immune deficiency disorders include congenital B cell immune deficiencies, e.g., Bruton's agammaglobulinemia, selective IgA deficiency, common variable immune deficiency, congenital T cell immune deficiencies, e.g., DiGeorge syndrome, autosomal dominant hyperimmunoglobulin E syndrome, IL-12 receptor deficiency, chronic mucocutaneous candidiasis, IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), congenital mixed immune deficiencies, e.g., severe combined immunodeficiency (SCID, Babies' These conditions include, but are not limited to, Leboy's disease, Glanzmann-Liniker syndrome, alymphocytosis), Wiskott-Aldrich syndrome, hyper-IgM syndrome, ataxia-telangiectasia, congenital neutrophil and phagocyte disorders such as chronic granulomatous disease (CGD), leukocyte adhesion deficiency type 1, Chediak-Higashi syndrome, myeloperoxidase deficiency, severe congenital neutropenia, congenital complement deficiencies such as terminal complement deficiency, C3 deficiency.

[0362] In certain embodiments, the subject has a defective immune system due to acquired immune deficiency. As used herein, the term "acquired immune deficiency" refers to an immune deficiency caused by a foreign factor that affects the subject's immune system. Acquired immune deficiency, also known as secondary immune deficiency, can be caused by various immunosuppressants, such as malnutrition, aging, certain medicines or treatments (e.g., chemotherapy (cytotoxic agents), disease-modifying antirheumatic drugs, immunosuppressants after organ transplantation, glucocorticoids, radiation therapy), and environmental toxins, such as mercury and other heavy metals, pesticides, and petrochemicals, such as styrene, dichlorobenzene, xylene, and ethylphenol. For medicines, the term "immunosuppression" generally refers to both the beneficial and potentially harmful effects of reducing the function of the immune system, while the term "immunodeficiency" generally refers only to the harmful effect of increased risk of infection.

[0363] Many specific diseases directly or indirectly cause immune suppression. These include many types of cancer, especially those of bone marrow and blood cells (leukemia, lymphoma, multiple myeloma), as well as certain chronic infectious diseases. Immunodeficiency is also a feature of acquired immune deficiency syndrome (AIDS), caused by the human immunodeficiency virus (HIV). HIV directly infects a small number of T-helper cells and also indirectly impairs other immune system responses. Various hormonal and metabolic disorders, including but not limited to anemia, hypothyroidism, diabetes, and hypoglycemia, can also cause immune deficiency. Smoking, alcoholism, and drug abuse also reduce immune responses.

[0364] Without wishing to be bound by any theory, the balanced T cell reconstitution and antigen-specific T cell responses provided by the compositions and methods of the present invention can modulate the immune system of a subject having an immune system deficiency.

[0365] IV. Kit Any of the compositions described herein can be included in a kit. In a non-limiting example, the kit includes a porous scaffold and a composition comprising a growth factor and a differentiation factor that induces differentiation of the recruited cells into T cell progenitors, the growth factor being present in an amount effective to induce formation of a tissue or organ in the scaffold and recruit cells to the scaffold. In a specific embodiment, the kit includes a porous scaffold and a composition comprising about 1 ng to about 1000 ng per scaffold, e.g., about 5 ng to about 500 ng, about 5 ng to about 250 ng, about 5 ng to about 200 ng, or about 200 ng, the growth factor being present in an amount effective to induce formation of a tissue or organ in the scaffold and recruit cells to the scaffold and a differentiation factor that induces differentiation of the recruited cells into T cell progenitors. In another specific embodiment, the kit includes a porous scaffold and a composition comprising a growth factor and a differentiation factor that induces differentiation of the recruited cells into lymphocytes, present in an amount of about 1 ng to about 1000 μg per scaffold, e.g., about 1 ng to about 500 μg, about 5 μg to about 250 μg, or about 10 μg to about 100 μg, effective to induce formation of a tissue or organ in the scaffold and recruit cells to the scaffold. In yet another embodiment, the kit includes a porous scaffold and a composition comprising about 0.03 ng / mm 3 (ratio of amount of growth factor by weight to volume of scaffold) ~ approx. 350ng / mm 3 , for example, about 0.1 ng / mm 3 ~about 300ng / mm 3 , about 0.5ng / mm 3 ~about 250ng / mm 3 , about 1ng / mm 3 ~about 200ng / mm 3 , about 2ng / mm 3 ~about 150ng / mm 3 , about 3ng / mm 3 ~ approx. 100ng / mm 3 , about 4ng / mm 3 ~about 50ng / mm 3 , or about 5 ng / mm 3 ~25ng / mm 3and the like, and present in an amount effective to induce formation of a tissue or organ in the scaffold and recruit cells to the scaffold, and a growth factor that induces differentiation of the recruited cells into lymphocytes.

[0366] In some embodiments, the kit includes a composition described elsewhere herein.

[0367] In certain embodiments, the kit comprises a syringe or alternative injection device for administering the composition, hi specific embodiments, a pre-filled syringe or injection device is pre-filled with the composition.

[0368] The kit may further include reagents or instructions for administering the composition of the invention to a subject. It may also include one or more reagents.

[0369] The components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, and preferably suitably aliquoted. If there is more than one component in the kit, the kit will also generally include a second, third, or other additional container into which the additional component may be separately placed. The kit may also include a second container means for storing a sterile pharma- ceutically acceptable buffer and / or other diluent. However, various combinations of components may be included in one vial. The kit of the present invention will also typically include a means for storing the composition of the present invention, e.g., a composition for modulating the immune system, and any other reagent containers in tightly sealed condition for commercial sale.

[0370] When the components of the kit are provided in one and / or multiple solutions, the solution is an aqueous solution, and a sterile aqueous solution is particularly preferred.However, the components of the kit may also be provided as dry powder.When the reagent and / or components are provided as dry powder, the powder can be reconstituted by adding a suitable solvent.It is also envisioned that the solvent can be provided in another container means.

[0371] The present invention is further illustrated by the following examples, which are not to be construed as limiting. All cited materials, such as references, publications, databases, database entries, and technologies cited herein are incorporated by reference into this application, even if not explicitly mentioned in the citation. In the event of any discrepancy between the statements in the cited materials and this application, the statements in this application shall prevail.

[0372] Section and table headings are not intended to be limiting. EXAMPLES

[0373] summary Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for numerous disorders, but T cell defects and dysregulation limit its utility. Herein, we report a biomaterial-based scaffold that mimics features of T cell lymphopoiesis in bone marrow. Bone marrow cryogels (BMCs) release bone morphogenetic protein-2 to recruit stromal cells and present the Notch ligand delta-like ligand-4 to facilitate T cell lineage specification of mouse and human hematopoietic progenitors. Subcutaneous injection of BMCs at the time of HSCT in mice enhanced thymic seeding of T cell precursors, T cell neogenesis, and diversification of the T cell receptor repertoire. Peripheral T cell reconstitution was increased approximately six-fold in mouse HSCT and approximately two-fold in human xenogeneic HSCT. Furthermore, BMCs promoted donor CD4+ regulatory T cell generation and improved survival after allogeneic HSCT. Compared with adoptive transfer of T cell precursors, BMCs increased donor chimerism, T cell generation, and antigen-specific T cell responses to vaccination. BMCs may provide an off-the-shelf approach to enhance T cell regeneration and reduce graft-versus-host disease in HSCT.

[0374] Introduction T cells are key helper, effector and regulatory cells of antigen-specific immunity that are essential for life. Reductions in numbers and functional defects of T cells are causally implicated in diseases ranging from congenital immune deficiencies to autoimmune and impaired immune surveillance disorders (Goronzy, JJ & Weyand, CM Successful and maladaptive T cell aging. Immunity 46, 364-378 (2017);Liston, A., Enders, A. & Siggs, OM Unravelling the association of partial T-cell 883 immunodeficiency and immune dysregulation. Nature Reviews Immunology 8, 545-558 884(2008)). In allogeneic HSCT, significant defects in T cell generation exist that predispose patients to infectious agents and may also contribute to graft-versus-host disease (GVHD) (Blazar, BR, Murphy, WJ & Abedi, M. Advances in graft-versus-host disease biology and therapy. Nature Reviews Immunology 12, 443-458 (2012)). These complications may be fatal and limit the use of HSCT in situations where it may be curative. Balanced reconstitution of naive helper and effector T cell subsets, together with restoration of the T cell receptor repertoire, remains a significant unmet clinical need (Krenger, W., Blazar, BR & Hollander, GA Thymic T-cell development in allogeneic stem cell transplantation. Blood 117, 6768-6776 (2011)).

[0375] New T cell regeneration from transplanted hematopoietic cells requires the availability of a sufficient pool of T cell precursors (Chaudhry, MS, Velardi, E., Dudakov, JA & Brink, MR Thymus: the next (re)generation. Immunological reviews 271, 56-71 (2016)) originating from the bone marrow and sufficient thymic function (Zlotoff, DA et al. Delivery of progenitors to the thymus limits T-lineage reconstitution after bone marrow transplantation. Blood 118, 1962-1970 (2011)). There is currently no clinical standard for enhancing T cell generation in vivo, but the majority of attempts have focused on the use of cytokines and cell-based therapies from the post-marrow phase of T cell lymphopoiesis. However, in clinical trials, the T cell-expanding cytokines IL-7 and IL-2 (Mohtashami, M., Shukla, S., Zandstra, P. & Zuniga-Pflucker, JC in Synthetic Immunology 95-120 (Springer, 2016)) were increased primarily in mature T cell subsets (Perales, M.-A. et al. Recombinant human interleukin-7 (CYT107) promotes T-cell recovery after allogeneic stem cell transplantation. Blood 120, 4882-4891 (2012)), and IL-2 was further limited by toxicity (Skrombolas, D. & Frelinger, JG Challenges and developing solutions for increasing the benefits of IL-2 treatment in tumor therapy. Expert review of clinical immunology 10, 207-217 (2014)).In contrast, administration of IL-22 has been shown to enhance early thymocyte recovery in preclinical mouse studies (Dudakov, JA et al. Interleukin-22 drives endogenous thymic regeneration in mice. Science 336, 91-95 (2012)). Alternatively, adoptive donor T cell infusions have been used to provide antigen-specific T cell protection from commonly encountered pathogens (Cobbold, M. et al. Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers. Journal of Experimental Medicine 202, 379-386 (2005);Rooney, CM et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92, 1549-1555 (2012)). (1998)), which are associated with transient responses, increased risk of GVHD, and T cell exhaustion. All the above strategies are limited by the availability of a sufficient pool of T cell precursors to drive thymus-dependent T cell generation.T cell precursors can be robustly generated ex-vivo by activation of Notch signaling, and co-administration of these cells with HSCT improves thymopoiesis and thymic architecture without exogenous co-administration of cytokines (Zakrzewski, JL et al. Tumor immunotherapy across MHC barriers using allogeneic T-cell precursors. Nature biotechnology 26, 453 (2008); Van Coppernolle, S. et al. Functionally mature CD4 and CD8 TCRαβ cells are generated in OP9-DL1 cultures from human CD34+ hematopoietic cells. The Journal of Immunology 183, 4859-4870 (2009); Awong, G. et al. Human proT-cells generated in vitro facilitate hematopoietic stem cell-derived T-lymphopoiesis in vivo and restore thymic architecture. Blood 122, 4210-4219 (2013). However, ex-vivo cell culture to generate sufficient precursors is laborious, and only a transient enhancement of thymopoiesis of donor cells has been demonstrated. Thus, widespread clinical translation of this technique is likely to be complicated.

