Methods and Compositions for Xenotransplantation

Abstract

Provided herein is a method of xenotransplantation, for example, pig-to-human transplantation. Also provided herein is an extracellular vesicle ("EV", e.g., exosome) and a composition comprising the same, for example, an EV expressing human CD47. Also provided herein is a method of producing EV and its use for xenotransplantation. In some aspects, the method of xenotransplantation comprises expressing human CD47 in the xenograft.

Description

[Technical field]

[0001] 1. Cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 240,637, filed September 3, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] 2. Sequence Listing This application contains a computer readable sequence listing submitted via the Patent Center in XML file format, the entire contents of which are incorporated herein by reference. The XML file of the sequence listing submitted via the Patent Center is entitled "14648-004-228_seqlist.xml", was created on August 30, 2022, and is 32,782 bytes in size.

[0003] 3.Government License Rights This invention was made with Government support under Grant No. P01 AI045897 awarded by the National Institute of Allergy and Infections Diseases (NIAID), National Institutes of Health (NIH). The Government has certain rights in this invention.

[0004] 4.Preface Provided herein is a method of xenotransplantation, e.g., pig-to-human transplantation. Also provided herein are extracellular vesicles ("EVs", e.g., exosomes) and compositions comprising same, e.g., EVs expressing human CD47. Also provided herein are methods of making EVs and their use for xenotransplantation. In some aspects, the method of xenotransplantation includes expressing human CD47 in the xenograft. In certain aspects, the method of xenotransplantation does not depend on the expression of signal regulatory protein alpha (SIRPα) on the target tissue. 5. [Background technology]

[0005] Currently, a severe shortage of allogeneic donors limits the number of organ transplants performed. This supply-demand gap can be filled by the use of organs from other species (xenografts). Given the ethical issues and impracticalities associated with the use of non-human primates, pigs are considered the most suitable donor species for humans. In addition to their organ size and physiological similarity to humans, pigs can be rapidly bred and inbred, making them particularly suitable for genetic modification to improve their ability to function as graft donors for humans (Sachs 1994, Path. Biol. 42:217-219; Piedrahita et al., 2004, Am. J. Transplant, 4 Suppl. 6:43-50).

[0006] Although the combination of nonspecific immunosuppressive therapy and transplantation is associated with high early graft tolerance, a significant limitation to the success of clinical organ transplantation is late graft loss, largely due to chronic rejection of the graft. Kidney transplantation provides an average of 4.4 years of life per recipient. See Rana et al. JAMA Surg. 2015;150(3):252-259. However, within 10 years after living donor kidney transplantation, more than 30% of grafts fail. See Department of Health and Human Services:2017 Annual Data Report:Kidney [Retrieved March 22, 2021]. Internet:<URL:srtr.transplant.hrsa.gov / annual_reports / 2017 / Kidney.aspx> Retrieved from.

[0007] Immune tolerance is further important for successful clinical xenotransplantation because the lifelong levels of immunosuppression required to prevent xenograft rejection can be unacceptably toxic. In addition, no markers have been identified that reliably indicate whether immunological tolerance has been achieved in a patient, and as a result, there are no laboratory parameters on which immunosuppression weaning can be based.

[0008] Therefore, goals in xenotransplantation include optimizing the durability of mixed chimeric cells derived from the donor animal after transplantation into a xenogeneic recipient, as well as maintaining the health and viability of the donor animal.

[0009] Mixed chimerism can induce tolerance to the donor at the level of T cells, B cells, and natural killer (NK) cells in the recipient. Griesemer A, Yamada K. and Sykes M., 2014, Immunol. Rev. 258: 241-258. Sachs DH, Kawai T. and Sykes M., 2014, Cold Spring Harb. Perspect. Med. 4: a015529. Hematopoiesis is a tightly regulated process involving the interaction of cytokines and adhesion molecules in the bone marrow microenvironment with receptors on hematopoietic cells. Since many of these receptor-ligand interactions are species-specific (e.g., IL-3 and IL-3R) or species-selective (e.g., SCF-cKIT, GM-CSF-GM-CSFR, VLA-5-fibronectin), mixed chimeric cells (e.g., from pigs) would be at a competitive disadvantage compared to endogenous hematopoietic cells (e.g., human cells), leading to gradual loss of function of the transplanted cells. This loss of function of chimerism is undesirable, since durable mixed chimerism can best ensure lifelong T, B, and NK cell tolerance.

[0010] Introducing human cytokine receptors and adhesion molecules into porcine donor animals would help overcome this competitive disadvantage and ensure lifelong chimerism and tolerance. Griesemer A, Yamada K. and Sykes M., 2014, Immunol.Rev., 258:241-258. Because hematopoiesis is tightly regulated, it may be desirable to insert these genes into their natural loci in the porcine genome so that they can function in a physiological manner under the control of native regulatory sequences. This can be achieved by disrupting the native porcine gene and replacing it with its human counterpart. However, this approach can have the problem of rendering porcine cells unresponsive or hyporesponsive to species-specific or species-selective porcine cytokines (or adhesion ligands), respectively. Thus, long-term expression of human transgenes may be detrimental to the health of the donor animal. Dwyer et al., J. Clin. Invest.2004 May;113(10):1440-6, and Crikis et al., 2010, Am. J. Transplant;10:242-50.

[0011] CD47, also known as integrin-associated protein (IAP), is a ubiquitously expressed 50 kDa cell surface glycoprotein that functions as a ligand for signal regulatory protein (SIRP) alpha (also known as CD172a and SHPS-1) (Brown, 2002, Curr. Opin. Cell. Biol., 14:603-7; Brown and Frazier, 2001, Trends Cell Biol., 111:130-5). CD47 and SIRPα constitute an intercellular communication system that plays an important role in diverse cellular processes, including cell migration, B cell adhesion, and T cell activation (Liu et al., 2002, J. Biol. Chem. 277:10028; Motegi et al., 2003, EMBO 122:2634; Yoshida et al., 2002, J. Immunol. 168:3213; Latour et al., 2001; J. Immunol. 167:2547). In addition, the CD47-SIRPα system is involved in the negative regulation of phagocytosis by macrophages. CD47 on the surface of several cell types (i.e., erythrocytes, platelets, or leukocytes) inhibited phagocytosis by macrophages. A role for CD47-SIRPα interactions in inhibiting phagocytosis is demonstrated by the observation that primary wild-type mouse macrophages rapidly phagocytose non-opsonized red blood cells (RBCs) obtained from CD47-deficient but not wild-type mice (Oldenborg et al., 2000; Science 288:2051). It has also been reported that CD47, through its receptor SIRPα, inhibits both Fcγ- and complement receptor-mediated phagocytosis (Oldenborg et al., J. 2001; Exp. Med. 193:855).

[0012] CD47 is ubiquitously expressed and acts as a ligand for signal transduction regulatory protein (SIRP) alpha, a key inhibitory receptor on macrophages and dendritic cells (DCs). Emerging evidence indicates that the CD47-SIRPα signaling pathway plays a key role in regulating macrophage and DC activation, providing a promising intervention target for immunological disorders. The CD47-SIRPα cell communication system is species-specific (e.g., porcine CD47 does not inhibit phagocytosis of porcine bone marrow cells). The lack of cross-reactivity between porcine CD47 and human SIRPα may also contribute to the rejection of other types of porcine cells (e.g., hepatocytes) by human macrophages, stimulating DC activation (see below), thus inducing anti-porcine T-cell responses.

[0013] CD47-deficient cells are vigorously rejected by macrophages after injection into syngeneic wild-type (WT) mice, demonstrating that CD47 provides a "don't eat me" signal to macrophages (Oldenborg PA,et al.,2000 Science,288:2051-4; Wang et al.,2007,Proc Natl Acad Sci USA.104:13744). Xenotransplantation using pigs as a graft source has the potential to overcome the severe shortage of human organ donors, a major limiting factor in clinical transplantation (Yang et al.,2007,Nature reviews Immunology.7:519-31). The robust rejection of xenogeneic cells by macrophages (Abe 2002, The Journal of Immunology 168:621) is largely caused by the lack of functional interaction between donor CD47 and recipient SIRPα (Wang et al., 2007, Blood;109:836-42; Ide et al., 2007, Proc Natl Acad Sci USA 104:5062-6; Navarro-Alvarez 2014, Cell Transplantation,23:345-54), which has led to the development of human CD47 transgenic pigs (Tena et al., 2017, Transplantation 101:316-21; Nomura et al., 2020, Xenotransplantation.2020;27:e12549). In addition to macrophages, a subpopulation of DCs also expresses SIRPα (Wang et al., 2007, Proc Natl Acad Sci USA. 104:13744-9; Guilliams et al., 2016, Immunity. 45:669-84).CD47-SIRPα signaling also inhibits DC activation and its ability to prime T cells, playing an important role in the induction of T cell tolerance by donor-specific transfusion (DST) or hepatocyte transplantation (Wang et al., 2007, Proc Natl Acad Sci USA. 104:13744-9; Wang et al., 2014, Cell transplantation 23:355-63; Zhang et al., 2016, Sci Rep. 6:26839). In addition to functioning as a "don't eat me" molecule that inhibits phagocytosis through interaction with SIRPα, upon ligation to its ligands (e.g., anti-CD47 antibodies, TSP-1, soluble SIRPα), CD47 signaling also induces cell senescence or death and suppresses cell proliferation. Summary of the Invention

[0014] 6. Summary of the Invention In one aspect, provided herein is a method of xenotransplantation, the method comprising: (a) obtaining an organ from a donor pig; (b) cross-dressing the organ with human CD47; and (c) transplanting the organ into a human recipient. In some embodiments, the cross-dressing step comprises exposing the organ to human CD47 containing extracellular vesicles (EVs). In some embodiments, the EVs are isolated from human cells. In some embodiments, the cells express recombinant human CD47. In some embodiments, the cells are transgenic cells.

[0015] In some embodiments, cross-dressing is achieved by incubating the organ with EVs expressing human CD47 for 2 hours. In some embodiments, cross-dressing is achieved by incubating the organ with EVs expressing human CD47 for 6 hours.

[0016] In some embodiments, cross-dressing is accomplished by ex vivo perfusion of the organ, hi some embodiments, cross-dressing is accomplished by in vivo perfusion of a donor pig, a human recipient, or a combination thereof.

