TWI806907B - Treating diabetes with genetically modified beta cells - Google Patents

Abstract

Described herein are human transgenic beta cells expressing fugetactic levels of CXCL12 to a subject in need thereof. Also described herein are beta cells comprising a transgene comprising a nucleic acid sequence encoding CXCL12.

Description

Treating diabetes with genetically modified beta cells

The present invention relates to genetically engineered human beta cells and methods of using such cells. Genetically engineered (transgenic) human beta cells express fugetactic amounts of the scavenger, thereby conferring protection against human mononuclear immune cells. In one embodiment, the chemoattractant is, for example, CXCL12 or CXCL13. In one embodiment, the transgenic β-cell comprises a vector, wherein the vector comprises a nucleic acid sequence encoding a keloid, and preferably a human keloid. In one embodiment, the transgenic beta cells are further engineered to be senescent cells. The methods of the invention include the use of such cells to express insulin in hyperglycemic environments, including insulin found in diabetic patients, especially type 1 diabetic patients.

Beta cells are responsible for producing insulin in the pancreas. In individuals with type 1 diabetes (T1D), the beta cells are attacked and destroyed by the immune system, and thus individuals with T1D cannot 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 (April 29, 2014) .

Type 2 diabetes (T2D) occurs when an individual's hyperglycemia persistently exceeds the capacity of the individual's beta cells and leads to beta cell dysfunction, dedifferentiation and death. Felicia W Pagliuca and Douglas A. Melton, "How to make a functional β-cell", Development 2013, 140(12), 2472-2483.

Allogeneic β-cell transplantation (also known as islet cell transplantation) from a normal donor to a diabetic recipient has been considered as a treatment for diabetes. However, the infiltration of mononuclear immune cells (T cells, B cells and NK cells) caused the 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/Cloning-technique- USED-Create-Pancreative-Cells-FORL-TYPE-DIABETES-94233303.html (April 29, 2014); Alan H. Cruickshank and EMYR W Benbow, "Recurrence of Diabetes", Patholo Gy of the Pancreas (2nd Edition 1995); Felicia W Pagliuca and Douglas A. Melton, "How To Make A Functional β-Cell", Development 2013, 140 (12), 2472-2483.

Current clinical practice is to transplant islets containing β cells into the liver via the portal vein, where the rationale is that most of the insulin released from the pancreas is used in the liver and this site is easily accessible by minimally invasive procedures. However, one-half of the beta 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. Furthermore, the blood-mediated transient inflammatory response (IBMIR) encapsulates transplanted islets in a fibrin clot and enhances the immune response to transplantation. Thus, several alternative sites for transplantation have been tested, including the bowel, renal capsule, omentum, and subcutaneously, which may be optimal for patient safety, but have not been adequately evaluated for systemic release of insulin.

To prevent their exposure to mononuclear immune cells, beta cells have been encapsulated in devices that reportedly serve the dual function of isolating these cells from immune destruction and protecting the host from the graft. Immune protection is required when non-autologous cells (eg, allogeneic or xenogeneic cells) are used for transplantation or if autologous cells are transplanted in an autoimmune environment, such as in type 1 diabetic patients. This can be achieved by blocking the cellular response by physically separating the cells using a semi-permeable membrane or scaffold. This approach reduces the need for immunosuppression and has been reviewed in detail elsewhere in recent years (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, if desired, allows removal. This is specifically associated with uncontrolled differentiation and growth, such as teratomas commonly 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 using cell purification (Kelly et al., 2011) or by modified differentiation methods to generate grafts consisting of pure mature β-cell populations. Encapsulation also prevents possible metastasis if the insulinoma/teratoma is formed by transplanted cells.

This method requires an efficient and reproducible microencapsulation protocol, and without the possibility of clearance or removal, some cells may die within the encapsulation material. Several reports have shown that microencapsulated β cells can continue to survive up to 6 months after implantation (Orlando et al., Diabetes. 2014;63:1433-1444). This means that when the device no longer functions, repeat surgery is required to replace the microencapsulated device.

In view of the above, there is a far unmet need to develop technologies to effectively treat diabetes, and especially type 1 diabetes.

The present invention relates to transgenic human beta cells, as well as transgenic senescent human beta cells, which express an effective amount of a scavenger to render these cells resistant to human immune cells. Removal agents are well known in the art and include "CXCL12". Also known as SDF-1, this cytokine is produced by the thymus and bone marrow stroma (see, eg, Honjo et al., US Patent No. 5,756,084, issued May 26, 1998, entitled: "Human Stromal-Derived Factor 1α. and 1β."). CXCL12 has been reported to repel effector T cells while recruiting immunosuppressive regulatory T cells to anatomical sites. See eg Poznansky et al., Nature Medicine 2000, 6:543-8. CXCL12 and its receptor CXCR4 have also been reported as components of angiogenesis.

Also disclosed are agents that repel 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.

One embodiment of the present invention is transgenic human beta cells expressing an effective amount of a culprit, preferably CXCL12 or CXCL13, to render the cells resistant to human immune cells. In one embodiment, such a phagocytic effective amount of a phagocytic agent is produced by introducing a human transgene directed against the agent (eg, CXCL12, CXCL13) into β cells or β cell precursors (eg, pluripotent stem cells). These human transgenic beta cells were further characterized by the expression of insulin in a hyperglycemic environment. Accordingly, these cells can be used in methods of treating diabetes in an individual. Transgenic human beta cells used in the methods described herein may be autologous or non-autologous, eg, allogeneic beta cells. In one embodiment, the patient suffers from T1D. In another example, transgenic human beta cells can be engineered into senescent cells (unable to divide) such that any further differentiation of these cells into cancer cells is abolished and induction of apoptosis due to inappropriate cell division is ineffective.

