WO2020167277A2

WO2020167277A2 - A next generation insulin secreting pancreatic tissue avoiding autoimmun attacks and production method thereof

Info

Publication number: WO2020167277A2
Application number: PCT/TR2020/050100
Authority: WO
Inventors: Ceren OZEL; Ayla EKER SARIBOYACI; Onur UYSAL
Original assignee: Eskisehir Osmangazi Universitesi
Priority date: 2019-02-11
Filing date: 2020-02-11
Publication date: 2020-08-20
Also published as: TR201902008A2; WO2020167277A9

Abstract

The present invention relates to a therapeutic method which enables reprogramming immune system that causes autoimmunity in T1D (Type 1 Diabetes) and also the production of glucose responsive insulin secreting tissues by means of developing new' techniques in endocrine pancreatic engineering. In the invention, in the new' generation insuring synthesizing tissue production method, by means of using decellularization and 3D printing techniques in combination, which are two different fields of tissue engineering, a hybrid printing technique is developed, and thus a bioactive matrix production re-printed by using stem cells of extracellular matrix of the pancreas is carried out. Insulin producing beta cells are obtained by means of differentiation of autologous mesenchymal stem cells and insulin secreting endocrine pancreas-like functional tissue pice production is provided. In the present invention, first the autoimmune immune system, which attacks the insulin-producing β-cel!s in T1D, is suppressed as the therapeutic method for the treatment of T1D. After autoimmune attacks are prevented in individual with T1D, the insulin producing tissue piece created within the scope of the invention is transplanted to individuals with T1D as subcutaneous biochips. Therefore, the said endocrine pancreas-like tissue called as biochip secretes insulin in response to glucose in diabetic individuals, and can bring the blood sugar· to a healthy level.

Description

A NEXT GENERATION INSULIN SECRETING PANCREATIC TISSUE AVOIDING AUTOIMMUNE ATTACKS AND PRODUCTION METHOD

THEREOF

Field of the Invention

The present invention relates to a therapeutic method which enables reprogramming immune system that causes autoimmunity in T1D (Type 1 Diabetes) and also the production of glucose responsive insulin secreting tissues by means of developing new techniques in endocrine pancreatic engineering.

Background of the Invention

Type 1 diabetes is an autoimmune disease, results from pancreatic beta cells death or the acummulation of damage of beta cells to the point that will stop insulin production from these cells, which is caused by the autoimmunity present in the pathogenesis of the disease [1]. Type 1 diabetes mellitus (T1D) is characterized by insufficient insulin production or complete lack of production of insulin as a result of autoimmune attacks caused by attacks of autoreactive T cells to b-cells producing insulin [2], is one of the diseases for the treatment of which intense efforts have been exerted. The discovery of insulin has been an important development for patients of diabetes. Insulin, which cannot be produced sufficiently from the pancreas, has begun to be compensated exogenously. For this reason, T1D1 is also called as Insulin Dependent Diabetes Mellitus=IDDM. Even though the T1DM patients can continue their lives by means of the insulin they take through injection, low quality of life has been an important problem despite the passing 80 years. Therefore, the production insulin again by the individuals with T1D has been seen as the solution of the disease. At first, pancreas transplant to patients, insulin producing beta-cell transplant and pancreatic islet have been tried. Thanks to the developing technology and increasing knowledge about stem cells, transplantation to individuals with T1D was attempted by performing beta cell differentiation especially from

SUBSTITUTE SHEETS (RULE 26) mesenchymal stem cells (MDGs). Gene cloning technologies were utilized in order to increase the differentiation efficacy of stem cells and exhibit better beta cell characteristics, and various genes were transferred to these cells [3]. 3D cell culture techniques that emerged later and provided the opportunity to mimic the body better have also influenced the T1D studies, and the researchers have attempted to obtain tissues that can produce insulin. Every tissue in the body has a special bio-composition which enables the cells to perform their functions perfectly. Even though the production of organ matrices with the 3D printing technique has become quite a phenomenon in recent years, the bio-composition of the tissue scaffold to be produced is substantially different from the natural composition existing in the tissues of the body. For this reason, capturing the said content in the tissues written with 3D bio-printers, the cells cultured in the used material fulfilling the correct biological functions and then transforming the produced tissues into clinical practice are important issues waiting to be solved.

One of the recent developments that have been made is culturing the islet-like materials on the decellularized liver extracellular matrix and attempting to generate insulin secreting 3D tissues [4]. Furthermore, reshaping the extracellular matrix obtained as a result of decellularization by being used as a raw material in the 3D bioprinter is a newly developed technique, whereby adipose, cartilage, heart tissue drafts were tried in vitro [5], and existed in a study about producing only heart tissue comprising transplant to the animals [6].

All these alternative T1D treatment approaches include beta-cell replacement, which can reverse the outcome of the disease by replacing damaged beta cells in the diabetic pancreas. However, if the aggressive autoimmunity still maintains its presence after the beta cell replacement and it is not prevented, it draws attention that specific immunity attacks will also continue against the newly transferred beta cells. Therefore, beta cell replacement which is performed without eliminating autoimmunity existing against beta cells alone is insufficient. In order to create Treg-based therapy approaches for autoimmune diseases, in vivo or in vitro inducing of Tregs has been performed in various studies. In these studies, Tregs were obtained as specific to antigen (monoclonal) or without being specific to any antigen (polyclonal). When they are transferred in order to target autoimmunity, it is shown that the therapeutic effects of polyclonal Tregs were weaker relative to the antigen- specific Tregs [7]; [8]; [9]; [10]; [11]; [12]; [13];

[14].

A promising approach about polyclonal Tregs is that Treg population is replicated by means of low dose IL-2 administration in vivo immune tolerance is provided

[15]; [16]; [17]. However, the use of polyclonal Tregs replicated in vitro or in vivo bears the potential to induce entire immune suppression, in other words, the risk of suppressing the beneficial immune system. On the contrary, autoantigen- specific Tregs provide an advantage of antigen specificity and offer a better approach for the treatment of autoimmune diseases, since they will not suppress the entire immune system [18]; [19]; [20]; [21]; [22]. Furthermore, various studies wherein autoantigen- specific Treg administration-mediated diseases, also comprising T1D, are regressed in mice with autoimmunity have taken their place in the literature [19]; [23]; [24]. Studies performed in the NOD mouse model have shown that islet antigen- specific Tregs are more effective than polyclonal Tregs in preventing onset of Type 1 diabetes disease. Furthermore, only the transfer of autoantigen- specific Tregs can stop diabetes that has started and been continuing [19]; [20]; [23]

Recently, approaches for the in vitro and in vivo production or induction of Tregs have emerged. One of the in vitro autoantigen-specific Treg production approaches is viral TCR gene transference to T cells. Stimulation of naive CD4 + T cells, to which TCR and/or FoxP3 gene is transferred, in the presence of TGF-b can transform these cells into Treg cells. Anti-CD3/anti-CD28-coated beads or antigen-primed dendritic cells can be used in in vitro replication of Tregs in presence of highly concentrated IL-2 [19]; [25]. However, difficulties in in vitro obtaining autoantigen-specific Treg and limitations in clinical practices have led researchers to in vivo induction of Treg. For this purpose, even though inducing Tregs from naive T cells in the periphery through TGF-beta has been a frequently used method, it has been determined that the suppression abilities of the Tregs obtained in this way are not permanent [26]; [27].

In preclinical studies carried out with animals with T1D, inducing autoantigen- specific Tregs was performed with antigen primed dendritic cells in vivo [28] or in vitro [29]. Upon various studies showing that antigen- specific tolerance (tolerance which is specific to antigen) of dendritic cells occurs through Tregs, clinical trials for the tolerance based treatment of autoimmune diseases, comprising T1D, have been conducted [30] and its safety against T1D has been tested in Phase I clinical trial [31].

Recently, a new therapeutic trend has emerged in which autoimmunity treatment and transplantation immunity can be targeted by means of conjugating autoantigens (creating a chemical bond) to splenocytes or nanoparticles [28]; [32];

[33]. In antigen- splenocyte or antigen-nanoparticle therapy, autoantigens are enabled to covalently bind to the surface of splenocytes or nanoparticles with ethylene carbodimide (ECDI). The said method has extensively been developed in animal models, and Phase I clinical study on multiple sclerosis has been conducted [34]; [35]; [36]. In this technique, poly(lactic-co-glycolide) PLG) is one of the most commonly used nanoparticles thanks to its biocompatible, biodegradable properties. When PLG or splenocytes conjugated with antigens via ECDI are transferred to a living thing, it is believed that ECDI induces apoptosis, and thus antigen-splenocyte or antigen-PLG therapy mechanism is dependent on TGF-beta production by phagocytes and apoptic cells [37]; [38]. It has been shown in several animal models that the said approach is significantly more effective than therapies wherein only soluble antigen or broad spectrum immunosuppressive drug is used without using splenocyte or PLG nanoparticles in prevention and treatment of autoimmunity [32]. Inducing autoantigen-specific Tregs in this way is a highly effective approach which has exciting preclinical results in multiple sclerosis models, Type 1 diabetes models [37].

Even if the damaged tissue is restored to its previous form as a result of targeting the Tregs with the aforementioned effective mechanisms, the autoreactive effector/memory T cells existing in the treated individuals may exhibit resistance and these remaining cells may cause the disease to relapse [39]. For this reason, for the long-term treatment of autoimmune diseases, first the autoreactive immune system existing in the individual with the disease should be destroyed and then immune tolerance should be provided.

In a study which offers a method for tolerating the immune system of a person who has been suffering from autoimmune disease, and in the patent of the related study, it has been shown that tolerance to autoantigens is regained first by breaking the impaired immune system in animal models with autoimmunity and then by stimulating the antigen- specific regulator T cells. In accordance with this, the immune cells were apoptosed with antibodies or low dose gamma irradiation for breaking the impaired immune system, followed by administration of phagocytes with autoantigens associated with the disease for inducing autoantigen- specific Treg [24]; [40]. However, immunotherapy which is to be applied to individuals with T1D will not be sufficient alone, since the deficiency in the number of insulin- secreting cells in response to glucose as a result of beta cell destruction will still exist as a problem requiring a solution.

Technical Problems in the Current Applications and Technical Solutions Proposed within the Scope of the Invention

Type 1 diabetes is a disease which causes beta cell deficiency and subsequent irregularities in blood sugar control as a result of a development of an autoimmune response against b-cells producing insulin in the pancreas. In terms of T1D treatment, immunotherapy regulates the balance between autoimmunity and regulatory mechanisms and deals with the main cause of T1D. T regulator cells play an important role in this immune intervention [41]. An alternative T1D treatment includes beta-cell replacement, which can reverse the outcome of the disease by replacing damaged beta cells in the diabetic pancreas. However, if the aggressive autoimmunity still maintains its presence after the beta cell replacement, it draws attention that specific immunity attacks will also continue against the newly transferred beta cells. Therefore, beta cell replacement which is performed without eliminating autoimmunity existing in the patient against beta cells alone is insufficient. On the other hand, immunotherapy which is to be applied to individuals with T1D will not be sufficient alone, since the deficiency in the number of insulin- secreting cells in response to glucose as a result of beta cell destruction will still exist as a problem requiring a solution [42]. Attempting to eliminate the autoimmunity problem of T1D with immunotherapy (dendritic cell or antigen-conjugated biodegradable nanoparticles-mediated Treg induction) is an approach that already exists [43]. However, these studies target only immunotherapy .