[0376] In the quest to develop a broadly applicable technique, the focus was on common lymphoid progenitors (CLPs) present in the bone marrow prior to thymus entry, which have the capacity to differentiate into naive T lymphocytes upon activation of Notch signaling and are the major source of thymopoiesis (Love, PE & Bhandoola, A. Signal integration and crosstalk during thymocyte migration and emigration. Nature Reviews Immunology 11, 469 (2011);Radtke, F., MacDonald, HR & Tacchini-Cottier, F. Regulation of innate and adaptive immunity by Notch. Nature Reviews Immunology 13, 427 (2013);Serwold, T., Ehrlich, LIR & Weissman, IL Reductive isolation from bone marrow and blood implicates common lymphoid progenitors as the major source of thymopoiesis. Blood 113, 807-815 (2009)). The stromal component of the bone marrow niche that enhances T cell lineage specification consists of osteocalcin-expressing bone marrow stromal cells that produce delta-like ligand-4 (DLL-4), providing a functional microenvironment crucial for generating T cell-competent CLPs (Vionnie, W. et al. Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow. Journal of Experimental Medicine, jem. 20141843 (2015)). These stromal cells are damaged by the process of preconditioning, which likely affects T cell lineage specification function.Furthermore, clinical experience with AIDS patients has shown that the adult thymus has the capacity to significantly improve cellular composition and T cell poiesis despite previous dysfunction and atrophy (Smith, KY et al. Thymic size and lymphocyte restoration in patients with human immunodeficiency virus infection after 48 weeks of zidovudine, lamivudine, and ritonavir therapy. The Journal of infectious diseases 181, 141-147 (2000). These previous findings support the development of a niche based on specific biological aspects of T cell lymphopoiesis in the bone marrow.

[0377] It is hypothesized that a T cell lymphopoietic bone marrow niche can be engineered to foster in vivo generation of T cell precursors that migrate to the native thymus, thereby subjecting them to host-driven selection to create a more balanced and broad immune repertoire. To test this hypothesis, an injectable, biomaterial-based cryogel (BMC) scaffold was created. The BMC scaffold promotes in vivo T cell development by incorporating molecular signals presented within the bone marrow niche. The BMC comprises a macroporous, hydrogel-based scaffold that allows cell infiltration. The BMC releases bone morphogenetic protein-2 (BMP-2) to facilitate recruitment of host stromal cells and their osteogenic differentiation, and presents the bioactive Notch ligand DLL-4 at a predefined density to infiltrating hematopoietic cells. These T lineage cues enhanced thymic precursor seeding following syngeneic (syn) and allogeneic (allo) HSCT in mice, enabling donor T cell reconstitution. BMC-reconstituted T cells were functional, had a diverse T cell receptor (TCR) repertoire, and showed reduced induction of GVHD.

[0378] Example 1 Bioactive macroporous bone marrow cryogel (BMC) differentiates hematopoietic precursors into precursor T cells in vitro The alginate-PEG BMC based scaffolds are macroporous hydrogels with interconnected pores of 50-80 μm diameter (Figures 1A-1C). To promote T cell lineage programming in hematopoietic progenitor cells, DLL-4 was incorporated into the polymer backbone (Radtke, F., MacDonald, HR & Tacchini-Cottier, F. Regulation of innate and adaptive immunity by Notch. Nature Reviews Immunology 13, 427 (2013)). To enable de novo bone formation (Wozney, JM et al. Novel regulators of bone formation: molecular clones and activities. Science 242, 1528-1534 (1988)), BMP-2 was added to the reaction mixture prior to low-temperature polymerization and then for release in a soluble form in vivo. These cryogels present both immobilized (DLL-4) and soluble (BMP-2) cues, unlike previous cryogels designed solely for sustained protein release (Koshy, ST, Zhang, DK, Grolman, JM, Stafford, AG & Mooney, DJ Injectable nanocomposite cryogels for versatile protein drug delivery. Acta biomaterialia 65, 36-43 (2018)).

[0379] In this study, BMC further supports the growth of bone and hematopoietic tissues. In vitro BMP-2 release (90% encapsulation efficiency) showed an initial burst of approximately 5% of the loaded amount, followed by a sustained release (Figure 1D). Less than 1% of the total amount of loaded DLL4 was detected in the supernatant, and the modified DLL-4 had similar binding kinetics to the unmodified protein (Figure 1E). In pooled release samples, the bioactivity of released BMP-2 was retained greater than 90% compared to freshly reconstituted BMP-2 (Figure 1F). BMP-2 bioactivity ranged from 95% at day 3 of release to approximately 85% at day 12, confirming that the released BMP-2 was highly active (Figure 1G). The highest in vitro bioactivity of DLL-4 was found at early time points, days 0 and 10 (Figures 1H and 1I). Bioactivity decreased at later time points, but was still above baseline after 3 months. To measure the ability of BMCs to induce differentiation of hematopoietic precursor mouse and human cells via Notch signaling, primary lineage-depleted bone marrow cells from mice and cord blood-derived human CD34+ hematopoietic cells were cultured in BMCs (Figure 1J).

[0380] Expansion of common lymphoid precursors was achieved by saturating the MA-COOH groups on the polymer backbone with 1% functionalization with MA-DLL4, corresponding to approximately 6 μg MA-DLL4 per gel, and this condition was selected for further evaluation. There were no significant differences in overall human or mouse cell expansion fold and viable numbers in any of the experimental conditions analyzed (Figures 1K and 1L). However, the lymphoid precursor fraction was only enhanced when DLL-4 was incorporated into BMCs, alone or in combination with BMP-2 (Figure 1M).

[0381] Example 2 BMCs form bone nodules with characteristics of hematopoietic tissue in vivo. Next, BMCs were analyzed for their ability to induce the trafficking of host and transplanted cells in a mouse model of HSCT. Following lethal total body irradiation (L-TBI), mice were injected with lineage-depleted hematopoietic cells (5 × 10 4 DLL-4 (approximately 93% lineage-depleted, Figure 2A) were implanted intravenously and BMCs (without cells) were simultaneously injected into the subcutaneous tissue of the dorsal flank (Figure 2B). To macroscopically quantify cell infiltration into BMCs, the size of each subcutaneous nodule was measured over a 6-week period (Figure 2C). BMCs with BMP-2 rapidly increased the size of the nodules to approximately three times their initial volume by 10 days after implantation, were substantially infiltrated with donor hematopoietic cells (Figure 2D), and formed localized bone nodules over a period of approximately 2 weeks (Figures 2E and 2F). Notably, bone formation was accompanied by vascularization, DLL-4 remained accessible, and bone was restricted to the BMC scaffold, demonstrating the regulation exerted by BMCs over this process at ectopic sites (Figures 2G-2L).

[0382] Hematopoietic tissue was visible in bone nodules near areas of lamellar bone on the inner surface of the BMC (Figure 2F). Capillary networks infiltrating the BMC were noted as early as 2 days after implantation (Figure 2G), and quantification began at day 10. Using histomorphometry, vascular density within the BMC was assessed at multiple time intervals up to 3 months after implantation (Figure 2F). Vascularity remained at approximately 25 vessels / mm by day 10. 2 The blood vessel density was 70 vessels / mm 2 DLL-4 distribution in BMCs increased to 100% and remained constant after day 30. To quantify the distribution and accessibility of alginate / DLL-4 in BMCs, histomorphometric analysis using Safranin-O staining was performed. At the earliest time point, day 10, approximately 85% of the alginate was accessible, which gradually decreased to 25% by day 90 (Figures 2I-2K). Alginate was not sequestered to any specific region within the BMCs at any time point.

[0383] Example 3 BMCs mobilize and expand host stromal cells and transplanted hematopoietic cells We assessed the infiltration and cellular composition of transplanted hematopoietic cells in BMCs at various time points after transplantation. + Cells expanded when BMP-2 was included, but not in the presence of DLL-4 alone (Figures 3A and 3B). Stromal cells present in BMC and native bone marrow were found to be similar, there was no difference in hematopoietic cell engraftment in native bone marrow between BMC-treated and untreated mice, and SDF-1α and interleukin-7 concentrations in BMC and native bone marrow were similar (Figures 3C-3G).

[0384] Stromal cells present in BMC were identified using immunophenotypic markers commonly associated with mouse mesenchymal stromal cells (Sca-1, CD29, CD44, CD73, CD105, CD106) and compared with bone marrow from transplanted 469 mice (Figure 3C). +Although stromal subsets were somewhat increased in BMC compared to bone marrow, the overall repopulation kinetics of these stromal subsets were similar in the two tissues. In BMC containing BMP-2, bone alkaline phosphatase (BAP) was comparable to native bone (Figure 3D). Adipose tissue was quantified using Oil Red O (ORO) and found to be less in BMC compared to whole native bone. More ORO was quantified in BMC containing BMP-2 compared to BMC without BMP-2 (Figure 3D). To determine whether BMC affected the engraftment of transplanted cells in endogenous bone marrow, colony formation assays were performed at three time points (Figure 3E). No differences were observed in the total number or type of CFUs arising from cells derived from bone marrow in mice treated with or without dual BMC. Concentrations of the homing factor stromal cell-derived factor-1 alpha (SDF-1α) and the lymphoid precursor supporting cytokine interleukin-7 (IL-7) (Brainard, DM et al. Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice. Journal of virology 83, 7305-7321 (2009)) in the recovered BMCs were similar to or higher than those in irradiated bone marrow from the same mice (Figure 2F). No ETP, DN2, or DN3-like cells were detected in the BMCs.

[0385] At later time points (>5 days post-transplant), more primitive donor hematopoietic cells (HSCs) and lymphoid-prestimulated multipotent progenitors (LMPPs) were quantified in BMCs. At day 14, between 6 and 7 million total GFP cells were detected in BMCs containing single factor BMP-2 or dual factors BMP-2 and DLL-4. + Cells were quantified. More than 80% of cells in both groups were CD11b +However, the CLP fraction in transplanted donor cells was only increased in dual-factor BMCs, resulting in an approximately 100-fold increase 2 weeks after transplantation compared to BMCs with BMP-2 alone (Figure 3A). Six weeks after transplantation, CLPs were approximately 10-fold more abundant in dual-factor BMCs, of which approximately 30% and 70%, respectively, were T-cell-competent Ly6D - was within the subset ( Fig. 3G ).

[0386] Bone marrow-derived precursor T cells migrate to the thymus and differentiate into naive T cells. To directly assess whether BMC-derived cells migrate to the thymus, dual factor BMCs were delivered in concert with stem cell therapy to the first set of lethally irradiated mice (Figures 3H and 3I). Dual BMCs were then explanted from these mice on day 10 and surgically transplanted subcutaneously into the dorsal flank of sublethally irradiated recipients. At day 20 after BMC transplantation, donor GFP in the thymus of these mice was detected. + and host cells were quantified. GFP in the thymus of recipient mice transplanted with BMCs + DP, SP CD4 + and SP CD8 + Cell quantification confirmed migration of T cell precursors from BMCs to the thymus. In another study, dual BMC treatment resulted in a greater enhancement in the number of ETPs in the thymus compared to a 10-fold increase with transplanted cell dose administered without BMC (Figure 3J). Dual BMCs also significantly outperformed bolus delivery of factors placed within BMCs and BMP-2 alone in generating thymocyte subsets over time (Figures 3K-3P).