[0017] In some embodiments, the method results in a decrease in phagocytosis by human macrophages of cross-dressed organ cells, as compared to non-cross-dressed organ cells, by about 5% to about 25%, as measured by FACS analysis of the percentage of CD14 positive cells that phagocytose the cross-dressed organ cells. In some embodiments, the method results in a decrease in phagocytosis by human macrophages of cross-dressed organ cells, as compared to non-cross-dressed organ cells, by about 25% to about 50%, as measured by FACS analysis of the percentage of CD14 positive cells that phagocytose the cross-dressed cells. In some embodiments, the method results in a decrease in phagocytosis by human macrophages of cross-dressed organ cells, as compared to non-cross-dressed organ cells, by about 50% to about 75%, as measured by FACS analysis of the percentage of CD14 positive cells that phagocytose the cross-dressed cells. In some embodiments, the method results in a reduction in phagocytosis by human macrophages of cross-dressed organ cells by about 75% to about 80% compared to non-cross-dressed organ cells, as measured by FACS analysis of the percentage of CD14 positive cells that phagocytose the cross-dressed cells. In some embodiments, the method results in a reduction in phagocytosis by human macrophages of cross-dressed organ cells by about 80% to about 85% compared to non-cross-dressed organ cells, as measured by FACS analysis of the percentage of CD14 positive cells that phagocytose the cross-dressed cells. In some embodiments, the method results in a reduction in phagocytosis by human macrophages of cross-dressed organ cells by about 85% to about 90% compared to non-cross-dressed organ cells, as measured by FACS analysis of the percentage of CD14 positive cells that phagocytose the cross-dressed cells.In some embodiments, the method results in a reduction in phagocytosis by human macrophages of cross-dressed organ cells compared to non-cross-dressed organ cells by about 90% to about 95%, as measured by FACS analysis of the percentage of CD14 positive cells that phagocytose the cross-dressed cells. In some embodiments, the method results in no detectable phagocytosis of the cross-dressed organ cells, as measured by FACS analysis of the percentage of CD14 positive cells that phagocytose the cross-dressed cells.

[0018] In some embodiments, the method results in increased organ viability due to protection from human macrophages, as measured by FACS analysis of the percentage of CD14 positive cells that phagocytose the cross-dressed cells. In some embodiments, the cross-dressed organ avoids phagocytosis without inducing apoptosis. In some embodiments, the cross-dressed organ avoids phagocytosis and does not exhibit detectable levels of apoptosis. In some embodiments, cells obtained from the cross-dressed organ exhibit about 5% to about 25% lower levels of apoptosis compared to cells obtained from a non-cross-dressed organ. In some embodiments, cells obtained from the cross-dressed organ exhibit about 25% to about 50% lower levels of apoptosis compared to cells obtained from a non-cross-dressed organ. In some embodiments, cells obtained from the cross-dressed organ exhibit about 50% to about 75% lower levels of apoptosis compared to cells obtained from a non-cross-dressed organ. In some embodiments, cells obtained from cross-dressed organs exhibit about 75% to about 80% lower levels of apoptosis compared to cells obtained from non-cross-dressed organs. In some embodiments, cells obtained from cross-dressed organs exhibit about 80% to about 85% lower levels of apoptosis compared to cells obtained from non-cross-dressed organs. In some embodiments, cells obtained from cross-dressed organs exhibit about 85% to about 90% lower levels of apoptosis compared to cells obtained from non-cross-dressed organs. In some embodiments, cells obtained from cross-dressed organs exhibit at least 90% lower levels of apoptosis compared to cells obtained from non-cross-dressed organs. In some embodiments, apoptosis is measured by propidium iodide staining.

[0019] In some embodiments, the cross-dressed organ exhibits reduced inflammation compared to a non-cross-dressed organ. In some embodiments, the cross-dressed organ exhibits about 5% to about 25% reduction in inflammation compared to a non-cross-dressed organ. In some embodiments, the cross-dressed organ exhibits about 25% to about 50% reduction in inflammation compared to a non-cross-dressed organ. In some embodiments, the cross-dressed organ exhibits about 50% to about 75% reduction in inflammation compared to a non-cross-dressed organ. In some embodiments, the cross-dressed organ exhibits about 75% to about 80% reduction in inflammation compared to a non-cross-dressed organ. In some embodiments, the cross-dressed organ exhibits about 80% to about 85% reduction in inflammation compared to a non-cross-dressed organ. In some embodiments, the cross-dressed organ exhibits about 85% to about 90% reduction in inflammation compared to a non-cross-dressed organ. In some embodiments, the cross-dressed organ exhibits at least a 90% reduction in inflammation compared to a non-cross-dressed organ.

[0020] In some embodiments, the human recipient exhibits a reduction in systemic inflammation after transplantation compared to transplantation with a non-cross-dressed organ. In some embodiments, the human recipient exhibits about a 5% to about 25% reduction in systemic inflammation after transplantation compared to transplantation with a non-cross-dressed organ. In some embodiments, the human recipient exhibits about a 25% to about 50% reduction in systemic inflammation after transplantation compared to transplantation with a non-cross-dressed organ. In some embodiments, the human recipient exhibits about a 50% to about 75% reduction in systemic inflammation after transplantation compared to transplantation with a non-cross-dressed organ. In some embodiments, the human recipient exhibits about a 75% to about 80% reduction in systemic inflammation after transplantation compared to transplantation with a non-cross-dressed organ. In some embodiments, the human recipient exhibits about an 80% to about 85% reduction in systemic inflammation after transplantation compared to transplantation with a non-cross-dressed organ. In some embodiments, the human recipient exhibits about an 85% to about 90% reduction in systemic inflammation after transplantation compared to transplantation with a non-cross-dressed organ. In some embodiments, the human recipient exhibits at least a 90% reduction in systemic inflammation after transplantation compared to transplantation with a non-cross-dressed organ.

[0021] In some embodiments, the organ is a kidney. In some embodiments, the organ is a lung. In some embodiments, the human recipient suffers from renal failure.

[0022] In some embodiments, the human recipient requires less immunosuppressive therapy than the standard of care in a comparable clinical environment. In some embodiments, the human recipient requires 10-20% less immunosuppressive therapy than the standard of care in a comparable clinical environment. In some embodiments, the human recipient requires 20-30% less immunosuppressive therapy than the standard of care in a comparable clinical environment. In some embodiments, the human recipient requires 30-40% less immunosuppressive therapy than the standard of care in a comparable clinical environment. In some embodiments, the human recipient requires 40-50% less immunosuppressive therapy than the standard of care in a comparable clinical environment. In some embodiments, the human recipient requires 50-60% less immunosuppressive therapy than the standard of care in a comparable clinical environment. In some embodiments, the human recipient requires 60-70% less immunosuppressive therapy than the standard of care in a comparable clinical environment. In some embodiments, the human recipient requires 70-80% less immunosuppressive therapy than the standard of care in a comparable clinical environment. In some embodiments, the human recipient requires 80-90% less immunosuppressive therapy than the standard of care in a comparable clinical environment. In some embodiments, the human recipient requires at least 90% less immunosuppressive therapy than the standard of care in a comparable clinical setting.

[0023] In some embodiments, the method results in a reduction in proteinuria. In some embodiments, proteinuria is reduced to less than 3 g per 24 hours. In some embodiments, proteinuria is reduced to 500 mg per 24 hours. In some embodiments, proteinuria is reduced to 300 mg per 24 hours. In some embodiments, proteinuria is reduced to 150 mg per 24 hours.

[0024] In some embodiments, the proteinuria disappears within 2 weeks of transplantation. In some embodiments, the proteinuria disappears within 1 month of transplantation. In some embodiments, the proteinuria disappears within 2 months of transplantation. In some embodiments, the proteinuria disappears within 4 months of transplantation.

[0025] In some embodiments, the method further comprises transplanting bone marrow tissue into the recipient. In some embodiments, the bone marrow is harvested from the same pig as the kidney. In some embodiments, the bone marrow is harvested from a different pig than the kidney. In some embodiments, the bone marrow is cross-dressed with human CD47 by exposure to EVs.

[0026] In some embodiments, the organ does not express human SIRPα. [Brief description of the drawings]

[0027] 7. Brief description of the drawings [Figure 1A-C] Cross-dressing of porcine LCLs (A), with transgenic hCD47 (B), and after co-culture with hCD47-Tg LCL cells (C). [Diagram 2] Cross-dressing of porcine LCL and with transgenic hCD47 after co-culture with hCD47-Tg LCL cells. [Figure 3A-B] (A) Cross-dressing of human Jurkat cells with transgenic hCD47 after coculture with hCD47-Tg LCL cells (B). [Figure 4A-C] Cross-dressing of hCD47KO Jurkat cells (A), with native hCD47 (B), and after coculture with WT Jurkat cells (C). [Figure 5A-B] Porcine LCLs (A) after coculture with WT Jurkat cells (B) and cross-dressing with native hCD47. [Figure 6]CD47 expression on WT Jurkat cells, porcine LCL / CD47p / h cells, CD47KO Jurkat cells, CD47KO cells mixed with WT Jurkat cells (mixed at time of staining), CD47KO Jurkat cells co-cultured with WT Jurkat or porcine hCD47-Tg LCL cells (24 h), porcine LCL cells, and LCL cells co-cultured with WT Jurkat cells (24 h). Numbers in the figure indicate the mean fluorescence intensity (MFI) of CD47 staining for gated CD47KO Jurkat and porcine LCL cells. [Figure 7A-D] Measurement of CD47 cross-dressing by extracellular vesicles (C) or exosomes (D) from CD47KO Jurkat cells (A), WT Jurkat cells (B) after 2 hours. [Figure 8A-D] Measurement of CD47 cross-dressing by extracellular vesicles (C) or exosomes (D) from CD47KO Jurkat cells (A), WT Jurkat cells (B) after 6 hours. [Figure 9A-D] Measurement of CD47 cross-dressing by porcine LCL cells (A), extracellular vesicles (C) or exosomes (D) from WT Jurkat cells after 2 hours. [Figure 10A-D] Measurement of CD47 cross-dressing by porcine LCL cells (A), extracellular vesicles (C) or exosomes (D) from WT Jurkat cells after 6 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] 8. Detailed Description of the Invention Provided herein are extracellular vesicles ("EVs", e.g., exosomes) bearing CD47 and compositions comprising same. Such CD47-bearing EVs can be used to cross-dress tissues, allowing such tissues to avoid phagocytic clearance by macrophages and other phagocytes. Such methods and compositions can be used in xenotransplantation. Methods for making EVs are described in Section 6.1. Compositions comprising the resulting EVs are described in Section 6.2. Methods for using EVs to cross-dress tissues are described in Section 6.3. Use of such tissues in xenotransplantation is described in Section 6.4.

[0029] As used herein, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where integers are required or expected, and in the case of percentages, the scope of the term is understood to include rounding up to the next integer, and rounding down to the next integer. For clarity, use herein of phrases such as "about X" and "at least about X" is understood to include "X" and specifically recite X.

[0030] As used herein, the term "extracellular vesicles (EVs)" generally refers to lipid membrane-encapsulated vesicles secreted by cells into the extracellular space, and includes, but is not limited to, exosomes and / or microvesicles.