Even though various applications have been tried to replace b-cells that were damaged in T1D before, three of these strategies stood out: (i) cell transplantation, (ii) islet transplantation and (iii) pancreatic transplantation [44]. However, pancreas and islet transplantation is limiting both in terms of the source that provides insulin due to the difficulties in finding a donor organ, and it also poses serious problems since it requires lifelong suppression of the immune system. Immunosuppressive drugs were used to overcome the second condition. However, the use of immunosuppressive drugs has seriously hindered the functioning of the immune system, and these individuals have experienced significant disadvantages in the fight against microbial diseases. Both problems were solved by beta cell differentiation from autologous MSCs taken from patients with T1D and the production of insulin secreting tissues. Insulin-producing cells were obtained by providing insulin-secreting tissues with the stem cells (autologous MSC) of the patient we use within the scope of the invention. Since the cells which construct the tissue transplanted to the patient are their own cells, an immune response does not develop against them, which eliminates the requirement to suppress the immune system.

Type 1 diabetes mellitus is a disease which does not currently have a cure and which has serious impacts worldwide. The discovery of insulin has been an important development for patients of diabetes. Insulin, which cannot be produced sufficiently from the pancreas, has begun to be compensated exogenously. For this reason, T1D1 is also called as Insulin Dependent Diabetes Mellitus=IDDM. Even though the T1DM patients can continue their lives by means of the insulin they take through injection, low quality of life has been an important problem despite the passing 80 years. This problem was solved by transplantation of tissue parts which can secrete insulin according to blood glucose level, to individuals with the disease. After the transplantation, the patients do not need exogenous insulin. Cell transplantation, islet transplantation, and pancreas transplantation has question marks in migration of cells delivered to the circulation to the desired region or how many of them will migrate, and hold questions that await answers such as which type of cell will be selected and where the administration area will be. However, the tissue scaffold that we have formed in the method we are using also functions as a vector for the cells, as well as it creates microenvironment for them. By this means, the cells can be transferred to the desired region in the patient.

The production of organ matrices has become quite popular in recent years with the 3D printing technique. However, the biocomposition of the tissue scaffold which is to be produced is quite different from the natural composition existing in the tissues of the body. Each tissue in the body has a specific biocomposition. This composition has a specific design which enables the cells in the tissues to perform their functions perfectly. However, capturing the said content in the tissues printed with 3D printers is a great problem. However, the use of the decellularized pancreatic matrix used within the scope of the invention as organ printing material in 3D printers preserves the natural biological content of the tissue. Extremely important problems are also overcome with this approach, which can play vital roles in the cells cultivated in the material to perform the correct biological functions.

Producing beta cells that can secrete insulin responsive to glucose from stem cells is a very difficult process due to low efficiency. It is also quite difficult to obtain human beta cell suitable for transplantation. Even though the highest efficiency can be obtained in viral methods in gene transfer to the cells in order to provide the desired phenotypic and functional feature or to stimulate in a certain direction, viral methods are not suitable for clinical use due to the safety problems such as the risk of creating mutagenesis and their immunogenicity. On the other hand, non-viral gene transfer vectors create a safer profile in tissue engineering. However, the expression of the gene transferred in non-viral methods in the target cell has not reached to the same level. This shows that vector design is quite important. An ideal gene transfer vector which is to be used in tissue engineering is biocompatible, biodegradable, and minimally cytotoxic and it should be able to effectively transfer DNA into the cell. Moreover, it should maintain the expression of the target protein in required time. The use of gene therapy strategies in combination with a tissue scaffold has led the emergence of a new field: Gene- Activated Matrix (GAM). GAM is the direct and continuous delivery of nucleic acids from a tissue scaffold to the cells to ensure non-viral gene transfection to the cell in an efficient and durable way. In general, the nucleic acids (a plasmid DNA, usually called pDNA carrying the target genes) which are desired to be transferred to the cells are comprised in cationic polymers or cationic lipids and they are mixed by adding to the solution in which the tissue scaffolds will be formed. Then 3D tissue scaffolds are formed from materials which have gained gene transfer ability and these constructs are called GAM. When the cells are cultivated on GAM and cultured, the cationic components loaded into the matrices combine with the cell membranes, and thus the genes in the cationic components are transferred to the cells by a non- viral method [45]. When all the properties which are desired related to biocompatibility and gene transfer are considered, chitosan which is a cationic polymer is thought to be one of the most suitable non-viral methods in accordance with this purpose. Especially the development of a water-soluble oligochitosan molecule with low molecular weight has significantly increased efficiency of gene transfer and has been tested in MSCs [46]. In the present invention, the problems related with producing beta cells that can secrete insulin responsive to glucose level from stem cells are solved by obtaining a gene activating matrix from the natural pancreatic extracellular matrix transferred with chitosan-pDNA nanoparticles and by creating a tissue scaffold by means of printing this matrix in 3D bio-printers. Therefore, Pdx-1 and MafA genes which play important roles during pancreatic development which will induce b-cell differentiation have been transferred to MSCs that were cultured on tissue scaffolds [47]. By means of enabling b-cell differentiation stimulation from MSCs with gene activated matrices created in the present invention, a new generation beta cell differentiation method for T1D has been developed and technical problems in beta cell differentiation in current methods have been eliminated by providing GAM-mediated 3D microenvironment that is produced within the scope of the invention.

One of the most difficult problems to overcome in T1D has been the autoimmunity developing against the insulin producing cells. In order to solve this problem, the cells are contained in a capsule with encapsulation techniques, and thus they are prevented from directly contacting autoreactive T lymphocytes. However these methods that were used did not allow a long-term treatment. Therefore, autoreactivity maintains its popularity on the agenda after encapsulation. However, by means of dendritic cells that we use within the scope of the invention- and antigen-conjugated biodegradable nanoparticles-mediated Treg programming specific tolerance was provided against the autoantigen and then biochip comprising insulin producing cells was transplanted. Therefore, this problem has been eliminated with immunotherapy realized by antigen-specific tolerance of autoreactive T lymphocytes.

The frequency of Treg being low in peripheral blood has been identified as one of the great challenges that must be eliminated in Treg-based immunotherapy in recent years [48]. For this reason, recently approaches for the in vitro and in vivo production or induction of Tregs have emerged. One of the in vitro autoantigen- specific Treg production approaches is viral TCR gene transference to T cells. However, these Tregs in vivo suppression properties of which are quite high have transgenic TCR manipulation, therefore they are not clinically applicable. In vitro autoantigen- specific Treg production and difficulties in obtaining antigen- specific Treg in sufficient numbers are other conditions limiting the clinic applications in determination of antigen specificity of Tregs. These limitations have directed the researchers towards in vivo Treg induction. In accordance with this, in vivo autoantigen- specific Tregs are induced from naive T cells in the periphery via TGF-beta [27]; [14] or with dendritic cells primed with antigen. However, they were tried when the autoimmune diseases had not been developed yet in the studies that were conducted, which causes not to be able to determine the therapeutic efficacy of these applications. It has been shown in several animal models that the antigen-PLG or splenocyte therapy approach is significantly more effective than therapies wherein only soluble antigen or broad spectrum immunosuppressive drug is used without using PLG nanoparticles or splenocyte in prevention and treatment of autoimmunity [32]. Inducing autoantigen- specific Tregs in this way is a highly effective approach which has exciting preclinical results in multiple sclerosis models, Type 1 diabetes models [37]. On the other hand, immune cells which respond to autoantigen in case of a disease become activated in an uncontrolled way with increasing pro-inflammatory cytokines, thereby causing Tregs to lose their suppression abilities. Tregs can even transform into effector cells such as Thl7 cells in this pro-inflammatory environment [24]. Even if the damaged tissue is restored to its previous form as a result of targeting the Tregs with the aforementioned effective mechanisms (such as antigen-PLG and dendritic cells), the autoreactive effector/memory T cells existing in the treated individuals may exhibit resistance and these remaining cells may cause the disease to relapse [39]. For the solution of this problem and for the long-term treatment of autoimmune diseases, first the autoreactive immune system existing in the individual with the disease should be destroyed and then immune tolerance should be provided.

According to the data from International Diabetes Federation, diabetes-related health-care cost were 673 billion dollars in 2015. It is expected that this figure will increase to 802 billion dollars in year of 2040. T1D is a disease which results in insufficient insulin production as a result of autoreactive T lymphocytes attacking insulin producing pancreatic b cells. Therefore, patients with T1D must take commercially available insulin daily to meet their insulin needs. In addition to this, chronic period T1D disease leads to serious secondary complications such as eye damage, neuropathy, nephropathy, cardiovascular diseases. When it is analyzed from this point of view, diabetes patients encounter quite serious economic burden for other diseases caused by diabetes.

South Korean Patent document no KR20080078204, an application known in the state of the art, discloses a method for preparing mesenchymal stem cells- mediated autologous dendritic cells. Mesenchymal stem cells are used as a stimulator in providing immunosuppression function to dendritic cells during obtaining dendritic cell which has immunosuppression capability. These two types of cells are co-cultured so that the dendritic cells stimulate the mesenchymal stem cells.

Chinese patent document no CN105670990, an application known in the state of the art, discloses preparation method and application of a tissue engineering material for promoting directional differentiation of mesenchymal stem cells. The invention is in field of biomedical engineering comprises preparation of a tissue engineering material for promoting differentiation of mesenchymal stem cells and the application of the said material. Poly(lactic-co-glycolic acid) called as PLGA is used, and this polymer is in class of unnatural polymers, in other words it is not produced in the body and does not exist in the body [49]. Even though it is biocompatible, the said polymer which is not produced in the body is a completely foreign medium for cells. As it is known, organs are comprised of two components: Cells and materials between cells called as extracellular matrix [50]. In decellularization technique used within the scope of the patent, the cells are detached from the extracellular matrix and only natural extracellular matrix of the organ or tissue is obtained. Therefore, the polymers that are used are a medium obtained after organ decellularization and belonging to organ itself that already exists, it is much more than PLGA comprised of a single polymer, and consists of natural biomaterial having a complex content comprising collagen typel, collagen type 4, fibronectin, glycosaminoglycans, and the like [51]. Replacing the dead beta cells is not a permanent and long term solution in T1D treatment. Because the autoimmunity against the beta cells still continues in the body. As a result, the permanent solution of T1D does not only complete the beta cell deficiency, but also the existing autoimmunity should be eliminated. However, patent no CN 105670990 targets only beta cell replacement. After the transplantation of beta cells produced in patent no CN105670990 to an individual with diabetes, it has not been shown that high blood glucose level was lowered to normal values, and there is no in vivo (realized within a living organism) study. Briefly, the function of the said product has not been shown in a living thing.

South Korean patent document no KR20150029280, an application known in the state of the art, discloses mesenchymal stem cell composition for treating diabetic wound which is originated from autologous and allogenic adipose tissue. Diabetic wounds are secondary vascular complications developing due to excessively increasing blood glucose as a result of the disease in individuals with diabetes. Invention no KR20150029280 relates to wound healing focused on repairing the vascular system (vessels) and inflammation occurring in the area (especially the foot) as a result of T1D. Autologous mesenchymal stem cell is used for wound healing and preventing inflammation. The mesenchymal stem cells inhibiting (stopping, preventing) the immune responses in the area where they are transplanted is a known characteristic of the mesenchymal stem cells [52]. Mesenchymal stem cells have a high potential for use in clinical treatments today, especially due to their ability to be obtained easily from many tissues, their anti- apoptotic and anti-inflammatory properties, not creating immune response in allogenic transplantations and the ability to differentiate into many somatic cells, including b-cells. The composition and a biodegradable tissue scaffold are transplanted into human body within the scope of the invention.