One embodiment of the invention uses beta cells, such as autologous or allogeneic beta cells, obtained from or derived from a non-diabetic human individual or a human individual with diabetes. These β cells include vectors expressing functional chemoattractants (eg, CXCL12, CXCL13). Such vectors are designed to express the agent in the transgenic β-cell in an amount sufficient to generate an efflux buffer around the β-cell. Without wishing to be bound by theory, it is expected that this buffer renders the beta cells resistant to immune cell attack, yet expresses the insulin that would be required to maintain proper blood glucose levels in diabetic individuals. The generation of autologous beta cells from patients with diabetes is known in the art. See, eg, Egli et al., EMBO J. 2015 Apr 1; 34(7): 841-855, which is incorporated herein by reference in its entirety. Allogeneic beta cells derived from stem cells are commercially available.

One aspect of the invention is the administration of transgenic human beta cells comprising a transgene encoding a scavenger (eg, CXCL12, CXCL13) to an individual in need thereof to regulate insulin levels in the individual and treat diabetes. In addition, the expression of a sufficient amount of the scavenger prevents the risk of destruction of the transgenic β-cells due to the infiltration of mononuclear immune cells. Transgenic human beta cells can be autologous or allogeneic. In one embodiment of the present invention, the transgenic β-cells are autologous β-cells derived from patients with diabetes. In another embodiment, the transgenic beta cells are allogeneic human beta cells. In another embodiment, the patient has type 1 diabetes.

Another aspect of the invention pertains to transgenic human beta cells capable of expressing an effective amount of a culprit (eg, CXCL12, CXCL13) to be resistant to immune disruption. A scavenger (eg, CXCL12, CXCL13) can be an endogenous agent, ie, one expressed by the individual to be treated; or an exogenous agent, such as an agent from a non-autologous source or an engineered scavenger. In one embodiment, a gene encoding a scavenger in beta cells is engineered to be overexpressed compared to an unengineered gene. Methods for engineering gene expression are known in the art, for example, performing site-directed gene editing to replace endogenous promoters with different promoters (eg, constitutive promoters, inducible promoters, etc.). In one embodiment, a recombinant polynucleotide encoding an agonist is inserted into a beta cell such that the agonist is expressed by the recombinant polynucleotide. Methods for inserting recombinant genes into cells (transduction, transfection, etc.) are well known in the art, as are methods for making vectors from recombinant polynucleotides for insertion into cells.

In some embodiments, the repellent is an engineered repellent. For example, the polypeptide sequence of a scavenger can be engineered to increase circulating half-life, incorporate conservative amino acid changes, enhance binding to the extracellular matrix, improve the activity of the agent, and the like. Thus, a gene encoding an engineered chemokine (eg, CXCL12 or CXCL13) can be engineered such that the gene has at least 95% sequence identity with the native gene, and preferably has 99% sequence identity with the native gene. Likewise, the amino acid sequence of the engineered keloid (eg, engineered CXCL12 or CXCL13) has at least 95% and preferably 99% sequence identity with the natural keloid.

In one embodiment, there is provided a human β-cell comprising a vector which itself comprises a nucleic acid sequence encoding human CXCL12 or engineered CXCL12, wherein the β-cell is rendered resistant to human immune cells.

In one embodiment, the human transgenic beta cells are autologous beta cells obtained from an individual with type 1 diabetes.

In one embodiment, the human beta cells are allogeneic beta cells.

In one embodiment, the human mononuclear immune cells comprise NK cells, T cells and B cells. In one embodiment, the T cells comprise cytotoxic T cells.

In one embodiment, the transgenic human beta cells express repellent amounts of human CXCL12.

In one embodiment, the human CXCL12 is selected from the group consisting of CXCL12α and CXCL12β.

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

In one embodiment, the human transgenic β-cell comprising a transgenic regulatory region upstream of the endogenous CXCL12 coding region, wherein the β-cell is resistant to human immune cells, is an autologous β-cell, and is preferably a β-cell obtained from a patient with diabetes.

In one embodiment, the human transgenic beta cell comprising a transgenic regulatory region upstream of an endogenous CXCL12 coding region, wherein the beta cell is resistant to a human immune cell, is an allogeneic beta cell.

In one embodiment, a human transgenic beta cell comprising an expressible human CXCL12 or CXCL13 gene is provided, wherein the cell expresses an effective amount of CXCL12 or CXCL13 to be resistant to human immune cells.

In one embodiment, the human transgenic beta cells comprise the human gene for CXCL12.

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

In one embodiment, the human transgenic β-cell comprises the human gene for CXCL12β.

In one embodiment, there is provided a human transgenic senescent beta cell comprising an expressible human CXCL12 or CXCL13 gene, wherein the cell expresses an effective amount of CXCL12 or CXCL13 to be resistant to human immune cells, and additionally, wherein the cell is senescent.

In one embodiment, human transgenic senescent beta cells comprise an expressible human gene for CXCL12 and are capable of expressing insulin in the presence of hyperglycemic mediators.

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

In one embodiment, the human transgenic β-cell comprises an expressible human gene which is CXCL12β.

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

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

In one embodiment, the β cell lines described herein are obtained by: (a) obtaining a population of human progenitor cells or human pluripotent stem cells from a human individual; (b) differentiating the individual's progenitor cells or pluripotent stem cells into β cells; and (c) introducing a nucleic acid molecule encoding an attractant into the β cells.

In one embodiment, the chemoattractant is an interleukin, a chemokine, a CXCR4-binding antibody, a CXCR4 ligand, a CXCR5-binding antibody, or a CXCR5 ligand.

In one embodiment, the chemoattractant is CXCL12, CXCL13, gp120 or IL-8.

In one aspect, a method is provided for increasing the survival of beta cells in a biological sample comprising immune cells by engineering the beta cells to express a scavenger in an amount sufficient to inhibit or block killing of the beta cells by immune cells.

Cross-References to Related Applications

This application asserts 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. 62/694,634, filed July 6, 8; U.S. Provisional Application No. 62/696,603, filed July 11, 2018; U.S. Provisional Application No. 62/717,587, filed August 10, 2018; U.S. Provisional Application No. 62/719,975, filed August 20, 2018; Priority of U.S. Provisional Application No. 62/734,910 filed; each of which is incorporated herein by reference in its entirety. sequence listing

This application contains a Sequence Listing, which was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Created on September 25, 2018, this ASCII copy is named 054610-501001WO_SL.txt and has a byte size of 8,668.

