Treating diabetes with genetically modified beta cells

Abstract

Described herein are human transgenic β cells expressing CXCL12 at depleted levels for use in a subject in need thereof. Also described herein are β cells comprising a transgene comprising a nucleic acid sequence encoding CXCL12.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 567,604, filed October 3, 2017; U.S. Provisional Application No. 62 / 568,117, filed October 4, 2017; U.S. Provisional Application No. 62 / 637,913, filed March 2, 2018; U.S. Provisional Application No. 62 / 662,651, filed April 25, 2018; U.S. Provisional Application No. 62 / 662,651, filed July 6, 2018 No. 62 / 694,634, filed on July 11, 2018; No. 62 / 696,603, filed on July 11, 2018; No. 62 / 717,587, filed on August 10, 2018; No. 62 / 719,975, filed on August 20, 2018; and No. 62 / 734,910, filed on September 21, 2018; each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on September 25, 2018, is named 054610-501001WO_SL.txt and is 8,668 bytes in size. Field of the Invention

[0005] The present invention relates to genetically modified human β cells and methods of using such cells. Genetically modified (transgenic) human β cells express a fugetactic amount of a fugetactic agent, thereby conferring protection against human mononuclear immune cells. In one embodiment, the fugetactic agent is, for example, CXCL12 or CXCL13. In one embodiment, the transgenic β cells comprise a vector, wherein the vector comprises a nucleic acid sequence encoding a fugetactic agent, and preferably a human fugetactic agent. In one embodiment, the transgenic β cells are further modified to be aging. The methods of the present invention include using these cells to express insulin in a hyperglycemic environment, including those found in diabetic patients, particularly type I diabetics. Background of the Invention

[0007] Beta cells are responsible for producing insulin in the pancreas. In subjects with type 1 diabetes (T1D), beta cells are attacked and destroyed by the immune system, and as a result, subjects with T1D are unable to effectively produce their own insulin. Cloning technique used to create pancreatic cells for type 1 diabetes, Diabetes.Co.Uk, www.diabetes.co.uk / news / 2014 / apr / cloning-technique-used-to-create-pancreatic-cells-for-type-1-diabetes-94233303.html (29 April 2014).

[0008] Type 2 diabetes (T2D) occurs when persistent hyperglycemia in a subject overwhelms the ability of the subject's beta cells to produce sufficient insulin to prevent hyperglycemia in the subject, and results in beta cell dysfunction, dedifferentiation, and death. Felicia W Pagliuca & Douglas A. Melton, “How to make a functional β-cell,” Development 2013, 140(12), 2472-2483.

[0009] Allogeneic β-cell transplantation (also known as islet cell transplantation) from normal donors to diabetic recipients has been considered as a treatment for diabetes. However, infiltration of mononuclear immune cells (T cells, B cells, and NK cells) leads to failure of β-cell transplantation. Cloning technique used to create pancreatic cells for type 1 diabetes , Diabetes.Co.Uk, http: / / www.diabetes.co.uk / news / 2014 / apr / cloning-technique-used-to-create-pancreatic-cells-for-type-1-diabetes-94233303.html (29 April 2014); Alan H. Cruickshank & Emyr W Benbow, "Recurrence of Diabetes," Pathology of the Pancreas (2nd ed., 1995); Felicia W Pagliuca & Douglas A. Melton, “How to make a functional β-cell,” Development 2013, 140(12), 2472-2483.

[0010] Current clinical practice is to transplant islets containing β cells into the liver via the portal vein, based on the principle that most of the insulin released from the pancreas is utilized in the liver and that this site is easily accessible via minimally invasive procedures. However, half of the β cells die shortly after transplantation, and this is thought to be due to low oxygen tension, active immune responses, and high levels of toxins and drugs in the liver. In addition, the immediate blood-mediated inflammatory response (IBMIR) encapsulates the transplanted islets in a fibrin clot and enhances the immune response against the graft. Therefore, several alternative transplantation sites have been tested, including the intestine, renal capsule, omentum, and subcutaneous, which may be optimal for patient safety, but have not yet been fully evaluated for systemic release of insulin.

[0011] To prevent their exposure to mononuclear immune cells, β cells have been encapsulated in devices that reportedly perform the following dual functions: separating these cells from immune destruction and protecting the host from the graft. When non-autologous cells (e.g., allogeneic or xenogeneic cells) are used for transplantation, or if autologous cells are transplanted into an autoimmune environment (e.g., patients with type 1 diabetes), immune protection is required. This can be achieved by physically isolating the cells using semipermeable membranes or scaffolds to block cellular responses. This approach reduces the need for immunosuppression and has recently been reviewed in detail elsewhere (Sakata et al., World J Gastrointest Pathophysiol. 2012; 3: 19–26; Qi, Adv Med. 2014; 2014: 429710). Encapsulation also prevents cells from escaping the site of the graft and allows removal when necessary. This is particularly relevant since uncontrolled differentiation and growth such as teratomas have been frequently observed in mice transplanted with stem cell-derived pancreatic precursors (Kroon et al., Nat Biotechnol. 2008;26:443–452; Kelly et al., Nat Biotechnol. 2011;29:750–756.). Teratoma formation can be prevented by generating transplants consisting of pure populations of mature beta cells, either by using cell purification (Kelly et al., 2011) or by improving differentiation methods. Encapsulation also prevents potential metastasis if insulinomas / teratomas form from transplanted cells.

[0012] This approach requires an efficient and reproducible microencapsulation protocol, and some cells may die within the encapsulated material without the possibility of being cleared or removed. Some reports have shown that microencapsulated beta cells can remain viable up to 6 months after implantation (Orlando et al., Diabetes. 2014;63:1433–1444). This means that repeat surgery is required to replace the microencapsulated device when the device is no longer functional.

[0013] In view of the above, there has long been an urgent need to develop technologies that effectively treat diabetes, and particularly type 1 diabetes. SUMMARY OF THE INVENTION

[0015] The present invention relates to transgenic human β cells and transgenic aged human β cells, which express an effective amount of chemotropes to render these cells resistant to human immune cells. Chemotropes are well known in the art and include "CXCL12". This cytokine, also known as SDF-1, is produced by the thymus and bone marrow stroma (see, e.g., U.S. Pat. No. 5,756,084, entitled: "Human stromal derived factor 1α. and 1β.", issued to Honjo et al. on May 26, 1998). It has been reported that CXCL12 repels effector T cells while recruiting immunosuppressive regulatory T cells to anatomical sites. See, e.g., Poznansky et al., Nature Medicine 2000, 6: 543-8. CXCL12 and its receptor CXCR4 are also reported as components of angiogenesis.

[0016] Also disclosed are agents for rejecting immune cells other than CXCL12, including but not limited to gp120, other CXCR4 ligands, IL-8, CXCR4 binding antibodies, CXCL13, CXCR5 ligands, CXCR5 binding antibodies, and the like.

[0017] One embodiment of the present invention is a transgenic human β cell that expresses an effective amount of an antagonist, preferably CXCL12 or CXCL13, so as to render the cell resistant to human immune cells. In one embodiment, such an effective amount of an antagonist is generated by introducing a human transgene for the agent (e.g., CXCL12, CXCL13) into a β cell or a precursor of a β cell (e.g., a pluripotent stem cell). These human transgenic β cells are further characterized as expressing insulin in a hyperglycemic environment. Therefore, these cells can be used in methods for treating diabetes in a subject. The transgenic human β cells used in the methods described herein can be autologous or non-autologous (e.g., allogeneic) β cells. In one embodiment, the patient suffers from T1D. In another embodiment, the transgenic human β cells can be modified to be senescent (unable to divide), so that any further differentiation of these cells into cancer cells is eliminated, and apoptosis induction due to inappropriate cell division is cancelled.