[0387] To analyze the effect of cell dose on the seeding of T cell precursors in the thymus, early thymic precursor (ETP) subsets were pre-treated with increasing doses of lineage-depleted cells for transplantation after L-TBI (5 × 10 4 ~5×10 5 A dose-dependent enhancement of ETP was found in this dose range (Figures 3A and 3H). Dual-functionalized BMCs were cultured at the lowest cell dose (5×10 4When double BMC was administered at 100x the dose of 100x the ETP in the thymus was increased 5-fold compared to the transplant-only group with the highest cell dose. Treatment with double BMC initially enhanced ETP (3-fold at days 12 and 42), followed by increases in DN2, DN3, DP and SP thymocyte subsets at later time points (Figures 3I-3N and 5Q). Between days 12 and 42, thymic cellularity and body weight in mice from double BMC treatment were significantly higher than mice from the transplant-only group. At day 22 post-HSCT, there were no significant differences in the numbers of thymic stromal subsets (mTECs, cTECs, fibroblasts and endothelial cells; Figures 3S and 3T).

[0388] Example 4 BMC enhances T cell regeneration and reduces GVHD after HSCT In the peripheral blood of mice treated with dual functionalized BMCs, accelerated T cell reconstitution was observed approximately 4 weeks after transplantation, but no significant differences were observed in B cell or bone marrow cell reconstitution (Figures 4A-4C). Analysis of T cell subsets in blood, spleen, and bone marrow revealed that mice with dual functionalized BMCs had homeostatic CD4 T cells in the spleen and bone marrow 30 days after transplantation and in the peripheral blood 40 days after transplantation. + :CD8 + The T cell ratio was shown to be restored (Figures 4D-4F). When BMC treatment was used in the context of HSCT after sublethal total body irradiation (SL-TBI), T cell reconstitution was enhanced (Figures 4G-4N). At 28 days post-transplant, mice treated with BMC had 1.5-fold and 2-fold higher absolute numbers of donor chimerism and DP donor thymocytes, respectively, compared to mice receiving only transplant (Figure 4G). Mice treated with BMC had more donor chimerism and donor-derived single positive CD4+ T cells than mice receiving only transplant. + Absolute thymocyte counts (2-fold and 3-fold, respectively) and a single positive CD8 + Thymocytes (1.7-fold and 15-fold, respectively) were also observed (Figures 4H and 4I). + T cells (3.5x) and CD8 +It was higher in T cells (2.5-fold). No differences were observed in chimerism or absolute numbers of B cells.

[0389] We next measured the kinetics of human T cell reconstitution using an established xenogeneic NSG-BLT mouse model (Brainard, DM et al. Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice. Journal of virology 83, 7305-7321 (2009)). In NSG-BLT mice treated with BMC, we observed an initial enhancement in the initial kinetics of T cell reconstitution and a somewhat transient decrease in the kinetics of B cell reconstitution (Figures 5A and 5B), and there were transiently fewer pre-B cell CFUs in the bone marrow of NSG-BLT mice treated with BMC (Figure 5C). Peripheral CD4 + :CD8 + The T cell ratio stabilized between days 50 and 60 post-transplant in mice treated with BMC, whereas the CD4 + compartment was not fully reconstituted (Figure 5D). Strikingly, the enhanced rate of T cell reconstitution did not accelerate the rate of GVHD-related death. Instead, NSG-BLT mice receiving dual functionalized BMCs survived longer than NSG-BLT mice (Figure 5F). In this model, BMC-treated NSG-BLT mice had significantly higher CD4+ T cells in the thymus and spleen than BMC-treated mice 50 days after transplantation. + FoxP3 + Regulatory T cells (T reg ) was two-fold higher (Figures 5G and 5H). A similar increase in survival was observed in an allogeneic MHC-mismatched HSCT mouse model receiving BMC (Figure 5I), and in this model, donor-derived CD4 + FoxP3 + T reg was 5-fold and 4-fold higher in the thymus and spleen of BMC-treated mice, respectively ( Fig. 5J ).

[0390] We next compared our BMC treatment to a widely tested T cell precursor infusion approach that consists primarily (>90%) of double negative (DN)2 and double negative (DN)3 precursors generated ex vivo using OP9-DL1 feeder cells (Figure 5K). At day 28 post-HSCT, donor chimerism was significantly higher in DP, SP4 and SP8 thymocyte populations in BMC-treated mice (Figures 5L-5N). In the spleen, donor chimerism was significantly higher in CD4 + T cells and CD8 + We found that both CD4+ and CD8+ T cells were abundant in donor mice treated with BMC, as well as in absolute numbers of donor CD4+ and CD8+ T cells (Figures 5O and 5P).

[0391] Example 5 BMCs enhance the diversity and function of regenerating T cells T cell receptor diversity (TCR diversity) was generated by stochastic somatic recombination of gene segments in the thymus. Mice treated with double BMC had significantly higher thymic cellularity and thymus weights than transplant-only controls until approximately 6 weeks after transplant (Figures 3Q and 3R). To characterize whether increased thymic cellularity also enhances the functionality and diversity of regenerating T cells in syngeneic transplants, TCR excision circles (TRECs) were quantified. TRECs are a signature of TCR rearrangement. TCR repertoire analysis was also performed. The number of TRECs in the thymus of mice treated with double BMC 1 month after transplant was similar to that in non-irradiated control mice, and both were higher than those in transplant-only mice and mice treated with BMP-2 BMC (Figure 6A). Although an overall lower number of TRECs was observed in the spleen compared to non-irradiated controls in the same groups, mice treated with dual BMCs still had a higher TREC count compared to the transplant-only and single factor BMP-2 BMC groups (Figure 6B). TCR V and J segment diversity in the CDR3 beta chain was assessed using the Simpson Index (SI), which takes into account the number of T cell clones present, as well as the relative abundance of each clone, 30 days after HSCT in mice treated with BMC and receiving transplants. The SI of mice with dual functional BMCs was 40% of the SI of non-irradiated control mice, whereas the SI of mice receiving transplants alone or BMP-2 BMCs was lower (16% and 8%, respectively) (Figure 6C). In HSCT, the lack of naive T cells with an extensive TCR repertoire has been associated with an increased risk of immunological complications and opportunistic infections (Douek, DC et al. Assessment of thymic output in adults after haematopoietic stem cell transplantation and prediction of T-cell reconstitution. The Lancet 355, 1875-1881 1076(2000)).A higher number of TRECs were recovered in the thymus and periphery of double BMC-treated mice, reflecting enhanced thymopoiesis. A relatively high frequency of specific TCR clones was also observed in double BMC-treated mice, but the overall greater diversity suggested a more balanced thymic-derived rearrangement.

[0392] To measure the ability of regenerated T cells to respond antigen-specifically, syngeneic transplants were vaccinated and then challenged 30 days after transplantation with the model protein ovalbumin (OVA) (Figure 6D). + CD8+ T cells were significantly higher in mice receiving dual functional BMCs compared to transplanted mice that received no BMC or only BMC with BMP-2 (Figure 6E). Similarly, donor antigen-specific T cell responses were approximately three-fold higher in mice treated with dual BMCs receiving allogeneic transplants compared to those resulting from the pro-T cell therapy approach (Figure 6F). Production of interferon (IFN)-γ and tumor necrosis factor-α (TNF-α) upon ex vivo stimulation was significantly higher in donor CD4+ T cells in BMC-treated and T cell precursor-treated groups at day 22. + and CD8 + We found that a significant proportion of the BMC-derived T cell-treated group had these factors at day 42, whereas a similar proportion of the BMC-derived T cell-treated group had these factors at day 42 (Figures 6G and 6H). + The robust antigen-specific generation of T cells suggests that BMC treatment has potential for use in combination with vaccination following HSCT.

[0393] Example 6 material and method The following materials and methods were used to carry out the invention.

[0394] General Methods and Statistics The sample size of the animal studies was based on previous studies without the use of additional statistical estimation (Brainard, DM et al. Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice. Journal of virology 83, 7305-7321 (2009);Bencherif, SA et al. Injectable cryogel-based whole-cell cancer vaccines. Nature communications 6, 7556 (2015);Palchaudhuri, R. et al. Non-genotoxic conditioning for hematopoietic stem cell 1066 transplantation using a hematopoietic-cell-specific internalizing immunotoxin. Nature biotechnology 34, 738 (2016)). Results were analyzed using one-way ANOVA with Tukey post-hoc test using GraphPad Prism software. When ANOVA was used, the variances between groups were found to be similar with the Bartlett test. Survival curves were analyzed using the log-rank (Mantel-Cox) test. Pathological samples and blood cell counts were blinded using alphanumeric coding.

[0395] material UP LVG sodium alginate with high guluronate content was purchased from ProNova Biomedical; 2-morpholinoethanesulfonic acid (MES), sodium chloride (NaCl), sodium hydroxide (NaOH), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), 2-aminoethyl methacrylate hydrochloride (AEMA) and acetone were purchased from Sigma-Aldrich. ACRL-PEG-NH2 (3.5 kDa) and 4arm PEG acrylate (10 kDa) were purchased from JenKem Technology.

[0396] Bone marrow cryogel (BMC) fabrication Bone marrow cryogels were made according to a previously described technique with some modifications (Bencherif, SA et al. Injectable preformed scaffolds with shape-memory properties. Proceedings of the National Academy of Sciences of the United States of America 109, 19590-19595). (2012). Methacrylated alginate (MA-alginate) was prepared by reacting alginate with AEMA. Sodium alginate was dissolved in 100 mM MES buffer (0.6% (wt / vol), pH 6.5). NHS and EDC were added to the mixture to activate the carboxylic acid groups of the alginate backbone, followed by AEMA (molar ratio of NHS:EDC:AEMA = 1:1.3:1.1) and the solution was stirred at room temperature (RT) for 24 h. The mixture was precipitated in acetone, filtered, and dried in a vacuum oven at RT overnight. A 2.5 wt% solution of MA-alginate and 4arm PEG acrylate macromonomer (MA-alginate:4arm PEG acrylate macromonomer) in deionized water was added. Alginate-PEG BMC was synthesized by preparing ACRYL-PEG-NH2 (molar ratio of PEG to acrylate = 4:1) followed by the addition of tetramethylethylenediamine (TEMED) (0.5% (wt / vol)) and ammonium persulfate (APS) (0.25% (wt / vol)). ACRYL-PEG-NH2 was conjugated with delta-like ligand 4 (DLL-4) (R&D Systems) using carbodiimide chemistry (molar ratio of NHS:EDC:DLL4 = 1:1.3:1.1). Low temperature polymerization was performed after adding BMP-2 (R&D Systems) to the polymer solution. All precursor solutions were pre-cooled to 4 °C to slow down the rate of polymerization and then frozen. After adding the initiator to the prepolymer solution, the solution was quickly transferred to a pre-cooled (-20 °C) Teflon mold. After overnight incubation, the gel was thawed and collected in a Petri dish on ice.

[0397] For scanning electron microscopy (SEM), BMCs were incubated in increasing concentrations of freshly prepared ethanol solutions (30, 50, 70, 90 and 100%) for 20 min each. BMCs were then incubated in hexamethyldisilazane (Electron Microscopy Sciences) for 10 min and dried in a desiccator / vacuum chamber for at least 1 h before being sealed for SEM. Dried BMCs were attached to sample stubs using carbon tape and coated with platinum / palladium in a sputter coater. Samples were imaged using secondary electron detection on a Carl Zeiss Supra 55 VP field emission scanning electron microscope (SEM).