[0031] As used herein, the term "exosomes" generally refers to a subset of EVs that are typically small in size (e.g., 30-150 nm in diameter) compared to other EVs, such as microvesicles.

[0032] As used herein, the term "cross-dressing" generally refers to the expression of a transgenic protein (e.g., CD47) in a cell, induced, for example, by incubating the cell with a cell or EV expressing the protein.

[0033] 8.1 How to generate EVs 8.1.1.EV Extracellular vesicles (EVs) are lipid bilayer encapsulated membranes released by cells into the extracellular environment. Examples of EVs include exosomes, microvesicles (MVs), and apoptotic bodies. See, for example, Carnino et al. Respiratory Research (2019) 20:240. In certain embodiments, the EVs comprise exosomes. In some embodiments, the EVs consist of exosomes. In certain embodiments, the EVs comprise MVs. In some embodiments, the EVs consist of MVs.

[0034] In certain embodiments, the EV is about 20 nm to about 2,000 nm. In some embodiments, the EV is about 20 nm to about 1,500 nm. In some embodiments, the EV is about 20 nm to about 1,000 nm. In some embodiments, the EV is about 20 nm to about 500 nm. In some embodiments, the EV is about 20 nm to about 250 nm. In some embodiments, the EV is about 20 nm to about 200 nm. In some embodiments, the EV is about 20 nm to about 150 nm. In some embodiments, the EV is about 50 nm to about 150 nm. In some embodiments, the EV is about 50 nm to about 1,500 nm. In some embodiments, the EV is about 50 nm to about 1,000 nm. In some embodiments, the EV is about 50 nm to about 500 nm.

[0035] Exosomes are one exemplary type of EV suitable for use in the present disclosure.Various markers for characterizing exosomes are known in the art, including but not limited to Alix, Tsg101, tetraspanins (e.g., CD63, CD81, CD82, CD53, and CD37), and flotillin.MVs are another exemplary type of EV suitable for use in the present disclosure, and the common protein markers used to define these vesicles include but are not limited to selectins, integrins, and CD40 ligand.

[0036] 8.1.2.EV Source Provided herein is an EV that comprises CD47, for example, human CD47. In some embodiments, the CD47 that the EV comprises is native to the cell that releases the EV. In other examples, the CD47 is not native to the cell that releases the EV (e.g., transgenic CD47). In a preferred embodiment, the CD47 is transgenic human CD47.

[0037] Many cell types release EVs, and EVs can carry various types of cargo, such as nucleic acids, proteins, and lipids, released by host cells. The EVs provided herein can be released by cell lines in culture or by primary cells in culture. In exemplary embodiments, the EVs are released from human cells, e.g., human primary cells in culture. In certain embodiments, the EVs provided herein are released from human cells expressing transgenic CD47. In other specific embodiments, the EVs provided herein are released from human cancer cells, e.g., human cancer cells that overexpress CD47 (e.g., Jurkat leukemia cells). The EVs provided herein can be released from cells that naturally express human CD47 or from cells that have been modified to recombinantly express human CD47, e.g., modified cells described in Section 6.1.3 below. In certain embodiments, the EVs provided herein are released from cells modified to overexpress human CD47, e.g., modified cells described in Section 6.1.3 below. In certain embodiments, the EVs provided herein are released from cells modified to inducibly express human CD47, such as cells modified as described in Section 6.1.3 below. In certain embodiments, the EVs provided herein are isolated from biological fluids, such as blood.

[0038] In some embodiments, cells releasing EVs provided herein can be treated with an agent that enhances EV release. Agents that enhance the release of EVs from cells are well known in the art, see, for example, Deng et al., Theranostics 2021, 11(9):4351-4362, Wang et al. Cells 2020, 9(3):660, and Nakamura et al., Molecular Therapy 28(10):2203-2219 October 2020. In certain embodiments, the agent that enhances EV release is ultrasound, adiponectin, norepinephrine, forskolin, fenoterol, methyldopamine, or mephenesin.

[0039] 8.1.3. Methods for generating transgenic cell lines releasing EVs Also provided herein are transgenic cells (e.g., primary cells or cell line cells) expressing CD47, which release EVs carrying CD47. The amino acid sequence of human CD47 can be found in the following NCBI Reference Sequence (RefSeq) Accession Numbers: NP_001768, NP_001369235.1, NP_942088, and XP_005247966.1. The nucleic acid sequence encoding human CD47 can be found in the following NCBI RefSeq Accession Numbers: NM_001777, NM_198793, XM_005247909.2, and NM_001382306.1. Any known splice variant of CD47 can be used to generate the transgenic cell lines provided herein. Non-limiting examples of amino acid and nucleotide sequences of human CD47 are provided in Table 1.

[0040] Provided herein is a vector (e.g., an expression vector) comprising a polynucleotide comprising a nucleotide sequence encoding CD47, e.g., human CD47. The vector can include a viral vector (e.g., adeno-associated virus (AAV), self-complementary adeno-associated virus (scAAV), adenovirus, retrovirus, lentivirus (e.g., simian immunodeficiency virus, human immunodeficiency virus, or modified human immunodeficiency virus), Newcastle disease virus (NDV), herpes virus (e.g., herpes simplex virus), alphavirus, vaccinia virus, etc.), plasmid, or other vector (e.g., non-viral vector, such as lipoplex, liposome, polymersome, or nanoparticle).

[0041] 8.1.3.1 Methods for generating transgenic cell lines Transgenic cells (including primary cells or cell line cells) can be produced using any method known in the art or provided herein.

[0042] The transgenic cell lines provided herein can be engineered to express CD47 (e.g., human CD47) using homologous recombination (HR) between cellular DNA and exogenous DNA (e.g., a DNA construct, vector, etc.) introduced into the cell. Alternatively, in some embodiments provided herein, the human CD47 transgene, along with all of its necessary regulatory sequences, is introduced into the cell line, e.g., as a human artificial chromosome.

[0043] Sequence-specific insertion (or knock-in) of a human CD47 transgene into the genome of a cell line can also be achieved by sequence-specific endonucleases combining constructs containing the human CD47 transgene with homologous recombination (HR) at the targeted chromosomal locus. Meyer et al., 2010, Proc. Natl. Acad. Sci. USA 107, 15022-15026. Cui et al., 2010, Nat. Biotechnol. 29:64-67. Moehle et al., 2007, Proc Natl Acad Sci USA 104:3055-3060.

[0044] Another example of sequence-specific endonucleases includes RNA-guided DNA nucleases, such as the CRISPR / Cas system. The Cas9 / CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats) system utilizes RNA-guided DNA binding and sequence-specific cleavage of target DNA. A guide RNA (gRNA) (e.g., containing 20 nucleotides) is complementary to the target genomic DNA sequence and constant RNA scaffold region upstream of the genomic PAM (protospacer adjacent motif) site (NNG). Cas (CRISPR-associated) protein binds to the gRNA and the target DNA to which the gRNA binds, and introduces a double-stranded break at a defined position upstream of the PAM site. Geurts et al.,2009,Science 325:433;Mashimo et al.,2010,PLoS ONE 5,e8870,Carbery et al.,2010,Genetics 186:451-459,Tesson et al.,2011,Nat.Biotech.29:695-696. Wiedenheft et al.Nature 2012,482:331-338, Jinek et al.Science,2012,337:816-821, Mali et al.,2013,Science 339:823-826, Cong et al.2012,Science 339:819-823.

[0045] The sequence-specific endonucleases of the methods and compositions described herein can be engineered, chimerized, or isolated from an organism. Endonucleases can be engineered to recognize specific DNA sequences, for example, by mutagenesis. Seligman et al. (2002) Nucleic Acids Research 30:3870-3879. Combinatorial assembly is a method by which protein subunits from different enzymes can be associated or fused. Amould et al. (2006) Journal of Molecular Biology 355:443-458. In certain embodiments, these two approaches, mutagenesis and combinatorial assembly, can be combined to produce engineered endonucleases with desired DNA recognition sequences.

[0046] The sequence-specific nuclease can be introduced into cells in the form of protein or in the form of nucleic acid encoding the sequence-specific nuclease, such as mRNA or cDNA.Nucleic acid can be delivered directly as part of a larger construct, such as a plasmid or viral vector, or by, for example, electroporation, lipid vesicle, viral transporter, microinjection, and biolistic methods.Similarly, the construct containing one or more transgenes can be delivered by any method suitable for introducing nucleic acid into cells.

[0047] In some embodiments, the transgenic cell lines provided herein inducibly express human CD47. Numerous inducible promoters and gene expression systems are known in the art. For example, promoters can be induced by chemicals, such as tetracycline, tamoxifen, or cumate. Gene expression can also be controlled by protein-protein interactions (e.g., the interaction between FKBP12 and mTOR, which is controlled by rapamycin). See, e.g., Kallunki et al. (2019), Cells 8:796.

[0048] In one embodiment, a sequence-specific recombination system can be used to achieve conditional knockout of a target gene. Recombinase is an enzyme that recognizes specific polynucleotide sequences (recombinase recognition sites) adjacent to an intervening polynucleotide, catalyzes mutual strand exchange, and results in inversion or excision of the intervening polynucleotide. Araki et al., 1995, Proc. Natl. Acad. Sci. USA 92:160-164.

[0049] In one embodiment, for conditional knockout of a target gene in a cell, the Cre-loxP system can be used, which involves targeted integration (knock-in) of loxP sites via homologous recombination (HR) and expression of an inducible Cre recombinase.

[0050] In another embodiment, conditional expression of a transgene (e.g., encoding a recombinase, or a human CD47 transgene) can be achieved by using a regulatory sequence that can be induced or inactivated by an exogenous stimulus. For example, a sequence-specific recombination system of a conditional knockout allele can be regulated, for example, by making the activity of the recombinase inducible by a chemical (drug). The chemical can activate transcription of the Cre recombinase gene or activate transport of the Cre recombinase protein to the nucleus. Alternatively, the recombinase can be activated by the absence of an administered drug, but not by the presence of the administered drug. Non-limiting examples of chemicals that regulate an inducible system (and thus induce, for example, a conditional knockout) include tetracycline, tamoxifen, RU-486, doxycycline, etc. Nagy A (Feb 2000), Cre recombinase: the universal reagent for genome tailoring, Genesis, 26(2):99-109. See, for example, the conditional knockout and knock-in constructs described in US Patent Application Serial No. 15 / 558,789.