Chinese patent document no CN104353115, an application known in the state of the art, discloses a kit for a pancreas decellularized scaffold and preparation method thereof and reseeding method of the scaffold. The tissue scaffold used in the invention is extracellular matrix obtained from decellularized pancreas. The permanent solution of T1D does not only complete the beta cell deficiency, but also the existing autoimmunity should be eliminated. Patent no CN104353115, same as patent no CN105670990, targets only beta cell replacement. Recellularization of tissue scaffold in patent no CN104353115 is performed with MIN-6 cell line. However, it is present in the literature that the said cell line is not a pure beta cell line [53]. Whereas, more importantly, this cell line is an insulinoma cell line. That is, the pancreas is a cell line obtained from a tumor originating from the B cells of Langerhans islets. This information is also included in the ATCC (American Type Culture Collection) where this cell line is sold [54]. Therefore, none of the tissues, chip, etc. produced with these cells is suitable for clinical use. In patent no CN104353115, as it is in CN105670990, after the transplantation of beta cells that are produced to an individual with diabetes, it has not been shown that high blood glucose level was lowered to normal values, and there is no in vivo (realized within a living organism) study. Briefly, the function of the said product has not been shown in a living thing. South Korean patent document no KR20060134264, an application known in the state of the art, discloses biochip of cell stimulation and detection of stem cell differentiation.

United States patent document no US2013017175, an application known in the state of the art, discloses activated mesenchymal stem cells for healing wound and impaired tissue regeneration. The invention is a tissue regeneration method which activates immunosuppressive properties of mesenchymal stem cells via inflammatory cytokines and thus has in vivo application in wound healing and inducing angiogenesis. The aim is to show how effective the use of mesenchymal stem cells having activated immunosuppression properties for graft versus host disease (GVHH), which develops as a result of tissue (transplant) rejection transferred in tissue transplants, can be in preventing inflammation caused by the said disease. In this direction, in vivo efficacy of the composition with activated mesenchymal stem cell created for preventing GVHH has been examined. In the invention no US2013017175, the objectives are forming a composition of inflammatory cytokine-activated mammalian mesenchymal stem cells for inducing angiogenesis, preventing inflammation and repairing tissue, and transplantation of cells to this area. This can be any tissue and it is the basic methodology of the invention using immunomodulating properties of mesenchymal stem cells in the inflammatory environment. It is claimed in the invention no US2013017175 that its use for preventing GVHD by supporting the transplanted tissue can also be functional in islet transplantation. In the invention no. US2013017175, the in vivo application of cell composition for tissue regeneration and organ repair consists of inflammatory cytokine-activated mammalian mesenchymal stem cells.

International patent document no W02015006519, an application known in the state of the art, comprises an immunotherapy and kit developed for treating or tolerating autoimmune diseases expressed with apoptose-antigen therapy. In the invention, the therapeutic steps are as follows: i) Detection of autoimmune subjects; ii) Guiding immune system cells to apoptosis; (a) by administering T cell antibodies (anti-CD3 and anti-CD8) or B cell antibodies (anti-CD20), (b) or by applying low-dose radiation (radiotherapy); iii) Administration of macrophage cells to the patient subject following radiotherapy application or antibody administration; iv) Finally, administration of autoantigens that cause autoimmunity to the patient subject. As a result of all these steps, in the part where radiotherapy was performed, the existing autoimmune cells were eliminated by apoptosis, and the macrophages that were administered were provided to support the immunotolerance more strongly when they encountered apoptotic cells. After this environment was prepared, an immunotherapy was presented, wherein Tregs were stimulated by giving autoimmune disease-related autoantigens to the patient, which is a commonly used method in antigen- specific therapy lately. When both patents are taken into consideration, the common aspect is the in vivo programming of Tregs specific to antigen in the immunotherapy section applied for T1D. However, in the invention no W02015006519 autoantigen and macrophages are transplanted to the individuals with the disease in combination, whereas in the present invention autoantigens are not administered to the individuals with the disease, first they are loaded (stimulated) to the dendritic cells in vitro and antigen specific dendritic cells are produced and/or conjugated to the biodegradable nanoparticles (PLGA- Antigen). Antigen specific dendritic cells and/or PLGA-Antigen nanoparticles produced as a result of this are administered to the patient, and thus the programming of Tregs in the immune system are programmed specific to the antigens that we have selected. The only objective in the invention is immunotherapy, immunotherapy which is to be applied to individuals with T1D will not be sufficient alone, since the deficiency in the number of insulin- secreting cells in response to glucose as a result of beta cell destruction will still exist as a problem requiring a solution.

Summary of the Invention

The present invention is a T1D treatment method which aims to eliminate the autoimmunity existing in T1 Diabetes patients by means of reprogramming and providing insulin-producing autologous cell replacement. In accordance with this objective, in the invention, on one hand autoimmune immunity system attacking insulin producing b-cells in T1D is prevented by means of in vivo programming of T regulator cells (Treg), and on the other hand the environment where the autoimmunity can be prevented is enabled to be created before the tissue transplantation so that biochip (insulin secreting tissue) that is produced as a result of the tissue engineering can maintain its function in the in vivo environment.

The invention also comprises the production method of endocrine pancreatic-like functional tissue piece that can bring blood glucose to a healthy level by secreting insulin as a response to glucose by developing new generation beta cell differentiation techniques in endocrine pancreatic engineering. With this aspect, the invention comprises both a product and a production method thereof. Preparation of elements forming the insulin secreting tissue ( mesenchymal stem cells, beta cell gene transfer system and decellularized pancreas matrix: dpESM), obtaining a tissue draft by means of printing these elements by using 3D bioprinters, and culture of this tissue draft under in vitro condition are aimed. Therefore, endocrine-pancreatic differentiation stimulation of mesenchymal stem cells was realized with the effect of various factors [(i) dpESM, (ii) b-cell differentiation-related gene transfer system and (iii) endocrine media culture].

The invention also comprises endocrine pancreas-like functional tissue piece product created with 3D bioprinter, Gene Activated Matrix comprised of decellularized pancreas integrated with cationic polymer-pDNA nanoparticular gene transfer system which is suitable for differentiating mesenchymal stem cells in its structure into beta cells (GAM; dpESM+cationic polymer-pDNA nanoparticles). GAM, stimulating differentiation in this endocrine direction, is a sub-product which is one of the topics of the invention, created within the scope of the invention. In the invention, on the other hand the biochip (insulin secreting tissue) that is produced is enabled to be transplanted to an individual with T1D the autoimmune attacks of which are prevented. After all these procedures, by means of the biochip which is transplanted to the patients with T1D the autoimmune episodes of which are prevented, lowering blood sugar to normal levels constitutes the final objective of the study.

Detailed Description of the Invention

One aspect of the invention is to provide a therapeutic method for T1D disease. The present invention provides a treatment method which enables b-cell regeneration in T1 Diabetes by means of the 3D culture medium, while also enabling to regulate the autoimmunity. While b-cell differentiation from MSCs is realized on the tissue scaffold produced in order to provide microenvironment very close to natural, it comprises the re-regulation of autoreactive T cells; dendritic cell-and/or antigen conjugated biodegradable nanoparticle-mediated Tregs against these cells. Therefore, the invention can target the treatment of T1D disease as a whole. Thus, a new strategy is provided allowing the treatment of T1D disease by means the production of individual- specific tissues and providing the tolerance of autoimmune T cells.

Another aspect of the invention is that it comprises a new generation beta cell differentiation method in pancreatic tissue engineering. It is the first presentation of printing tissue scaffold designed suitable for b-cell differentiation by using natural decellularizes pancreas extracellular matrix comprising gene transfer system and the applicability of this method. By means of the present invention, the natural polymer structure of the matrix (dpESM) obtained from the pancreas is enhanced via non-viral b-cell gene transfer systems in tissue produced using 3D bioprinters. With the gene activated matrices obtained in this way, a 3D microenvironment suitable for beta cell differentiation is provided, thereby developing a new generation beta cell differentiation method.

The invention essentially comprises the following inventive steps as a tissue engineering application and preparation method:

a) Making the product obtained by lyophilization of decellularized pancreas (dp ESM) printable in 3D bioprinters

b) Preparation of bioink (bioink: dpESM+chitosan-pDNA nanoparticles+MSC composition) o Integration of the nanoparticular gene (genes related with b-cell differentiation) transfer system that will be created by using a non-viral, that is cationic polymer instead of virus to dpESM

o Obtaining Gene Activated Matrix (GAM) which enables to stimulate stem cells in endocrin pancreas direction with chitosan-pDNA nanoparticular b-cell genes transfer system and dpESM composition

o Adding MSC composition to the dpESM mixture containing chitosan-pDNA nanoparticular b-cell genes transfer system in certain concentration and under ambient conditions

c) Printing Insulin Secreting Tissue (Biochip) with 3D bioprinter from the bioink

Another aspect of the present invention is to provide a treatment product for T1D. It is the production of endocrine pancreatic-like functional tissue piece (biochip) which is transplanted in order to bring blood sugar to a healthy level in individuals with T1D whose autoimmune attacks are prevented. This "product" formed by the development of various techniques creates a new concept in itself.

After autoimmune attacks are prevented in patient subjects with T1D (Type 1 Diabetes), the outlines and content of the method used within the scope of the invention in which the production of endocrine pancreas-like tissue grafts suitable for lowering glucose sensitive blood sugar and transplantation as subcutaneous biochips are aimed are as follows:

- After identifying a subject as suffering from T1D, patient specific selection of T1D autoantigen (b-cell antigens) combinations and dosage to be administered as intravenous injection to the subjects with T1D by using antibody test,

- Obtaining immature/naïve dendritic cells from bone marrow progenitors or blood mononuclear cells of subjects with T1D, in order to provide antigen specific tolerance in subjects with T1D,

The autologous immature/naive dendritic cells, which will induce tolerance to program Tregs present in the patient as antigens-specific, are obtained from the bone marrow progenitors of individuals with T1D with culture containing GM-CSF cytokines. Briefly, bone marrow (BM) cells are obtained from femur and tibia sources and erythrocytes are subjected to hypotonic lysis. BM cells (10⁷) are cultured in 10 ml medium added with GM-CSF (lOOU/ml) cytokine. Half of the medium of the cells are replaced daily with fresh media containing cytokines. In order to determine the purity of the dendritic cells, the cells are marked with CD11c, which is a dendritic cell marker, on the 7th day of culture, and analyzed in flow cytometry.

The resulting dendritic cells are co-cultured with the previously prepared lysate (apoptotic bodies of b-cells) and GAD65 peptide at the next specified stage. When the dendritic cells phagocytose the apoptotic bodies and peptides, they meet the term primed/pulsed with the targeted antigens.

- Loading the identified T1D autoantigens in vitro to the dendritic cells obtained from the subjects or conjugating to synthetic biodegradable nanoparticles***, in order to achieve antigen specific tolerance in subjects with T1D,

Preparation of lysate (apoptotic bodies of b-cells)

For the apoptosis of b-cells, first ready to use b-cell line or 3D bioship the physical homogenization of which is carried out is cultivated in each well of 24 well petri dish comprising 500 ml media therein such that it will be 3x10⁵ cell/well. After it is irradiated with ultraviolet B (UVB, 10mJ/m2) for 45 minutes, it is cultured overnight in medium of %5 CO2 37°C. The characterization of apoptose is verified in flow cytometry by dying the annex with V-fluorescein isothiocyanate (FITC) and propidium iodide.

Co-culture and characterization of dendritic cells with autoantigens (b-cell antigens)

For co-culture of the resulting dendritic cells with apoptotic bodies and GAD65 peptide as b-cell specific antigens; 10⁶ cells/well dendritic cell: and 3x10⁵ cells/well apoptotic beta cells (in ratio of ratio 3:1) are cultured together in 24- well containers. It is continued for 2 hours by adding 5mM synthetic GAD65 peptide to the prepared culture. It is characterized by flow cytometry analysis, wherein dendritic cells are primed with diabetogenic antigens (beta cell apoptotic bodies and GAD65 peptide). As a result of the analysis of dendritic cells marked with CFSE before co-culture and with CD11 after co-culture, Dendritic cells primed with apoptotic cell are determined as CD 11c- and CFSE positive, whereas Dendritic cells that are not primed are determined as CD11C+ and CFSE negative.