The invention provides autologous/allogeneic human beta cells that are transgenic and comprise a transgene encoding a human scavenger (e.g., CXCL12, CXCL13), or are genetically engineered to express or overexpress an endogenous (human) scavenger (e.g., CXCL12, CXCL13) in invasive amounts. In a preferred embodiment, the transgenic β cells described herein are further engineered to be senescent cells. In another aspect of the method, β cells are engineered or treated to express an effective amount of an attractant (eg, CXCL12, CXCL13) to inhibit immune destruction of transgenic human β cells and produce insulin in response to a hyperglycemic environment.

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

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

The term "fugetaxis" or "fugetactic" refers to the ability of an agent to repel (or chemically repel) eukaryotic cells that have the ability to migrate. The attractant amount of CXCL12 or CXCL13 (or other attractant) exhibited by the cell is an amount sufficient to block or inhibit the migration of immune cells towards the cell, or in some aspects, cause the cell to repel the immune cell.

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

The terms "anti-immune cells" or "stealth to the immune system" indicate that the β-cells exhibit an amount of a scavenger sufficient to block or inhibit the migration of immune cells towards the cell, or in some aspects, cause the β-cells to repel the immune cells. In a preferred embodiment, such blocking or inhibition is measured by the degree of cell death following exposure of the β-cells of the invention to human mononuclear immune cells (eg, PBMC). Cell death can be assessed by lactate dehydrogenase (LDH) released from cells that have undergone lysis. Preferably, the anti-immune cell β-cells of the present invention can be assessed by cells showing a 50% lower LDH amount relative to a control at a ratio of about 30:1 immune cells to β-cells of the present invention during a two-day incubation period. More preferably, the anti-immune beta cells exhibit 60% lower amounts of LDH relative to controls; and even more preferably, 75% lower amounts of LDH relative to controls; and optimally, 95% lower amounts of LDH relative to controls. The procedure used to assess the amount of LDH is described in Example 2 herein.

Repellents are agents with repellent activity. Repellants may include, but are not limited to, CXCL12, CXCL13, gp120, IL-8, CXCR4-binding antibodies, CXCR4 ligands, CXCR5-binding antibodies, or CXCR5 ligands.

The term "effector T cells" refers to differentiated T cells capable of eliciting a specific immune response by releasing cytokines.

The term "regulatory T cell" refers to a T cell that reduces or suppresses the immune response of a B cell or other T cell to an antigen.

The term "CXCL12" or "SDF-1 polypeptide" refers to cytokines well known in the art (see eg Table 1). In one embodiment, the term refers to a protein or fragment thereof that binds a CXCL12-specific antibody and has chemotactic or cytotoxic activity. Chemotactic or cytotoxic activity is determined by analyzing the direction of T cell migration (eg, toward or away from the relevant agent). See, eg, 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", Transplantation 2007, 83(2), 174-183. "Dress" or "dress migration" is the movement of migratory cells away from the source of the agent (ie, towards lower concentrations of the agent). It should be understood that the term "CXCL12" refers to all known isoforms thereof, including alpha, beta, gamma, delta, epsilon, phi and theta isoforms. Preferred CXCL12 isoforms are α and β. CXCL12 is known to induce angiogenesis.

The terms "type 1 diabetes" and "type 2 diabetes" refer to two serious pathophysiologies associated with increased blood glucose. Type 1 diabetes is characterized by an autoimmune attack on the insulin-producing beta cells of the pancreas, while type 2 diabetes is associated with beta cell dysfunction and increased peripheral insulin resistance. Similar to type 1, beta 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 typically characterized by loss of insulin-producing beta cells in the islets of the pancreas, resulting in insulin deficiency. This type of diabetes can be further classified as immune-mediated or idiopathic. Most type 1 diabetes is immune mediated, in which beta cell loss is due to T cell mediated autoimmune attack. Type 2 diabetes is characterized by beta cell dysfunction and insulin resistance. It is believed that defective responsiveness of body tissues to insulin involves the insulin receptor and downstream cell signaling. Similar to type 1 diabetes, deficiencies in β-cell populations are also a pathogenic factor in many types of type 2 diabetes. In the early stages of type 2 diabetes, hyperglycemia can be reversed by a variety of measures and drugs that improve insulin secretion and lower glucose production by the liver. As the disease progresses, insulin secretion decreases, and therapeutic replacement of insulin may sometimes become necessary in some patients.

"Individual" or "patient" refers to a mammal, preferably a human individual.

A "subject in need" or "patient in need" is a subject with Type 1 or Type 2 diabetes.

As used herein, the terms "treat", "treating", "treatment" and similar terms mean to reduce or ameliorate a disorder and/or symptoms associated therewith. It is to be understood that, although not exclusive, treating a disorder or condition does not require complete elimination of the disorder, condition or symptoms associated therewith.

In the present invention, "comprises", "comprising", "containing" and "having" and similar terms may have the meanings assigned to them in the US Patent Law and may mean "includes", "including (including)" and similar terms; Recited, but excluding prior art embodiments, so long as the essential or novel features of the recited would not be altered by the presence of more than recited.

The term "about" when used in front of a numerical designation such as temperature, time, amount, concentration and such other designations (including ranges) indicates that it may vary (+) or (-) 10%, 5%, 1%, or any subrange or approximation of a subvalue therebetween. Other definitions also appear throughout the context of the present invention.

One aspect of the invention is transgenic β cells, such as human autologous β cells or non-autologous β cells, such as allogeneic β cells, comprising a nucleic acid encoding a chemoattractant (e.g., CXCL12, CXCL13) operably linked to a promoter such that the chemoattractant (e.g., CXCL12, CXCL13) is expressed in a chemoattractant amount in the microenvironment of the β cell. The promoter may be endogenous to the beta cell or a heterologous but functional promoter in the beta cell. Preferably, the nucleic acid encoding the chemoattractant (eg, CXCL12, CXCL13) is endogenous to the individual treated with the transgenic beta cells. In one embodiment, the allogeneic beta cells are derived from a non-T1D donor.