[0051] 8.1.3.2 Extrachromosomal expression of EVs In certain embodiments, provided herein are methods of producing EVs in which CD47 is expressed from extrachromosomal DNA. Extrachromosomal DNA is DNA that is not integrated into host chromosomal DNA. Non-limiting examples of extrachromosomal DNA include plasmids and circular extrachromosomal DNA. In eukaryotic cells, extrachromosomal DNA can be found inside or outside the nucleus. For example, a host cell can be transfected with a vector encoding human CD47 (e.g., a vector described in section 6.1.3 above), and the human CD47 protein is expressed from the vector without being integrated into the host DNA.

[0052] 8.1.3.3 Isolation of EVs from cells EVs can be isolated from cells (e.g., transgenic cells expressing CD47) using any method known in the art or described herein. See, e.g., Carnino et al. Respiratory Research (2019) 20:240.

[0053] For example, EVs can be isolated by fractional centrifugation of cell culture supernatant. In an exemplary protocol, the cell supernatant is centrifuged at 2,000g (3,000 rpm) for 20 minutes to remove cell debris and dead cells. The EVs are then purified by centrifugation at 16,500g (9,800 rpm) for 45 minutes. Exosomes can be obtained by a similar protocol, where the cell supernatant is centrifuged at 2,000g (3,000 rpm) for 20 minutes to remove cell debris and dead cells, and then the exosomes are isolated by centrifugation at 100,000g (26,450 rpm) for approximately 2 to 16 hours.

[0054] EVs, including exosomes, can also be purified using gradient density centrifugation. In this method, EVs are separated based on their buoyant density in a solution of either sucrose, iohexol, or iodixanol. Other examples of methods used to isolate EVs, such as exosomes, include precipitation with organic solvents (e.g., polyethylene glycol, sodium acetate, or protamine), immunoprecipitation, separation using antibody-coated magnetic beads (e.g., anti-CD63-coated magnetic beads), microfluidic devices, and ultrafiltration. For example, see Carnino et al. Respiratory Research (2019) 20:240, and Momen-Heravi et al. Biol. Chem. 2013;394(10):1253-1262 for exemplary protocols. Further exemplary methods are isolation using heparin-conjugated agarose beads (see, e.g., Balaj et al. (2015) Sci Rep 5, 10266) and purification using Tim4 affinity purification (see, e.g., Nakai et al. (2016) Sci Rep 6, 33935).

[0055] Additionally, commercially available kits for isolation of EVs are available and can be used to isolate the EVs provided herein. Non-limiting examples include the exoEasy Kit (Qiagen), ExoQuick® Kit (Systems Bioscience), Total Exosome Isolation Reagent (ThermoFisher Scientific), and EasySep™ Human Pan-Extracellular Vesicle Positive Selection Kit (Stem Cell Technologies).

[0056] In certain embodiments, the EVs provided herein are isolated or purified. The EVs provided herein can be purified using any method known in the art or provided herein. As used herein, "isolated" or "purified" EVs are substantially free of cellular material, particulates, or other contaminants (e.g., organelles, lipids, cholesterol) from the cell or tissue source from which the EVs are derived. In certain embodiments, the EVs provided herein are of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% purity. In certain embodiments, the EVs provided herein are of greater than 99% purity. Purity can be determined, for example, by measuring particle size using dynamic light scattering or single particle tracking analysis, or by techniques such as flow cytometry, ELISA, or electron microscopy. See, e.g., Balaj et al. (2015) Sci Rep 5, 10266, Nakai et al. (2016) Sci Rep 6, 33935, and Carnino et al. (2019) Respiratory Research 20:240.

[0057] 8.1.3.4 Assays for detecting mRNA levels Several methods of detecting or quantifying mRNA levels are known in the art. Exemplary methods include, but are not limited to, Northern blot, ribonuclease protection assay, PCR-based methods (e.g., quantitative PCR), RNA sequencing, Fluidigm® analysis, and the like. Human CD47 mRNA sequence can be used to prepare a probe that is at least partially complementary to the mRNA sequence. The probe can then be used to detect mRNA in a sample using any suitable assay, such as PCR-based methods, Northern blotting, dipstick assay, TaqMan™ assay, and the like.

[0058] In other embodiments, a nucleic acid assay can be provided for testing human CD47 expression in a biological sample. The assay typically contains a solid support and at least one nucleic acid contacted to the support, the nucleic acid corresponding to at least a portion of an mRNA. The assay can also have a means for detecting changes in expression of the mRNA in the sample. The assay method can vary depending on the type of mRNA information desired. Exemplary methods include, but are not limited to, Northern blots and PCR-based methods (e.g., qRT-PCR). Methods such as qRT-PCR can also accurately quantify the amount of mRNA in a sample.

[0059] A typical mRNA assay method can include the steps of (1) obtaining a surface-bound probe of interest, (2) hybridizing a population of mRNA to the surface-bound probe under conditions sufficient to provide specific binding, (3) washing after hybridization to remove nucleic acids that do not specifically bind to the surface-bound probe, and (4) detecting the hybridized mRNA. The reagents used in each of these steps and the conditions used can vary depending on the particular application.

[0060] Other methods, such as PCR-based methods, can also be used to detect the expression of human CD47. An example of a PCR method can be found in U.S. Patent No. 6,927,024, which is incorporated herein by reference in its entirety. An example of a RT-PCR method can be found in U.S. Patent No. 7,122,799, which is incorporated herein by reference in its entirety. A method of in situ fluorescent PCR is described in U.S. Patent No. 7,186,507, which is incorporated herein by reference in its entirety.

[0061] In some embodiments, quantitative reverse transcription-PCR (qRT-PCR) can be used to both detect and quantify RNA targets (Bustin et al., Clin. Sci. 2005, 109:365-379). In some embodiments, qRT-PCR-based assays can be useful for measuring mRNA levels in cell-based assays. Examples of qRT-PCR-based methods can be found, for example, in U.S. Patent No. 7,101,663, which is incorporated herein by reference in its entirety.

[0062] In contrast to conventional reverse transcriptase-PCR and agarose gel analysis, qRT-PCR gives quantitative results. An additional advantage of qRT-PCR is its relative ease and convenience of use. Instruments for qRT-PCR, such as the Applied Biosystems 7500, are commercially available, as are reagents, such as TaqMan® sequence detection chemistry. For example, TaqMan® Gene Expression Assays can be used according to the manufacturer's instructions. These kits are pre-formulated gene expression assays for rapid and reliable detection and quantification of human, mouse, and rat mRNA transcripts. An exemplary qRT-PCR program is, for example, 50° C. for 2 minutes, 95° C. for 10 minutes, 40 cycles of 95° C. for 15 seconds, then 60° C. for 1 minute.

[0063] 8.1.3.5 Assays for detecting polypeptide or protein levels Several protein detection and quantification methods can be used to measure the level of human CD47. Any suitable protein quantification method can be used. In some embodiments, an antibody-based method is used. Exemplary methods that can be used include, but are not limited to, immunoblotting (Western blot), ELISA, immunohistochemistry, immunofluorescence, flow cytometry, cytometric bead array, mass spectrometry, and the like. Several types of ELISA are commonly used, including direct ELISA, indirect ELISA, and sandwich ELISA.

[0064] 8.2 EV composition Provided herein are compositions comprising EVs described herein, e.g., CD47-bearing EVs, such as CD47-bearing exosomes ("EV compositions"). Purified EVs can be cryopreserved, e.g., by freezing the EVs in the presence of a cryoprotectant, lyophilized or spray-dried. EVs can be stabilized by using hydrophilic polymers (e.g., polyethylene glycol) or scaffolds (e.g., scaffolds that include components of the extracellular matrix to which EVs bind in vivo). See, e.g., Kusuma et al. (2018) Front. Pharmacol., 9:1199.

[0065] The EV composition provided herein may vary in CD47 content. In certain embodiments, the EV comprises CD47 mRNA. The level of human CD47 mRNA can be determined by any suitable method known in the art, for example, the method described in section 6.1.3.4. In certain embodiments, the EV comprises CD47 polypeptide or protein. The level of human CD47 protein can be determined using any suitable method known in the art, for example, the method described in section 6.1.3.5.

[0066] Thus, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the EVs present in one unit of the EV composition express human CD47. Similarly, human CD47 may account for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the total membrane-associated protein in the EV composition.

[0067] The EV compositions provided herein may further comprise a suitable carrier, e.g., a pharma- ceutically acceptable carrier. In general, a "pharma- ceutically acceptable" carrier is a material that is not biological or undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects, such as toxicity. Exemplary pharma- ceutically acceptable carriers include, but are not limited to, aqueous solvents (e.g., water; balanced salt solutions, such as phosphate-buffered saline (PBS), Hank's balanced salt solution (HSB), Earle's balanced salt solution (EBSS); and cell culture media), as well as non-aqueous solvents (e.g., fats, oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), vegetable oils, and injectable organic esters, such as ethyl oleate). Carriers include liquid, semi-solid, e.g., pastes, or solid carriers. In addition, if necessary, the composition may contain minor amounts of auxiliary substances, such as wetting agents, or emulsifying agents, stabilizing agents, or pH buffering agents. The pH and exact concentration of various components in the pharmaceutical composition are adjusted according to well-known parameters. Thus, the EV of the present invention can be formulated according to known techniques for administration in a pharma-ceutically acceptable carrier. See, for example, Remington, The Science And Practice of Pharmacy (22nd Ed. 2012).

[0068] In some embodiments, the composition may contain 0.01% to 99% w / v EVs. In some embodiments, the composition may contain 0.05% to 99% w / v EVs. In some embodiments, the composition may contain 0.1% to 99% w / v EVs. In some embodiments, the composition may contain 0.5% to 99% w / v EVs. In some embodiments, the composition may contain 1% to 99% w / v EVs. In some embodiments, the composition may contain 5% to 99% w / v EVs. In some embodiments, the composition may contain 10% to 99% w / v EVs. In some embodiments, the composition may contain 15% to 99% w / v EVs. In some embodiments, the composition may contain 20% to 99% w / v EVs. In some embodiments, the composition may contain 25% to 99% w / v EVs. In some embodiments, the composition may contain 30% to 99% EVs by weight / volume. In some embodiments, the composition may contain 35% to 99% EVs by weight / volume. In some embodiments, the composition may contain 40% to 99% EVs by weight / volume. In some embodiments, the composition may contain 50% to 99% EVs by weight / volume. In some embodiments, the composition may contain 55% to 99% EVs by weight / volume. In some embodiments, the composition may contain 60% to 99% EVs by weight / volume. In some embodiments, the composition may contain 70% to 99% EVs by weight / volume. In some embodiments, the composition may contain 75% to 99% EVs by weight / volume. In some embodiments, the composition may contain 80% to 99% EVs by weight / volume. In some embodiments, the composition may contain 85% to 99% EVs by weight / volume. In some embodiments, the composition may contain 90% to 99% w / v EVs, In some embodiments, the composition may contain 95% to 99% w / v EVs.In some embodiments, the composition may comprise EVs, such as 100% lyophilized EVs.