For the characterization if dendritic cells which will show immune tolerance, they are activated by means of adding lipo saccharide to their culture, and then CD40 and CD86 second co-stimulant expression and release of pro-inflammatory cytokines are analyzed. Dendritic cells that are tolerant to co- stimulant expression increase depending on resistance against pro-inflammatory cytokines are characterized by flow cytometric analysis and analyzing the medium by ELISA.

Production of b-cell Specific Antigen-Conjugated Nanoparticle Biodegradable PLG nanoparticle which allow conjugating autoantigens and possible donor MHC antigens are conjugated with isolated b-cell apoptotic bodies and commercially available GAD65 as b-cell specific antigens by means of ethylene carbodiimide (ECDI) as it is mentioned in previous steps, and therefore biodegradable antigen- conjugated-nanoparticle (antigen-ECDI-PLG) is created. Briefly, ECDI [(l-Ethyl-3-(3' dimethylaminopropyl) carbodiimide HC1 is used to conjugate in vitro b-cell specific antigens to biodegradable PLG nanoparticles. PLG particles (3mg) are washed 3 times with PBS to remove sugar from lyophilization and mixed with 30mg/ml ECDI and 1200mg/ml lysate (apoptotic bodies of b cells) and 5 mM GAD65 peptide for each dose. Paired particles are washed twice with PBS to remove excess ECDI and filtered with a 40 mih cell sieve. Lysate and peptide conjugation efficiency is characterized by measuring the amount of protein remaining in the supernatant with Protein assay. Before becoming a lysate, it is characterized by flow cytometry analysis wherein GAD65 marked with CFSE and b cell apoptotic bodies marked with CFSE- are conjugated with PLG nanoparticles.

- Apoptosis of autoimmune system cells existing in the T1D subjects with low dose (in sublethal level) radiation in order to provide antigen specific tolerance,

- Then injecting dendritic cell and/or antigen-conjugated biodegradable nanoparticles to the subjects with T1D intravenously (IV),

In induction of in vitro stimulated dendritic cells-mediated tolerance, for dendritic cell administration primed with b-cell specific antigens (GAD65 peptide and lysate), approximately 2-3 million/injection Macrophage/Dendritic for each one of the mice, approximately 8-12 million/injection Macrophage/Dendritic cell injection for humans are carried out, and number of doses and injections can be increased depending on the immune response of the patient.

In antigen-conjugated-nanoparticle-mediated tolerance induction, each patient (nanoparticle/injection containing at least 10 mg peptide for human, total at least 30 mg, 1-2 mg/injection for mouse) is transplanted with the antigen primed nanoparticles, and the number of doses and injections can be increased according to the immune response of the patient.

- Obtaining pancreas extracellular matrix (dpESM) purified from the cell by means of performing decellularization process of pancreas while performing the aforementioned process steps,

The process of obtaining decellularized pancreas extracellular matrix (dpESM) by means of perfusion decellularization from pancreas is realized under sterilized conditions in an airflow container (Heraeus Instruments, Hanau, Germany). To disintegrate the cells of the isolated pancreas (dpESM that does not develop immune response can be allogeneically or xenogenically isolated from the pancreas of rodents and other mammals such as rabbit, rat, mouse, etc. and the pancreas of the human cadaver), and to remove from the organ extracellular matrix, a retrograde (reverse) perfusion method, which is connected to the perfusion system and mediated by the hepatic portal vein, is used. For this, first, it is is perfused from the 1% TritonX-100 portal vein for 60 minutes as it will be 10 ml/minute. Then 0.5% sodium dodecyl sulfate (SDS) is perfused for 120 minutes. The again, it is perfused with the 1% TritonX-100 for 15 minutes as it will be 10 ml/minute. The sterilization of the matrix is provided with 0.1% 300 ml peracetic acid. The enzyme and chemicals are removed from the tissue by washing with perfusion in phosphate buffer (PBS) with a pH of 7.4 for 2 hours as it will be 2ml/minute.

For dpESM characterization, first, in dpESM, whether the DNA content is left is determined by PCR method. After decellularization process, histochemical and immunofluorescence analyzes are performed to determine whether the parenchymal and stromal integrity of the pancreas is maintained in dpESM, as well as whether there are any cell remaining therein and also to determine glucosaminoglycan (GAG) content and the result is compared with natural pancreatic tissue.

- Then transforming the product obtained by lyophilization and powdering of the decellularized pancreatic matrix (dpESM) into a material in the form of a printable solution in 3D bioprinters,

For lyophilization and powdering of the dpESM;

A lyophilizer (FTS Systems Bulk FreezeDryer Model 8-54) is used to completely remove the water retained by dpESM. Decellularized pancreatic tissue, which is kept wrapped in aluminum foil so that it does not freeze at -200 ^º C, all samples that are lyophilized in the lyophilizer for 20 hours (± 2 hours) between 0 and 100 °C are stored in air-tight sealed packages. Then it is sterilized by e-beam (electron- beam) irradiation at 22 kGy. Lyophilized layers are immersed in 0.9% saline for 5 minutes. In order to powderize the dpESM, the lyophilised layers are crushed in a mill compressed with liquid nitrogen. dpESM powder is prepared by acid extraction of the said particles in 0.5 N HCL (per 50 mL/g of powder) for 3 hours at room temperature. Then, it is washed with sterilized distilled water by being centrifuged at 4 ^º C for 10 minutes at lOOOOg. dESM powder is extracted with ethanol and ether, and the ether is evaporated under a chemical decanter.

The protein content of dpESM is determined with LCMS-IT-TOF analysis.

The dried dp-ESM is disintegrated in liquid nitrogen with the help of a mortar; it is mixed with 10% pepsin (w/w) prepared with 0.5 M acetic acid for 48 hours at room temperature in order to become soluble. Its ion balance is provided by using lOx PBS. The solution is filtered through filters having pore diameters of 40 pm to avoid undissolved matrix particles. The final concentration of the dpESM which is become soluble is adjusted with 0.5 M acetic acid. Then mycoplasma screenign is performed with commercially available (such as MycoAlert™ Mycoplasma Detection Kit) kits. The pH of dpESM solution is expected to between 2.8-3 at this stage. This pH value is adjusted to 7.4 by using 10 M NaOH. All these processes are carried out at 10 °C, so that the solution does not gelate It is stored in liquid form for obtaining bioink.

- In the meantime, using a cationic polymer (chitosan or other cationic polymers which are not toxic for the subjects) enabling the transfer of genes related to the b-cell differentiation with a non-viral method, and forming chitosan-pDNA nanoparticular b-cell gene transfer system**** with ionic gelation method,

For Chitosan-pDNA nanoparticles to be used as gene transfer system, oligochitosan (Mw 7.3kDa; DD >97%) molecule (as present in Novamatrix, FMC Biopolymer, Norway) is commercially supplied.

Ionic gelation formula for chitosan-pDNA nanoparticles is created by electrostatic interaction between cationic (+) chitosan and anionic (-). In summary, chitosan is dissolved in 1%-2% acetic acid and added dropwise into aqueous solution containing pDNA and Sodium tripolyphosphate (TPP), and thus creating crosslink between chitosan- pDNA particles. The chitosan-pDNA nanoparticles precipitated as a result of ionic gelation are kept for 30 minutes at room temperature for stabilization before use. - Obtaining Gene Activated Matrix (GAM; dpESM+chitosan pDNA nanoparticles) by combining gene transfer system with decellularized pancreas matrix (dpESM) in order to differentiate mesenchymal stem cells into beta cells in the tissue to be printed in 3D bioprinter, - Obtaining autologous mesenchymal stem cell (MSC) from bone marrow or other tissues and making MSCs ready for the production of endocrine pancreas-like tissue (biochip) as number and phenotype for endocrine differentiation,

Bone marrow aspirates are taken into tubes comprising heparin under proper conditions for the isolation of bone marrow (BM) derived mesenchymal stem cells (MSC) of individuals with T1D. Bone marrow aspirates are washed with PBS or HBSS buffer (Hans Balanced Salt Solution) comprising penicillin, streptomycin and amphotericin B in ratio of 10% two times by centrifugation in a sterilized cabin at 300g. It is taken into 0.8% ammonium chloride and kept at + 4C° for 15 minutes. After being centrifuged and washed with PBS, it is diluted with DMEM-F12 (Dulbecco’s modified Eagle’s medium-low glucose and F12 addition) culture media comprising %1 PSA, %10 Human AB (as it is commercially available from Seralab), 1 ng/mL bFGF, % 1 glutamax, it is cultivated into a flask, and cultured in an incubator providing an environment at 37 °C comprising %5 CO2.

The medium of the cells is replaced every 3 days and when the cells are 70-80% confluent, the cells that are adhered by trypsinization process are removed and passaged in a ratio of 1: 3. The characterization of cells obtained of 3^rd passage is carried out.

MSCs obtained from bone marrow tissue of individuals with T1D are are isolated thanks to their adhesion ability to culture container, and the morphologic properties of the adhered cells are analyzed with the phase-contrast microscope during the study. Flow cytometric analysis (CD73, CD90, CD105 positive and CD45 CD34 negativity in terms of surface markers of MSCS) and immunocytochemical marking (Immunhistochemical Marking (IHC) and Immunofluorescent Marking (IF)) studies are performed in order to determine the immunophenotypic properties. Gene expressions are determined with RT-PCR.

- Preparation of bioink by means of adding chitosan-pDNA nanoparticles and MSCs into printable dpESM form-by providing proper conditions wherein its gelation is prevented,

Following the aforementioned technique performed at 10°C to prevent the gelation of the dpESM solution, the following elements are added to the dpESM, which is stored in liquid form, for the production of Bio Ink:

Chitosan-pDNA nanoparticles:

Chitosan-pDNA nanoparticles that ate prepared as it is mentioned before and the dpESM solution which is prepared are mixed gently. MSCs:

To prevent cells and Nanoparticles containing DNA from being damaged during bioprinting, they are coated with a protective gel that can degrade immediately after printing and will not interfere with the interaction of the matrix and cells. For this, before printing, to the solution of gelatin methacryloyl (GelMA) (10% (weight/volume) and 0.5% (weight/volume) 1-[4-(2-hydroxyethoxy),phenil]-2-hydroxy-2- methyl-l-propanone photoinitiator (PI), it is prepared by adding cells such that it will be 5x10⁵ cell/ml. Bone marrow-derived MSCs in the 3^rd passage (P3) are removed from the culture dish and added to the mixture containing dpESM + chitosan pDNA nanoparticles at a concentration of 6xl0⁶ cells/ml and mixed gently. With the addition of cells, the dpESM + chitosan pDNA nanoparticles + MKH composition is called as bioink.

Printing biochip from the bioink with a 3D bioprinter,

High resolution (-1.25-50 micron), multi-headed 3D-bio-printer, extrusion-based bio-printer with adjustable heating and cooling properties is used in printing the 3D tissue scaffolding designed to differentiate MSCs into pancreatic b-cells. In order to optimize the cell viability and the printability temperature of the material bioprinting is carried out at 22.5 °C, 25 °C, 27.5 °C and 30 °C with temperature control, and the results are evaluated and the optimum conditions for each gel are determined and then printed. The tissue scaffolds created after bioprinting process is enabled to gelate by crosslinking by means of applying UV light for 17 seconds at 850 mW from a distance of approximately 8.5 cm to the tissue product printed in order to realize a fast crosslinking (photo-polymerization with photocuring process). When cross-linking cannot be performed at the desired degree, Alginat is added in the same amount of GelMA added to the bioink, and it is left to the 100mM CaCl₂ solution for 3 minutes after printing (V/V). Cell proliferation/viability analysis of biochip obtained from the bioink with 3D printer

Determination of cell viability in the biochip is realized with two different analyses, namely Fluorescent Live/Dead Staining (as in commercial product of Live/Dead Cell Double Staining Kit/Sigma- Aldrich) and [2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium] 3D cell culture viability analysis kits (as in commercial product of 3D Cell Culture HTS Cell Viability Complete Assay Kit, Biovision).