One aspect of the invention is a human beta cell comprising a genetically engineered endogenous human gene encoding a chemoattractant (e.g., CXCL12, CXCL13), wherein the gene has been engineered to include a heterologous promoter operably linked to a sequence encoding the chemoattractant, such that the chemoattractant is expressed by the endogenous gene in a chemoattractant amount in the microenvironment of the beta cell. The promoter can be introduced into the beta cell to be operably linked to the sequence encoding the dispersant using genome editing techniques known in the art. CXCL12 is well known to have several isoforms, including alpha, beta, gamma and theta. In a preferred embodiment, the isoform used is CXCL12β. The transgenic human beta cells described herein produce insulin in response to a hyperglycemic environment. The term "insulin" is meant to encompass both proinsulin and insulin.

In general, the invention provides β cells, and preferably human β cells, that exhibit an attractant (e.g., CXCL12, CXCL13) in an amount sufficient to block or inhibit migration of immune cells (e.g., human immune cells) to β cells, or to repel immune cells. The terms immune cells and monocytes (T cells, B cells and NK cells) are used interchangeably. The ability of an attractant (eg, CXCL12, CXCL13) polypeptide to repel immune cells (eg, effector T cells) can be assessed in vitro using a boyden chamber assay. See eg, as previously described in Poznansky et al., Journal of Clinical Investigation, 109, 1101 (2002). Alternatively, the survival of transgenic human beta cells is assessed by combining such cells with human PBMCs. The rate of cell death can be assessed by measuring one or more cell death markers over time. One commonly used such marker is lactate dehydrogenase (LDH), which is released during necrosis.

Without wishing to be bound by any theory, applicants contemplate that, in one aspect of the invention, the amount of occult (e.g., CXCL12, CXCL13) produced by the transgenic β-cells is sufficient to provide phagocytosis in the β-cell microenvironment, but not in an amount sufficient to increase the systemic amount of the agent and upset the balance between beneficial effects of the agent in one process and adverse consequences in another. Furthermore, CXCL12 is known to induce angiogenesis when it binds to its receptor CXCR4. Furthermore, without being bound by any theory, it is expected that the microenvironment of the implanted transgenic beta cells expressing CXCL12 will induce an angiogenic response that increases the survival of the implanted cells.

A cytotoxic effective amount of a chemoattractant (eg, CXCL12, CXCL13) is any amount sufficient to block killing of transgenic β cells by immune cells. For example, an effective amount of a dispersant (eg, CXCL12, CXCL13) in the microenvironment of transgenic β cells may be at least about 100 ng/mL and preferably at least 100 nM. In some embodiments, the amount of the scavenger (eg, CXCL12, CXCL13) in the microenvironment of the transgenic β-cell is at least about 1000 ng/mL. For example, the following specific ranges are suitable for use in 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 M or about 100 nM to about 1 µM.

In various embodiments, the effective amount of the extinction agent (eg, CXCL12, CXCL13) in the microenvironment of the transgenic β cells is in the range of 20 ng/mL to about 5 μg/mL. In various embodiments, the repelling effective amount is in the range of 20 ng/mL to about 1 µg/mL. In various embodiments, the amount of the expelling agent (e.g., CXCL12, CXCL13) in the microenvironment of the β-cell is sufficient for expelling in the range of 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, the transgenic β-cells may express sufficient amounts of culverts such that the microenvironment produces an invasive effect that extends to adjacent non-transgenic β-cells. The effective amount of the dispersing agent (eg, CXCL12, CXCL13) in the microenvironment of the transgenic β-cell can be any value or sub-range (inclusive) within the stated range.

Although immunologists commonly use mice and mouse DNA to further understand how the human immune system works, there are significant differences between humans and mice. Therefore, the vector-encoded chemoattractant (eg, CXCL12, CXCL13) is preferably a human agent. Javier Mestas and Christopher CW 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 (2015 August 21).

CXCL12 polypeptides are known in the art. See, eg, Poznansky et al., Nature Medicine 2000, 6:543-8 and US Patent Publication No. 20170246250, both of which are incorporated herein by reference in their entirety. The terms CXCL12 and SDF-1 are used interchangeably. Exemplary CXCL12/SDF-1 isoforms are provided in Table I of US Publication 20170246250. Exemplary CXCL12/SDF1 isoforms are also provided in Table 1 (below): Table 1: Human CXCL12/SDF1 isoforms

In one embodiment, the CXCL12 polypeptide has at least about 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to NP 001029058 and has chemoattractant or chemoattractant activity. In one embodiment, CXCL12 polypeptide and 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 have at least 85%, 90%, 95%, 96%, 98%, 99%, or 100%of aminic acid. Sequence consistency and have cytopenin or eliminate activity. Such sequence identity is based on the substitution of a first amino acid by a known conserved second amino acid. Such conservative substitutions are well-recognized in the art, and testing for the cytotoxic properties of the resulting engineered CXCL12 polypeptides is also well known in the art. See eg, Poznansky, supra.

CXCL13 peptides are known in the art. CXCL13 is also known as B-lymphocyte chemoattractant (BLC) or B-cell-attracting chemoattractant 1 (BCA-1), and these terms are used interchangeably. For example, human CXCL13 can be found in accession number Q53X90. In one embodiment, the CXCL13 polypeptide has an amino acid sequence comprising: MKFISTSLLLMLLVSSSLSPVQGVLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGCPRKEIIVWKKNKSIVCVDPQAEWIQRMMEVLRKRSSSTLPVPVFKRKIP (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 to Q53X90 and has chemoattractant or cytotoxic 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 chemoattractant or cytotoxic activity.

Transgenic beta cells used in the methods described herein may be autologous or non-autologous, eg, allogeneic beta cells. "Autologous cells" are cells from the same individual. "Allogeneic" cells are cells from a genetically similar but not identical donor of the same species. Allogeneic cells suitable for use in the methods of the invention are preferably from human individuals. Allogeneic cells suitable for use in the methods of the invention may be from a relative, such as a sibling, cousin, parent or child; or a non-relative. Criteria for selecting an allogeneic donor are well known in the art, see, eg, Tatum et al., Diabetes Metab Syndr Obes 2017: 10 73-78. Human allogeneic β-cells are commercially available, and autologous β-cell lines are generated by the method described by Egli et al., supra.