[0398] Quantification of biomolecule release The stock concentration of BMP-2 was known from the manufacturer and was verified using ELISA. To determine the release kinetics of BMP-2 encapsulation efficiency and to confirm stable conjugation of DLL-4, BMCs were incubated in 1 ml of sterile PBS at 37° C. with shaking. The medium was changed periodically. The released agent in the supernatant was detected by ELISA (Peprotech). Samples were released until no more BMP-2 was detectable in the release medium. Cryogels were then digested using at least 1000 U of the enzyme alginate lyase. The digestion products were analyzed for BMP-2 using ELISA. The amount of BMP-2 and DLL-4 in the cryogels and release medium was compared to the known amounts of loaded BMP-2 and DLL-4 to calculate the encapsulation efficiency.

[0399] Biomolecular Activity Assays Alkaline phosphatase activity assay for BMP-2 bioactivity

[0400] Alkaline phosphatase assays were performed using MC3T3-E1 subclone 4 cells as previously described. Macdonald, ML et al. Tissue integration of growth factor-eluting layer-by-layer polyelectrolyte multilayer coated implants. Biomaterials 32, 1446-1453 (2011). Cells were cultured under different experimental conditions. The experiments were carried out in the following conditions: (1) growth medium, (2) differentiation medium supplemented with BMP-2 released from BMCs, and (3) native BMP-2.

[0401] Notch activation assay for DLL-4 bioactivity

[0402] To quantify the in vitro bioactivity of DLL-4 following exposure to serum proteins that can inactivate this morphogen in vivo, we used a previously characterized Notch reporter cell line, CHO-K1+2xHS4-UAS-H2B-Citrine-2xHS4 cH1+hNECD-Gal4esn c9, a kind gift from M. Elowitz (Caltech) (Sprinzak, D. et al. Cis-interactions between Notch and Delta generate mutually exclusive signaling states. Nature 465, 86 (2010); Nandagopal, N. et al. Dynamic ligand discrimination in the Notch signaling pathway. Cell 172,869-880. e819 (2018)). These cells were cultured in Alpha MEM Earle's Salts (Irvine Scientific) supplemented with 10% Tet System Approved FBS (Clontech), 100 U / ml penicillin-100 μg / ml streptomycin-0.292 mg / ml L-glutamine (Gibco) at 37 °C for 24 h at 5% CO. 2The cells were grown at 37 °C in a humidified atmosphere in the presence of DLL-4. Both BMCs with and without DLL-4 were incubated in 96-well plates with complete cell culture medium and without cells. At predefined time intervals (up to 3 months), 20,000 Notch reporter cells were seeded on top of the BMCs in the wells. After 24 h, confocal microscopy was performed using a Zeiss LSM 710 confocal system. The colorimetric output in response to binding with the Notch ligand DLL-4 was quantified and used as an indicator of DLL-4 bioactivity in the scaffolds (Figure 1H). In particular, the total YFP fluorescence of each cell in the field (50–100; 4–5 fields encompassing more than 80% of the gel surface) was calculated and background fluorescence was subtracted. The median YFP fluorescence was calculated and divided by the median fluorescence of cells seeded on BMCs without DLL-4 and reported.

[0403] Affinity determination by surface plasmon resonance

[0404] The dissociation constants of wild-type DLL4 and MA-DLL4 for Notch1 were determined by surface plasmon resonance using a BIAcore T200 instrument (GE Healthcare) as previously described (Heliotis, M., Lavery, K., Ripamonti, U., Tsiridis, E. & Di Silvio, L. Transformation of a prefabricated hydroxyapatite / osteogenic protein-1 implant into a vascularised pedicled bone flap in the human chest. International journal of oral and maxillofacial surgery 35, 265-269 (2006). Briefly, biotinylated recombinant Notch1 was attached to a streptavidin-coated sensor chip (GE Healthcare). The binding curves were immobilized on a 1:1 Langmuir model (Blox Healthcare). Increasing concentrations of wild-type methacrylated DLL4 protein in buffer were flowed over the chip at 20°C. The association and dissociation phases were carried out for 120 and 60 seconds, respectively, at 10 μl per minute. The steady-state binding curves were fitted to a 1:1 Langmuir model using the BIAcore evaluation software to determine the K d It was decided.

[0405] In vitro cell culture on bone marrow cryogel (BMC)

[0406] Mouse BM cells were harvested from the limb. The ground tissue and cells were filtered through a 70 micron mesh. A single cell suspension was prepared by passing the cells once through a 20 gauge needle. Total cellularity was determined by counting the cells using a hemocytometer. BM cells were depleted of mature immune cells (expressing CD3-ε, CD45R / B220, Ter-119, CD11b or Gr-1) by magnetic selection (BD Biosciences). Cells were incubated with a mixture of lineage antibodies conjugated with Pacific Blue (antibodies against CD3, NK1.1, Gr-1, CD11b, CD19, CD4 and CD8), as well as with Sca-1 and c-kit specific antibodies. Hematopoietic cells (Lin - Sca-1 hi c-kit hi ) were isolated. The sorted cells were ≥95% pure. Human cord blood-derived CD34 + Cells were purchased (Allcells) and expanded for 7 days using expansion supplements (Stemcell Technologies). +Cells were isolated using a positive selection kit (StemCell Technologies). 96-well plates were pre-coated with Pluronic F127 (Sigma). Each BMC was placed individually in a well of a 96-well plate. Ten thousand mouse or human cells isolated as described above were added to the same well in a volume of 200 μl of RPMI (with L-glutamine) 1640 with 10% fetal bovine serum (FBS) and 1% antibiotic and antimycotic solution (containing penicillin, streptomycin and amphotericin B). For mouse cells, the medium was supplemented with 10 ng / ml stem cell factor (SCF; R&D Systems), 10 ng / mL FMS-like tyrosine kinase 3 ligand (Flt3L; R&D Systems) and 1 ng / mL interleukin-7 (IL-7; R&D Systems) with 50% medium change steps on days 2, 4 and 6. For human cells, the medium was supplemented with 100ng / mL SCF, 100ng / mL Flt3L, 100ng / mL TPO (R&D Systems), and 100ng / mL IL-7 (R&D Systems). After one week of culture, cells were isolated by digesting BMC with 1mg / ml alginate lyase (Sigma). The solution was passed through a 70 micron filter, and cells were processed for FACS analysis as described below.

[0407] BM transplantation and blood analysis All animal studies were approved by the Harvard Institutional Animal Care and Use Committee and followed National Institutes of Health guidelines and relevant ethical regulations. b ), BALB / c(H-2 d ), C57BL / 6(CD 45.1 +), CByJ.B6-Tg(UBC-GFP)30Scha / J(GFP) and NSG mice (Jackson Laboratories) were female and between 6 and 8 weeks of age at the start of the experiment. All mice within each experiment were age-matched and no randomization was performed. Pre-established criteria for animal attrition were inability to inject the desired cell dose into transplanted mice and death due to post-operative complications in humanized mice. Health concerns unrelated to the procedure (e.g., malocclusion, severe dermatitis) were criteria for attrition and euthanasia.

[0408] Transplantation model All TBI experiments were performed using a Cs-137 γ-radiation source. Sublethal TBI (SL-TBI, B6 recipients), 1 × 500 cGy + 5 × 10 5 lineage-depleted bone marrow cells; syngeneic HSCT (syn-HSCT, B6 recipient) 1×1000cGy+5×10 4 ~5×10 5 (indicated in figure) lineage-depleted GFP BM cells; Allogeneic-HSCT (BALB / c MHC-mismatched recipient) with GVHD 1×850cGy+5×10 5 lineage-depleted GFP BM cells + 1 x 10 6 GFP splenocytes; Allogeneic HSCT (BALB / c MHC-mismatched recipient) without GVHD: 1×850cGy+5×10 5 or 5 x 10 lineage-depleted GFP BM cells 6 in vitro generated GFP T cell precursors + 10 3syngeneic HSCs. Humanized BLT (bone marrow-liver-thymus) mouse studies were performed by the MGH and Ragon Institute Human Immune System Mouse Program with institutional IACUC approval as previously described (Brainard, DM et al. Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice. Journal of virology 83, 7305-7321 (2009)). BM cells for transplantation or analysis were harvested by crushing all limbs or one femur, respectively, and processed as described above. While mice were anesthetized, they received subcutaneous injections of two BMCs suspended in 0.2 ml of sterile PBS via a 16-gauge needle in the dorsal flank. One BMC was injected on each side of the spine and positioned approximately midway between the hind and forelimbs. The size of the subcutaneous nodules was quantified over time by measuring the length, width, and height of the nodules using calipers. All cohorts of mice (typically 10 mice / group) were serially bled. White blood cells, hemoglobin, red blood cells, platelets, and hematocrit levels were quantified by CBC analysis (Abaxis VetScan HM5).

[0409] To directly assess whether BMC-derived cells migrate to the thymus, dual-factor BMCs were delivered in concert with stem cell therapy to the first set of lethally irradiated mice (B6 recipients were treated with 1000 cGy L-TBI and 5 × 10 5 10 days after HSCT, the bifactorial BMCs were explanted and immediately surgically implanted into subcutaneous pockets in a second set of B6 mice that had received 500 cGy of SL-TBI without any additional cell transfer 48 hours prior. 20 days after surgery, the mice were sacrificed and thymocytes in these mice were analyzed.

[0410] Flow cytometry (FACS) analysis Anti-mouse antibody against CD8-α (53-6.7), anti-mouse antibody against CD3-ε (145-2C11), anti-mouse antibody against B220 (RA3-6B2), anti-mouse antibody against CD11b (M1 / 70), anti-mouse antibody against CD25 (PC61), anti-mouse antibody against CD117 (2B8), anti-mouse antibody against Sca-1 (D7), anti-mouse antibody against CD127 (A7R34), and anti-human antibody against CD45 (H130 ), anti-human antibody against CD3 (HIT3a), anti-human antibody against CD4 (SK3), anti-human antibody against CD19 (HIB19), anti-human antibody against CD34 (581), anti-human antibody against CD38 (HB-7) and anti-human antibody against CD7 (CD7-6B7), anti-human antibody against IFN-γ (XMG1.1), anti-human antibody against TNF-α (MP6-XT22) and corresponding isotype antibodies were purchased from BioLegend. Anti-human CD8 (RPA-T8) was purchased from BD Biosciences. CD44 (IM7) was purchased from eBioscience. SIINFEKL tetramer (Alexa Fluor 647 H-2K b OVA) was obtained from the NIH Tetramer Core Facility. All cells were gated based on forward and side scatter characteristics to limit debris including dead cells. Antibodies were diluted according to the manufacturer's recommendations. Cells were gated based on fluorescence minus one control and the frequency of cells staining positive for each marker was recorded. To quantify T, B and myeloid cells, blood samples were lysed and stained with anti-CD45, anti-B220, anti-CD3, anti-CD4, anti-CD8 and anti-CD11b antibodies and absolute numbers of T, B and myeloid cells were calculated using flow cytometry frequencies and white blood cell values ​​obtained by CBC analysis. Analysis was based on donor events within the CD45+ for blood cells and CD45- for stromal cells gates.