- Obtaining insulin secreting beta-like cells from MSCs present in the tissue by culturing tissue printed in 3D bioprinter from the bioink in endocrine differentiation medium,

Differentiation of MSCs into b-cells in the biochip is physically induced by tissue scaffold obtained from the pancreatic extracellular matrix, and it is also induced at the gene level by means of the MafA and Pdx-1 transcription factors carried by the gene activating matrix present in the biochip design; and it is further induced by adding inducing factors to the medium. Therefore, the b-cell differentiation of MSCs is supported more strongly. MSCs are induced with a four stage protocol for pancreatic b-cell differentiation. Differentiation is initially initiated by culturing on the LG-DMEM medium supplemented with 10% Human AB Serum (if the patient is human, for other mammalian patients Fetal calf serum is used), 10 mM nicotinamide and 4 nM activin A, 25 ng/ml recombinant EGF and 0.5 mM b-mercaptoethanol for 3 days. Then in the second step the cells are incubated for 5 days in the LG-DMEM medium containing 10% Human AB Serum, 10 mM nicotinamide, 4 nM activin A and 25 ng / ml recombinate EGF. In the third induction stage, the cells are cultured for 7 days with 2% Human AB Serum, 10 mM nicotinamide 10 nM Exendin-4, 10 mg/ml INGAP- pp and IX ITS. In the final induction stage, the cells are cultured with H-DMEM comprising 10 mM nicotinamide, 10 nM Exendin-4, 10 mg/ml INGAP-pp and IX ITS and recombinant bFGF, and thus the differentiation is completed. Cells are incubated at 37 °C, 5% C02 under controlled culture conditions. The media of the biochips are refreshed by being replaced once in every two days.

In order to evaluate differentiation of MSCs into b cells in gene expression level, Gata-4, Hnf3b, Hnf4a, insulin I, insulin II, islet amyloid polypeptide (IAPP), glucose transporter-2 (Glut 2), C-peptid, Pdx-1, Nkx2.2, MafA, Ngn3, MafA and Isl-1 genes are evaluated in Real Time-PCR. In order to determine the level of secreted insulin, INSULIN and C-PEPTIDE ELISA analysis (Human C-Peptide ELISA Enzyme-Millipore) is performed, and in order to see protein level GATA-4, HNF3B, HNF4A, INSULIN I, INSULIN II, islet amyloid polypeptide (IAPP), glucose transporter-2 (GLUT 2), C-PEPTID, PDX-1, NKX2.2, MAFA, NGN3, MAFA and ISL-1 immunofluorescent markings are performed. For characterization of biochip scaffolding’s similarity to the parenchymal and stromal construct of the natural pancreas, Hematoxylin-Eosin (H-E), Alcian Blue and Sirius Red stainings are analyzed by histochemical method. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) marking is performed to determine status of the apoptotic damage of the cells in the biochip by using the apoptose detection kits as it is the In-Situ Cell Death Detection Kit, F. Hoffmann-La Roche). TEM and SEM images are captured for morphological analysis of the biochip.

To determine the function of the biochip by in vitro glucose responsive insulin secretion analysis;

The insulin level in the medium of the biochip is determined by an ELISA-based method using a commercial kit (as in the Millipore Insulin ELISA Kit). In order to determine glucose-dependent insulin secretion, b-cells in the biochip are exposed to two different glucose concentrations in vitro to test whether they secrete insulin into the medium depending on the glucose added to the medium, and the insulin determination is carried out in the collected medium. Before starting insulin secretion analysis, differentiated b-cells are cultured on insulin-free medium for 48 hours, and periodically washed with PBS until insulin is completely removed from the medium. Serum-free LG- DMEM (low glucose content; 5.5 mmol/L) comprising 0.5% BSA is added to the wells and the cells are incubated at 37 °C for 1 hour. The supernatant is collected and frozen at -70 °C for basal insulin secretion analysis. Then the cells are cultured on H-DMEM (high glucose content; 25 mmol /L) medium again for 1 hour. At the end of the period, the supernatants are collected and frozen to determine glucose- stimulated insulin secretion b-cells differentiated from MSCs (trial group), non-differentiated MSCs (negative control) and commercially available b-cell line (positive control), the amount of insulin stimulated with glucose in the supernatants (protein levels) is determined with microplate reader.

- It is comprised of the steps of transplantation of pancreas-like functional tissue grafts to the subjects with T1D as subcutaneous biochips and bringing the blood sugar to a healthy level by means of secretion of insulin as a response to glucose in the subject with T1D thanks to biochip.

In order to determine whether there is bacterial contamination in the biochip after differentiation by means of the Contamination Analysis with Bioburden of the biochip before transplantation, 5 ml differentiation medium is taken in a 15 ml conical bottom tube and mixed with a 7ml SOC medium and incubated at 37 °C. Its absorbance is determined at 425 and 600 nm 24, 48, 72 and 96 hours after the incubation.

Following immunotherapy, biochips prepared under in vitro conditions are transplanted subcutaneously to the individuals with T1D under anesthesia.

Evaluation of therapeutic effect of the transferred biochip

In individuals who were followed up for 23 weeks after the transplantation, fasting blood glucose level (fasting blood sugar, FBS) is measured with the blood sample taken periodically. FBS³ 126 mg/ dl is considered as diabetic values.

Elisa analyses of C-peptide and insulin

On the 30^th and 140^th days, serum samples are taken and the human- specific C-peptide level and the human nonspecific insulin level are analyzed with the ELISA assay kit (Wako Osaka, Japan and Shibayagi, Gunma, Japan).

Oral glucose tolerance test

The function of the biochip to be transferred is measured with OGTT (Oral Glucose Tolerance Test) in 30^th day. The way of performing the said test: glucose solution was orally given to the individual with T1D fasted for 8-12 hours (adults: solution comprising 75g glucose, children: 1.75 g/kg body weight) and measurements were performed at various hours (0, 30, 60, and 120. minutes). FBS ³ 126 mg/ dl, postprandial blood glucose³ 200 mg/dl are considered as diabetic values.

Blood glucose measurements in diabetic individuals

It was used to confirm the presence of hyperglycemia (fasting blood glucose level ³ 126 mg / dl, postprandial blood glucose 200 mg / dl) in individuals with T1D. For this, measurements are performed on blood samples taken from individuals to determine blood glucose levels after 72 hours with a portable glucometer device (Medisense Precision QID; Abbott Laboratories, Bedford, MA, USA). Glycosylated hemoglobin test

HbAlc (Glycosylated Hemoglobin) test is valuable in the follow-up of the diabetic patient and gives an idea about the three-month blood sugar average. < 6.5% is considered as ideal level for HbAlc in diabetic patients.

Consequently, as a result of tolerance based immunotherapy, a technique has been developed in which the autoimmune attacks existing in T1D can be prevented, and furthermore the blood glucose level is brought to normal level and the long-term normal blood glucose level can be maintained thanks to the transplantation of glucose-responsive insulin-secreting tissue grafts that have been produced. The most important characteristic of this technique in its therapeutic use is that the transplanted tissue grafts prevent the autoimmune attacks and it can lower blood sugar in glucose sensitive diabetic individuals.

* The diagnosis test or evaluation of individuals with T1D is performed with oral glycose tolerance test (OGTT), glycolysated hemoglobin test or fasting plasma glucose test. Furthermore, the diagnosis of T1D patients having an immune system reactive to which type of diabetes antigens is performed by conducting antibody test. The information obtained here is used for selecting individual specific autoantigens.

** In the method aiming individual specific treatment, instead of GAD65 used as autoantigen or in addition to this as biomarker of b cell damage in individuals with T1D, antigen/antigens detected in the highest level with antibodies and defined in diabetes development can be used. For example, such as nonspecific islet cell antibodies (ICAs), insulin, insulinoma antigen-2 (IA-2) and transmembrane protein tyrosine phosphatase (ICA512), islet- specific glucose-6- phosphatase catalytic subunit related protein (IGRP) (Wenzlau and Hutton, 2013). Which antigen will be chosen will be determined by subjecting the individual with T1D to an antibody test used for the diagnosis of T1 Diabetes by a doctor or veterinarian with a standard experience with this technique.

*** In order to achieve antigen specific tolerance in subjects with T1D both methods (Loading the determined T1D autoantigens in vitro to the dendritic cells obtained from the subjects or conjugating to synthetic biodegradable nanoparticles) can be used alternatively. However, if they are insufficient in prevention of autoimmune attacks, they can be used in combination in order to increase the effect of immuno tolerance. The choice of which method will be used in alternative use can be determined by a doctor or veterinarian who has a standard experience in this field depending on whether dendritic cells can be obtained from patient subjects.

For achieving immunotolerance of autoreactive T cells which are the cause of T1D against b-cells, tolerating antigen-conjugated nanoparticles are obtained by means of in vitro conjugation of biocompatible and biodegradable nanoparticles [such as polymeric nanoparticles or lyposomal nanoparticles like PLG poly(lactic- co-glycolyte) also called as PLGA poly(lactic-co-glycolic acid] with T1D autoantigens. After the injection of antigen-conjugated nanoparticles to the subjects with T1D, the Tregs existing in the patient are enabled to be reprogrammed in terms of tolerance.

****“pDNA” in the chitosan-pDNA nanoparticular b-cell genes transfer system is the short name of the plasmid DNA comprising beta cell differentiation genes, and Pdx-1 and MafA genes or transcription factors playing role in b cell development are present in pDNA. By combining chitosan and pDNAs with ionic gelation, nano-sized chitosan-pDNA particles are obtained as the gene transfer vector.

The term of “individual” or“subject” mentioned as subject or individual with T1D corresponds to human or any one of the mammalian animals. The dosage regimens in immunotherapy method carried out as intravenous injection 24 hours after radiation irradiation to the individuals with T1D can be adjusted in order to targeted optimum response (for example tolerance and/or therapeutic effect created in a subject). For example, a single dose expressed as an injection may be administered, several divided doses may be administered over time, or the dose may be reduced or increased proportionally according to the requirements of the state of the disease. T1D autoantigens (b-cell antigens) are the antigens obtained by lysate of prepared synthetic antigens defined as autoantigen in T1D with GAD65 and/or 3D insulin secreting tissue/ from apoptotic bodies of b-cells.

Programming of Tregs specific to in vivo antigen is carried out by the steps of i) Detection of autoimmune subjects;

ii) Apoptosis of immune system cells by means of low dose radiation (radiotherapy);

iii) Loading the determined T1D autoantigens in vitro to the dendritic cells obtained from the subjects or conjugating to synthetic biodegradable nanoparticles in order to achieve antigen specific tolerance in subjects with

T1D,

iv) Administration of autoantigen primed dendritic cells and/or antigen- conjugated biodegradable nanoparticles to the subject with T1D following radiotherapy.

An endocrine pancreas-like tissue piece (biochip) created with a 3D bioprinter obtained with the method of the present invention comprises pancreas extracellular matrix obtained by decellularized pancreas lyophilisation, chitosan- pDNA nanoparticular gene (Pdx-1 and MafA genes or other transcription factors playing a role in b cell development) transfer system, and autologous (derived from bone marrow or obtained from other tissues) mesenchymal stem cells in its construct. The product created with 3D bioprinter comprises Gene Activated Matrix formed of decellularized pancreas integrated with chitosan-pDNA nanoparticular gene transfer system suitable for differentiation of mesenchymal stem cells into beta cells (GAM; dpESM+chitosan pDNA nanoparticles) in its construct. GAM, stimulating differentiation in this endocrine direction, is a sub product which is one of the topics of the invention, created within the scope of the invention. Chitosan-pDNA nanoparticular b-cell gene transfer system is created by using a cationic polymer; chitosan or other cationic polymers which are not toxic for the subjects enabling the transfer of genes related to the b-cell differentiation with a non-viral method.