In one embodiment, the transgenic human beta cells used in the methods of the invention are autologous transgenic beta cells, which can be prepared by pluripotent progenitor or pluripotent stem cell-derived beta cells obtained from a patient using methods known in the art. These derived β-cells may comprise (eg, be transfected or infected with, etc.): an expression vector comprising a nucleic acid sequence encoding an attractant (eg, CXCL12, CXCL13).

Alternatively, transgenic beta cells used in the methods of the present invention can be prepared by isolating pancreatic islet beta cells from an individual in need thereof. These isolated islet β-cells may comprise (eg transfected or infected with, etc.): an expression vector comprising a nucleic acid sequence encoding an attractant (eg CXCL12, CXCL13). Alternatively, beta cells can be genetically engineered to express an endogenous scavenger (eg, CXCL12, CXCL13) gene such that they constitutively produce a scavenger (eg, CXCL12, CXCL13) in an effective amount.

In one embodiment of the invention, the β-cell comprises (e.g., transfected or infected with, etc.): an expression vector comprising a nucleic acid molecule encoding an attractant (e.g., CXCL12, CXCL13) operably linked to a promoter suitable for expression in the β-cell. The vector may integrate into the genome of the beta cell or it may exist episomally and not integrate into the genome.

The transgenic β-cells of the present invention can also be prepared from adult stem cells by isolating the adult stem cells from the individual, culturing the stem cells under appropriate conditions to expand the cell population and induce differentiation into β-cells. Cells can be engineered to express an effective amount of a chemoattractant (eg, CXCL12, CXCL13) by introducing an expression vector encoding an effective amount of a chemoattractant (eg, CXCL12, CXCL13) into the cell; or editing the genome to express an effective amount of a chemoattractant (eg, CXCL12, CXCL13). The vector can be introduced into the stem cell prior to differentiation into beta cells or the genome of the stem cell can be edited to contain a heterologous promoter. Alternatively, the vector can be introduced into the resulting beta cells or the genome of the resulting beta cells can be edited to contain a heterologous promoter.

The transgenic β-cells of the present invention can also be prepared by: generating induced pluripotent stem (iPS) cells from individual somatic cells, such as β-cells, fibroblasts or keratinocytes; treating the iPS cells to induce differentiation into β-cells; and introducing an expression vector comprising a nucleic acid sequence encoding a chemoattractant (such as CXCL12, CXCL13) into the differentiated β-cells.

The transgenic β cells of the present invention can also be prepared by: preparing induced pluripotent stem (iPS) cells generated from individual somatic cells; introducing an expression vector comprising a nucleic acid sequence encoding a chemoattractant (such as CXCL12, CXCL13) into the iPS cells; and treating the iPS cells before or after introducing the transgene to induce differentiation into β cells.

The transgenic β-cells of the present invention can also be produced using methods known in the art by obtaining progenitor cells or progenitor-like cells, such as pancreatic β-cell progenitor cells; introducing a vector comprising a nucleic acid sequence encoding an attractant (e.g., CXCL12, CXCL13) into the cells; and treating the cells before or after introducing the vector to induce differentiation into β-cells, or insulin-releasing cells that respond to the amount of glucose in vivo; see, for example, Millman et al. Nature Communications (May 2016 10) 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 (27 February 2018). Progenitor cells and progenitor-like cells may be autologous or non-autologous to the individual being treated with the transgenic cells, eg, allogeneic cells. Insulin producing cells that respond to the amount of glucose in vivo (see eg Qadir et al., supra) can be genetically engineered to express a decimator (eg CXCL12, CXCL13) as described herein and is also an embodiment of the invention. Such genetically engineered insulin-producing cells may also be used in the methods of the invention to treat diabetes as described herein.

Any suitable somatic cells from an individual 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 der ived 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 C ell 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, e.g., US Patent Publication No. 20170081641 and US 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 betes Reports 2017 Nov, 17: 116; US 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).

Preferably, the sequence encoding the chemoattractant 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, β-actin promoter, muscle creatine kinase promoter and human elongation factor promoter (EF1α), GAPDH promoter, actin promoter and ubiquitin promoter as well as viral promoters (including SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse milk Adenotumor virus promoter, Rous sarcoma 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 related promoters, such as viral and eukaryotic promoters, are also well known in the art (see, eg, Sambrook and Russell (Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Press)). The regulatory region operably linked to the sequence encoding the keloid may be any constitutive promoter suitable for expression in the individual cell.

Transgenic cells of the present invention expressing a phenotypic agent (eg, CXCL12, CXCL13), whether autologous or non-autologous, eg, allogeneic, can be administered to an individual in need by any means known in the art for administering β cells. Transgenic cells of the invention can be administered in an amount sufficient to provide an amount of insulin capable of alleviating at least some of the symptoms associated with low insulin amounts.

Another aspect of the present invention is a method of treating diabetes in an individual in need thereof, comprising the steps of: (a) obtaining or deriving beta cells or insulin-producing beta cells from the individual; (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 individual.

Various vectors suitable for transferring exogenous genes into mammalian cells, such as β cells, including vectors integrated into the genome and vectors not integrated into the genome but present in the form of episomes; and methods for introducing such vectors into cells are available, and each vector and method are known in the art. For example, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated (AAV) type vectors and EBV type vectors can be used. See, eg, US 20110280842; 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, Vol. 6, No. 3 Issue 1, pp. 30675-30703.

Another aspect of the present invention is a method for increasing the survival period of β-cells in a biological sample containing immune cells, which comprises introducing an expression vector encoding a chemoattractant (such as CXCL12, CXCL13) into the β-cell; or editing the genome of the β-cell so that the β-cell expresses the chemoattractant (such as CXCL12, CXCL13). In one aspect of the invention, the chemoattractant (eg, CXCL12, CXCL13) is expressed by β cells in an amount sufficient to block or inhibit migration of immune cells (eg, T cells, B cells and/or NK cells) to β cells. In one aspect of the invention, an attractant (eg, CXCL12, CXCL13) is expressed by a beta cell in an amount sufficient for the beta cell to repel the immune cell. In one aspect of the invention, the genetically engineered beta cells are in an individual, such as a human individual with type 1 or type 2 diabetes. In one embodiment, the beta cells are autologous beta cells of the individual.