[0069] The amount of EVs contained in the compositions provided herein can be easily determined by those skilled in the art. In some embodiments, the amount of EVs is sufficient to cross-dress xenografts. In certain embodiments, the amount of EVs is sufficient to cross-dress xenografts and reduce organ cell phagocytosis. In some embodiments, the amount of EVs is sufficient to cross-dress xenografts and reduce systemic inflammation in the recipient after transplantation.

[0070] In certain embodiments, the amount of EV is a quantified amount.Various methods for quantifying EV, including MV and exosome, are known in the art.For example, non-limiting exemplary methods for quantifying EV include electron microscopy (EM), surface plasmon resonance (SPR), flow cytometry, tunable resistive pulse sensing (TRPS), nanosite nanoparticle tracking analysis, protein-based methods, and enzyme-linked immunosorbent assay.

[0071] In some embodiments, the compositions provided herein comprise about 1.0×10 6 ~Approx. 1.0×10 15 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 7 ~Approx. 1.0×10 14 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 8 ~Approx. 1.0×10 13 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 9 ~Approx. 1.0×10 12 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 10 ~Approx. 1.0×10 11 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs.6 ~Approx. 1.0×10 10 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 6 ~Approx. 1.0×10 8 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 8 ~Approx. 1.0×10 15 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 8 ~Approx. 1.0×10 12 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 10 ~Approx. 1.0×10 15 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 12 ~Approx. 1.0×10 15 Includes EVs.

[0072] In some embodiments, the compositions provided herein comprise about 1.0×10 6 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 7 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 8 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 9 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 10 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 11 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 12 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 13 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 14 In some embodiments, the compositions provided herein comprise about 1.0×10 EVs. 15Includes EVs.

[0073] In some embodiments, the compositions provided herein comprise about 1.0 micrograms (μg) to about 100 grams (g) of EV protein. In some embodiments, the compositions provided herein comprise about 5.0 μg to about 50 g of EV protein. In some embodiments, the compositions provided herein comprise about 10.0 μg to about 10 g of EV protein. In some embodiments, the compositions provided herein comprise about 50.0 μg to about 5 g of EV protein. In some embodiments, the compositions provided herein comprise about 100.0 μg to about 1 g of EV protein. In some embodiments, the compositions provided herein comprise about 1.0 μg of EV protein. In some embodiments, the compositions provided herein comprise about 5.0 μg of EV protein. In some embodiments, the compositions provided herein comprise about 10.0 μg of EV protein. In some embodiments, the compositions provided herein comprise about 25.0 μg of EV protein. In some embodiments, the compositions provided herein comprise about 50.0 μg of EV protein. In some embodiments, the compositions provided herein comprise about 100.0 μg of EV protein. In some embodiments, the compositions provided herein comprise about 250.0 μg of EV protein. In some embodiments, the compositions provided herein comprise about 500.0 μg of EV protein. In some embodiments, the compositions provided herein comprise about 1.0 mg of EV protein. In some embodiments, the compositions provided herein comprise about 5.0 mg of EV protein. In some embodiments, the compositions provided herein comprise about 10.0 mg of EV protein. In some embodiments, the compositions provided herein comprise about 25.0 mg of EV protein. In some embodiments, the compositions provided herein comprise about 50.0 mg of EV protein. In some embodiments, the compositions provided herein comprise about 100.0 mg of EV protein. In some embodiments, the compositions provided herein comprise about 250.0 mg of EV protein.In some embodiments, the compositions provided herein comprise about 500.0 mg of EV protein. In some embodiments, the compositions provided herein comprise about 1.0 g of EV protein. In some embodiments, the compositions provided herein comprise about 5.0 g of EV protein. In some embodiments, the compositions provided herein comprise about 10.0 g of EV protein. In some embodiments, the compositions provided herein comprise about 25.0 g of EV protein. In some embodiments, the compositions provided herein comprise about 50.0 g of EV protein. In some embodiments, the compositions provided herein comprise about 100.0 g of EV protein. In some embodiments, the compositions provided herein comprise about 250.0 g of EV protein. In some embodiments, the compositions provided herein comprise about 500.0 g of EV protein.

[0074] In some embodiments, the amount of EVs included in the composition is relative to the amount of cells from which the EVs are generated. For example, in some embodiments, the amount of EVs is about 1.0×10 cells cultured for more than 48 hours. 6 ~Approx. 1.0×10 10 In some embodiments, the amount of EVs is about 1.0 x 10 cells cultured for about 48 hours. 6 ~Approx. 1.0×10 10 In some embodiments, the amount of EVs is about 1.0 x 10 cells cultured for about 24 hours. 6 ~Approx. 1.0×10 10 In some embodiments, the amount of EVs is about 1.0 x 10 cells cultured for about 16 hours. 6 ~Approx. 1.0×10 10 In some embodiments, the amount of EVs is about 1.0 x 10 cells cultured for about 12 hours. 6 ~Approx. 1.0×10 10 In some embodiments, the amount of EVs is about 1.0 x 10 cells cultured for less than 12 hours. 6 ~Approx. 1.0×10 10This is the amount harvested from cells.

[0075] In some embodiments, the amount of EVs is about 1.0 x 10 8 ~Approx. 1.0×10 10 In some embodiments, the amount of EVs is about 1.0 x 10 cells cultured for about 24 hours. 8 ~Approx. 1.0×10 10 In some embodiments, the amount of EVs is about 1.0 x 10 cells cultured for about 16 hours. 8 ~Approx. 1.0×10 10 In some embodiments, the amount of EVs is 1.0 x 10 cells cultured for about 12 hours. 6 ~Approx. 1.0×10 10 In some embodiments, the amount of EVs is 1.0 x 10 cells cultured for less than 12 hours. 8 ~Approx. 1.0×10 10 This is the amount harvested from cells.

[0076] In some embodiments, the amount of EVs is about 1.0 x 10 6 ~Approx. 1.0×10 8 In some embodiments, the amount of EVs is 1.0 x 10 cells cultured for about 24 hours. 6 ~Approx. 1.0×10 8 In some embodiments, the amount of EVs is 1.0 x 10 cells cultured for about 16 hours. 6 ~Approx. 1.0×10 8 In some embodiments, the amount of EVs is 1.0 x 10 cells cultured for about 12 hours. 6 ~Approx. 1.0×10 8 In some embodiments, the amount of EVs is 1.0 x 10 cells cultured for less than 12 hours. 6 ~Approx. 1.0×10 8 This is the amount harvested from cells.

[0077] In some embodiments, the amount of EVs is about 1.0×10 5 In some embodiments, the amount of EVs is about 1.0 x 10 6 In some embodiments, the amount of EVs is about 1.0 x 10 7 In some embodiments, the amount of EVs is about 1.0 x 10 8 In some embodiments, the amount of EVs is about 1.0 x 10 9 In some embodiments, the amount of EVs is about 1.0 x 10 10 In some embodiments, the amount of EVs is about 1.0 x 10 11 In some embodiments, the amount of EVs is about 1.0 x 10 12 In some embodiments, the amount of EVs is about 1.0 x 10 13 In some embodiments, the amount of EVs is about 1.0 x 10 14 In some embodiments, the amount of EVs is about 1.0 x 10 15 This is the amount harvested from cells.

[0078] 8.3 How to use EV to cross-dress organizations As used herein, the term "cross-dressing" describes the expression of a transgenic protein (e.g., CD47) in a cell, e.g., induced by incubating the cell with a cell or EV expressing the protein. For example, porcine cells can be cross-dressed with human CD47 by exposing them to cells expressing human CD47, e.g., by co-incubation. In certain embodiments, the EVs provided herein are incubated with bone marrow tissue from a donor pig. In some embodiments, the EVs provided herein are incubated with a kidney from a donor pig. In some embodiments, the EVs provided herein are incubated with both bone marrow tissue and kidney from the same donor pig. In some embodiments, the EVs provided herein are incubated with a kidney from a first donor pig and bone marrow tissue from a second donor pig.

[0079] 8.4 Methods for using EVs in xenotransplantation Provided herein is a method of xenotransplantation, comprising cross-dressing the xenograft with CD47 (e.g., human CD47) before transplanting the xenograft into the target tissue. In certain embodiments, the target tissue is kidney tissue. In other embodiments, the target tissue is lung tissue. In some embodiments, the target tissue is human. In some embodiments, the target tissue does not express SIRPα (e.g., human SIRPα) as determined by methods known in the art (e.g., Western blotting, flow cytometry, or quantitative polymerase chain reaction). In certain aspects, CD47 cross-dressing is independent of signal regulatory protein alpha (SIRPα) expression on the target tissue.

[0080] 8.4.1. Methods for exposing xenografts to EVs In certain embodiments, cross-dressing is achieved by exposing the xenograft to EVs containing CD47. In other embodiments, cross-dressing is achieved by co-culturing the xenograft with a cell line (e.g., a transgenic cell line) expressing human CD47. In some embodiments, the xenograft is exposed to EVs in vitro. In some embodiments, the xenograft is exposed to EVs in vivo. For example, EVs such as exosomes can be injected via the retro-orbital sinus, tail vein, or intracardiac, or similar approaches to deliver EVs in vivo. In some embodiments, the xenograft is exposed to EVs ex vivo. In certain embodiments, the xenograft blood vessels are perfused with EVs in vivo. For example, the xenograft can be perfused in vivo in either the donor or the recipient, or both. In some embodiments, the xenograft blood vessels are perfused with EVs ex vivo.

[0081] In some embodiments, xenografts are exposed to EVs more than once. In some embodiments, xenografts are exposed to EVs 2, 3, 4, 5, 6, 7, 8, 9, or 10 times over the course of 1, 2, or 3 days. In some embodiments, xenografts are repeatedly exposed to the same EV composition. In some embodiments, xenografts are exposed to different EV compositions.

[0082] As provided herein, exposure of xenografts to EVs can occur before transplantation, after transplantation, or both. In some embodiments, xenografts are exposed to EVs before transplantation. In some embodiments, xenografts are exposed to EVs after transplantation. In some embodiments, xenografts are exposed to EVs before and after transplantation.

[0083] In some embodiments, the xenograft is exposed more than once after transplantation. For example, after transplantation, the recipient may be infused (e.g., intravenously) with EVs one or more times. In some embodiments, the recipient is infused with EVs daily. In some embodiments, the recipient is infused with EVs two or more times a day. In some embodiments, the recipient is infused.