[0018] One embodiment of the present invention uses β cells, such as autologous or allogeneic β cells, obtained or derived from a non-diabetic human subject or a human subject with diabetes. These β cells include a functional chemotactic agent (e.g., CXCL12, CXCL13) expression vector. Such vectors are designed to express the agent in transgenic β cells at a level sufficient to produce a chemotactic buffer around the β cells. Without wishing to be bound by theory, it is expected that this buffer allows the β cells to resist attack by immune cells but still express insulin, as is necessary to maintain appropriate blood glucose levels in diabetic subjects. The generation of autologous β cells from patients with diabetes is known in the art. See, for example, Egli et al., EMBO J. 2015Apr 1; ​​34(7): 841–855, which is incorporated herein by reference in its entirety. Allogeneic β cells derived from stem cells are commercially available.

[0019] One aspect of the invention is to administer transgenic human β cells (which contain a transgene encoding a chemoattractant (e.g., CXCL12, CXCL13)) to a subject in need thereof to regulate insulin levels and treat diabetes in the subject. In addition, the expression of a sufficient amount of the chemoattractant protects against the risk of destruction of the transgenic β cells by infiltration of mononuclear immune cells. The transgenic human β cells can be autologous or allogeneic. In one embodiment of the invention, the transgenic β cells are autologous β cells derived from a patient with diabetes. In another embodiment, the transgenic β cells are allogeneic human β cells. In another embodiment, the patient suffers from type 1 diabetes.

[0020] Another aspect of the present invention relates to transgenic human β cells, which can express an effective amount of chemotropic agents (e.g., CXCL12, CXCL13) to resist immune destruction. The chemotropic agent (e.g., CXCL12, CXCL13) can be an endogenous agent, i.e., an agent expressed by the subject to be treated, or an exogenous agent, such as an agent from a non-autologous source or a modified chemotropic agent. In one embodiment, the gene encoding the chemotropic agent in the β cell is modified to be overexpressed compared to the unmodified gene. Methods for modifying gene expression are known in the art, such as site-directed gene editing to replace endogenous promoters with different promoters (e.g., constitutive promoters, inducible promoters, etc.). In one embodiment, a recombinant polynucleotide encoding a chemotropic agent is inserted into a β cell so that the chemotropic agent is expressed by the recombinant polynucleotide. Methods (transduction, transfection, etc.) for inserting recombinant genes into cells are well known in the art, such as methods for preparing vectors with recombinant polynucleotides for insertion into cells.

[0021] In some embodiments, the chemotropic agent is a modified chemotropic agent. For example, the polypeptide sequence of the chemotropic agent can be modified to increase the circulation half-life, to incorporate conservative amino acid changes, to enhance the binding to the extracellular matrix, to improve the activity of the agent, etc. Accordingly, the gene encoding the modified chemotropic agent (e.g., CXCL12 or CXCL13) can be modified so that the gene has at least 95% sequence identity with the native gene, and preferably has 99% sequence identity with the native gene. Similarly, the amino acid sequence of the modified chemotropic agent (e.g., modified CXCL12 or CXCL13) has at least 95% and preferably 99% sequence identity with the native agent.

[0022] In one embodiment, a human β cell is provided comprising a vector, the vector itself comprising a nucleic acid sequence encoding human CXCL12 or a modified CXCL12, wherein the β cell is made resistant to human immune cells.

[0023] In one embodiment, the human transgenic β cells are autologous β cells obtained from a subject suffering from type 1 diabetes.

[0024] In one embodiment, the human β cells are allogeneic β cells.

[0025] In one embodiment, human mononuclear immune cells include NK cells, T cells and B cells. In one embodiment, T cells include cytotoxic T cells.

[0026] In one embodiment, the transgenic human β cells express efferotropic amounts of human CXCL12.

[0027] In one embodiment, the human CXCL12 is selected from CXCL12α and CXCL12β.

[0028] In one embodiment, the human transgenic β cell comprises a transgenic regulatory region upstream of the endogenous CXCL12 coding region, wherein the β cell is resistant to human immune cells. Preferably, the endogenous CXCL12 coding region regulatory region comprises a constitutive promoter. In some embodiments, the endogenous CXCL12 coding region regulatory region comprises an inducible promoter.

[0029] In one embodiment, the human transgenic β cells comprising a transgene regulatory region upstream of the endogenous CXCL12 coding region, wherein the β cells are resistant to human immune cells, are autologous β cells, and preferably β cells obtained from a patient suffering from diabetes.

[0030] In one embodiment, the human transgenic β cell comprising a transgene regulatory region upstream of the endogenous CXCL12 coding region, wherein the β cell is resistant to human immune cells, is an allogeneic β cell.

[0031] In one embodiment, a human transgenic β cell is provided, comprising an expressible human CXCL12 or CXCL13 gene, wherein the cell expresses an apoptotic amount of CXCL12 or CXCl13 so as to be resistant to human immune cells.

[0032] In one embodiment, the human transgene β comprises a human gene for CXCL12.

[0033] In one embodiment, the human transgenic β cell comprises a human gene selected from the group consisting of CXCL12α and CXCL12β.

[0034] In one embodiment, the human transgenic β cells comprise a human gene for CXCL12β.

[0035] In one embodiment, a human transgenic senescent β cell is provided, comprising an expressible human CXCL12 or CXCL13 gene, wherein the cell expresses an apoptotic amount of CXCL12 or CXCL13 so as to be resistant to human immune cells, and further wherein the cell is senescent.

[0036] In one embodiment, the human transgenic senescent β cells comprise an expressible human gene for CXCL12 and are capable of expressing insulin in the presence of hyperglycemic medium.

[0037] In one embodiment, the human transgenic senescent β cells comprise an expressible human gene selected from the group consisting of CXCL12α and CXCL12β.

[0038] In one embodiment, the expressible human gene contained in the human transgenic β cells is CXCL12β.

[0039] In one embodiment, a method for producing insulin in response to a hyperglycemic environment is provided, the method comprising contacting the environment with a population of human transgenic β cells as described above.

[0040] In one embodiment, the human transgenic β cells are resistant to human immune cells selected from the group consisting of T cells, B cells, NK cells, and mixtures thereof.

[0041] In one embodiment, the beta cells described herein are obtained by:

[0042] (a) obtaining a population of human progenitor cells or human pluripotent stem cells from a human subject;

[0043] (b) differentiating the subject's progenitor cells or pluripotent stem cells into β cells; and

[0044] (c) Introducing a nucleic acid molecule encoding the chemotactic agent into the β cell.

[0045] In one embodiment, the chemoattractant is a cytokine, a chemokine, a CXCR4 binding antibody, a CXCR4 ligand, a CXCR5 binding antibody, or a CXCR5 ligand.

[0046] In one embodiment, the antagonist is CXCL12, CXCL13, gp120, or IL-8.

[0047] In one aspect, methods are provided for promoting survival of β cells in a biological sample comprising immune cells by modifying the β cells to express an agonist at a level sufficient to inhibit or block killing of the β cells by the immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a photograph of a Western blot showing the relative amounts of CXCL12α, CXCL12β, CXCL12γ, CXCL12θ, CXCL12δ, and CXCL14 when each is overexpressed in β cells.