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 virions, and agent-enhanced uptake of DNA. Lipofectamine reagents are commercially available (eg Transfectam ^™ and Lipofectin ^™ ). Cationic and neutral lipids suitable for efficient receptor-recognizing liposome transfection of polynucleotides are known. Nucleic acids can be delivered to cells (administration ex vivo) or tissues of interest (administration in vivo). 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, e.g., Oumard et al., Cytotechnology (2006) 50: 93-108), which can mediate directed insertion of transgenes.

Vectors suitable for use in the present invention include expression vectors comprising a nucleic acid encoding an attractant (eg, CXCL12, CXCL13) operably linked to a promoter to direct 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). The promoter used to direct the expression of a chemoattractant (e.g., CXCL12, CXCL13) can be, for example, the SV40 early promoter, the SV40 late promoter, the metallothionein promoter, the murine mammary tumor virus promoter, the Rous sarcoma virus promoter, or other promoters shown to be effective for expression in mammalian cells.

Vectors suitable for use in the methods of the present invention include, for example, SV40 vectors, papilloma virus vectors, Epstein-Barr virus vectors, retroviral vectors, and lentiviral vectors.

Vectors used in the present invention may contain regulatory elements from eukaryotic viruses, such as SV40, papillomavirus, and Epstein-Barr virus, including, for example, signals for efficient polyadenylation of transcripts, transcription termination, ribosome binding and/or translation termination. Additional elements of the vector may include, for example, enhancers and heterologous splicing intron signals.

In one embodiment of the present invention, the genome of β cells can be genetically modified to increase the expression of endogenous chemokine (eg CXCL12, CXCL13) genes. Such increased expression can be achieved by: introducing a heterologous promoter operably linked to the endogenous chemoattractant (e.g., CXCL12, CXCL13) gene; or altering the endogenous chemoattractant (e.g., CXCL12, CXCL13) promoter such that the beta cells express a chemoattractant (e.g., CXCL12, CXCL13). Such increased expression can be achieved by introducing a promoter into the genome of the beta cell such that it is operably linked to a sequence encoding an endogenous qualifier, and thereby expresses or overexpresses the qualifier in invasive amounts.

Gene editing technologies for modifying genomes are well known in the art and include, for example, CRISPR/CAS 9, Piggybac, Sleeping Beauty genome editing systems (see, e.g., Zhang et al. Molecular Therapy Nucleic Acids, Vol. 9, Dec. 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); 2014; 157(6): 1262-78.); zinc finger nuclease-like systems (see for example, Porteus and Carroll, Nat Biotechnol. 2005; 23(8): 967-73; Urnov et al., Nat Rev Genet. 2010; 11(9): 636-46); TALEN-like systems (transcription activator-like effector nucleases) (See eg, 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).

In one embodiment, the transgenic beta cells described herein are treated with an agent that renders the cells viable and capable of controlling the patient's blood glucose but unable to replicate (ie, induce cellular senescence). One such reagent is mitomycin C, a known DNA cross-linking agent. After treatment, the DNA in these cells is cross-linked, thereby making it impossible to form the single-stranded DNA required for replication. Such treatments prevent cells, especially those arising from stem cells, from dividing, so that if a cell becomes 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 is incorporated herein by reference in its entirety. By way of example only, such agents include agents that cause telomere dysfunction due to replication-associated telomere shortening, secondary cytotoxic stress, such as exposure to UV, gamma irradiation, hydrogen peroxide, and hypoxia. The particular means by which the β-cells of the invention are rendered non-replicating is not critical, but the proviso is that these cells can be implanted without the risk of cell division. The adult pancreas has very little beta-cell turnover, the study showed, suggesting that limiting the ability of the cells to divide would have little effect on insulin production by the implanted cells. . See eg, Perl et al., The Journal of Clinical Endocrinology & Metabolism , Vol. 95, No. 10, Oct. 1, 2010, pp. E234-E239.

Another aspect of the invention is a method of modulating the amount of insulin in an individual comprising administering to an individual in need thereof a β-cell of the invention, wherein the β-cell expresses insulin and produces a scavenger (eg, CXCL12, CXCL13) in a depressant amount. 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 a tactic agent, either integrated into the beta cell genome or present episomally. In one embodiment of the invention, the transgenic β-cells can be genetically engineered to overexpress endogenous scavenger agents (eg, CXCL12, CXCL13) in scavenging amounts.

Methods of introducing transgenic beta cells described herein into an individual are well known to those skilled in the art and include, but are not limited to, injection, intravenous, portal vein or parenteral administration. Single, multiple, continuous or intermittent administration can be performed. See, eg, Schuetz and Markmann, Curr Transplant Rep. 2016 Sep; 3(3): 254-263.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of 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 may be prepared to contain 250 ml of sterile Ringer's solution and 100 mg of combination. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in some detail, for example, in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985) and 18th and 19th ed., which are incorporated herein by reference.

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

Furthermore, in order to avoid any possible transformation of transgenic beta cells into cancerous cells that would raise the possibility of the patient developing a tumor, the transgenic beta cells can be rendered senescent by exposure to known agents such as mitomycin C or exposure to secondary toxic stress from ionizing radiation, hypoxia, hydrogen peroxide, etc. Cells derived from pluripotent stem cells typically undergo apoptosis during inappropriate cell division or as a result of immune cell clearance. The senescent transgenic beta cells described herein are unable to divide, thereby eliminating the apoptosis triggering event that occurs during cell division. In addition, the senescent transgenic beta cells described herein are immune cell resistant, thereby providing protection against the induction of apoptosis due to immune cell clearance. Accordingly, it is expected that the transgenic beta cells described herein will have an up to significantly longer lifespan than non-senescent transgenic beta cells.