In the method of the present invention, the said endocrine pancreas-like tissue piece (biochip) is used as therapeutic in treatment of T1D patients. The product is administered subcutaneously, and enables the blood sugar to be lowered by secreting glucose responsive insulin. The product which is obtained within the scope of the invention is suitable for clinical use since a non-viral system is used for gene transfer. Furthermore, can be produced according to the desired suitable micro-patterns with 3D bioprinters by designing via software in electronic media after powderizing the matrix obtained with decellularization of the organ, and in this way it is suitable for integrating the non-viral gene transfer system to the matrix. The product, if needed, is a matrix which allows addition of various polymer, growth factors, medium stabilizers into the solution obtained by making the decellularized pancreas matrix soluble.

The method of the present invention allows to give their final 3D shape to the biochips in accordance with the re-design of the matrix in the computer medium, and to design the matrix according to the target area where it is to be transferred while being transferred to a living thing. In case the cell viability in the sample tissue printed from bioink used as cell matrix solution is low, the solution obtained my mixing the created gene transfer system with the matrix in solution form can be used as bioink, and printing in the 3D bioprinter can be carried out with these two components. The MSCs can be added to the tissue scaffold after printing during culture. With this technique a doctor or a veterinarian with a standard experience can easily determine the effective dose of components required in immunotherapy (dendritic cells, antigen conjugated nanoparticles or antigens). For example, doctor or veterinarian first administers a level lower than the required level in order to achieve the desired therapeutic effect through the immuno therapeutic components that are used, and increases the dosage in time until the desired effect is achieved.

In the new generation insulin synthesizing tissue production method which is one of the subjects of the invention, by means of using decellularization and 3D printing techniques in combination, which are two different fields of tissue engineering, a hybrid printing technique is developed, and thus a bioactive matrix production re-printed by using stem cells of extracellular matrix of the pancreas is carried out. Insulin producing beta cells are obtained by means of differentiation of autologous mesenchymal stem cells and insulin secreting endocrine pancreas like functional tissue piece production is provided. In the therapeutic method for the treatment of T1D which is another subject of the present invention, first the autoimmune immune system, which attacks the insulin-producing b-cells in T1D is suppressed. After autoimmune attacks are prevented in individual with T1D, the insulin producing tissue piece created within the scope of the invention is transplanted to individuals with T1D as subcutaneous biochips. Therefore, the tissue can secrete insulin in response to glucose in diabetic individuals and bring the blood sugar to a healthy level.

Within the scope of the project, first subjects with T1D are determined, and the autoantigens and their dosages to be administered to the subjects with T1D are determined. In subjects developing T1D, antigen-primed dendritic cells and/or antigen-conjugated biodegradable nanoparticles are created in order to induce antigen- specific tolerance against T1D. Both methods can be used alternatively in order to provide antigen specific tolerance in subjects with T1D. However, if they are insufficient in prevention of autoimmune attacks, they can be used in combination in order to increase the effect of immuno tolerance. The choice of which method will be used in alternative use can be determined by a doctor or veterinarian who has a standard experience in this field depending on whether a sufficient number of dendritic cells can be obtained from patient subjects. After the cells of autoimmune system existing in the subjects with T1D are subjected to apoptosis with low dose (sublethal level) radiation irradiation, induction/reprogramming of T-regulator cells (Treg) which will provide antigen specific tolerance against T1D in the subject is carried out after intravenous (IV) injection of the created dendritic cell and/or antigen-conjugated biodegradable nanoparticles to the subject with T1D. On the other hand, for insulin secreting tissue production, which is another aspect of the invention, the matrix which will create the said tissue scaffold: the cell-free pancreatic extracellular matrix (dpESM) is obtained by pancreatic decellularization and lyophilization of this is performed. The dpESM, which is completely removed from water by means of lyophilization process, is transformed into a solution in a suitable concentration in order to support the cell viability in the best way and to carry out the bio-printing in the most ideal conditions in 3D printers after being powderized. In the meantime, a matrix which transfers b-cell genes allowing the tissue scaffold to perform gene transfer is created- in order to perform a stronger differentiation from mesenchymal stem cells (MSC) used as cellular component in the tissue into insulin secreting beta cell- as second step in obtaining insulin secreting tissue. Chitosan-pDNA nanoparticles used as b-cell genes transfer system are prepared by combining chitosan which is a cationic polymer with pDNA vector carrying b- cell genes. Mesenchymal stem cells (MSC) which are the last component of the endocrine pancreas-like tissue desired to be created are also obtained in this stage, and they are made ready in terms of number&phenotype for endocrine differentiation. To prevent cells from being damaged during bioprinting, they are coated with a protective material that can degrade immediately after printing and will not interfere with the interaction of the matrix and cells. Chitosan-pDNA nanoparticles and MSCs are added into the dpESM preserved in liquid phase in order to obtain bioink, and a pre-designed 3D tissue printing is carried out a 3D bioprinter. In case the cell viability in the sample tissue printed from bioink used as cell matrix solution is low, the solution obtained my mixing the created gene transfer system with the matrix in solution form is used as bioink, and printing in the 3D bioprinter is carried out with these two components. The MSCs are added to the tissue scaffold after printing during culture. In both cases, the tissue scaffold created as a result of printing comprises Gene Activated Matrix (GAM: dpESM + chitosan-pDNA nanoparticles) By means of culturing the 3D tissue obtained in both ways in the endocrine differentiation medium, the mesenchymal stem cells in the tissue are differentiated in the endocrine direction and insulin secreting beta-like cells are obtained. In this stem cell differentiation, a multidirectional endocrine differentiation stimulation is provided by means of GAM and endocrine culture medium. After the transplantation of endocrine pancreas-like functional tissue grafts (biochip) to the subjects with T1D as subcutaneous biochips, it is tested that the biochip can bring the blood sugar to a healthy level by secreting insulin in response to glucose.

The term of “individual” or“subject” mentioned as subject or individual with T1D corresponds to human or any one of the mammalian animals which develop T1D. The element for the treatment of which a product is created in the invention and a therapeutic method is presented is the subjects with T1D.

T1D autoantigens (b-cell antigens) are the antigens that are determined to be primed to the dendritic cells in vitro in order to provide antigen specific tolerance and/or to be conjugated to biodegradable nanoparticles. These are th antigens produced ready, synthetically (such as GAD65) produced antigens from b-cell antigens defined as autoantigen in T1D, and the antigens obtained by lysate of the 3D tissue produced within the scope of the invention and from the apoptotic bodies of b-cells. Antigen specific tolerating dendritic cell; tolerating dendritic cells are obtained by in vitro loading of dendritic cells obtained from subjects with T1D with T1D autoantigens. Antigen primed dendritic cells are created in order to reprogram the Tregs present in the tlD patients in tolerance direction. Dendritic cells are co cultured with beta cell antigens causing autoimmunity, and they are designed as antigen- specific which causes the autoimmunity of TlD, and therefore immunosuppression is achieved specific for only the said antigens. Within the scope of the invention, antigen- specific designed dendritic cells are administered in vitro, and thus T regulator cells (Treg) are stimulated. Therefore, antigen specific immunosuppression for preventing autoimmunity in TlD is realized by means of antigen specific in vivo programming of T regulator cells (Treg). Dendritic cells are transferred to the patient after being primed with autoantigens in vitro and making them antigen specific. In other words, before transferring dendritic cell to the subject with TlD, dendritic cells stimulated in vitro with the autoantigens are created. These antigen specific stimulated tolerating dendritic cells are administered to the patients. Within the scope of the invention, biodegradable nanoparticles to which antigen is conjugated (PLGA-Antigen) are used together with dendritic cells or as an alternative to the dendritic cells. PLGA- Antigen nanoparticles are prepared with antigens (beta-cell antigens) selected related with the TlD. By means of administering PLGA-Antigen particles to the patient after radiotherapy, Tregs in the immune system are programmed specific to the antigens that we have selected, thereby presenting antigen- specific tolerance based immunotherapy application. Antigens obtained from beta-cell apoptotic bodies (they can be used in combination when necessary depending on the patient) are used in the invention as antigens (for example GAD65).

Antigen-conjugated biodegradable nanoparticles; For achieving immunotolerance of autoreactive T cells which are the cause of TlD against b- cells, tolerating antigen-conjugated nanoparticles are obtained by means of in vitro conjugation of biocompatible and biodegradable nanoparticles [such as polymeric nanoparticles or lyposomal nanoparticles like PLG poly(lactic-co- glycolyte) also called as PLGA poly(lactic-co-glycolic acid] with T1D autoantigens. After the injection of antigen-conjugated nanoparticles to the subjects with T1D, the Tregs existing in the patient are enabled to be reprogrammed in tolerance direction.

Low dose radiotherapy; Apoptosis of autoimmune system cells existing in the T1D subjects are subjected to apoptosis with low dose (in sublethal level) radiation. Radiotherapy is applied for both eliminating the existing autoimmune cells by apoptosis, and antigen primed dendritic cell and nanoparticles administered following the radiotherapy supporting the immunotolerance better when they encounter apoptotic cells.

T regulator cells induced to in vivo antigen specific tolerance (with antigen primed dendritic cell and antigen-conjugated biodegradable nanoparticles- mediated Treg induction); in order to achieve immunotolerance of autoreactive T cells which are the cause of the disease against b-cell antigens, antigen primed dendritic cell and antigen-conjugated biodegradable nanoparticles are injected to subjects with T1D at a certain dosage and concentrations. Therefore, it enables i) in vivo programming of T regulator cells (Treg), which play a major role in immunosuppression in order to achieve immunotolerance of autoimmune immune cells attacking insulin-producing b-cells in individuals with T1D, and eventually preventing autoimmune attacks, and ii) creating a medium wherein autoimmunity can be prevented in order that tissue engineering product (biochip) that we have created as the basis of the invention can continue its function before the transplantation of biochip.

Decellularized pancreas extracellular matrix (dpESM); pancreas extracellular matrix (dpESM) which is purified from cell with pancreas decellularization is obtained to be used as bioink, i.e. printing material (material to be loaded to the cartridge) for 3D bioprinter. Since decellularized material is used in tissue scaffolds produced by the technique within the scope of the invention, it is also quite important in differentiation of mesenchymal stem cells which is a component of pancreatic tissue and cultured in the said tissue scaffold that we will produce as well as it allows bioprinting with natural extracellular matrix of the organ. Extracellular matrix components are structures that are generally protected between species, and they either create no immune response even in xenogeneic transplantations, or the immune response they create can easily be tolerated. In order to make the matrix obtained after the pancreas is decellularized ready to be printed in 3D printers, matrix is lyophilized and powderized, and then it is transformed into soluble form, thereby giving it a gelling ability. This form of the matrix which is produced is well suited to support cell viability and to create a designable microenvironment. The polymers that are used within the scope of the invention are a medium obtained after organ decellularization and belonging to organ itself that already exists, it is much more than ready to use synthetic polymers comprised of a single polymer, and consists of natural biomaterial having a complex content comprising collagen typel, collagen type 4, fibronectin, glycosaminoglycans, and the like [31].