[0411] Bone and fat quantification and histological examination After euthanasia, BMCs and tissues were explanted. For bone quantification using bone alkaline phosphatase (BALP), BMCs and femurs were ground, homogenized, and strained through a 70 micron filter. BALP was then quantified using a BALP ELISA kit (Creative Diagnostics) according to the manufacturer's protocol. An Oil Red O staining kit (Biovision) was used for lipid quantification. Harvested BMCs and bones were washed, fixed, processed, and stained according to the manufacturer's protocol. BMCs and bones were then ground, strained, and resuspended in equal volumes before measuring optical density (OD 492 ). For histological staining, tissues were fixed in 4% paraformaldehyde (PFA). PFA-fixed specimens were partially decalcified using a rapid decalcifying formic acid / hydrochloric acid mixture (Decalcifying Solution, VWR) for approximately 4 hours and embedded in paraffin wax. Sections (5 μm) of specimens were stained with routine trichrome, Safranin-O or Verhoeff-Van Gieson stains.

[0412] Quantification of thymic T cell receptor excision circles (TRECs) TREC quantification was performed as previously described (Warnke, P. et al. Growth and transplantation of a custom vascularised bone graft in a man. The Lancet 364, 766-770 (2004)). Briefly, thymuses were harvested from non-irradiated C57BL / 6 mice, transplanted and transplanted mice injected with BMC (30 days after conditioning). Total DNA was extracted using TRIZOL followed by tissue homogenization in a Bullet Blender Storm BBX24 instrument (Next Advance, Inc.). DNA was quantified by UV-Vis and 1 μg DNA per sample was used as input for real-time PCR. Absolute numbers of single joint TRECS (sjTREC) per sample were calculated using a standard curve of mouse sjTREC plasmid.

[0413] TCR analysis Extracted lymphocyte RNA was quantified using UV-Vis. Equimolar amounts of RNA from each sample were submitted to iRepertoire for sequencing and bioinformatics analysis, where samples were reverse transcribed and amplified using a primer set that specifically amplifies beta TCR RNA. Sequencing results provided a range of total reads and unique CDR3 numbers for each sample.

[0414] Vaccination and nonspecific T cell-stimulation testing Thirty days after transplantation, animals were immunized with a bolus vaccine containing 100 μg ovalbumin (OVA), 100 μg CpG-ODN, and 1 μg GM-CSF. Ten days later, animals were challenged with an intravenous injection of ovalbumin. On day 12, spleens were collected from mice euthanized in the vaccination study. Splenocytes were isolated by mechanically disrupting the spleen with a 70 μm cell strainer. Red blood cells in the collected tissue were lysed and white blood cells were prepared for analysis. For nonspecific stimulation, cells were incubated with PMA (10 ng / mL) + ionomycin (2 μM) for 5 hours. Brefeldin A (10 μg / mL) was added after 2 hours of incubation. Cells were then collected, washed, and stained with fluorescent dye-conjugated antibodies against T cell surface antigens. The cells were then fixed and permeabilized using a fixation / permeabilization solution kit reagent (BD), and stained with IFN-γ-specific and TNF-α-specific antibodies.

[0415] Consideration Here, we demonstrate that acellular biomaterial-based BMCs mimic key features of the T cell-lymphoid bone marrow niche after hematopoietic stem cell transplantation and promote the regeneration of immune-competent T cells. Subcutaneously administered BMCs interfaced with the host vasculature in vivo to form a host-device interface and present lineage instruction cues to donor-mobilized progenitor cells. It is well established that BMP-2 induces osteogenic differentiation of recruited mesenchymal cells and indirectly promotes rapid angiogenesis (Smadja, DM et al. Bone morphogenetic proteins 2 and 4 are selectively expressed by late outgrowth endothelial progenitor cells and promote neoangiogenesis. Arteriosclerosis, thrombosis, and vascular biology 28, 2137-2143 (2008)). In this study, we observed early angiogenesis followed by maturation of the vasculature to a density consistent with that observed in endogenous bone marrow (Lafage-Proust, M.-H. et al. Assessment of bone vascularization and its role in bone remodeling. BoneKEy reports 4 (2015)).The finding of different hematopoietic and stromal progenitor populations quantified within BMCs is consistent with previous observations that hematopoiesis occurs in ectopic bone nodules (Kuznetsov, SA et al. The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH / PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow. J Cell Biol 167, 1113-1122 (2004));Song, J. et al. An in vivo model to study and manipulate the hematopoietic stem cell niche. Blood 115, 2592-2600 (2010)). Incorporation of the bioactive Notch ligand DLL-4 into the polymer scaffold promoted early enhancement of T cell precursor generation in BMCs and led to a significant increase in the number of thymic precursors compared to controls receiving lineage-depleted bone marrow grafts, consistent with established models of syngeneic and allogeneic HSCT (Wils, E.-J. et al. Flt3 ligand expands lymphoid progenitors prior to recovery of thymopoiesis and accelerates T cell reconstitution after bone marrow transplantation. The Journal of Immunology 178, 3551-3557 (2007);Maillard, I. et al. Notch-dependent T-lineage commitment occurs at extrathymic sites following bone marrow transplantation. Blood 107, 3511-3519 (2006).This finding is supported by the observation of enhanced generation of TRECs, increased complexity of the TCR repertoire and increased vaccination efficacy.

[0416] The BMC approach is conceptually and practically distinct from other strategies to promote T cell regeneration after HSCT, and its relevance in HSCT is supported by preclinical studies in this study. The BMC approach, used at 10-fold lower doses compared to T cell precursor infusion, resulted in higher numbers of T cell precursors and functional T cells in the thymus and periphery. BMC treatment differs from other methods in that it can be administered at the time of HSCT. In contrast, T cell precursors are generated in vitro over 2–4 weeks from donor hematopoietic cells, with complex cell culture requirements in preclinical models and are patient-specific. By providing T cells that promote cues for transplanted HSCs in vivo without the need for ex-vivo culture, the BMC approach could be an off-the-shelf product, avoiding the considerations of infrastructure required for cell manufacturing (Garber, K. (Nature Publishing Group, 2018) and complementing the activity of cytokine treatments. When lower doses were used before HSCT, BMCs somewhat enhanced T cell reconstitution in the periphery, but significantly increased donor-derived T cell generation in the thymus and donor T cell chimerism. Findings suggest that the application of BMCs is relevant in the context of low-intensity HSCT.

[0417] Enhanced human T cell reconstitution in xenogeneic NSG-BLT mice was accompanied by a small, transient decrease in B cell reconstitution, which coincided with a corresponding decrease in pre-B CFUs. Because this humanized mouse model is broadly tolerant to human immune cells, important mouse cytokines are expressed in the human CD34+ / CD4+ cells in this model. +It has also been found to be inefficient at inducing hematopoiesis, including the development of human B cells, from NOD-scid IL2Rγ null mice (Jangalwe, S., Shultz, LD, Mathew, A. & Brehm, MA Improved B cell development in humanized NOD-scid IL2Rγ null mice transgenically expressing human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3. Immunity, inflammation and disease 4, 427-440 (2016)). Notch activation of a fraction of the transplanted cells that seed the BMCs likely enhances T cell specification at the expense of B cell specification. However, the transient reduction in B cell production is small and unlikely to be of clinical significance.

[0418] The formation of bone nodules is restricted by the geometry of the scaffold, which has been demonstrated in non-human primates (Ripamonti, U. Bone induction by recombinant human osteogenic protein-1 (hOP-1, BMP-7) in the primate Papio ursinus with expression of mRNA of gene products of the TGF-β superfamily. Journal of cellular and molecular medicine 9, 911-928 (2005)) and humans (Heliotis, M., Lavery, K., Ripamonti, U., Tsiridis, E. & Di Silvio, L. Transformation of a prefabricated hydroxyapatite / osteogenic protein-1 implant into a vascularised pedicled bone flap in the human chest. International journal of oral and maxillofacial surgery 35, 265-269 (2006);Warnke, P. et al. Growth and transplantation of a custom vascularised bone graft in a man. The This is consistent with previous reports of scaffold-induced bone formation, which was well tolerated in many species, including the US (Lancet 364, 766-770 (2004)). Previous clinical experience with other scaffold-based systems has shown that device size can remain constant between species (Hodi, S. (2012)).Even with the larger growth factor doses that may be required for human use, the sustained release provided by this polymer-based hydrogel system is expected to allow the use of BMP-2 at doses several orders of magnitude lower than the large doses currently used in the clinic, delivered as a bolus release and associated with undesirable side effects (Carragee, EJ, Hurwitz, EL & Weiner, BK A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. The Spine Journal 11, 471-491 (2011)). After T cell regeneration, BMCs can be easily removed, like other devices often used for HSCT, or made of biodegradable materials to be resorbed (Biffi, R. et al. Use of totally implantable central venous access ports for high-dose chemotherapy and peripheral blood stem cell transplantation: results of a monocentre series of patients. Annals of oncology 15, 296-300 (2004)).

[0419] CD4 after allogeneic HSCT +T cell recovery is usually delayed, leading to a reversal of the normal CD4 / CD8 ratio (Li, MO & Rudensky, AY T cell receptor signalling in the control of regulatory T cell differentiation and function. Nature Reviews Immunology 16, 220 (2016)). Mice treated with BMC showed a more balanced reconstitution of T cells in the thymus and spleen of humanized and allografted mice, as well as a higher proportion of donor CD4 + Regulatory T cells (T reg ) was observed. reg Considering the important role of donor-type CD4 in suppressing GVHD (Hoffmann, P., Ermann, J., Edinger, M., Fathman, C. G. & Strober, S. + CD25 + regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. Journal of Experimental Medicine 196, 389-399 (2002), Donner T reg BMC-mediated enhancement of T cell generation contributes to the alleviation of GVHD-like pathology, potentially regIt is likely that BMP-2 enhanced survival in mice through regulation by TGF-beta family proteins, which are key regulators of proliferation (Wan, YY & Flavell, RA 'Yin-Yang' functions of transforming growth factor-β and T regulatory cells in immune regulation. Immunological reviews 220, 199-213 (2007)). Furthermore, the time course in the allogeneic GVHD model is consistent with at least part of the role of BMC resulting from influencing pre-existing T committed or mature T cells.

[0420] Altogether, these findings suggest that BMCs represent an easy-to-administer, off-the-shelf system capable of enhancing T cell regeneration after HSCT, which, if similarly implemented in the human setting, may offer a means of combating the immunological complications and opportunistic infections that currently limit the clinical application of potentially curative HSCT.