Transgenic, and preferably senescent, engineered beta cells can be transplanted into an individual via a graft. The ideal site for beta cell transplantation will be one that supports engraftment, long-term function and survival of the transplanted cells in an individual and is readily accessible for maximum patient safety. Implantation sites include liver, intestinal tract, subcutaneous and pancreatic sites.

The following abbreviations used herein have the following meanings and, if not defined, have their generally accepted scientific meanings. Amino acids are described herein using established one-letter abbreviations. FLAG = DYKDDDDK protein tag (SEQ ID NO: 10) g/L = grams per liter HRP = horseradish peroxidase LDH = lactate dehydrogenase iBLOT = semi-dry protein delivery device (Invitrogen) MES = 2-(N-morpholino)ethanesulfonic acid mL = milliliters N/A = not applicable nM = nanomoles PBMC = peripheral blood mononuclear cells PBS = phosphate buffered salineTM B = 3,3',5,5'-tetramethylbenzidine mL = microliter mg = microgram × g = double gravity Example Example 1 : Model cells used to evaluate the expression of CXCL12-a and CXCL12-b isoforms

HEK293 cells were transfected with 2 different isoforms of CXCL12 (α and β) using commercially available plastids for each isoform (plastids purchased from GenScript). Transfected cells were selected with 250 μg/mL G418 (available from ThermoFisher) and a stable pool was generated for each isoform. Cells were kept in appropriate medium for 3 days. Modified media from transfected HEK293 cells expressing CXCL12α and CXCL12β were diluted 1:1 with Assay Dilution Buffer. Two independent cells were established for each isoform, and the concentration of each isoform in solution was then obtained by using the absorbance of the normalized concentration curve. This experiment was repeated twice and the results were as follows: CXCL12α CXCL12β 1. 310 nM 1410 nM 2. 274 nM 1330 nM

The above results show that, compared with the transgenic model cells expressing CXCL12α, the transgenic model cells express a significantly higher amount of CXCL12β. Example 2 - Model Cells for Assessing the Expression of Other Isoforms of CXCL12 HEK293 cells were transfected with 5 different isoforms of CXCL12 (α and β) using commercially available plastids for each isoform (plastids purchased from GenScript). Transfected cells were selected with 250 μg/mL G418 (available from ThermoFisher) and a stable pool was generated for each isoform. Cells were kept in appropriate medium for 3 days. Modified medium was separated on 4% to 8% NuPage gels (available from ThermoFisher) with MES buffer and transferred to nitrocellulose (iBLOT).

The expression level was detected by Western blot method with HRP-labeled anti-FLAG tag antibody/TMB chromogen (purchased from GenScript), as shown in FIG. 1 . The results demonstrate that the gamma, delta and theta isoforms of CXCL12 have higher concentrations than the alpha or beta isoforms. Example 3 : Preparation of transgenic β cells

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

Cells were transduced with a lentiviral vector (pLenti-C-Myc-DDK, OriGene Technologies, Rockville, MD) containing a human CXCL12 isotype (CXCL12a/SDF-1α or CXCL12b/SDF-1β) or a control. Lentiviral vectors were used at a ratio of approximately 10:1 per beta cell. Sequences including tags (underlined) are provided below. Concentrations of CXCL12 isoforms were determined by ELISA (RayBioTech, Norcross, GA) (Table 1). CXCL12a (also known as SDF1a) deposit number NM_199168 CXCL12b (also known as SDF1b) deposit number NM_000609 Example 4 : Transgenic β Cells Reject PBMCs

Transgenic β cells from Example 3 were contacted with human peripheral blood mononuclear cells (PBMCs, Innovative Research, Novi, MI) at a ratio of 30:1 (PBMCs to β cells). Briefly, PBMCs were resuspended in complete beta medium, counted and adjusted by adding 100 μL of PBMCs (to minimize dilution of expressed CXCL12) to achieve a 30:1 PBMC:β cell ratio. The final volume was 1.1 mL. Background controls for β cells in the absence of PBMCs and PBMCs in the absence of β cells were also generated. Next, 150 μL of medium was removed from each sample and centrifuged at 1200×g for 10 minutes. The supernatant was removed and stored at 4°C (hour zero). Cells were returned to the incubator and sampled 24 and 48 hours later in a similar manner to the time zero sample.

LDH release was tested at 24 and 48 hours after exposure using the Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific) according to the manufacturer's instructions. Increased LDH is an indicator of cytotoxicity (cytolysis).

Data (background subtracted) from representative experiments are provided in Table 1 and Figure 2A. Data from a second representative experiment are presented in Figure 2B. Table 1. LDH and CXCL12 levels

These data suggest that CXCL12 expressed by β-islet cells protects β-islet cells from attack by immune cells, thereby rendering them resistant. Beta cells expressing SDF1b/CXCL12b in higher amounts than SDF1a/CXCL12a in this experiment showed essentially no cytotoxicity in the presence of PBMCs. Example 5 : Alternative Preparation of Transgenic Beta Cells

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 not encoding CXCL12. Transgenic β cells carrying a retroviral vector encoding CXCL12 were analyzed for the expression of excitatory amounts of CXCL12 using Boyden chamber assay as previously described in Poznansky et al., Journal of Clinical Investigation, 109, 1101 (2002). It was expected that transgenic β cells expressing at least 100 nM CXCL12 would reject immune cells in this assay. Example 6 : Effect of forced senescence of transgenic β cells on expression of transgenic cytokines

Beta cells were prepared as described in Example 3. Baseline expression ("before") was determined by ELISA analysis of the expression levels of SDF1a/CXCL12a and SDF1b/CXCL12b prior to mitomycin C (purchased from Santa Cruz Biotechnology) treatment. The medium was replaced with fresh medium containing 10 μg/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. Cells were washed twice with PBS. After the second wash, the cells were supplemented with fresh complete medium. SDF1a/CXCL12a or SDF1b/CXCL12b expression was determined by ELISA analysis.

Data from two representative experiments are shown in Table 2 and Figure 3A. SDF1a/CXCL12a and SDF1b/CXCL12b expression was not affected by forced senescence of transgenic β cells.