Chitosan-pDNA nanoparticles b-cell genes transfer system are formed in order to enable the mesenchymal stem cells present in the produced pancreatic tissue to differentiate into insulin secreting b-cell like cells. These non-viral nanoparticles, that is to be formed by means of using a cationic polymer (chitosan or other nontoxic cationic polymers for subjects) instead of virus, are gene (genes related with b-cell differentiation) transfer system. “pDNA” in the chitosan-pDNA nanoparticular b-cell genes transfer system is the short name of the plasmid DNA comprising beta cell differentiation genes, and Pdx-1 and MafA genes or transcription factors playing role in b cell development are present in pDNA. By combining chitosan and pDNAs with ionic gelation, nano-sized chitosan-pDNA particles are obtained as the gene transfer vector.

Gene Activated Matrix (GAM) is comprised ofdpESM+chitosan-pDNA nanoparticles composition. In order to obtain Gene Activated Matrix (GAM) which enables the stimulation of stem cells in endocrine pancreas direction, the integration of dpESM form printable in 3D bioprinters with chitosan-pDNA nanoparticles b-cell gene transfer system is provided. In the meantime, bioink is prepared by means of combining two elements. By means of GAM technology prepared with this technique, beta-cell differentiation from mesenchymal stem cells is supported strongly with the effect of both gene transfer system and natural dpESM.

Mesenchymal stem cells (MSC) are used as an origin stem cell in order to create insulin secreting cells in the pancreas, in other words mesenchymal stem cells are differentiated into beta cells with a series of methods. It is also the third element of the bioink. Within the scope of the invention, autologous mesenchymal cells (taken from the patient, taken from the same individual) are obtained from the bone marrow or other tissues of the patient and they are directed to differentiate/transform into b-cells. Even though the characteristics of MSCs such as not creating an immune response in anti-apoptotic, anti-inflammatory, allogenic transplantations is one of the reasons for selecting these cells, their immunosuppression characteristic is not the primary objective.

Immunosuppression is realized by means of autoimmunity in T1D, in vivo programming of t regulator cells (Treg) specific to antigen. In the invention, 3 dimensional tissue scaffold formed with autologous and bone marrow derived mesenchymal stem cells (Bioink; dpESM+chitosan pDNA nanoparticles+ MSC composition) and forming method is presented as novelty.

Bioink (Bioink; dpESM+chitosan pDNA nanoparticles+MSC composition); bioink for 3D bioprinter, that is elements to be used as printing material (the material to be loaded to the cartridge), are integrated to be suitable for printing in a bioprinter, and thus the preparation of bioink is completed. 3D tissue scaffold is produced by using bioinks and 3D bioprinters to create the 3D tissue piece previously designed in computer medium. The said 3D construct creates a structural environment and provides porous biocompatible niches for mesenchymal stem cells so that they have micro patterns previously designed relative to the natural tissue and mechanical properties specific to the tissue, thereby transforming into insulin secreting pancreatic tissue. The response of the cell against the material provides important factors such as adhesion to the material, reproduction on the material, differentiation, protein synthesis profile.

Endocrine pancreas-like functional tissue piece (biochip) is both a new product created within the scope of the invention and a product used in treatment method of T1D which is another aspect of the invention. Within this context, following the immunotherapy, it is the biochip that is subcutaneously transplanted in order to bring blood sugar to a healthy level by secreting insulin in response to glucose in the T1D subject.

The invention is a therapeutic method for T1D treatment as a whole as endocrine pancreas-like functional tissue piece production and transplantation which can bring blood sugar to a healthy level by secreting insulin as response to glucose after enabling the reprogramming of the immune system causing autoimmunity in Type Diabetes (T1D). At the same time, it is also the production of glucose responsive insulin secreting natural tissues by means of developing new generation beta cell differentiation techniques in endocrine pancreas engineering. This invention, which aims the treatment of T1D in every aspect, consists of two main sub-studies in detail: Immunotherapy and b-cell replacement (replacement of the lost tissue). Within the scope of the invention, all aspects for the treatment of T1D are presented, namely immunotherapy and beta-cell replacement.

Immunoregulation approaches are reorganized within the scope of the invention, and two important points are aimed: 1 )In vivo programming of T regulator cells (Treg) playing a primary role in immunosuppression in order to provide immunotolerance of autoimmune immunity cells (especially the autoreactive cytotoxic T cells) attacking insulin producing b-cells in individuals with T1D and eventually preventing autoimmune attacks 2) being able to create the medium in which the autoimmunity can be prevented before the transplantation of the biochip in order that the biochip which is the created tissue engineering product (insulin secreting tissue) can continue its function in vivo medium. For beta cell replacement, the production and transplantation of endocrine pancreatic-like functional tissue piece that can bring blood glucose to a healthy level by secreting insulin as a response to glucose by developing new generation beta cell differentiation techniques in endocrine pancreatic engineering is achieved.

The steps wherein specific methodological innovations are provided in the invention are about creating tissue engineering product which is enriched with gene transfer for b-cell replacement in T1D and wherein a plurality of techniques that have not been tried before are developed. Here, by means of using decellularization and 3D bioprinting techniques which are two separate fields of tissue engineering together, a hybrid printing technique is developed, and therefore are written bioactive matrix production is provided by using stem cells of pancreas’s extracellular matrix.

Within the light of all these information, the present invention comprises the following innovations:

i. Therapeutic approach of the invention as a whole for T ID,

ii. Production of endocrine pancreas-like functional tissue piece iii. Tissue engineering method that is used

iv. Transplantation of the said tissue In the invention, autoimmune system cells existing in the individuals with T1D are subjected to apoptosis with low dose radiotherapy, and then the tolerance (immune tolerance against b-cells) of autoreactive T cells in T1D is enabled by induction of dendritic cell and antigen-conjugated biodegradable nanoparticle- mediated regulator T cell (Treg). The problems about the insulin requirement in the studies targeting only autoimmunity are overcome in the present as follows: Following the reprogramming the autoimmunity, the biochip secreting insulin as a response to glucose produced in order to eliminate insulin deficiency originating from b-cells lost upon the development of T1D is transplanted to the individuals with T1D. Consequently, it is a technical improvement wherein autoimmunity existing in tlD is eliminated, blood glucose level is brought to normal level and a long term normal blood glucose level can be maintained.

Production technique for endocrine pancreas-like tissue scaffold to be used for beta-cell differentiation from MSCs is a technique specifically designed for the treatment of TlD. The said technique which allows the solution produced by making the extracellular matrix obtained from pancreas decellularization soluble being printed according to micro patterns previously designed in 3D printers is a method used for the first time for treatment of TlD. Therefore, strong aspects of both decellularization and 3D bioprinting technique are combined, and a new and strong hybrid technique for endocrine pancreas tissue engineering is created.

Differentiation simulation of mesenchymal stem cells printed in 3D bioprinters from bioink and present in 3D tissue cultured in endocrine differentiation medium in endocrine pancreas direction is enabled by means of i. gene transfer system related to b-cell differentiation, ii. dpESM, and iii. endocrine medium.

In the invention, decellularized pancreas extracellular matrix (dpESM) is obtained to be used as bioink, i.e. printing material (material to be loaded to the cartridge) for 3D bioprinter. Since decellularized material is used in tissue scaffolds produced by the said technique, it is also quite important in differentiation of mesenchymal stem cells which is cultured in this produced tissue scaffold and is an element of the created tissue into beta-cell as well as it allows bioprinting with natural extracellular matrix of the organ. Extracellular matrix components are structures that are generally protected between species, and they either create no immune response even in xenogeneic transplantations can easily be tolerated. After dpESM is obtained, the product which is powderized after lyophilisation is made solution for printing in order to obtain the bio-ink form that optimizes cell viability and the printability of the material in the 3D bioprinter. Therefore, a homogenous composition is acquired in the produced tissue while a form allowing the printing of the matrix is obtained.

Endocrine pancreas-like functional tissue piece (biochip) is obtained, which is subcutaneously transplanted so that it can bring the blood level to a healthy level by secreting insulin in response to glucose in a subject with T1D after the immunotherapy. Biochip is living and completely biological system which is a biocompatible and a living system, does not comprise materials such as plastic or metal and which is designed to be transplanted into a living body. By means of the biochip produced within the scope of the invention, both beta cell replacement is provided, and it is aimed to regulate and eliminate autoimmune response with interactions of nanoparticles and dendritic cells, T regulator cell that are used. In the invention, it is not approached to T1D from a single point and the disease is treated as a whole and it is dealt with in a complex and multi-stepped way.

The tissue scaffolding used within the scope of the invention is designed via the software in electronic media after powderizing the matrix obtained by organ decellularization (the similarity between the said patent and our invention in terms of matrix is up to this point) and produced according to the desired suitable micro patterns with 3D bioprinters, and therefore it is produced with an integrated method wherein nonviral gene transfer system can also be integrated to the matrix. Using this system brings many advantages;

i. The solution obtained by making the decellularized pancreas matrix that is used soluble constitutes the main material of the tissue scaffold.

ii. By means of being able to re-design the matrix in a computer medium, being able to give final forms to the chips to be produced allows the matrix to be designed depending on the area where it is to be transferred while transferring it to a living thing.

iii. The content of the matrix allows, if needed, adding various polymer, growth factors, medium stabilizers, etc. to the solution obtained by making the decellularized pancreas matrix soluble. In other words, the system that is used is suitable for enriching the medium.

iv. Integrating the gene transfer system which will facilitate the differentiation of cells cultivated after the recellularization of the matrix into the insulin secreting beta cells to the decellularized pancreas extracellular matrix solution is also possible thanks to the method used within the scope of the invention. Furthermore, gene transfer system is preferred as a nonviral system for producing biochips suitable for clinic.

In summary, the advantages and innovations provided by the invention are as follows:

In various studies carried out about T1D, allogenic beta cell, islet or pancreas transplantation is tried, and this kind of transplantations cause serious immune responses since they are allogenic. The patients have to use immunosuppressive drugs. Within the scope of the invention, the autologous mesenchymal stem cells collected from the patients are used, and beta cell differentiation is carried out from these cells. Therefore the produced tissues are specific to individuals, and they do not cause an immune response when transplanted to the patient.

Individuals with T1D have to use insulin taken exogenously throughout their life. By means of the produced tissue, the insulin requirement of the body will be produced according to the blood glucose level, and individuals with the disease can continue their lives without using exogenous insulin.

The insulin purchased to be used exogenously creates an important burden for the economy of our country, considering that the number of patients with T1D is quite high. This economic loss is avoided by means of the produced tissue.

Beta cells created by differentiation from MSCs are produced in 3D culture media unlike the 2D culture methods. This case allows the opportunity to imitate the body better unlike many previous studies. The invention is a technique for insulin secreting endocrine pancreas-like tissue scaffold to be used for beta-cell differentiation from MSCs is a technique specifically designed for the treatment of T1D. The said technique which allows the solution produced by making the extracellular matrix obtained from pancreas decellularization soluble being printed according to micro patterns previously designed in 3D printers is a method used for the first time for treatment of T1D. Therefore, strong aspects of both decellularization and 3D bioprinting technique are combined, and a new and strong hybrid technique for endocrine pancreas tissue engineering is created. Gene activated matrices (GAM: dpESM+chitosan-pDNA nanoparticles) are obtained by means of strengthening the nonviral gene transfer systems (chitosan pDNA nanoparticles) of natural (non- synthetic) polymer structure of the matrix (dpESM) obtained from pancreas printed in 3D bioprinters. In the tissue scaffold printed from the said matrix, a 3D microenvironment that is unique for beta cell differentiation from mesenchymal stem cells is provided, thereby developing a new generation beta cell differentiation method.

3D bioprinting has advantages such as allowing the cells to be placed into the printed tissue while the tissue scaffold is in printing stage, being able adjust the produced tissue scaffold in the desired composition, and being able to design according to the desired micro patterns, and the method also provides niches more biocompatible than the matrices created with other methods. However, the material to be used in 3D bioprinting and the composition that is formed are important.