[0421] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0422] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims. In certain embodiments, for example, the following are provided: (Item 1) 1. A composition for modulating the immune system in a subject, comprising: A porous scaffold; A growth factor present in an amount of about 1 ng to about 1000 ng per scaffold and effective to induce formation of a tissue or organ within the scaffold and to recruit cells to the scaffold; a differentiation factor that induces differentiation of the mobilized cells into T cell precursors; A composition comprising: (Item 2) The growth factor is about 0.03 ng / mm based on the volume of the scaffold. 3 ~about 350ng / mm 3 2. The composition according to claim 1, wherein (Item 3) 1. A composition for modulating the immune system in a subject, comprising: A porous scaffold; Approximately 0.03ng / mm based on the volume of the scaffold 3 ~about 350ng / mm 3 a growth factor, present in an amount effective to induce tissue or organ formation within the scaffold and to recruit cells to the scaffold; a differentiation factor that induces differentiation of the mobilized cells into T cell precursors; A composition comprising: (Item 4) 4. The composition of claim 1, wherein the scaffold comprises a hydrogel. (Item 5) 5. The composition according to any one of claims 1 to 4, wherein the scaffold comprises a cryogel. (Item 6) 6. The composition of any one of items 1-5, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyric acid, poly-epsilon-caprolactone, polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic® polyols, poloxamers, poly(uronic acids), poly(anhydrides), poly(vinylpyrrolidone), and any combination thereof. (Item 7) 7. The composition of any one of items 1 to 6, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of alginate, alginate derivatives, and combinations thereof. (Item 8) 8. The composition according to any one of items 1 to 7, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and combinations thereof. (Item 9) Item 9. The composition of any one of items 1 to 8, wherein the scaffold comprises pores having a diameter of about 1 μm to 100 μm. (Item 10) 9. The composition according to any one of items 1 to 8, wherein the scaffold comprises macropores. (Item 11) Item 12. The composition according to item 10, wherein the macropores have a diameter of about 50 μm to 80 μm. 12. The composition of claim 10 or 11, wherein the scaffold comprises macropores of different sizes. (Item 13) 13. The composition of any one of items 1 to 12, wherein the scaffold is injectable. (Item 14) 14. The composition of any one of claims 1 to 13, wherein the scaffold comprises methacrylated alginate (MA-alginate). (Item 15) 15. The composition according to any one of items 1 to 14, wherein the scaffold comprises hyaluronic acid or a hyaluronic acid derivative. (Item 16) 16. The composition of any one of items 1 to 15, wherein the scaffold comprises a click hydrogel or a click cryogel. (Item 17) Item 17. The composition of item 16, wherein the scaffold comprises click alginate, click gelatin, or click hyaluronic acid. (Item 18) Item 18. The composition of any one of items 1-17, wherein the scaffold comprises porogen hydrogel microbeads and bulk hydrogel, and the porogen hydrogel microbeads degrade at least 10% more rapidly than the bulk hydrogel polymer scaffold after administration of the scaffold to a subject. (Item 19) 19. The composition of claim 18, wherein the porogen hydrogel microbeads comprise oxidized alginate. (Item 20) 20. The composition according to any one of items 1 to 19, wherein the cells are stem cells. (Item 21) 21. The composition of item 20, wherein the stem cells are hematopoietic stem cells. (Item 22) 20. The composition according to any one of items 1 to 19, wherein the cells are progenitor cells. (Item 23) 23. The composition according to any one of items 1 to 22, wherein the tissue or organ comprises bone tissue or hematopoietic tissue. (Item 24) 24. The composition according to any one of items 1 to 23, wherein the tissue or organ is formed about 7 to 21 days after administration of the composition to the subject. (Item 25) 25. The composition of claim 24, wherein the tissue or organ is formed about 14 days after administration of the composition to the subject. (Item 26) 26. The composition according to any one of items 1 to 25, wherein the cells are stromal cells. (Item 27) The scaffold has a diameter of about 100 μm. 3 ~about 10cm 3 27. The composition according to any one of items 1 to 26, wherein the size of the composition is 0.01 to 0.25. (Item 28) The scaffold is about 10 mm 3 ~ approx. 100mm 3 28. The composition according to item 27, wherein the size of the composition is (Item 29) The scaffold is about 30 mm 3 29. The composition according to item 28, wherein the size of (Item 30) 30. The composition according to any one of items 1 to 29, wherein the growth factor comprises a protein belonging to the transforming growth factor protein beta (TGF-β) superfamily. (Item 31) 31. The composition of claim 30, wherein the growth factor comprises a protein selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, growth differentiation factor (GDF)-1, GDG-2, GDF-3, GDF-5, GDF-6, GDF-8, GDF-9, GDF-10, GDF-11, GDF-15, anti-Mullerian hormone (AMH), activin, Nodal, TGF-β1, TGF-β2, TGF-β3, TGF-β4, and any combination thereof. (Item 32) 32. The composition of claim 30 or 31, wherein the growth factor comprises BMP-2. (Item 33) 32. The composition of claim 30 or 31, wherein the growth factor comprises TGF-β1. (Item 34) 34. The composition according to any one of items 1 to 33, wherein the growth factor is present at about 5 ng to about 500 ng. (Item 35) 35. The composition according to item 34, wherein the growth factor is present at about 5 ng to about 250 ng. (Item 36) 36. The composition according to item 35, wherein the growth factor is present at about 5 ng to about 200 ng. (Item 37) 37. The composition of claim 36, wherein the growth factor is present at about 200 ng. (Item 38) The growth factor is about 6 ng / mm 3 ~about 10ng / mm 3 Present in item 37 Composition of. (Item 39) The growth factor is about 6.5 ng / mm 3 ~ approx. 7.0ng / mm 3 39. The composition according to item 38, wherein (Item 40) 40. The composition according to any one of items 1 to 39, wherein the growth factor retains its bioactivity for at least 12 days after the growth factor is incorporated into the scaffold. (Item 41) 41. The composition according to any one of items 1 to 40, wherein the T cell precursor cells are capable of differentiating into T cells. (Item 42) Item 43. The composition of item 41, wherein the T cells comprise cells selected from the group consisting of CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), and any combination thereof. 43. The composition of claim 41 or 42, wherein the T cells comprise Tregs. (Item 44) 44. The composition according to any one of items 1 to 43, wherein the differentiation factor binds to a Notch receptor. (Item 45) 45. The composition of claim 44, wherein the Notch receptor is selected from the group consisting of Notch-1 receptor, Notch-2 receptor, Notch-3 receptor, Notch-4 receptor, and any combination thereof. (Item 46) 46. ​​The composition of claim 44 or 45, wherein the differentiation factor is selected from the group consisting of Delta-like 1 (DLL-1), Delta-like 2 (DLL-2), Delta-like 3 (DLL-3), Delta-like 3 (DLL-3), Delta-like 4 (DLL-4), Jagged 1, Jagged 2, and any combination thereof. (Item 47) 47. The composition according to any one of items 1 to 46, wherein the differentiation factor is covalently linked to the scaffold. (Item 48) 48. The composition of claim 47, wherein the differentiation factor is covalently linked to the scaffold using click chemistry. (Item 49) 49. The composition of claim 47 or 48, wherein the differentiation factors are covalently linked to the scaffold using N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocyclooctyne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or disulfide chemistry. (Item 50) 50. The composition according to any one of items 1 to 49, wherein the differentiation factor is present in an amount of about 1 ng to about 1000 μg per scaffold. (Item 51) 51. The composition of claim 50, wherein the differentiation factor is present in an amount of about 1 μg to about 100 μg per scaffold. (Item 52) 52. The composition of claim 51, wherein the differentiation factor is present in an amount of 1 μg to about 10 μg per scaffold. (Item 53) 53. The composition of claim 52, wherein the differentiation factor is present at about 6 μg per scaffold. (Item 54) 54. The composition according to any one of items 1 to 53, wherein the differentiation factor retains its bioactivity for at least about 3 months after the differentiation factor is incorporated into the scaffold. (Item 55) 55. The composition according to any one of items 1 to 54, wherein the mobilized cells are transplant cells. (Item 56) 55. The composition according to any one of items 1 to 54, wherein the mobilized cells are not transplant cells. (Item 57) 56. The composition of item 55, wherein the mobilized cells are autologous. (Item 58) 56. The composition of claim 55, wherein the mobilized cells are allogeneic. (Item 59) 56. The composition of claim 55, wherein the mobilized cells are xenogeneic. (Item 60) Item 60. The composition according to any one of items 1 to 59, wherein the differentiated cells are capable of migrating out of the scaffold. (Item 61) 61. The composition of claim 60, wherein the differentiated cells are capable of homing to a tissue in a subject after administration of the composition to the subject. (Item 62) 62. The composition of any one of items 1 to 61, further comprising a homing factor capable of promoting the recruitment of the cells to the scaffold. (Item 63) 63. The composition of claim 62, wherein the homing factor comprises stromal cell-derived factor (SDF-1). (Item 64) 64. A method of modulating the immune system in a subject, comprising administering to the subject a composition according to any one of items 1 to 63, thereby modulating the immune system in the subject. (Item 65) 64. A method for reducing immune hyper-reactivity in a subject, comprising administering to the subject a composition according to any one of items 1 to 63, thereby reducing immune hyper-reactivity in the subject. (Item 66) 64. A method for increasing donor chimerism in a subject receiving a transplant, comprising administering to the subject a composition according to any one of items 1 to 63, thereby increasing donor chimerism in the subject. (Item 67) 64. A method for promoting balanced T cell reconstitution in a subject, comprising administering to the subject a composition according to any one of items 1 to 63, thereby promoting balanced T cell reconstitution in the subject. (Item 68) 68. The method according to any one of items 64 to 67, further comprising the step of administering hematopoietic stem cells or hematopoietic progenitor cells to the subject. (Item 69) 70. The method of claim 68, wherein the composition is administered to the subject simultaneously with or following administration of the hematopoietic stem cells or hematopoietic progenitor cells to the subject. (Item 70) Approximately 1 x 10 per kilogram of the subject's body weight 5 ~About 50×10 6 Item 71. The method according to item 68 or 69, wherein a single hematopoietic stem cell and / or hematopoietic progenitor cell is administered to the subject. Approximately 1 x 10 per kilogram of the subject's body weight 5 ~Approx. 1×10 6 71. The method of claim 70, wherein a single hematopoietic stem or progenitor cell is administered to the subject. (Item 72) 72. The method according to any one of items 64 to 71, wherein the method enhances T cell reconstitution in the subject. (Item 73) The method of item 72, which enhances T cell neogenesis. (Item 74) 74. The method of claim 73, wherein the enhanced T cell neogenesis is characterized by enhanced T cell receptor excision circles (TRECs). (Item 75) 75. The method according to any one of items 64 to 74, wherein T cell diversity is enhanced in the subject. (Item 76) 76. The method of claim 75, wherein the T cell diversity is characterized by an enhanced T cell receptor (TCR) repertoire. (Item 77) Controllability T(T reg 77. The method according to any one of items 64 to 76, wherein the level of .gamma.- ... (Item 78) 78. The method according to any one of items 64 to 77, wherein the subject is a human having a defective immune system. (Item 79) 79. The method of claim 78, wherein the subject has a defective immune system due to immunosenescence. (Item 80) 80. The method of claim 79, wherein the subject is over 30 years old, over 40 years old, over 50 years old, over 60 years old, over 70 years old, or over 80 years old. (Item 81) 81. The method according to any one of items 78 to 80, wherein the subject has a defective immune system due to a congenital immune deficiency. (Item 82) The method according to any one of Items 78 to 81, wherein the subject has an acquired immune deficiency. 