[0050] Figure 2A and 2B is a bar graph showing the relative amount of lactate dehydrogenase (LDH, a marker of cell lysis) released from β cells expressing CXCL12α or CXCL12β that were incubated with PBMCs at a PBMC:β ​​cell ratio of 30:1 for 24 and 48 h.

[0051] Figure 3 Shown are the expression levels of CXCL12α and CXCL12β in the two groups of β cells expressing each cytokine.

[0052] Figure 4A and 4B is a bar graph showing the relative amount of LDH released from β cells expressing CXCL12α or CXCL12β that were incubated with PBMCs at a PBMC:β ​​cell ratio of 30:1 for 24 and 48 h with or without senescence induction of β cells (by mitomycin C treatment).

[0053] Figure 5 Shown is the induction of insulin by hyperglycemic challenge of β-cells expressing CXCL12α or CXCL12β, with or without treatment with mitomycin C. DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention provides autologous / allogeneic human β cells, which are transgenic and contain a transgene encoding a human antagonist (e.g., CXCL12, CXCL13), or have been genetically modified to express or overexpress an endogenous (human) antagonist (e.g., CXCL12, CXCL13) in an antagonist amount. In a preferred embodiment, the transgenic β cells described herein are further modified to be senescent. In another of its method aspects, the β cells are modified or treated so as to express an effective amount of an antagonist (e.g., CXCL12, CXCL13) so as to inhibit immune destruction of the transgenic human β cells and produce insulin in response to a hyperglycemic environment.

[0056] Before disclosing the present invention in further detail, the following terms will first be defined. If a term is not defined, it has its generally accepted scientific meaning as understood in the art.

[0057] The term "CXCL13" refers to all known isoforms thereof. Since CXCL13 is known to mediate proliferation of certain cancer cells, it is not preferred. See, e.g., https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3839818 / .

[0058] The term "antagonism" or "antagonistic" refers to the ability of an agent to repel (or chemoexclude) eukaryotic cells that have the ability to migrate. The antagonistic amount of CXCL12 or CXCL13 (or other antagonistic agent) expressed by a cell is an amount sufficient to block or inhibit the migration of immune cells toward the cell, or in some aspects to repel immune cells from the cell.

[0059] The term "human immune cell" is used interchangeably with the term "human mononuclear immune cell" and includes NK cells, T cells, and B cells.

[0060] The term "immune cell resistant" or "hidden from the immune system" indicates that the β cell expresses an amount of an antagonist sufficient to block or inhibit immune cell migration toward the cell, or in some aspects exclude immune cells from the β cell. In a preferred embodiment, such blocking or inhibition is measured by the extent of cell death after the β cells of the present invention are exposed to human mononuclear immune cells (e.g., PBMC). Cell death can be assessed by lactate dehydrogenase (LDH) release from cells that have undergone lysis. Preferably, the immune cell resistant β cells of the present invention can be assessed by cells that demonstrate LDH levels less than 50% relative to a control at a ratio of about 30:1 immune cells to the β cells of the present invention over a two-day incubation period. More preferably, the immune resistant β cells demonstrate LDH levels less than 60% relative to a control; and even more preferably, LDH levels less than 75% relative to a control; and most preferably, LDH levels less than 95% relative to a control. The procedure for assessing LDH levels is set forth in Example 2 herein.

[0061] The chemotropic agent is an agent having chemotropic activity. The chemotropic agent may include, but is not limited to, CXCL12, CXCL13, gp120, IL-8, CXCR4 binding antibody, CXCR4 ligand, CXCR5 binding antibody or CXCR5 ligand.

[0062] The term "effector T cell" refers to a differentiated T cell that is capable of generating a specific immune response by releasing cytokines.

[0063] The term "regulatory T cells" refers to T cells that reduce or inhibit the immune response of B cells or other T cells to an antigen.

[0064] The term "CXCL12" or "SDF-1 polypeptide" refers to a cytokine well known in the art (see, e.g., Table 1). In one embodiment, the term refers to a protein or fragment thereof that binds to a CXCL12-specific antibody and has chemotactic or chemotactic activity. Chemotactic or chemotactic activity is determined by measuring the direction of T cell migration (e.g., toward or away from a target agent). See, e.g., Poznansky et al., Nature Medicine 2000, 6:543-8; N. Papeta et al., "Long-term survival of transplanted allogeneic cells engineered to express a T Cell chemorepellent," Transplantation2007, 83(2), 174-183. "Cellular tropism" or "cell tropism" is the movement of migratory cells away from the source of an agent (i.e., toward a lower concentration of the agent). It should be understood that the term "CXCL12" refers to all known isoforms thereof, including α, β, γ, δ, ε, φ, and θ isoforms. Preferred CXCL12 isoforms are α and β. CXCL12 is known to induce angiogenesis.

[0065] The terms "type 1 diabetes" and "type 2 diabetes" refer to two major pathophysiologies associated with elevated blood sugar. Type 1 diabetes is characterized by an autoimmune attack against the insulin-producing β cells of the pancreas, while type 2 diabetes is associated with weak β cell function and increased peripheral insulin resistance. Similar to type 1 diabetes, β cell death is also observed in type 2 diabetes. Type 1 and often type 2 diabetes require individuals to inject insulin. Type 1 diabetes is usually characterized by the loss of insulin-producing β cells of the islets of Langerhans in the pancreas, resulting in insulin deficiency. This type of diabetes can be further classified as immune-mediated or idiopathic. Most type 1 diabetes has an immune-mediated nature, in which β cell loss is due to an autoimmune attack mediated by T cells. Type 2 diabetes is characterized by β cell dysfunction combined with insulin resistance. It is believed that the defective responsiveness of body tissues to insulin involves insulin receptors and downstream cell signaling. Similar to type 1 diabetes, insufficient β cell mass is also a causative factor in many type 2 diabetes patients. In the early stages of type 2 diabetes, hyperglycemia can be reversed by various measures and agents that improve insulin secretion and reduce glucose production in the liver. As the disease progresses, impairment of insulin secretion occurs, and therapeutic replacement of insulin may occasionally become necessary in some patients.

[0066] "Subject" or "patient" refers to a mammal, preferably a human subject.

[0067] A "subject in need thereof" or a "patient in need thereof" is a subject suffering from type 1 or type 2 diabetes.

[0068] As used herein, the terms "treat," "treating," "treatment," and the like refer to the reduction or amelioration of a disorder and / or symptoms associated therewith. It should be understood that treating a disorder or condition does not require, although not exclusive of, complete elimination of the disorder, condition, or symptoms associated therewith.

[0069] In the present disclosure, “comprises,” “comprising,” “containing,” and “having,” and the like, may have the meaning ascribed thereto in U.S. patent law, and may mean “includes,” “including,” and the like; “consisting essentially of,” or “consists essentially of,” likewise have the meaning ascribed thereto in U.S. patent law, and the term is open-ended, allowing the existence of more than what is recited, as long as the basic or novel features of what is recited are not altered by the existence of more than what is recited, but excluding prior art embodiments.

[0070] When used before numerical designations such as temperature, time, amount, concentration, and such others (including ranges), the term "about" indicates an approximate value, which may differ by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Throughout this disclosure, other definitions appear in context.

[0071] One aspect of the present invention is a transgenic beta cell, such as a human autologous beta cell or a non-autologous beta cell, such as an allogeneic beta cell, comprising a nucleic acid encoding a chemotropic agent (e.g., CXCL12, CXCL13) operably linked to a promoter, such that the chemotropic agent (e.g., CXCL12, CXCL13) is expressed at a chemotropic level in the beta cell microenvironment. The promoter can be a promoter endogenous to the beta cell, or a promoter heterologous to the beta cell but functional in the beta cell. Preferably, the nucleic acid encoding the chemotropic agent (e.g., CXCL12, CXCL13) is endogenous to the subject treated with the transgenic beta cell. In one embodiment, the allogeneic beta cell is derived from a non-T1D donor.