[0084] In certain embodiments, the organ is directly infused with EVs in vivo before transplantation, after transplantation, or a combination thereof. For example, the organ may be directly perfused (e.g., via the hepatic portal vein) before transplantation. In some embodiments, the organ is directly perfused in vivo before transplantation. In some embodiments, the organ is directly perfused in vivo after transplantation. In some embodiments, the organ is directly perfused in vivo before and after transplantation. In some embodiments, the organ is directly perfused in vivo two or more times before transplantation. In some embodiments, the organ is directly perfused in vivo two or more times after transplantation. In some embodiments, the organ is directly perfused in vivo two or more times before and after transplantation.

[0085] Exposure of the xenograft to EVs can be for any time sufficient to allow the xenograft to cross-dress. In some embodiments, the xenograft is exposed to EVs for about 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, 7-8 hours, 8-9 hours, 9-10 hours, 10-11 hours, or 11-12 hours. In some embodiments, the xenograft is exposed to EVs for about 12-16 hours. In some embodiments, the xenograft is exposed to EVs for about 16-24 hours. In some embodiments, the xenograft is exposed to EVs for more than 24 hours.

[0086] In some embodiments, EVs are engineered to improve their delivery to specific organs or cell types. Various techniques for engineering EVs with targeting properties are known in the art (see, e.g., Murphy, DE, et al. Exp Mol Med 51, 1-12 (2019)). By way of example, in some embodiments, EVs express specific targeting peptides that improve targeting of the EV to its intended effector cells. Another exemplary targeting technique involves engineering EVs to express a combination of specific integrins that improve targeting of the EV to its intended effector cells. In some embodiments, the engineering is performed after EV production.

[0087] 8.4.2. Effect of exposure to EVs in xenografts In some embodiments, cross-dressing CD47 on a cell causes the cell to avoid phagocytosis. In certain embodiments, cross-dressing CD47 on a cell causes the cell to avoid phagocytosis without inducing apoptosis.

[0088] In certain embodiments, cells cross-dressed with CD47 generated according to the present disclosure, such as by the methods described in Section 6.4, express CD47 with reduced ligation to CD47 ligands (e.g., thrombospondin (TSP-1)) compared to cells expressing CD47 that have not been cross-dressed with CD47 from EVs. In some embodiments, cells cross-dressed with CD47 generated according to the present disclosure, such as by the methods described in Section 6.4, express CD47 with no or undetectable levels of ligation to CD47 ligands (e.g., thrombospondin (TSP-1) or SIRPα) compared to cells expressing CD47 that have not been cross-dressed with CD47 from EVs.

[0089] For example, binding of CD47 to TSP-1 can cause inflammation and death in cells expressing CD47. However, the present disclosure is based in part on the finding that cells cross-dressed with CD47 do not transmit apoptotic signaling. Conversely, cells that are not cross-dressed with CD47 and that endogenously or exogenously express CD47 undergo cell death and increased inflammation. Thus, in some embodiments, CD47 cross-dressed cells generated according to the present disclosure exhibit reduced cell death compared to cells expressing CD47 that are not cross-dressed with CD47 from EV. In certain embodiments, CD47 cross-dressed cells generated according to the present disclosure exhibit reduced cell death upon exposure to SIRPα, or fragments, chimeras, and / or fusions thereof, compared to cells expressing CD47 that are not cross-dressed with CD47 from EV. In certain embodiments, cells cross-dressed with CD47 generated according to the present disclosure exhibit reduced cell death upon exposure to approximately 50 nM human SIRPα-Fc for 1 hour compared to cells expressing CD47 that are not cross-dressed with CD47 from EVs and exposed to the same amount of SIRPα-Fc.

[0090] Phagocytosis can be determined by any method known in the art or described herein, for example, as described in Example 4. For example, cells can be labeled with Celltrace Violet and incubated with human macrophages. The level of phagocytosis can then be measured by using flow cytometry to determine the percentage of macrophages (CD14 positive cells) that phagocytose the labeled target cells. Provided herein is a method of cross-dressing a xenograft, comprising exposing the xenograft to EVs containing human CD47 prior to transplantation, wherein the method reduces the phagocytosis of the xenograft by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to a non-cross-dressed xenograft.

[0091] In certain embodiments, the xenograft methods provided herein result in a reduction in phagocytosis without inducing apoptosis. Apoptosis can be measured using any method known in the art or described herein. For example, apoptosis can be measured by staining cells with propidium iodide (PI) or PI and Annexin V. Apoptosis can be undetectable or reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to xenografts expressing allogeneic CD47.

[0092] Avoidance of phagocytosis can result in longer survival of cells, which can lead to extended chimerism. For example, cross-dressing of human CD47 on porcine bone marrow cells can allow porcine bone marrow cells to avoid phagocytosis after transplantation into human recipients, which leads to extended survival of porcine bone marrow cells. The extended survival of porcine bone marrow cells can then lead to extended chimerism, which can be beneficial in avoiding transplant rejection.

[0093] As provided herein, cross-dressing xenografts with EVs containing human CD47 prior to transplantation reduces xenograft inflammation. According to some embodiments, xenograft inflammation may be undetectable or may be reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to xenografts expressing allogeneic CD47.

[0094] Also provided herein is cross-dressing a xenograft with EVs containing human CD47 prior to transplantation to reduce systemic inflammation in the recipient. According to some embodiments, systemic inflammation in the recipient following xenograft transplantation may be undetectable or reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to following transplantation with a xenograft expressing allogeneic CD47.

[0095] Transplant rejection is a major problem for many recipients of xenografts, often requiring long-term administration of immunosuppressive therapy, and avoidance of phagocytosis may reduce xenograft rejection in the recipient. Thus, in one aspect, provided herein is a method for reducing xenograft rejection in a recipient, comprising exposing the xenograft to EVs containing human CD47 prior to transplantation.

[0096] In some embodiments, the methods result in a reduction in administration of immunosuppressive therapy to the recipient (e.g., about a 10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or greater than 90% reduction in administration) compared to xenograft recipients that have not been exposed to EVs containing human CD47 prior to transplantation. In certain embodiments, the methods result in a reduction in the administration of immunosuppressive therapy to the recipient (e.g., about a 10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or greater than 90% reduction in administration) compared to the amount of immunosuppressive therapy typically administered to a comparable recipient (e.g., a person of the same sex and comparable age, height, and / or weight who has received the same type of tissue or organ as the recipient) receiving a xenograft that has not been exposed to EVs containing human CD47. In certain embodiments, the methods result in a reduction in the administration of immunosuppressive therapy to the recipient (e.g., about a 10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or greater than 90% reduction in administration) compared to the standard of care in a comparable clinical setting. In some embodiments, the methods result in a reduction in the administration of immunosuppressive therapy to the recipient (e.g., about a 10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or greater than 90% reduction in administration) compared to the amount of immunosuppressive therapy required by such recipient after receiving a previous xenograft that was not exposed to EVs containing human CD47 prior to transplantation. In other embodiments, the methods result in a reduction in the administration of immunosuppressive therapy to the recipient (e.g., about a 10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or greater than 90% reduction in administration) compared to the required amount of immunosuppressive therapy in a comparable clinical setting where the xenograft in the comparable clinical setting has not been exposed to EVs containing human CD47 prior to transplantation. In some embodiments, the methods result in the recipient not requiring further administration of immunosuppressive therapy, e.g., the immunosuppressive therapy described in Section 6.4.3 below.

[0097] In some embodiments, the methods result in increased xenograft survival compared to xenografts that have not been exposed to EVs comprising human CD47 prior to transplantation. In some embodiments, the methods result in increased xenograft survival (e.g., increased survival of about 10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-75%, 75-100%, 100-200%, 200-300%, or greater than 300%, or an increase of 1-2 years, 2-3 years, 3-4 years, 4-5 years, 5-6 years, 6-8 years, 8-10 years, 10-15 years, or 15-20 years) compared to a comparable xenograft (e.g., a tissue or organ of the same type as the recipient) that has not been exposed to EVs comprising human CD47 prior to transplantation, transplanted into a comparable recipient (e.g., a patient of the same sex and comparable age, height, and / or weight). In some embodiments, the methods result in an increase in xenograft survival (e.g., an increase in survival of about 10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-75%, 75-100%, 100-200%, 200-300%, or greater than 300%, or an increase of 1-2 years, 2-3 years, 3-4 years, 4-5 years, 5-6 years, 6-8 years, 8-10 years, 10-15 years, or 15-20 years) compared to the survival of a xenograft previously received by the recipient that was not exposed to EVs comprising human CD47 prior to transplantation.

[0098] In some embodiments, the methods result in an increase in survival (e.g., an increase in survival of about 10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-75%, 75-100%, 100-200%, 200-300%, or greater than 300%, or an increase of 1-2 years, 2-3 years, 3-4 years, 4-5 years, 5-6 years, 6-8 years, 8-10 years, 10-15 years, or 15-20 years) compared to the survival of the xenograft in a comparable clinical setting in which the xenograft has not been exposed to EVs comprising human CD47 prior to transplantation.

[0099] In some embodiments, the method results in a better health-related quality of life for the recipient compared to a recipient of a xenograft that has not been exposed to EVs comprising human CD47 prior to transplantation. In other embodiments, the method results in a better health-related quality of life for the recipient compared to a comparable recipient (e.g., a person of the same sex and comparable age, height, and / or weight who has received the same type of tissue or organ as the recipient) who has received a xenograft that has not been exposed to EVs comprising human CD47 prior to transplantation. In other embodiments, the method results in a better health-related quality of life for the recipient compared to the health-related quality of life experienced by the recipient after a previous xenograft. In other embodiments, the method results in a better health-related quality of life for the recipient compared to a comparable clinical setting in which the xenograft has not been exposed to EVs comprising human CD47 prior to transplantation. Health-related quality of life refers to the overall impact of health aspects on an individual's quality of life and includes physical symptoms, functional status, psychological status, and social relationships. Health-related quality of life can be assessed by any means known in the art, including, for example, the 36-Item Short Form Survey (SF-36), EuroQol-5 Dimensions (EQ-5D), and the Kidney Disease Quality of Life Instrument (KDQOL). See, e.g., Parizi et al. The Patient-Patient-Centered Outcomes Research (2019) 12:171-181.

[0100] In some embodiments, the methods result in longer (e.g., 10-20%, 20-30%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% longer, or 2-3 fold, 3-5 fold, 5-7 fold, 7-10 fold, or 10-15 fold longer) survival of transplant recipients compared to xenograft recipients that have not been exposed to EVs containing human CD47 prior to transplantation. In other embodiments, the methods result in longer survival (e.g., 10-20%, 20-30%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% longer, or 2-3 times, 3-5 times, 5-7 times, 7-10 times, or 10-15 times longer) of a transplant recipient compared to the survival of a comparable recipient (e.g., a person of the same sex and comparable age, height, and / or weight who has received the same type of tissue or organ as the recipient) who has received a xenograft that has not been exposed to EVs containing human CD47. In other embodiments, the methods result in longer survival of the transplant recipient (e.g., 10-20%, 20-30%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% longer, or 2-3 fold, 3-5 fold, 5-7 fold, 7-10 fold, or 10-15 fold longer) compared to survival of the transplant recipient in a comparable clinical setting in which the xenograft is not exposed to EVs containing human CD47 prior to transplantation.