1. A method of reducing immune hyperresponsiveness in a subject, comprising administering to the subject: A porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and to recruit cells to the scaffold; a differentiation factor that induces differentiation of the mobilized cells into T cell precursors; administering a composition comprising thereby reducing immune hyper-reactivity in said subject. (Item 84) 1. A method for increasing donor chimerism in a subject receiving a transplant, comprising administering to the subject: A porous scaffold; Inducing the formation of a tissue or organ within the scaffold and infusing cells into the scaffold. a growth factor present in an amount effective to mobilize a differentiation factor that induces differentiation of the mobilized cells into T cell precursors; thereby increasing donor chimerism in said subject. (Item 85) 1. A method for promoting balanced T cell reconstitution in a subject, comprising administering to the subject: A porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and to recruit cells to the scaffold; a differentiation factor that induces differentiation of the mobilized cells into T cell precursors; thereby resulting in balanced T cell reconstitution in said subject. (Item 86) 1. A method of modulating the immune system of a human having an immune system deficiency, comprising administering to said human: A porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and to recruit cells to the scaffold; a differentiation factor that induces differentiation of the mobilized cells into T cell precursors; administering to said human a composition comprising the human has a defective immune system due to immunosenescence, congenital immune deficiency, or acquired immune deficiency; method. (Item 87) 87. The method of any one of items 83 to 86, wherein the scaffold comprises a hydrogel. (Item 88) 88. The method of any one of items 83 to 87, wherein the scaffold comprises a cryogel. (Item 89) 89. The method of any one of items 83-88, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyric acid, poly-epsilon-caprolactone, polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic® polyols, poloxamers, poly(uronic acids), poly(anhydrides), poly(vinylpyrrolidone), and any combination thereof. (Item 90) 90. The method of any one of items 83-89, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of alginate, alginate derivatives, and combinations thereof. (Item 91) Item 92. The method of any one of items 83-91, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and combinations thereof. (Item 92) 92. The method of any one of items 83 to 91, wherein the scaffold comprises macropores. (Item 93) Item 93. The method of item 92, wherein the macropores have a diameter of about 1 μm to about 100 μm. (Item 94) Item 94. The method of item 93, wherein the macropores have a diameter of about 50 μm to 80 μm. (Item 95) 95. The method of any one of items 92 to 94, wherein the scaffold comprises macropores of different sizes. (Item 96) 96. The method of any one of items 83 to 95, wherein the scaffold is injectable. (Item 97) 97. The method of any one of items 83 to 96, wherein the scaffold comprises methacrylated alginate (MA-alginate). (Item 98) Item 98. The method according to any one of items 83 to 97, wherein the scaffold comprises hyaluronic acid or a hyaluronic acid derivative. (Item 99) 99. The method of any one of items 83 to 98, wherein the scaffold comprises a click hydrogel or a click cryogel. (Item 100) Item 99. The method of item 99, wherein the scaffold comprises click alginate, click gelatin, or click hyaluronic acid. (Item 101) 101. The method of any one of items 83 to 100, wherein the scaffold comprises porogen hydrogel microbeads and bulk hydrogel, and the porogen hydrogel microbeads degrade at least 10% more rapidly than the bulk hydrogel after administration to a subject. (Item 102) Item 102. The method of item 101, wherein the porogen hydrogel microbeads comprise oxidized alginate. (Item 103) 103. The method according to any one of items 83 to 102, wherein the cells are stem or progenitor cells. (Item 104) 104. The method of claim 103, wherein the cells are selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, recombinant hematopoietic stem cells, recombinant hematopoietic progenitor cells, and any combination thereof. (Item 105) 103. The method of any one of items 83-102, wherein said cells are selected from the group consisting of hematopoietic bone marrow cells, mobilized peripheral blood cells, recombinant hematopoietic bone marrow cells, recombinant mobilized peripheral blood cells, and any combination thereof. (Item 106) 106. The method according to any one of items 83 to 105, wherein the tissue or organ comprises bone tissue or hematopoietic tissue. (Item 107) 107. The method of claim 106, wherein the tissue or organ is formed about 7 to 21 days after administering the composition to the subject. (Item 108) 108. The method according to any one of items 83 to 107, wherein the tissue or organ is formed about 14 days after administering the composition to the subject. (Item 109) 109. The method of claim 107 or 108, wherein at least two compositions are administered to the subject. (Item 110) 110. The method of claim 109, wherein the compositions are of similar size. (Item 111) 111. The method according to any one of items 83 to 110, wherein the cells are stromal cells. (Item 112) The scaffold has a diameter of about 100 μm. 3 ~about 10cm 3 The method according to any one of items 83 to 111, wherein the size is (Item 113) The scaffold is about 10 mm 3 ~ approx. 100mm 3 Item 113. The method according to item 112, wherein the size of the (Item 114) The scaffold is about 30 mm 3 Item 114. The method according to item 113, wherein the size of the (Item 115) 15. The method according to any one of items 83 to 114, wherein the growth factor comprises a protein belonging to the transforming growth factor protein beta (TGF-β) superfamily. (Item 116) 116. The method of claim 115, wherein the growth factor comprises a protein selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, growth differentiation factor (GDF)-1, GDG-2, GDF-3, GDF-5, GDF-6, GDF-8, GDF-9, GDF-10, GDF-11, GDF-15, anti-Mullerian hormone (AMH), activin, Nodal, TGF-β1, TGF-β2, TGF-β3, TGF-β4, and any combination thereof. (Item 117) 117. The method of claim 115 or 116, wherein the growth factor comprises BMP-2. (Item 118) 117. The method of claim 115 or 116, wherein the growth factor comprises TGF-β1. (Item 119) 19. The method of any one of items 83 to 118, wherein the growth factor retains its bioactivity for at least 12 days after the growth factor is incorporated into the scaffold. (Item 120) 120. The method according to any one of items 83 to 119, wherein the T cell precursor cells are capable of differentiating into T cells. (Item 121) Item 122. The method of item 120, wherein the T cells comprise cells selected from the group consisting of CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), and any combination thereof. 121. The method of item 119 or 120, wherein the lymphocytes comprise Tregs. (Item 123) 123. The method of any one of items 83 to 122, wherein the differentiation factor binds to a Notch receptor. (Item 124) 124. The method of item 123, wherein the Notch receptor is selected from the group consisting of Notch-1 receptor, Notch-2 receptor, Notch-3 receptor, Notch-4 receptor, and any combination thereof. (Item 125) The differentiation factor is selected from the group consisting of Delta-like 1 (DLL-1), Delta-like 2 (DLL-2), Delta-like 3 (DLL-3), Delta-like 3 (DLL-3), Delta-like 4 (DLL-4), Jagged 1, Jagged 2, and any combination thereof. 125. The method according to item 123 or 124, selected from the group consisting of (Item 126) 126. The method of any one of items 83 to 125, wherein the differentiation factor is covalently linked to the scaffold. (Item 127) Item 127. The method of item 126, wherein the differentiation factor is covalently linked to the scaffold using click chemistry. (Item 128) 128. The method of claim 126 or 127, wherein the differentiation factors are covalently linked to the scaffold using N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, avidin-biotin reaction, azide and dibenzocyclooctyne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or disulfide chemistry. (Item 129) 129. The method according to any one of items 83 to 128, wherein the differentiation factor retains its bioactivity for at least about 3 months after the differentiation factor is incorporated into the scaffold. (Item 130) 130. The method according to any one of items 83 to 129, wherein the mobilized cells are transplant cells. (Item 131) 130. The method according to any one of items 83 to 129, wherein the mobilized cells are not transplant cells. (Item 132) 131. The method of item 130, wherein the mobilized cells are autologous. (Item 133) 131. The method of item 130, wherein the mobilized cells are allogeneic. (Item 134) 131. The method of claim 130, wherein the mobilized cells are xenogeneic. (Item 135) 135. The method according to any one of items 83 to 134, wherein the differentiated cells are capable of migrating out of the scaffold. (Item 136) 136. The method of claim 135, wherein the differentiated cells are capable of homing to a tissue in a subject after administration of the composition to the subject. (Item 137) 137. The method of any one of items 83 to 136, wherein the composition further comprises a homing factor capable of promoting the recruitment of the cells to the scaffold. (Item 138) 138. The method of claim 137, wherein the homing factor comprises stromal cell-derived factor (SDF-1). (Item 139) 139. The method according to any one of items 83 to 138, wherein the growth factor is present at about 1 ng to about 1000 μg. (Item 140) 139. The method according to any one of items 83 to 139, wherein the growth factor is present at about 1 ng to about 1000 ng. (Item 141) Item 141. The method of item 140, wherein the growth factor is present at about 5 ng to about 500 ng. (Item 142) Item 142. The method of item 141, wherein the growth factor is present at about 5 ng to about 250 ng. (Item 143) Item 143. The method of item 142, wherein the growth factor is present at about 5 ng to about 200 ng. (Item 144) 144. The method of claim 143, wherein the growth factor is present at about 200 ng. (Item 145) The growth factor is about 0.03 ng / mm based on the volume of the scaffold. 3 ~about 350ng / mm 3 The method according to any one of items 83 to 144, wherein (Item 146) The growth factor is about 6 ng / mm 3 ~about 10ng / mm 3 Item 146. The composition according to item 145, wherein (Item 147) The growth factor is about 6.5 ng / mm 3 ~ approx. 7.0ng / mm 3 147. The composition according to item 146, wherein (Item 148) Approximately 5×10 per kilogram of the subject's body weight 5 ~About 50×10 6 148. The method according to items 83 to 147, wherein a single hematopoietic stem cell and / or hematopoietic progenitor cell is administered to the subject. (Item 149) Approximately 5×10 per kilogram of the subject's body weight 5 149. The method of claim 148, wherein a number of hematopoietic stem and / or progenitor cells are administered to the subject. (Item 150) 150. The method of claim 148 or 149, wherein the hematopoietic cells are selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, recombinant hematopoietic stem cells, recombinant hematopoietic progenitor cells, and any combination thereof. (Item 151) 150. The method of claim 148 or 149, wherein the hematopoietic cells are selected from the group consisting of hematopoietic bone marrow cells, mobilized peripheral blood cells, recombinant hematopoietic bone marrow cells, recombinant mobilized peripheral blood cells, and any combination thereof. (Item 152) The method of any one of items 83 and 87-151, wherein the method reduces autoimmunity in the subject. (Item 153) The method according to any one of items 83 and 87 to 152, for preventing or treating an autoimmune disease. (Item 154) Item 154. The method of item 153, wherein the autoimmune disease is a disease selected from the group consisting of type 1 diabetes, rheumatoid arthritis, psoriasis, arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, and allergies. (Item 155) The method of any one of items 83, 84, and 87-151, which reduces graft-versus-host disease (GVHD). (Item 156) 156. The method of claim 155, wherein the GVHD is associated with hematopoietic stem cell transplantation (HSCT) to the subject. (Item 157) 157. The method of item 156, wherein the composition is administered simultaneously with or after hematopoietic stem cell transplantation (HSCT). (Item 158) 158. The method of claim 157, wherein the GVHD is associated with a solid organ transplant. (Item 159) 159. The method of claim 158, wherein the composition is administered to the subject prior to the transplantation. (Item 160) 160. The method of any one of items 155 to 159, wherein the GVHD is acute GVHD. (Item 161) 160. The method of any one of items 155 to 159, wherein the GVHD is chronic GVHD. (Item 162) The method of any one of items 155 to 161, wherein the method reduces GVHD-related morbidity, GVHD-related mortality, or GVHD-related loss of long-term survival. (Item 163) The method of any one of items 84 and 87 to 151, wherein the transplant is a hematopoietic stem cell transplant (HSCT). (Item 164) 164. The method of any one of items 84, 87-151, and 163, wherein the increased donor chimerism comprises T cell chimerism. (Item 165) 165. The method of claim 164, wherein the T cells comprise CD4+ T cells, CD8+ T cells, or Treg cells. (Item 166) 152. The method of any one of items 85 and 87-151, wherein the balanced T cell reconstitution is characterized by a homeostatic CD4+:CD8+ T cell ratio of about 0.9 to about 2.5 in peripheral blood. (Item 167) 152. The method according to any one of items 86 to 151, wherein the human has a defective immune system due to immunosenescence. (Item 168) 168. The method of claim 167, wherein the human is over 30 years old, over 40 years old, over 50 years old, over 60 years old, over 70 years old, or over 80 years old. (Item 169) 169. The method of any one of items 86-151, 167, and 168, wherein the human has a defective immune system due to a congenital immune deficiency. (Item 170) The method according to any one of items 86 to 151 and 167 to 169, wherein the human has a defective immune system due to acquired immune deficiency. (Item 171) Controllability T(T reg 171. The method of any one of items 83 to 170, wherein the level of .gamma.- ... (Item 172) 172. The method of any one of items 83 to 171, wherein the composition is administered by injection. (Item 173) Item 173. The method of item 172, wherein the injection is a subcutaneous injection. (Item 174) Needle and A reservoir comprising the composition according to any one of items 1 to 63; Plunger and A syringe comprising: (Item 175) The composition according to any one of items 1 to 63, instructions for administering the composition; Including the kit. (Item 176) 174. The method of any one of items 83 to 173, further comprising administering to the subject a stem cell or progenitor cell mobilizing agent in an amount effective to induce migration of stem cells or progenitor cells from bone marrow to blood. (Item 177) 177. The method of claim 176, wherein the stem cell mobilizing agent or progenitor cell mobilizing agent is selected from the group consisting of IL-1, IL-2, IL-3, IL-6, GM-CSF, G-CSF, plerixafor, PDGF, TGF-beta, NGF, IGF, growth hormone, erythropoietin, thrombopoietin, and combinations thereof. (Item 178) 178. The method of claim 176 or 177, wherein the stem cell mobilizing agent or the progenitor cell mobilizing agent is administered before, simultaneously with, or after administration of the composition. (Item 179) 179. The method of any one of items 176 to 178, further comprising administering to the subject a therapeutically effective amount of electromagnetic radiation.