[0072] One aspect of the present invention is a human β cell comprising a genetically modified endogenous human gene encoding an antagonist (e.g., CXCL12, CXCL13), wherein the gene is modified to include a heterologous promoter operably linked to an antagonist coding sequence, so that the antagonist is expressed by the endogenous gene at an antagonist level in the β cell microenvironment. Genome editing techniques known in the art can be used to introduce a promoter into β cells to be operably linked to an antagonist coding sequence. As is well known, CXCL12 has several isoforms, including α, β, γ, and θ. In a preferred embodiment, the isoform used is CXCL12β. The transgenic human β cells described herein produce insulin in response to a hyperglycemic environment. The term "insulin" is intended to cover both proinsulin and insulin.

[0073] In general, the present invention provides beta cells, preferably human beta cells, expressing chemotaxis agents (e.g., CXCL12, CXCL13) at a level sufficient to block or inhibit immune cells (e.g., human immune cells) from migrating to beta cells or sufficient to repel immune cells. The terms immune cells and monocytes (T cells, B cells, and NK cells) can be used interchangeably. The ability of chemotaxis agents (e.g., CXCL12, CXCL13) polypeptides to repel immune cells (e.g., effector T cells) can be evaluated in vitro using a Boyden chamber assay. See, for example, as previously described in Poznansky et al., Journal of Clinical Investigation, 109, 1101 (2002). Alternatively, the viability of transgenic human beta cells is evaluated by combining such cells with human PBMCs. Cell mortality can be assessed by measuring one or more cell death markers over time. One such marker commonly used is lactate dehydrogenase (LDH) released during cell necrosis.

[0074] Without wishing to be bound by any theory, applicants anticipate that in one aspect of the invention, the amount of chemotropic agent (e.g., CXCL12, CXCL13) produced by transgenic β cells is sufficient to provide a chemotropic effect in the β cell microenvironment, but the amount produced is insufficient to increase the systemic level of the agent and disturb the balance between the beneficial effect of the agent in one process and the harmful consequences in another process. In addition, CXCL12 is known to induce angiogenesis when bound to its receptor CXCR4. Again, without being bound by any theory, it is expected that the microenvironment of implanted transgenic β cells expressing CXCL12 will induce an angiogenic response that enhances the viability of the implanted cells.

[0075] The effective amount of the chemotropic agent (e.g., CXCL12, CXCL13) is any amount sufficient to block immune cells from killing transgenic β cells. For example, the effective amount of the chemotropic agent (e.g., CXCL12, CXCL13) in the microenvironment of transgenic β cells can be at least about 100 ng / mL, and preferably at least 100 nM. In some embodiments, the amount of the chemotropic agent (e.g., CXCL12, CXCL13) in the microenvironment of transgenic β cells is at least about 1000 ng / mL. For example, the following specific ranges are suitable for the present invention: about 100 nM to about 200 nM, about 100 nM to about 300 nM, about 100 nM to about 400 nM, about 100 nM to about 500 nM, about 100 nM to about 600 nM, about 100 nM to about 700 nM, about 100 nM to about 800 nM, about 100 nM to about 900 nM, or about 100 nM to about 1 μM.

[0076] In embodiments, the effective amount of the chemoattractant (e.g., CXCL12, CXCL13) in the transgenic β cell microenvironment ranges from 20 ng / mL to about 5 μg / mL. In embodiments, the effective amount of the chemoattractant ranges from 20 ng / mL to about 1 μg / mL. In embodiments, the amount of the chemoattractant (e.g., CXCL12, CXCL13) in the β cell microenvironment is a sufficient amount for chemoattractant, ranging from about 100 ng / mL to about 500 ng / mL, about 500 ng / mL to 5 μg / mL, about 800 ng / mL to about 5 μg / mL, or about 1000 ng / mL to about 5000 ng / mL. Without wishing to be bound by theory, it is expected that when transgenic and non-transgenic β cells are used together, transgenic β cells can express a sufficient amount of the chemoattractant so that the microenvironment producing the chemoattractant effect extends to adjacent to the non-transgenic β cells. The effective amount of the chemotropic agent (eg, CXCL12, CXCL13) in the microenvironment of transgenic β cells can be any value or subrange within the range, including the endpoints.

[0077] Although mice and mouse DNA are widely used by immunologists to understand the human immune system, there are significant differences between humans and mice. Accordingly, the chemokines (e.g., CXCL12, CXCL13) encoded by the vector are preferably human agents. Javier Mestas & Christopher C.W. Hughes, "Of Mice and Not Men: Differences between Mouse and Human Immunology," Journal of Immunology 2004, 172(5), 2731-2738; O. Cabrera et al., "The unique cytoarchitecture of human pancreatic islets has implications for islet cell function," PNAS 2006, 103(7), 2334-2339; M. Votey, “Of mice and men: how the nPOD program is changing the way researchers study type 1 diabetes,” diaTribe, Network for Pancreatic Organ Donors with Diabetes (August 21, 2015).

[0078] CXCL12 polypeptides are known in the art. See, for example, Poznansky et al., Nature Medicine 2000, 6:543-8 and U.S. Patent Publication No. 20170246250, both of which are incorporated herein by reference in their entirety. The terms CXCL12 and SDF-1 can be used interchangeably. Exemplary CXCL12 / SDF1 isoforms are provided in Table 1 of U.S. Publication No. 20170246250. Exemplary CXCL12 / SDF1 isoforms are also provided in Table 1 (below):

[0079]

[0080] In one embodiment, the CXCL12 polypeptide has at least about 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with NP 001029058 and has chemokine or chemotactic activity. In one embodiment, the CXCL12 polypeptide has at least about 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 and has chemokine or chemotactic activity. Such sequence identity is based on replacing the first amino acid with a known conservative second amino acid. Such conservative substitutions are well established in the art, and the resulting modified CXCL12 polypeptides are well known in the art for testing their chemotactic properties. See, e.g., Poznansky supra.

[0081] CXCL13 peptides are known in the art. CXCL13 is also known as B lymphocyte chemotactic factor (BLC) or B cell attracting chemokine 1 (BCA-1), and these terms can be used interchangeably. For example, human CXCL13 can be found at accession number Q53X90. In one embodiment, the CXCL13 polypeptide has an amino acid sequence comprising MKFISTSLLLMLLVSSLSPVQGVLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGCPRKEIIVWKKNKSIVCVDPQAEWIQRMMEVLRKRSSSTLPVPVFKRKIP (SEQ ID NO: 7). In one embodiment, the CXCL13 polypeptide has at least about 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with Q53X90 and has chemokine or chemotactic activity. In one embodiment, the CXCL13 polypeptide has at least about 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:7 and has chemokine or chemotactic activity.