[0101] In one aspect, the method of transplantation described herein results in a reduction in the risk, severity, or duration of proteinuria. Protein excretion of more than 150 mg per day is generally used as a diagnosis of proteinuria. Dipstick analysis is often used to measure protein concentration in urine. It is a semi-quantitative method, and the results are expressed as negative, trace, 1+, 2+, 3+, or 4+. See, for example, Carroll and Temte, Am Fam Physician 62(6):1333-1340(2000). Only total protein or albumin levels may be measured to provide a quantitative test. Results may be expressed in total protein or albumin levels, or in albumin to creatine ratios or protein to creatine ratios.

[0102] In certain embodiments, the transplantation method described herein results in a reduction in the severity of proteinuria.In certain embodiments, the transplantation method described herein results in a reduction in the duration of proteinuria.For example, the severity of proteinuria in patients treated according to the method herein can be reduced compared to the severity of proteinuria observed in patients who receive donor kidneys that are not cross-dressed with human CD47.

[0103] In some embodiments, the severity of proteinuria, as measured by urinary protein levels, is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%. In some embodiments, patients treated according to the methods provided herein do not experience proteinuria, defined as excretion of more than 150 mg of protein per day in the urine. In some embodiments, patients treated according to the methods provided herein may experience transient proteinuria that resolves after 1, 2, 3, 3-7, 7-10, 10-14 days, or after 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8 weeks, or after 1, 2, 3, 4, 5, 6 months after transplantation.

[0104] In some embodiments, recipients treated with the methods described herein who develop proteinuria have a concentration of total protein in the urine of less than about 60 mg per day, less than about 80 mg per day, less than about 100 mg per day, less than about 120 mg per day, less than about 140 mg per day, less than about 160 mg per day, less than about 200 mg per day, less than about 220 mg per day, less than about 240 mg per day, less than about 260 mg per day, less than about 280 mg per day, less than about 300 mg per day, less than about 320 mg per day, less than about 340 mg per day, less than about 360 mg per day, less than about 380 mg per day, or less than about 400 mg per day.

[0105] In some embodiments, recipients treated with the methods described herein who develop proteinuria have a concentration of albumin in the urine of less than about 5 mg per day, less than about 10 mg per day, less than about 20 mg per day, less than about 30 mg per day, less than about 40 mg per day, less than about 50 mg per day, less than about 60 mg per day, less than about 70 mg per day, less than about 80 mg per day, less than about 90 mg per day, or less than about 100 mg per day.

[0106] In some embodiments, the ratio of protein to creatinine in a 24 hour urine sample of a patient treated according to the methods described herein is less than about 0.2, less than about 0.4, less than about 0.6, less than about 0.8, or less than about 1. In some embodiments, the ratio of albumin to creatinine in a 24 hour urine sample of a patient treated according to the methods described herein is less than about 0.02, less than about 0.04, less than about 0.06, less than about 0.08, or less than about 0.1.

[0107] In some embodiments, the risk of a recipient treated with the methods described herein developing proteinuria is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to the risk in a recipient of a donor kidney that has not been cross-dressed with human CD47.

[0108] 8.4.3. Additional Actions In certain embodiments, the patient treated according to the methods described herein receives additional treatment. The patient may receive additional treatment by one or more different methods. The additional treatment may be performed before, at the same time, or after the treatment method provided herein.

[0109] In certain embodiments, the patient receiving the xenograft according to the methods described herein undergoes intraosseous bone marrow transplantation (IBBM). In certain embodiments, the bone marrow is from the same source as the xenograft. In certain embodiments, the bone marrow expresses human CD47.

[0110] In some embodiments, patients receiving xenografts according to the methods described herein receive immunosuppressive therapy. Immunosuppressive therapy may be any FDA approved treatment shown to reduce transplant rejection and / or ameliorate xenograft outcomes. Non-limiting examples of immunosuppressive therapy include calcineurin inhibitors (e.g., tacrolimus or cyclosporine), antiproliferative agents (e.g., antimetabolites such as mycophenolate, 6-mercaptopurine or its prodrug azathioprine), inhibitors of mammalian target of rapamycin (mTOR) (e.g., sirolimus, rapamycin), steroids (e.g., prednisone), cell cycle inhibitors (azathioprine or mycophenolate mofetil), lymphocyte depleting agents (e.g., antithymocyte globulin, or antibodies such as alemtuzumab, cyprism, or basiliximab), and costimulatory blockers (e.g., belatacept). See, e.g., Chung et al. (2020)., Ann Transl Med. Mar;8(6):409, van der Mark et al. (2020), Eur Respir Rev;29:190132, and Benvenuto et al. (2018), J Thorac Dis 10:3141-3155.

[0111] Immunosuppressive therapy can be administered as induction therapy (perioperatively or immediately after surgery), as a maintenance dose, or for acute rejection. Induction therapy typically includes basiliximab, antithymocyte globulin, or alemtuzumab. Immunosuppressive therapy can also be administered as maintenance therapy, which is often required to continue for the recipient's lifetime. Maintenance immunosuppressive therapy typically includes calcineurin inhibitors (tacrolimus or cyclosporine), antiproliferative agents (mycophenolate or azathioprine), and corticosteroids. Immunosuppressive therapy for acute rejection typically includes thymoglobulin or mycophenolate. See, e.g., Chung et al. (2020), Ann Transl Med. Mar; 8: 409 and Benvenuto et al., (2018) J Thorac Dis 10: 3141-3155.

[0112] Non-limiting examples of immunosuppressants include: (1) antimetabolites, such as purine synthesis inhibitors (e.g., inosine monophosphate dehydrogenase (IMPDH) inhibitors, e.g., azathioprine, mycophenolate, and mycophenolate mofetil), pyrimidine synthesis inhibitors (e.g., leflunomide and teriflunomide), and antifolates (e.g., methotrexate); (2) calcineurin inhibitors, such as tacrolimus, cyclosporine A, pimecrolimus, and voclosporin; (3) thalides. (4) IL-1 receptor antagonists, such as anakinra; (5) mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin (sirolimus), deforolimus, everolimus, temsirolimus, zotarolimus, and biolimus A9; (6) corticosteroids, such as prednisone; and (7) antibodies against any one of a number of cellular or serum targets, including antilymphocyte globulin and antithymocyte globulin.

[0113] Non-limiting exemplary cellular targets and their respective inhibitor compounds include complement component 5 (e.g., eculizumab), tumor necrosis factor (TNF) (e.g., infliximab, adalimumab, certolizumab pegol, afelimomab, and golimumab), IL-5 (e.g., mepolizumab), IgE (e.g., omalizumab), BAYX (e.g., nerelimomab), interferon (e.g., faralimomab), IL-6 (e.g., ersilimomab), IL-12 and IL-13 (e.g., lebrikizumab and ustekinumab), CD3 (e.g., muromonab-CD3, otelixizumab, tepelizumab, visilizumab), CD4 (e.g., clenoliximab, keliximab, and zanolimumab), CDI la (e.g., efalizumab), CD18 (e.g., erlizumab), CD20 (e.g., afutuzumab, ocrelizumab, pascolizumab), CD23 (e.g., lumiliximab), CD40 (e.g., teneliximab, toralizumab), CD62L / L-selectin (e.g., acerizumab), CD80 (e.g., galiximab), CD147 / basigin (e.g., gavilimomab), CD154 (e.g., ruplizumab) , BLyS (e.g., belimumab), CTLA-4 (e.g., ipilimumab, tremelimumab), CAT (e.g., bertilimumab, lerdelimumab, metelimumab), integrins (e.g., natalizumab), IL-6 receptor (e.g., tocilizumab), LFA-1 (e.g., ozlimomab), and IL-2 receptor / CD25 (e.g., basiliximab, dacilizumab, inolimomab).

[0114] Patient population In preferred embodiments, the patient treated according to the methods described herein (e.g., a recipient of a xenograft cross-dressed with human CD47) is a human patient. As used herein, the terms "subject" and "patient" are used interchangeably and include any human or non-human mammal. Non-limiting examples include members of the human, horse, pig, cow, rat, mouse, dog, and cat species. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human. In certain embodiments, the subject is a human adult. In some embodiments, the subject is a human child. In certain embodiments, the subject is a human and receives one or more donor grafts from a porcine donor. In other specific embodiments, the subject is a non-human primate (e.g., a baboon, a cynomolgus monkey, or a rhesus monkey) and receives one or more grafts from a porcine donor.

[0115] In one embodiment, the patient treated according to the methods described herein requires a kidney transplant. The patient may require a kidney transplant due to kidney failure or rejection of the donor kidney. Renal failure can have many causes, including but not limited to high blood pressure (hypertension), physical injury, diabetes, kidney disease (polycystic kidney disease, glomerular disease), and autoimmune diseases such as lupus. Renal failure can be acute or chronic. Renal failure can also be diagnosed by clinical tests such as glomerular filtration rate, blood urea nitrogen, and serum creatinine, imaging tests (ultrasound, computed tomography), or kidney biopsy.

[0116] In some embodiments, patients treated according to the methods described herein have stage 1 kidney disease. In some embodiments, patients treated according to the methods described herein have stage 2 kidney disease. In some embodiments, patients treated according to the methods described herein have stage 3 kidney disease. In some embodiments, patients treated according to the methods described herein have stage 4 kidney disease. In some embodiments, patients treated according to the methods described herein have stage 5 kidney disease.

[0117] In some embodiments, patients treated according to the methods described herein have a glomerular filtration rate (GFR) of about 90 or greater. In some embodiments, patients treated according to the methods described herein have a GFR of about 60-90. In some embodiments, patients treated according to the methods described herein have a GFR of about 30-60. In some embodiments, patients treated according to the methods described herein have a GFR of about 15-30. In some embodiments, patients treated according to the methods described herein have a GFR of about 15 or less. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] EXAMPLES

[0118] 9. Working Example The examples in this section (ie, Section 7) are provided by way of illustration, not limitation.

[0119] 9.1 Example 1: Cross-dressing of porcine LCL cells and human Jurkat cells with transgenic hCD47 after co-culture with hCD47-Tg LCL cells This example demonstrates that heterologous human CD47 can be normally expressed in cell lines that do not express human CD47 following co-culture.