Claims

1. 1. A composition for modulating the immune system in a subject, comprising: a porous scaffold comprising a click hydrogel or a click cryogel; a growth factor present at 5 ng to 250 ng per scaffold or 0.03 ng / mm to 350 ng / mm based on the volume of the scaffold and in an amount effective to induce formation of a tissue or organ within said scaffold and to recruit hematopoietic stem and / or progenitor cells to said scaffold, wherein said tissue or organ formed comprises bone tissue or hematopoietic tissue; a differentiation factor that induces differentiation of the mobilized cells into T cell precursors; A composition comprising:

2. (i) the scaffold comprises a click hydrogel; (ii) the scaffold comprises a click cryogel; (iii) the scaffold comprises a polymer or copolymer selected from the group consisting of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid or hyaluronic acid derivatives, poly(lysine), polyhydroxybutyric acid, poly-epsilon-caprolactone, polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic® polyols, poloxamers, poly(uronic acids), poly(anhydrides), poly(vinylpyrrolidone), and any combination thereof; (iv) the scaffold comprises a polymer or copolymer selected from the group consisting of alginate, alginate derivatives, and combinations thereof; (v) the scaffold comprises a polymer or copolymer selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and combinations thereof; (vi) the scaffold comprises pores having a diameter between 1 μm and 100 μm; (vii) the scaffold comprises macropores; Optionally, the macropores have a diameter of from 50 μm to 80 μm; Optionally, the scaffold comprises macropores of different sizes; (viii) the scaffold is injectable; (ix) the scaffold comprises methacrylated alginate (MA-alginate); (x) the scaffold comprises click alginate, click gelatin, or click hyaluronic acid; (xi) the scaffold comprises porogen hydrogel microbeads and bulk hydrogel, and the porogen hydrogel microbeads degrade at least 10% more rapidly than the bulk hydrogel polymer scaffold after administration of the scaffold to a subject; Optionally, the porogen hydrogel microbeads comprise oxidized alginate; and / or (xii) the scaffold has a thickness of 100 μm ３ ~10cm ３ , 10 mm ３ ~100mm ３ or 30 mm ３ The composition of claim 1 , wherein the size of the composition is

3. The composition of claim 1 or 2, wherein the tissue or organ is formed 7 to 21 days or 14 days after administration of the composition to the subject.

4. (i) the growth factor comprises a protein belonging to the transforming growth factor protein beta (TGF-β) superfamily; (ii) the growth factor comprises a protein selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, growth differentiation factor (GDF)-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-8, GDF-9, GDF-10, GDF-11, GDF-15, anti-Mullerian hormone (AMH), activin, Nodal, TGF-β1, TGF-β2, TGF-β3, TGF-β4, and any combination thereof; (iii) the growth factor comprises BMP-2; (iv) the growth factor comprises TGF-β1; (v) the growth factor is present at 5 ng to 500 ng, 5 ng to 250 ng, 5 ng to 200 ng, or 200 ng; (vi) the growth factor is at a concentration of 0.03 ng / mm based on the volume of the scaffold ３ ~350ng / mm ３ and optionally the growth factor is present at 6 ng / mm ３ ~10ng / mm ３ or 6.5 ng / mm ３ ~7.0ng / mm ３ and / or (vii) The composition of any one of claims 1 to 3, wherein the growth factor retains its bioactivity for at least 12 days after the growth factor is incorporated into the scaffold.

5. (i) the T cell precursor cells are capable of differentiating into T cells, and optionally the T cells comprise cells selected from the group consisting of CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), and any combination thereof; (ii) the differentiation factor binds to a Notch receptor, and optionally The Notch receptor is selected from the group consisting of Notch-1 receptor, Notch-2 receptor, Notch-3 receptor, Notch-4 receptor, and any combination thereof; (iii) The differentiation factor is Delta-like 1 (DLL-1), Delta-like 2 (DLL-2), Delta-like 3 (DLL-3), Delta-like 3 (DLL-3), Delta-like 4 (DLL-4), Jagged 1, Jagged 2, and any combination thereof; (iv) the differentiation factor is covalently linked to the scaffold; Optionally, the differentiation factor is covalently linked to the scaffold using click chemistry; Optionally, the differentiation factors are covalently linked to the scaffold using N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocyclooctyne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or disulfide chemistry; (v) the differentiation factor is present in an amount between 1 ng and 1000 μg per scaffold, between 1 μg and 100 μg, between 1 μg and 10 μg, or between 6 μg per scaffold; and / or (vi) The composition of any one of claims 1 to 4, wherein the differentiation factor retains its bioactivity for at least 3 months after the differentiation factor is incorporated into the scaffold.

6. the mobilized cells are transplant cells; the mobilized cells are not transplant cells; the mobilized cells are autologous; the mobilized cells are allogeneic; the mobilized cells are xenogeneic; The differentiated cells are capable of migrating out of the scaffold; and / or teeth The composition of any one of claims 1 to 5, wherein the differentiated cells are capable of homing to a tissue in a subject after administration of the composition to the subject.

7. 7. The composition of any one of claims 1 to 6, further comprising a homing factor capable of promoting the recruitment of the cells to the scaffold, optionally wherein the homing factor comprises stromal cell-derived factor (SDF-1).

8. 8. The composition of any one of claims 1 to 7, for use in modulating the immune system in said subject, reducing immune hyper-reactivity in said subject, increasing donor chimerism in said subject, and / or promoting balanced T cell reconstitution in said subject.

9. The composition is administered to the subject in combination with hematopoietic stem or progenitor cells, and optionally the composition is administered to the subject simultaneously with or following administration of the hematopoietic stem cells or hematopoietic progenitor cells to the subject, and optionally 1×10 per kilogram of the subject's body weight ５ ~50 x 10 ６ pcs or 1 x 10 ５ ~1×10 ６ The composition for use according to claim 8, characterized in that a single hematopoietic stem cell and / or hematopoietic progenitor cell is administered to the subject.

10. the composition enhances T cell reconstitution in the subject; The composition enhances T cell neogenesis, Optionally, the enhanced T cell neogenesis is characterized by enhanced T cell receptor excision circles (TREC); the composition enhances T cell diversity in the subject; Optionally, the T cell diversity is characterized by an enhanced T cell receptor (TCR) repertoire; and / or The composition has controllability T (T ｒｅｇ 10. The composition for use according to claim 8 or 9, which increases the level of .

11. (i) the subject is a human having a compromised immune system, and optionally The subject has an immune system defect due to immunosenescence, and optionally The subject is over 30 years old, over 40 years old, over 50 years old, over 60 years old, over 70 years old, or over 80 years old, and optionally The subject has a defective immune system due to a congenital immune deficiency; and / or (ii) The composition for use according to any one of claims 8 to 10, wherein the subject has an acquired immune deficiency.

12. 1. A composition for use in a method of reducing immune hyper-reactivity in a subject, increasing donor chimerism in a subject receiving a transplant, promoting balanced T cell reconstitution in a subject, or modulating the immune system of a subject having an immune system deficiency, said composition comprising: a porous scaffold comprising a click hydrogel or a click cryogel; a growth factor present in an amount between 5 ng and 250 ng, or between 0.03 ng / mm and 350 ng / mm based on the volume of the scaffold, and effective to induce formation of a tissue or organ within said scaffold and to recruit hematopoietic stem and / or progenitor cells to said scaffold, wherein said tissue or organ formed comprises bone tissue or hematopoietic tissue; a differentiation factor that induces differentiation of the mobilized cells into T cell precursors; A composition comprising:

13. (i) the composition reduces autoimmunity in the subject; (ii) the composition prevents or treats an autoimmune disease; Optionally, the autoimmune disease is a disease selected from the group consisting of type 1 diabetes, rheumatoid arthritis, psoriasis, arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, and allergies; (iii) the composition reduces graft-versus-host disease (GVHD), and optionally the GVHD is associated with hematopoietic stem cell transplant (HSCT) to the subject; Optionally, the composition is administered simultaneously with or after hematopoietic stem cell transplantation (HSCT); Optionally, the GVHD is associated with solid organ transplant; Optionally, the composition is administered to the subject prior to the transplantation; Optionally, the GVHD is acute GVHD; Optionally, the GVHD is chronic GVHD; and / or Optionally, the method reduces GVHD-associated morbidity, GVHD-associated mortality, or reduced long-term survival rates.

14. Needle and A reservoir comprising the composition of any one of claims 1 to 7; Plunger and A syringe comprising:

15. A composition according to any one of claims 1 to 7, instructions for administering the composition; Including the kit.

16. The composition is administered in combination with a stem cell or progenitor cell mobilizing agent in an amount effective to induce mobilization of stem cells or progenitor cells from bone marrow to blood, and optionally: the stem cell mobilizing agent or the progenitor cell mobilizing agent is selected from the group consisting of IL-1, IL-2, IL-3, IL-6, GM-CSF, G-CSF, plerixafor, PDGF, TGF-beta, NGF, IGF, growth hormone, erythropoietin, thrombopoietin, and combinations thereof, and optionally The stem cell mobilizing agent or the progenitor cell mobilizing agent is administered prior to, simultaneously with, or after administration of the composition, and optionally The composition for use according to any one of claims 8 to 13, further characterized in that said composition is administered in combination with a therapeutically effective amount of electromagnetic radiation.

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