[0082] The transgenic β cells used in the methods described herein can be autologous or non-autologous (e.g., allogeneic) β cells. "Autologous" cells are cells from the same individual. "Allogeneic" cells are cells from a donor of the same species that are genetically similar but not identical. Allogeneic cells that can be used in the methods of the present invention are preferably from a human subject. Allogeneic cells that can be used in the methods of the present invention can be from a relative, such as a sibling, cousin, parent, or child, or from a non-relative. Criteria for selecting allogeneic donors are well known in the art, see, e.g., Tatum et al., Diabetes Metab Syndr Obes 2017: 10 73-78. Human allogeneic β cells are commercially available, and autologous β cells are obtained by Egli et al., and the method described above was used to produce

[0083] In one embodiment, the transgenic human β cells used in the method of the present invention are autologous transgenic β cells, which can be prepared by methods known in the art by deriving β cells from multipotent progenitor cells or pluripotent stem cells obtained from patients. These derived β cells can contain an expression vector (e.g., transfected, infected, etc.) containing a nucleic acid sequence encoding a chemotactic agent (e.g., CXCL12, CXCL13).

[0084] Alternatively, transgenic β cells for use in the methods of the present invention can be prepared by isolating pancreatic β cells from a subject in need thereof. These isolated pancreatic β cells can contain an expression vector (e.g., transfected, infected, etc.), the expression vector comprising a nucleic acid sequence encoding an antagonist (e.g., CXCL12, CXCL13). Alternatively, the β cell can be genetically modified to express an endogenous antagonist (e.g., CXCL12, CXCL13) gene so that it constitutively produces an antagonist (e.g., CXCL12, CXCL13) in an effective amount.

[0085] In one embodiment of the invention, the β cell comprises an expression vector (e.g., transfected, infected, etc.) comprising a nucleic acid molecule encoding an antagonist (e.g., CXCL12, CXCL13) operably linked to a promoter suitable for expression in the β cell. The vector may be integrated into the genome of the β cell, or it may exist in episomal form without integration into the genome.

[0086] The transgenic β cells of the present invention can also be prepared from adult stem cells by isolating adult stem cells from a subject, culturing the stem cells under appropriate conditions to expand the population and induce differentiation into β cells. Cells can be modified to express an effective amount of chemotropic agents (e.g., CXCL12, CXCL13) by introducing an expression vector encoding a chemotropic amount of chemotropic agents (e.g., CXCL12, CXCL13) into cells, or by editing the genome to express chemotropic amounts of chemotropic agents (e.g., CXCL12, CXCL13). The vector can be introduced into stem cells before differentiation into β cells, or the genome of the stem cells can be edited to contain a heterologous promoter. Alternatively, the vector can be introduced into the resulting β cells, or the genome of the resulting β cells can be edited to contain a heterologous promoter.

[0087] The transgenic β cells of the present invention can also be prepared by: generating induced pluripotent stem (iPS) cells from a subject's somatic cells, such as β cells, fibroblasts or keratinocytes; treating the iPS cells to induce differentiation into β cells; and introducing an expression vector into the differentiated β cells, wherein the expression vector comprises a nucleic acid sequence encoding a chemokine (e.g., CXCL12, CXCL13).

[0088] The transgenic β cells of the present invention can also be prepared by: preparing induced pluripotent stem (iPS) cells generated from somatic cells of a subject; introducing an expression vector comprising a nucleic acid sequence encoding a chemotactic agent (e.g., CXCL12, CXCL13) into the iPS cells; and treating the iPS cells before or after the introduction of the transgene to induce differentiation into β cells.

[0089] The transgenic β cells of the present invention can also be generated by obtaining progenitor cells or progenitor-like cells, such as pancreatic β cell progenitor cells, introducing a vector containing a nucleic acid sequence encoding a chemoattractant (e.g., CXCL12, CXCL13) into the cell, and treating the cell before or after the introduction of the vector by methods known in the art to induce differentiation into β cells or insulin-releasing cells that respond to glucose levels in the body, see, for example, Millman et al. Nature Communications (May 10, 2016) pp. 1-8); Baek et al. Curr Stem Cell Rep (2016) 2: 52-61; Russ et al., EMBO. J. 34, 1759-1772 (2015); and Qadir et al., Cell Reports 22, 2408-2420 (February 27, 2018). Progenitor cells and progenitor-like cells can be autologous or non-autologous, such as allogeneic, for subjects treated with transgenic cells. Insulin producing cells that respond to glucose levels in vivo (see, e.g., Qadir et al., supra) can be genetically modified as described herein to express efflux levels of efflux agents (e.g., CXCL12, CXCL13), and are also an embodiment of the present invention. Such genetically modified insulin producing cells can likewise be used in the methods of the present invention to treat diabetes as described herein.

[0090] Any suitable somatic cell from a subject can be reprogrammed into iPS cells by methods known in the art, see, for example, Pagliuca and Melton (2013) How to make a functional β-cell, Development (3013) 140(12); 2472-2483; Yu et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920; Takahashi and Yamanaka, 2006, Cell 126(4): 663-676; Wernig et al., 2007, Nature 448: 7151; Okita et al., 2007 Nature 448: 7151; Maherali et al., 2007 Cell Stem Cell 1: 55-70; Lowry et al., 2008 PNAS 105: 2883-2888; Park et al., 2008 Nature 451: 141-146.; Takahashi et al., 2007 Cell 131, 861-872; U.S. Patent No. 8,546,140; U.S. Patent No. 7,033,831; and U.S. Patent No. 8,268,620. iPS cells can be differentiated into beta cells using methods known in the art, see, for example, U.S. Patent Publication No. 20170081641 and U.S. Patent Publication No. 20130164787, and Millette and Georgia, "Gene Editing and Human Pluripotent Stem Cells: Tools for advancing Diabetes Disease Modeling and Beta Cell Development", Current Diabetes Reports November 2017, 17: 116; U.S. Patent Application No. 20130273651; Shi, Y. et al. Stem Cells., 25: 656-662 (2005); or Tateishi, K. et al., J Biol Chem., 283: 31601-31607 (2008).

[0091] Preferably, the antagonist coding sequence is operably linked to a regulatory region suitable for expression in beta cells. Suitable regulatory regions are known in the art and include promoters, such as mammalian promoters, including, for example, hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, beta-actin promoter, muscle creatine kinase promoter and human elongation factor promoter (EF1α), GAPDH promoter, actin promoter and ubiquitin promoter, and viral promoters, including SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, human immunodeficiency virus (HIV) promoter, cytomegalovirus (CMV) promoter, adenovirus promoter, adeno-associated virus promoter or herpes simplex virus thymidine kinase promoter. Other relevant promoters, such as viral and eukaryotic promoters, are also well known in the art (see, for example, Sambrook and Russell (Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Press). The regulatory region operably linked to the chemokine coding sequence may be any constitutive promoter suitable for expression in the cells of the subject.

[0092] The transgenic cells (whether autologous or non-autologous, such as allogeneic) expressing antagonists (e.g., CXCL12, CXCL13) of the present invention can be administered to a subject in need thereof by any means known in the art for administering β cells. The transgenic cells of the present invention can be administered in an amount sufficient to provide such insulin levels that can alleviate at least some of the symptoms associated with low levels of insulin.

[0093] Another aspect of the present invention is a method for treating diabetes in a subject in need thereof, comprising the following steps: (a) obtaining or deriving β cells or insulin-producing β-like cells from the subject; (b) introducing a suitable expression vector encoding a chemoattractant (e.g., CXCL12, CXCL13) into the cells to form autologous transgenic cells expressing the introduced chemoattractant (e.g., CXCL12, CXCL13); and (c) transplanting the autologous transgenic cells into the subject.

[0094] Many vectors that can be used to transfer exogenous genes into mammalian cells (e.g., β cells), including vectors that integrate into the genome, and vectors that are not integrated into the genome but exist as episomes, and methods for introducing such vectors into cells are available and known in the art. For example, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV)-based vectors, and EBV-based vectors can be used. See, for example, US20110280842, Narayanavari and Izsvák, Cell Gene Therapy Insights 2017; 3 (2), 131-158; Hardee et al., Genes 2107, 8, 65; Tipanee et al., Bioscience Reports (2017) 37, and Chira et al. Oncotarget, Vo. 6, No. 31, pp. 30675-30703.