Since decellularized (nonsynthetic, natural) material is used in the tissue scaffolds to be produced with the said technique, it is quite important to provide bioprinting opportunity with the natural extracellular matrix of the organ. Because, both the ratios of extracellular matrix proteins such as collagen, fibronectin, laminin, etc. relative to each other are exactly preserved, and every area of the tissue allows cellularization easily by means of the cells existing in the bioink. Extracellular matrix components are structures that are generally protected between species, and they either create no immune response even in xenogeneic transplantations can easily be tolerated. By means of the said technique, while the natural construct is preserved, decellularized extracellular matrices obtained from different species can be used for treatment. Therefore, the problem experienced in allogenic, even in xenogeneic tissue/organ transplantations is overcome.

The invention is also a cellular treatment type for T1D. It comprises the production method of endocrine pancreatic-like functional tissue piece (biochip) that can bring blood glucose to a healthy level by secreting insulin as a response to glucose by developing new generation beta cell differentiation techniques in endocrine pancreatic engineering. This "product" formed by the development of various techniques creates a new concept in itself. The problems about the insulin requirement in the studies targeting only autoimmunity are overcome in the present as follows: Within the scope of the invention, whole approach for the treatment of T1D is presented, both immunotherapy and beta-cell replacement; 1) prevention of autoimmune attacks by means of in vivo programming of T regulator cells (Treg) in individuals developing T1D, 2) being able to create the environment where autoimmunity can be prevented before the transplantation of the biochip so that the biochip (insulin secreting tissue), a tissue engineering product which is the basis of the invention, can maintain its function in vivo, 3) With the development of new generation beta cell differentiation techniques in endocrine pancreatic engineering, it provides endocrine pancreas-like functional tissue piece production and transplantation that can bring blood sugar to a healthy level by secreting insulin in response to glucose. In the invention, while b-cell differentiation from MSCs is realized on the tissue scaffold providing microenvironment very close to natural, the re-regulation of dendritic cell-and/or antigen conjugated biodegradable nanoparticle- mediated Tregs is provided in order to create immunotolerance of autoreactive immunity cells in T1D against beta cells. Therefore, the invention can target the treatment of T1D disease as a whole. Thus, a new strategy is provided allowing the treatment of T1D disease by means the production of individual-specific tissues and providing the tolerance of autoimmune T cells.

The Industrial Applicability of the Invention

The product produced within the scope of the invention has a potential to significantly avoid costs for commercially available insulin since it is a live and insulin secreting tissue that can be produced specific to patient. In addition to this, since the other complications seen in chronic period in diabetes patients can be prevented, the expenses for the treatment of these diseases will be enabled to be reduced or even eliminated. Consequently, this product we have produced has great potential to prevent a significant economic loss spent for diabetes. The production of the product is a method suitable for biofabrication. Tissues specific to individuals can be produced by only changing the cells of the patient for each patient.

In case the aims of clinical use of the method presented in the invention are achieved as a treatment, this method will not only eliminate the economic burden required for the treatment of diabetes, but also prevent many secondary complications that occur in the chronic period before they develop. Therefore, it will provide high added value to our country. The target population of the invention is Tld patients, but even though the invention is a product intended for the treatment of T1D disease, it will also create a model for the treatment of many other autoimmune diseases.

The invention is the product of a quite complex approach which combines both beta cell replacement and immunotherapy, among the important approaches developed up to today, in current techniques of tissue engineering, and rearranges the existing tissue engineering techniques to be suitable for clinical use, and which consists of brad new and various steps. It is a multidisciplinary study that can be given as example fir studies in both the stem cell field, the immunotherapy field, the tissue engineering field, and the T1D field.

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Claims

1. A method developed for the production of endocrine pancreas-like tissue grafts suitable for lowering glucose sensitive blood sugar and transplantation as subcutaneous biochips after autoimmune attacks are prevented in patient subjects with T1D (Type 1 Diabetes), characterized in that it comprises the process steps of

- Obtaining immature/naive dendritic cells from bone marrow progenitors or blood mononuclear cells of subjects with T1D, in order to provide antigen specific tolerance in subjects with T1D,

- Loading the identified T1D autoantigens in vitro to the dendritic cells obtained from the subjects or conjugating to synthetic biodegradable nanoparticles, in order to achieve antigen specific tolerance in subjects with T1D,

- Low dose (in sublethal level) radiation-induced apoptosis of autoimmune system cells primarily existing in the T1D subjects in order to provide antigen specific tolerance,

- Then intravenous (IV) injection of dendritic cell and/or antigen-conjugated biodegradable nanoparticles to the subjects with T1D

- Obtaining decellularized pancreas extracellular matrix (dpESM) purificaiton of pancreas tissue from the cell by performing decellularization process while performing the aforementioned process steps,

- Then preparation of dpESM solution after lyophilization/powdering process to form of a printable solution in 3D bioprinters,

- In the meantime, using a cationic polymer (chitosan or other cationic polymers which are not toxic for the subjects) enabling the transfer of genes related to the b-cell differentiation with a non-viral method, and forming chitosan-pDNA nanoparticular b-cell gene transfer system with ionic gelation method,

- Obtaining Gene Activated Matrix (GAM; dpESM+chitosan pDNA nanoparticles) by integrating gene transfer system with decellularized pancreas matrix (dpESM) in order to differentiate mesenchymal stem cells into beta cells within the tissue to be printed in 3D bioprinter,

- Isolation of autologous mesenchymal stem cell (MSC) from bone marrow or other tissues and preperation of the cells ready as number and phenotype for endocrine differentiation and subsequently the production of endocrine pancreas-like tissue (biochip),

- Preparation of bioink via adding chitosan-pDNA nanoparticles and MSCs into printable dpESM form by providing proper conditions wherein its gelation is prevented.

- Printing biochip from the bioink with a 3D bioprinter,

- Obtaining insulin secreting beta-like cells from MSCs present in the 3D bioprinted tissue from bioink, after culturing of the tissue within endocrine differentiation medium,

- The transplantation of pancreas-like functional tissue grafts as subcutaneous biochips to the subjects with T1D, and thanks to biochip which is able to bring the blood sugar to a healthy level via glucose responsive insulin secretion in the subject with T1D

2. The method of claim 1, characterized in that the dosage regimens in immunotherapy method performed as intravenous injection (IV) 24 hours after radiation irradiation to the subjects with T1D can be adjusted in order to provide optimum targeted tolerance response in those subjects.

3. The method of claim 2, characterized in that the dosage regimens in immunotherapy method performed as intravenous injection 24 hours after radiation irradiation to the subjects with T1D are administered as a single dose injection.

4. The method of claim 2, characterized in that the dosage regimens in immunotherapy method performed as intravenous injection 24 hours after radiation irradiation to the subjects with T1D are administered as several divided dosages in time.

5. The method of claim 2, characterized in that the dosage regimens in immunotherapy method performed as intravenous injection 24 hours after radiation irradiation to the subjects with T1D can be decreased or increased in line with the requirements of the status of the disease.

6. The method of claim 2, characterized in that the technique of injection in immunotherapy method performed as intravenous injection 24 hours after radiation irradiation to the subjects with T1D, can be administered to different organ or tissue parts according to the requirements of the status of the disease (for example intradermal injection to periumbilical region of intraperitoneal or abdomen area).

7. The method of claim 2, characterized in that a couple of injection methods can be used in combination for injection administration in immunotherapy method carried out 24 hours after the radiation irradiation to the subjects with T1D stated in claim 6.

8. The method of claim 1, characterized in that T1D autoantigens (b-cell antigens) are the antigens obtained by lysate of prepared synthetic antigens defined as autoantigen in T1D with GAD65 and/or 3D insulin secreting tissue/ from apoptotic particles of b-cells.

9. The method of claim 8, characterized in that the antigen/antigens used as biomarker of b cell damage in subjects with T1D detected in the highest level with antibodies and defined in diabetes development can be used instead of GAD65 or used in addition to GAD65.

10. The method of claim 8 or 9, characterized in that the antigen selection is identified with application of an antibody test used for the diagnosis of T1 Diabetes to the subjects with T1D by a doctor or veterinarian qualifed with this technique.

11. The method according to claim 1, characterized in that in vivo antigen specific programming of Tregs comprises the steps of:

i) Identitification of autoimmune subjects;

ii) Apoptosis of immune system cells with low dose radiation (radiotherapy);

iii) Priming in vitro the dendritic cells obtained from the subjects with the determined T1D autoantigens to or conjugating to synthetic biodegradable nanoparticles with the determined T1D autoantigens in order to achieve antigen specific tolerance in subjects with T1D,

iv) Administration of autoantigen primed-dendritic cells and/or antigen- conjugated biodegradable nanoparticles to the subjects with T1D subsequent radiotherapy process.

12. The method of claim 1, and in vivo antigen specific programming of Tregs stated in claim 11 comprising a method of priming in vitro the dendritic cells obtained from the subjects with the determined T1D autoantigens to or conjugating to synthetic biodegradable nanoparticles with the determined T1D autoantigens, wherein the synthetic biodegradable nanoparticles used in that technique can be any molecule having a biocompatible biodegradable structure (as it is in polymeric or liposomal nanoparticles).

13. An endocrine pancreas-like tissue (biochip) constructed with 3D bioprinter obtained with a method of Claim 1, characterized in that it comprises pancreas extracellular matrix obtained with decellularized pancreas lyophilization, chitosan-pDNA nanoparticles-gene transfer system containing b cell differentiation genes, and autologous mesenchymal stem cells in its construct.

14. The Chitosan-pDNA nanoparticles b cell gene transfer systemexisting the structure of the biochip of claim 13, characterized in that it comprises Pdx-1 and MafA genes as b cell differentiation genes, or genes of other transcription factors playing a role in b cell development.

15. Autologous mesenchymal stem cells present in the construct of the biochip according to claim 13, characterized in that it can be obtained from bone marrow or other tissues.

16. The product obtained with a method of claim 13, characterized in that it decreases the blood glucose healthy levels by secreting insulin in response to blood glucose levels after transplanted subcutaneously.

17. The product constructed with 3D bioprinter obtained with a method of claim 1, characterized in that it comprises Gene Activated Matrix formed of decellularized pancreas integrated with chitosan-pDNA nanoparticular gene transfer system suitable for differentiation of mesenchymal stem cells into beta cells (GAM; dpESM+chitosan pDNA nanoparticles) in its construct.

18. The product of claim 17, characterized in that a nonviral system is used for gene transfer which is a method suitable for clinical use.

19. The product of claim 18, characterized in that the endocrine pancreas-like tissue (biochip) is therapeutically used for treatment of T1D patients.

20. The product obtained with a method of claim 10 and therapeutically used with a method of claim 16, characterized in that it provides personalized immunotherapy .

21. The product constructed with 3D bioprinter obtained with a method of claim 1, characterized in that after the matrix obtained with decellularization of the organ is powdered, it is designed via software for electronics which enables to produce desired micro patterns with 3D bioprinters, and in this way it is suitable for integrating the nonviral gene transfer system to the matrix.

22. The product constructed with 3D bioprinter obtained with a method of claim 1, wherein the decellularized pancreas matrix is a matrix that allows adding, if required, various polymer, growth factors, and medium stabilizers into the solution obtained by solubilizing the decellularized pancreatic matrix if needed.

23. The method of claim 1, characterized in that it is suitable for re-designing the matrix in the computer environment to form 3D final shape of biochips and it can be designed to the target region to be transferred while being transferred to the living organism.

24. The method according to claim 1, characterized in that in case the cell viability is low in the sample tissue printed from bioink used as a cellularized matrix solution, the solution obtained by mixing the developed gene transfer system with the matrix in solution form can be used as bioink and 3D bioprinting can be carried out with these two components in the bioprinter, and the MSCs are added into the tissue scaffold during culture after bioprinting.