Table 2: CXCL12a and CXCL12b levels before and after mitomycin C treatment Example 7 : Effect of Forced Senescence of Transgenic β Cells on PBMC Challenge

Beta 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.

Data from two representative experiments are shown in Figure 4A and Figure 4B. LDH levels were not affected by forced senescence of transgenic β cells. Example 8 : Effect of Forced Senescence of Transgenic β Cells on Insulin Production

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

Complete growth medium was replaced with 1 mL of 2nd medium and maintained on 2nd medium with medium replacement every 24 hours for 2 days. On day 3, β cells were challenged with high blood glucose medium (4.5 g/L glucose). Samples of conditioned medium were taken 24 hours after hyperglycemic challenge and insulin expression was measured by sandwich ELISA.

Data from two representative experiments are shown in Table 3 and Figure 5. The results showed that transgenic β cells and transgenic senescent β cells produced substantially equal amounts of insulin as control β cells in response to a hyperglycemic challenge. Table 3: Insulin performance in response to hyperglycemic challenge Example 9 : In Vivo Evaluation of Transgenic β Cells

Humanized mice with a humanized immune system, see e.g., N. Walsh, "Humanized mouse models of clinical disease", Annu Rev Pathol 2017, 12, 187-215; E. Yoshihara et al., were administered transgenic human beta cells expressing a depletion of CXCL12 or control transgenic human beta cells and analyzed insulin production and migration at various time points after initial administration Genetic β-cell survival in mice. It is expected that transgenic human beta cells expressing a depleted amount of CXCL12 will survive for a longer period of time than control transgenic human beta cells. It is also expected that mice receiving transgenic human β cells expressing depletion amounts of CXCL12 will also have higher amounts of human insulin than mice receiving control transgenic human β cells, and that higher amounts of human insulin will persist for longer periods of time compared to the amount in mice administered control transgenic human β cells.

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., were administered CXCL12-overexpressing human beta cells genetically engineered with the endogenous CXCL12 gene or control human beta cells, and humans were analyzed at various time points after initial administration Insulin production and β-cell survival in mice. It is expected that genetically engineered human beta cells that overexpress CXCL12 will survive for a longer period of time than control human beta cells that are not genetically engineered to overexpress CXCL12. It is also expected that mice receiving genetically modified human β cells overexpressing CXCL12 will also have higher amounts of human insulin than mice receiving control human β cells, and that higher amounts of human insulin will persist for a longer period of time compared to the amount in mice administered control human β cells.

Optionally, cells are treated with an agent that cross-links DNA within the cell (eg, mitomycin C) to prevent cell division.

The foregoing description has been set forth in illustration of the invention only and is not meant to be limiting. Since those skilled in the art can make modifications to the described embodiments that incorporate the spirit and gist of the present invention, the present invention should be regarded as broadly including all changes and equivalents within the scope of the patent application.

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

2A and 2B are bar graphs showing the relative amount of lactate dehydrogenase (LDH, a marker of cell lysis) released from CXCL12α- or CXCL12β-expressing β-cells incubated with PBMCs at a 30:1 PBMC:β-cell ratio for 24 and 48 hours.

Fig. 3 shows the expression levels of CXCL12α or CXCL12β in two β cell groups expressing each cytokine.

4A and 4B are bar graphs showing the relative amount of LDH released from CXCL12α- or CXCL12β-expressing β-cells incubated with PBMCs at a 30:1 PBMC:β-cell ratio for 24 and 48 hours in the presence or absence of induced senescence of β-cells by mitomycin C treatment.

Figure 5 shows insulin induction by hyperglycemic challenge of beta cells expressing CXCL12α or CXCL12β in the presence or absence of treatment with mitomycin C.

Claims (21)

A human beta cell expressing or overexpressing a human fugetactic agent in an amount sufficient to render the beta cell resistant to human immune cells, wherein the beta cell is capable of expressing insulin and unable to undergo cell division. A 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. The human β cell according to claim 1 or 2, wherein the cell is: a) an autologous β cell obtained from an individual suffering from type 1 diabetes, or b) an allogeneic β cell. The human β-cell according to claim 1 or 2, wherein the cell expresses a suppressed amount of human CXCL12. The human β cell according to claim 4, wherein the CXCL12 is selected from the group consisting of CXCL12α and CXCL12β. A human beta cell comprising a transgenic regulatory region upstream of an endogenous CXCL12 coding region, wherein the beta cell is resistant to human immune cells. The human β-cell according to claim 6, wherein the cell is an autologous β-cell. The human β-cell according to claim 6, wherein the cell is an allogeneic β-cell. The human β cell according to claim 7, wherein the cell is an autologous β cell obtained or derived from an individual suffering from type 1 diabetes. The human beta cell according to claim 8, wherein the cell is an allogeneic beta cell obtained or derived from an individual without type 1 diabetes. The human β cell according to any one of claims 6 to 10, wherein the transgene regulatory region is an exogenous constitutive or inducible promoter. The human beta cell according to any one of claims 6 to 10, wherein the cell expresses CXCL12 in a depressive amount. The human β cell according to claim 12, wherein the CXCL12 is selected from the group consisting of CXCL12α and CXCL12β. The human β cell according to any one of claims 2 and 6 to 10, wherein the β cell cannot perform cell division. The human β cell according to any one of claims 1, 2 and 6 to 10, wherein the human CXCL12 is selected from the group consisting of CXCL12α, CXCL12β, CXCL12δ and CXCL12γ. The human β cell according to any one of claims 1, 2 and 6 to 10, wherein the human immune cells comprise NK cells, cytotoxic T cells and B cells. An in vitro method of producing insulin in response to a hyperglycemic environment comprising contacting the environment with a population of human beta cells according to any one of claims 1 to 16. The method according to claim 17, wherein the hyperglycemic environment comprises human immune cells. The method according to claim 18, wherein the human immune cells comprise NK cells, T cells and B cells. A composition for treating diabetes, the composition comprising the human β-cell according to any one of claims 1-16. A use of the human beta cell according to any one of claims 1 to 16, which is used for preparing a drug for treating diabetes.