[0095] Another aspect of the present invention is a method for promoting the survival of β cells in a biological sample containing immune cells, which includes introducing an expression vector encoding a chemoattractant (e.g., CXCL12, CXCL13) into a β cell, or by editing the genome of the β cell so that the β cell expresses a chemoattractant (e.g., CXCL12, CXCL13) in a chemoattractant amount. In one aspect of the present invention, the chemoattractant (e.g., CXCL12, CXCL13) is expressed by the β cell at a level sufficient to block or inhibit the migration of immune cells (e.g., T cells, B cells, and / or NK cells) to the β cell. In one aspect of the present invention, the chemoattractant (e.g., CXCL12, CXCL13) is expressed by the β cell at a level sufficient to exclude immune cells from the β cell. In one aspect of the present invention, the genetically modified β cell is in a subject, such as a human subject suffering from type 1 or type 2 diabetes. In one embodiment, the β cell is an autologous β cell of the subject.

[0096] Methods for delivering viral and non-viral vectors to mammalian cells are well known in the art and include, for example, lipofection, microinjection, ballistics, virosomes, liposomes, immunoliposomes, polycations or lipid-nucleic acid conjugates, naked DNA, artificial virus particles, and agents that enhance DNA uptake. Lipofection agents are commercially available (e.g., Transfectam TM and Lipofectin TM). Cationic lipids and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides are known. Nucleic acids can be delivered to cells (ex vivo administration) or target tissues (in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes, such as immunolipid complexes, is well known to those skilled in the art. Recombination-mediated systems can be used to introduce vectors into cells. Such recombination methods include, for example, the use of site-specific recombinases such as Cre, Flp or PHIC31 (see, for example, Oumard et al., Cytotechnology (2006) 50: 93-108), which can mediate the directional insertion of transgenes.

[0097] Vectors suitable for use in the present invention include expression vectors comprising a nucleic acid encoding an ectotropic agent (e.g., CXCL12, CXCL13) operably linked to a promoter that directs transcription. Suitable promoters are well known in the art and are described, for example, in Sambrook and Russell (Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Press). Promoters used to direct expression of ectotropic agents (e.g., CXCL12, CXCL13) can be, for example, SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, or other promoters shown to be effective for expression in mammalian cells.

[0098] Vectors that can be used in the methods of the present invention include, for example, SV40 vectors, papilloma virus vectors, Epstein-Barr virus vectors, retrovirus vectors, and lentivirus vectors.

[0099] The vector used in the present invention can comprise regulatory elements from eukaryotic viruses, such as SV40, papilloma virus and Epstein-Barr virus, including, for example, signals for efficient polyadenylation, transcription termination, ribosome binding and / or translation termination of the transcript. Additional elements of the vector can include, for example, enhancers and intronic signals for heterologous splicing.

[0100] In one embodiment of the present invention, the genome of the β cell can be genetically modified to increase the expression level of the endogenous chemoattractant (e.g., CXCL12, CXCL13) gene. Such increased expression can be achieved by introducing a heterologous promoter operably connected to the endogenous chemoattractant (e.g., CXCL12, CXCL13) gene, or changing the endogenous chemoattractant (e.g., CXCL12, CXCL13) promoter so that the β cell expresses the chemoattractant (e.g., CXCL12, CXCL13) at the chemoattractant level. Such increased expression can be achieved by introducing a promoter into the genome of the β cell so that it is operably connected to the endogenous chemoattractant coding sequence, and thereby expressing or overexpressing the chemoattractant in chemoattractant amounts.

[0101] Gene editing techniques for modifying genomes are well known in the art and include, for example, CRISPR / CAS9, Piggybac, Sleeping Beauty genome editing systems (see, e.g., Zhang et al. Molecular Therapy Nucleic Acids, Vol. 9, December 2017, pp. 230-241; systems (see, e.g., Cong et al., Science. 2013; 339(6121): 819-23; Mali et al., Science. 2013; 339(6121): 823-6; González et al., Cell Stem Cell. 2014; 15(2): 215-26); He et al., Nucleic Acids Res. 2016; 44(9); Hsu et al., Cell. 2014; 157(6): 1262-78.), zinc finger nuclease-based systems (see, e.g., Porteus and Carroll, Nat Rev. 2014; 157(6): 1262-78.), Biotechnol. 2005;23(8):967–73; Urnov et al., Nat Rev Genet. 2010;11(9):636–46), TALEN-based systems (transcription activator-like effector nucleases) (see, e.g., Cermak et al., Nucleic Acids Res. 2011;39(12); Hockemeyer et al., Nat Biotechnol. 2011;29(8):731–4; Joung and Sander JD, Nat Rev Mol Cell Biol. 2013;14(1):49–55; Miller et al., Nat Biotechnol. 2011;29(2):143–8, and Reyon et al., Nat Biotechnol. 2012;30(5):460–5).

[0102] In one embodiment, the transgenic beta cells described herein are treated with an agent that renders the cells viable and capable of controlling blood glucose in a patient, but incapable of replicating (i.e., induced cellular senescence). One such agent is mitomycin C, which is a known DNA cross-linking agent. Upon treatment, the DNA in these cells is cross-linked, thereby making it impossible to form single-stranded DNA necessary for replication. Such treatment prevents cells, particularly cells generated from stem cells, from dividing, so that if the cell transforms into a cancer cell, it cannot divide. Other known agents capable of inducing cellular senescence include those described by Petrova et al., "Small Molecule Compounds that Induce Cellular Senescence" Aging Cell, 15(6): 999-1017 (2016), which reference is incorporated herein in its entirety. By way of example only, such agents include agents that cause telomere dysfunction due to replication-related telomere shortening, subtoxic stresses (e.g., exposure to UV, gamma radiation, hydrogen peroxide, and hypoxia). The specific means by which the beta cells of the present invention are rendered non-replicative is not critical, provided that the cells can be implanted without the risk of cell division. Studies have shown that the adult pancreas has very little beta cell turnover, suggesting that limiting the ability of cells to divide has little to no effect on insulin production by implanted cells. See, e.g., Perl et al., The Journal of Clinical Endocrinology & Metabolism , Vol. 95, No. 10, October 1, 2010, pp. E234-E239.

[0103] Another aspect of the invention is a method of regulating insulin levels in a subject, comprising administering to a subject in need thereof a beta cell of the invention, wherein the beta cell expresses insulin and produces a chemotropic amount of an antagonist (e.g., CXCL12, CXCL13). The beta cell may be an autologous beta cell or a non-autologous beta cell, such as an allogeneic beta cell, and may have a vector expressing the antagonist, which may be integrated into the beta cell genome or present in episomal form. In one embodiment of the invention, the transgenic beta cell may be genetically modified to overexpress an endogenous antagonist (e.g., CXCL12, CXCL13) at a chemotropic level.

[0104] Methods for introducing the transgenic β cells described herein into an individual are well known to those skilled in the art and include, but are not limited to, injection, intravenous, intraportal, or parenteral administration. Single, multiple, continuous, or intermittent administration may be performed. See, e.g., Schuetz and Markmann, Curr Transplant Rep. 2016 Sep; 3(3): 254–263.