[0120] To determine whether human CD47 can be transferred from one cell to another by EVs, cells from a parental porcine B lymphoma cell line (LCL) that does not express human CD47 (hCD47) were co-cultured with LCL cells transgenically expressing hCD47. As shown in Figure 1A and Figure 2, the parental porcine LCL cell line did not express hCD47 as measured by FACS (Figure 1A, Figure 2, and Table 2). In contrast, porcine LCL cells expressing transgenic human CD47 (hCD47-Tg LCL cells) expressed high levels of CD47 (Figure 1B, Figure 2, and Table 2). Of note, detection of huCD47 in the parental LCL cell line after co-culture with hCD47-Tg LCL cells showed a strong increase in hCD47 expression (Figure 1C, Figure 2, and Table 2). [Table 2]

[0121] Similar results were observed using human T-cell leukemia Jurkat cells in which CD47 was knocked out by CRISPR-Cas9 and co-cultured with hCD47-Tg LCL cells. Briefly, CD47 knockout (KO) Jurkat cells were co-cultured with hCD47-Tg LCL cells, and hCD47 expression was measured by FACS. As shown in Figure 3B, co-culture led to a strong increase in hCD47 expression in CD47KO Jurkat cells (Figure 3B and Table 2).

[0122] These data demonstrated that porcine cells could be cross-dressed with human CD47 by co-culturing with cells expressing human CD47.

[0123] 9.2 Example 2: Cross-dressing of porcine LCL and hCD47 knockout Jurkat cells with native hCD47 after co-culture with wild-type Jurkat cells This example demonstrates that cross-dressing of hCD47 from one cell line to another following co-culture was reproducible in additional exemplary cell line models.

[0124] Briefly, CD47 knockout (KO) cells were co-cultured with parental (wild type, WT) Jurkat cells or porcine LCL cells for 24 h and analyzed by FACS for hCD47 cross-dressing on gated CD47KO Jurkat cells using anti-hCD47-BV786 mAb. Cells cultured alone were used as staining controls, which were stained separately or mixed immediately before staining. Shown are representative histograms (numbers in the figure indicate the mean fluorescence intensity (MFI) of gated CD47KO Jurkat cells).

[0125] As shown in Figure 4A, the levels of human CD47 in CD47KO Jurkat cells determined by FACS were almost completely undetectable compared to wild-type (WT) Jurkat cells (Figures 4 and 4B, Figure 6, and Table 3, respectively). After co-culture of CD47KO Jurkat cells with WT Jurkat cells, a strong increase in hCD47 was observed in CD47KO Jurkat cells (Figure 4C, Figure 6, and Table 3). [Table 3]

[0126] Similar results were observed when porcine LCL cells were co-cultured with WT Jurkat cells. As shown in Figures 5A-5B, co-culture of porcine LCL cells with WT Jurkat cells resulted in a strong increase in hCD47 expression compared to non-co-cultured porcine LCL cells (Figures 5A and 5B, respectively, and Table 3).

[0127] These results also demonstrated that human CD47 cross-dressing can be achieved on human cells and can be induced not only by hCD47 transgenic cells but also by cells expressing only native CD47.

[0128] 9.3 Example 3: CD47 Cross-dressing of CD47KO Jurkat Cells with Extracellular Vesicles or Exosomes from WT Jurkat Cells This example demonstrates that EVs isolated from cells expressing CD47 can be used to cross-dress cells that do not express CD47.

[0129] Briefly, MVs and exosomes were isolated from supernatants collected from WT Jurkat cells cultured in 10% exosome-depleted FBS. Extracellular vesicles (EVs) and exosomes (Exos) from cell culture supernatants were purified by standard differential centrifugation protocols. Supernatants collected from 48-h cell cultures were centrifuged at 2,000g (3,000 rpm) for 20 min to remove cell debris and dead cells. Extracellular vesicles were pelleted after centrifugation at 16,500g (9,800 rpm) for 45 min (Beckman Coulter, Optima XE-90) and resuspended in PBS. Pelleted exosomes from the supernatants were further centrifuged at 100,000g (26,450 rpm) for 2 h at 4°C (Beckman Coulter, Optima XE-90) and resuspended in PBS.

[0130] The isolated MVs and exosomes were then co-cultured with Jurkat cells in which CD47 was knocked out (CD47 KO Jurkat cells) for 2 or 6 hours. As shown in Figure 7C, co-culture with MVs for 2 hours led to an increase in CD47 expression in CD47 KO Jurkat cells (Figure 7C and Table 4). After 6 hours of co-culture with MVs or exosomes, CD47 expression was increased in CD47 KO Jurkat cells (Figures 8C and 8D, respectively, and Table 4). [Table 4]

[0131] These data indicate that EVs (e.g., MVs or exosomes) from cells expressing hCD47 can be used to cross-dress cells that do not express hCD47.

[0132] 9.4 Example 4: CD47 cross-dressing of porcine LCL cells with extracellular vesicles or exosomes from WT Jurkat cells This example demonstrates that after co-culture, porcine cells can be cross-dressed with hCD47 from human cells.

[0133] Briefly, MVs and exosomes were isolated from WT Jurkat cells as described above. The isolated MVs and exosomes were then co-cultured with porcine LCL cells that do not express hCD47 for 2 or 6 hours. As shown in Figure 9C, co-culture with MVs for 2 hours led to an increase in CD47 expression in porcine LCL cells (Figure 9C and Table 5). After 6 hours of co-culture with MVs or exosomes, CD47 expression was increased in porcine LCL cells (Figures 10C and 10D, respectively, and Table 5). [Table 5]

[0134] These data indicate that porcine LCL cells (expressing porcine CD47) can be cross-dressed with human CD47 by exposure to EVs or exosomes isolated from wild-type human Jurkat cells.

[0135] 10. Equivalents Although the present invention has been described in detail with reference to specific embodiments thereof, it will be understood that functionally equivalent variations are within the scope of the present invention. Indeed, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. 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. Such equivalents are intended to be encompassed by the scope of the following claims.

[0136] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1. 1. Use of organs from a donor pig in the manufacture of a medicine for xenotransplantation, comprising: The use, wherein the preparation comprises cross-dressing the organ with human CD47, and the organ is transplanted into a human recipient.

2. 2. The use of claim 1, wherein the cross-dressing step comprises exposing the organ to human CD47 containing extracellular vesicles (EVs).

3. The use according to claim 2, wherein the EVs are isolated from human cells.

4. The use according to claim 3, wherein the cells express recombinant human CD47.

5. The use according to claim 3 , wherein the cell is a transgenic cell.

6. The use described in claim 4, wherein the cells are transgenic cells.

7. The use according to any one of claims 2 to 6, wherein the cross-dressing is achieved by incubating the organ with EVs expressing human CD47 for 2 hours or 6 hours.

8. The use according to any one of claims 2 to 6, wherein the cross-dressing is achieved by ex vivo perfusion of the organ.

9. The method comprises: (a) resulting in a decrease in phagocytosis by human macrophages; (b) resulting in a decrease in phagocytosis of the cross-dressed organ cells by human macrophages of about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, or about 90% to about 95%, as compared to non-cross-dressed organ cells, as measured by FACS analysis of the percentage of CD14-positive cells that phagocytose the cross-dressed cells; (c) the percentage of CD14 positive cells that phagocytose the cross-dressed cells is measured by FACS analysis, resulting in no detectable phagocytosis of the cross-dressed organ; or (d) The percentage of CD14 positive cells that phagocytose the cross-dressed cells is measured by FACS analysis, resulting in increased survival of the organ due to protection from human macrophages.

2. The use according to claim 1.

10. 2. The use of claim 1, wherein the cross-dressed organ (a) avoids phagocytosis without inducing apoptosis, or (b) avoids phagocytosis and does not exhibit any detectable level of apoptosis.

11. 2. The use of claim 1, wherein cells obtained from the cross-dressed organ exhibit about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, or at least about 90% lower levels of apoptosis compared to cells obtained from a non-cross-dressed organ.

12. 12. The use according to claim 10 or 11, wherein apoptosis is measured by propidium iodide staining.

13. 10. The use of claim 1, wherein the cross-dressed organ (a) exhibits reduced inflammation compared to a non-cross-dressed organ, or (b) exhibits about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, or at least about 90% reduction in inflammation compared to a non-cross-dressed organ.

14. 2. The use of claim 1, wherein the human recipient (a) exhibits reduced systemic inflammation after transplantation compared to transplantation with a non-cross-dressed organ, or (b) exhibits about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, or at least about 90% reduction in systemic inflammation after transplantation compared to transplantation with a non-cross-dressed organ.

15. The use according to claim 1, wherein the organ is a kidney or a lung.

16. 16. The use of claim 15, wherein the human recipient is suffering from renal failure.

17. 10. The use of claim 1, wherein the human recipient (a) requires less immunosuppressive therapy than standard of care in a comparable clinical setting, or (b) requires 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or at least 90% less immunosuppressive therapy than standard of care in a comparable clinical setting.

18. 2. The use of claim 1, wherein the method (a) results in a reduction in proteinuria, or (b) proteinuria is (i) reduced to less than 3 g per 24 hours, (ii) reduced to 500 mg per 24 hours, (iii) reduced to 300 mg per 24 hours, or (iv) reduced to 150 mg per 24 hours.

19. 19. The use of claim 18, wherein proteinuria (a) disappears within two weeks of said transplantation, (b) disappears within one month of said transplantation, (c) disappears within two months of said transplantation, or (d) disappears within four months of said transplantation.

20. The use according to claim 1, wherein the medicament is further administered in conjunction with transplantation of bone marrow tissue into the recipient.

21. 21. The use of claim 20, wherein the bone marrow is (a) harvested from the same pig as the kidney, (b) harvested from a different pig than the kidney, or (c) cross-dressed with human CD47 by exposure to EV.

22. The use of claim 1 , wherein the organ does not express human SIRPα. (a) an organ from a donor pig; and (b) Extracellular vesicles (EVs) containing human CD47 A composition comprising:

24. The composition described in claim 23, wherein the EV is isolated from a human cell.

25. The composition of claim 24, wherein the cells express recombinant human CD47.

26. A composition described in any one of claims 23 to 25, wherein the organ is a kidney or a lung.

27. An ex vivo method for expressing human CD47 in an organ from a donor pig, comprising: (a) contacting the organ from the donor pig with extracellular vesicles (EVs) containing human CD47; and (b) incubating the organ with the EVs for a time sufficient to increase the expression of human CD47. A method comprising:

28. The method described in claim 27, wherein the EV is isolated from a human cell.

29. The incubating step comprises incubating for about (a) 1 to 12 hours; (b) 12 to 16 hours; (c) 16 to 24 hours, or (d) More than 24 hours 29. The method of claim 27 or 28, 30. The method of claim 27, wherein the method is achieved by perfusing the organ ex vivo.

31. The method described in claim 27, wherein the organ is a kidney or a lung.

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