[0105] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of culture media and reagents for pharmaceutically active substances (including cells) is well known in the art. A typical pharmaceutical composition for intravenous infusion of beta cells can be prepared to contain 250 ml of sterile Ringer's solution and 100 mg of combination. Actual methods for preparing parenterally administrable compounds are known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 17th edition, Mack Publishing Company, Easton, Pa. (1985), and the 18th and 19th editions thereof, which references are incorporated herein by reference.

[0106] The transgenic and genetically modified beta cells of the invention can be introduced into any of several different sites well known in the art including, but not limited to, the pancreas, abdominal cavity, kidney, liver, portal vein, or spleen of a subject.

[0107] In addition, in order to avoid any possible transformation of transgenic β cells into cancer cells (which may lead to the possibility of tumor development in patients), transgenic β cells can be caused to age by contact with known agents such as mitomycin C, or exposure to subtoxic stress from ionizing radiation, hypoxia, hydrogen peroxide, etc. Cells derived from pluripotent stem cells usually undergo apoptosis during inappropriate cell division or due to immune cell clearance. The aged transgenic β cells described herein cannot divide, thereby eliminating the apoptotic trigger that occurs during cell division. In addition, the aged transgenic β cells described herein are immune cell resistant, thereby providing protection against apoptosis induction due to immune cell clearance. Accordingly, it is expected that the transgenic β cells described herein have a longer lifespan to a significantly longer lifespan compared to non-aging transgenic β cells.

[0108] Transgenic and preferably aged modified β cells can be transplanted into a subject via a graft. An ideal β cell transplant site would be one that supports engraftment, long-term function and survival of the transplanted cells in the subject and is easily accessible for maximum patient safety. Sites for implantation include the liver, intestine, subcutaneous, and pancreatic sites.

[0109] As used herein, the following abbreviations have the following meanings, and if abbreviations are not defined, they have their generally recognized scientific meaning.Amino acids are described herein using their established single-letter abbreviations.

[0110] FLAG = DYKDDDDK protein tag (SEQ ID NO: 10)

[0111] g / L = grams per liter

[0112] HRP = horseradish peroxidase

[0113] LDH = lactate dehydrogenase

[0114] iBLOT = Semi-dry protein transfer apparatus (Invitrogen)

[0115] MES = 2-(N-morpholino)ethanesulfonic acid

[0116] mL = milliliters

[0117] N / A = Not Applicable

[0118] nM = nanomolar concentration

[0119] PBMC = peripheral blood mononuclear cells

[0120] PBS = Phosphate Buffered Saline

[0121] TMB = 3,3',5,5'-Tetramethylbenzidine

[0122] μL = microliter

[0123] μg = microgram

[0124] xg = gravity multiple. Example

[0125] Example 1: Model cells for evaluating the expression levels of CXCL12-a and -b isoforms

[0126] HEK293 cells were transfected with 2 different CXCL12 isoforms (α and β) using commercially available plasmids for each isoform (plasmids available from GenScript). Transfected cells were selected with 250 ug / mL of G418 (commercially available from ThermoFisher) and stable pools were prepared for each isoform. Cells were allowed to acclimate to conditions for 3 days in the appropriate media. Conditioned media from transfected HEK293 cells expressing CXCL12α and CXCL12β were diluted 1:1 with assay dilution buffer. Two separate pools were established for each isoform, and then the concentration of each isoform in solution was obtained by absorbance using a standardized concentration curve. This experiment was repeated twice, and the results are as follows:

[0127]

[0128] The above results demonstrate that the transgenic model cells express CXCL12β at a significantly higher level than the transgenic model cells expressing CXCL12α.

[0129] Example 2 - Model cells for evaluating the expression levels of other isoforms of CXCL12

[0130] HEK293 cells were transfected with 5 different CXCL12 isoforms (α and β) using commercially available plasmids for each isoform (plasmids available from GenScript). Transfected cells were selected with 250 ug / mL of G418 (commercially available from ThermoFisher) and stable pools were prepared for each isoform. Cells were allowed to acclimate for 3 days in the appropriate media. Conditioned media were separated in 4-8% NuPage gels (commercially available from ThermoFisher) with MES buffer and transferred to nitrocellulose (iBLOT).

[0131] like Figure 1 The expression levels were detected on western blots using HRP-labeled anti-FLAG tag antibody / TMB chromogen (available from GenScript) as shown in Figure 1. The results demonstrate that the gamma, delta, and theta isoforms of CXCL12 have higher concentrations than the alpha or beta isoforms.

[0132] Example 3: Preparation of transgenic β cells

[0133] Pancreatic β cells derived from human induced pluripotent stem cells were purchased from Takara Bio USA, Inc. (Mountain View, CA) and cultured according to the instructions provided.

[0134] Cells were transduced with lentiviral vectors (pLenti-C-Myc-DDK, OriGene Technologies, Rockville, MD) containing human CXCL12 isoforms (CXCL12a / SDF-1α or CXCL12b / SDF-1β) or controls. Lentiviral vectors were used at a ratio of approximately 10:1 per β cell. Sequences including the tag (underlined) are provided below. The concentration of CXCL12 isoforms was determined by ELISA (RayBioTech, Norcross, GA) (Table 1).

[0135] CXCL12a (also known as SDF1a)

[0136] Accession number NM_199168

[0137]

[0138] CXCL12b (also known as SDF1b)

[0139] Accession number NM_000609

[0140]

[0141] Example 4: Transgenic β cells reject PBMCs

[0142] Transgenic β cells from Example 3 were contacted with human peripheral blood mononuclear cells (PBMC, Innovative Research, Novi, MI) at a ratio of 30:1 (PBMC:β ​​cells). Briefly, PBMCs were resuspended in β complete medium, counted and adjusted with the addition of 100 uL PBMCs to allow a 30:1 PBMC:β ​​cell ratio (to minimize dilution of expressed CXCL12). The final volume was 1.1 mL. Background controls of β cells without PBMCs and PBMCs without β cells were also prepared. 150 uL of culture medium was immediately removed from each sample and centrifuged at 1200 xg for 10 minutes. The supernatant was removed and stored at 4°C (time 0). The cells were returned to the incubator and sampled in a similar manner to the time 0 sample after 24 and 48 hours.

[0143] LDH release was tested at 24 and 48 h post-exposure using the Pierce LDH Cytotoxicity Assay Kit (ThermoScientific) according to the manufacturer's instructions. Increased LDH is an indicator of cytotoxicity (cell lysis).

[0144] Table 1 and Figure 2A Data from a representative experiment (background subtracted) are provided in . Data from a second representative experiment are provided in Figure 2B Available in.

[0145] Table 1. LDH and CXCL12 levels

[0146]

[0147] These data indicate that CXCL12 expression by β islet cells protects β islet cells from attack by immune cells, rendering them resistant. β cells expressing SDF1b / CXCL12b, which were expressed at higher levels than SDF1a / CXCL12a in this experiment, showed essentially no cytotoxicity in the presence of PBMCs.

[0148] Example 5: Alternative preparation of transgenic β cells

[0149] β cells isolated from subjects with type 1 diabetes were transfected or infected in vitro with a retroviral expression vector encoding CXCL12, or a control retroviral vector that does not encode CXCL12. Transgenic β cells with a retroviral vector encoding CXCL12 were assayed for expression of efflux amounts of CXCL12 using a Boyden chamber assay as previously described in Poznansky et al., Journal of Clinical Investigation, 109, 1101 (2002). Transgenic β cells expressing at least 100 nM CXCL12 are expected to exclude immune cells in this assay.

[0150] Example 6: Effect of forced senescence of transgenic β cells on transgenic cytokine expression

[0151] β cells were prepared as described in Example 3. Expression levels of SDF1a / CXCL12a and SDF1b / CXCL12b were measured by ELISA prior to treatment with mitomycin C (available from Santa Cruz Biotechnology) to determine baseline expression ("before"). The medium was replaced with fresh medium containing 10 ug / mL mitomycin C, an agent known to induce senescence. The cells were returned to the incubator for 2 hours. The medium containing mitomycin C was removed by gentle pipetting. The cells were washed twice with PBS. After the second wash, the cells were fed with fresh complete medium. The expression of SDF1a / CXCL12a or SDF1b / CXCL12b was determined by ELISA assay.

[0152] Data from two representative experiments are shown in Tables 2 and Figure 3 A. Expression of SDF1a / CXCL12a and SDF1b / CXCL12b is not affected by forced senescence of transgenic β cells.

[0153] Table 2: CXCL12a and –b levels before and after mitomycin C treatment

[0154] .

[0155] Example 7: Effects of forced senescence of transgenic β cells on PBMC attack

[0156] β cells were prepared as described in Example 3. Cells were treated with mitomycin C or control as described in Example 4. Cells were contacted with PBMCs as described in Example 2.

[0157] Data from two representative experiments are shown in Figure 4A and 4B Middle. LDH levels are not affected by forced senescence of transgenic β cells.

[0158] Example 8: Effects of forced senescence of transgenic β cells on insulin production

[0159] Beta cells were prepared as described in Example 3. Cells were treated with mitomycin C or a control as described in Example 4.

[0160] The complete growth medium was replaced with 1 mL of medium 2 and maintained on medium 2 for 2 days with medium changes every 24 hours. On day 3, β cells were challenged with high glucose medium (4.5 g / L glucose). Samples of conditioned medium were taken 24 hours after the high glucose challenge and insulin expression was measured by sandwich ELISA.

[0161] Data from two representative experiments are shown in Tables 3 and Figure 5 The results demonstrate that transgenic β cells and transgenic aged β cells produce essentially equivalent amounts of insulin as control β cells in response to a hyperglycemic challenge.

[0162] Table 3: Insulin expression in response to hyperglycemic challenge

[0163] .

[0164] Example 9: In vivo evaluation of transgenic β cells

[0165] Humanized mice with humanized immune systems (see, e.g., N. Walsh, "Humanized mouse models of clinical disease," Annu Rev Pathol 2017, 12, 187-215; E. Yoshihara et al.) are administered transgenic human β cells expressing efflux amounts of CXCL12 or control transgenic human β cells, and the production of insulin and the survival of the transgenic β cells in the mice are determined at various time points after the initial administration. It is expected that transgenic human β cells expressing efflux amounts of CXCL12 will survive for a longer period of time compared to control transgenic human β cells. It is also expected that mice receiving transgenic β cells expressing efflux amounts of CXCL12 will also have higher amounts of human insulin than mice receiving control transgenic human β cells, and that the higher levels of human insulin will persist for a longer period of time compared to the levels in mice administered control transgenic human β cells.

[0166] Humanized mice with humanized immune systems (see, e.g., N. Walsh, "Humanized mouse models of clinical disease," Annu Rev Pathol 2017, 12, 187-215; E. Yoshihara et al.) are administered with genetically modified human β cells or control human β cells that overexpress CXCL12 from an endogenous CXCL12 gene, and the production of human insulin and the survival of β cells in the mice are determined at various time points after the initial administration. It is expected that genetically modified human β cells that overexpress CXCL12 will survive for a longer period of time compared to control human β cells that are not genetically modified to overexpress CXCL12. It is also expected that mice that receive genetically modified human β cells that overexpress CXCL12 will also have higher amounts of human insulin than mice that receive control human β cells, and that higher levels of human insulin will last for a longer period of time compared to the levels in mice that are administered control human β cells.

[0167] Optionally, the cells are treated with an agent that crosslinks DNA within the cells to prevent cell division (eg, mitomycin C).

[0168] The foregoing description is merely set forth to illustrate the present invention and is not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the present invention may occur to those skilled in the art, the present invention should be broadly interpreted as including all changes within the scope of the claims and their equivalents.

Claims

1. A human β cell expressing or overexpressing a human chemotropic agent in an amount sufficient to render the β cell resistant to human immune cells, wherein the β cell is capable of expressing insulin and incapable of cell division, and further wherein the chemotropic agent is selected from the human α or β isoforms of CXCL12.

2. A human β cell comprising a vector which itself comprises a nucleic acid sequence encoding a human CXCL12 α or β isoform, wherein the β cell is resistant to human immune cells.

3. The human β cell of claim 1 or 2, wherein the cell is an autologous β cell obtained from a subject suffering from type 1 diabetes.

4. The human β cell of claim 1 or 2, wherein the cell is an allogeneic β cell. The human β cells of claim 1 , wherein the human immune cells comprise NK cells, cytotoxic T cells and / or B cells. The human β cell of claim 1 , wherein the cell expresses an efferotropic amount of human CXCL12.

7. A human β cell comprising a transgenic regulatory region upstream of an endogenous CXCL12 α or β isoform coding region to achieve sufficient overexpression of said α or β isoform of CXCL12 α or β to render said β cell resistant to human immune cells.

8. The human β cell of claim 7, wherein the cell is an autologous β cell.

9. The human β cell of claim 7, wherein the cell is an allogeneic β cell.

10. The human β cell of claim 8, wherein the cell is an autologous β cell obtained or derived from a subject suffering from type 1 diabetes. The human β cell of claim 9 , wherein the cell is an allogeneic β cell obtained or derived from a subject without type 1 diabetes.

12. The human β cell of any one of claims 7-11, wherein the transgene regulatory region is an exogenous constitutive or inducible promoter.

13. The human β cell of claim 7, wherein the cell expresses CXCL12β. The human β cell of claim 2 , wherein the β cell is incapable of cell division.

15. The human β cell of any one of claims 7-11 or 13-14, wherein the human immune cells comprise NK cells, cytotoxic T cells, and B cells.

16. Use of human transgenic beta cells according to any one of claims 1, 2 or 7 for the preparation of a medicament for producing insulin in response to a hyperglycemic environment.

17. The use of claim 16, wherein the hyperglycemic environment comprises human immune cells.

18. The use of claim 17, wherein the human immune cells include NK cells, T cells and B cells.

19. A composition for use in treating diabetes, comprising the human transgenic beta cells according to any one of claims 1, 2 or 7.

20. An allogeneic human β cell genetically modified ex vivo to express or overexpress human CXCL12 in an amount sufficient to render the β cell resistant to human immune cells, wherein the β cell is capable of expressing insulin and is obtained by differentiation from human stem cells, wherein the human CXCL12 is an α or β isoform.

21. An allogeneic human β cell comprising a vector which itself comprises a nucleic acid sequence encoding human CXCL12, wherein the β cell is resistant to human immune cells and is obtained by differentiation from human stem cells, wherein the human CXCL12 is an α or β isoform.

22. The human β cell of claim 20 or 21, wherein the human immune cell comprises a NK cell, a cytotoxic T cell and / or a B cell.

23. The human β cell of claim 20 or 21, wherein the cell expresses CXCL12β.

24. The human β cell of claim 20 or 21, wherein the β cell is incapable of cell division.

25. Use of human beta cells according to claim 20 or 21 for the preparation of a medicament for producing insulin in response to a hyperglycemic environment.

26. A pharmaceutical composition for producing insulin in response to a hyperglycemic environment, comprising human beta cells according to claim 20 or 21.

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