DESC:A METHOD FOR ANALYSING MOLECULAR SEMAPHORE BEFORE AND AFTER ISLET TRANSPLANTATION

Field of the Invention
[0001] The present invention relates generally to the fields of diagnosis and molecular science, and more particularly to method for analyzing molecular biosignatures associated with islet transplantation. The molecular biosignatures are relevant to predicting, diagnosing and determining risk of islet transplant rejection.

Background
[0002] Islet cell transplantation is a treatment for type 1 diabetes. It includes transplantation of isolated islet cells from a donor pancreas, which are digested, purified and then injected into portal vein of liver of a recipient. However, islet transplantation has its own limitations, as most of the recipients lose insulin independence over time.
[0003] There are several factors which contribute to the limitations of islet transplantations. One of the factors is insufficient cell number or quality of islet cells which may impair initial cell function and can lead to beta cell exhaustion over time. Cellular and immune reactions to the transplanted cells, such as instant blood mediated immune reaction, recurrent autoimmunity or allograft rejection is another factor which can lead to destruction of cells. A third factor is usage of immunosuppressive drugs which can affect engraftment and beta-cell function or cause insulin resistance. To overcome the limitations of islet transplantation, processes that affect islet function, islet rejection needs to be studied.
[0004] Currently, suspected graft rejection episodes are confirmed only by invasive biopsy. The usage of serial biopsies for repeatedly assessing the graft integrity to adjust immunosuppressive drugs treatment and individualize treatment to a patient is often clinically impossible, impractical, cost-prohibitive, and a major burden for patients. Moreover, biopsies have limited sensitivity, specificity and turn-around times which restrict their usefulness for making rapid immunosuppressive dosing decisions.
[0005] Therefore, there is a need for a non-invasive procedure to improve functioning and increase longevity of transplanted islet cells. Moreover, there is a need for an economical, non-invasive method to monitor cell integrity, detect rejection of transplanted islet cells for providing customized treatment.

Objective of the Invention
[0006] The main objective of the present disclosure is to provide a method for defining functionality of islet cells before and after the transplantation to enable customised treatment.
[0007] Another objective of the present disclosure is to provide a non- invasive method for identifying rejected transplanted islet cells.
[0008] Still another objective of the present disclosure is to provide a method for diagnosis of cell functioning, cell rejection, death of rejected cells, and expression of insulin from the cells in islet transplantation.
[0009] Yet another objective of the present disclosure is to provide a method for early detection of rejection of transplanted islet cells.
[00010] The other objectives and advantages of the present disclosure will be apparent from the following description when read in conjunction with the accompanying drawings, which are incorporated for illustration of preferred embodiments of the present disclosure and are not intended to limit the scope thereof.

Summary of the Invention
[00011] In view of the foregoing, an embodiment herein provides a method for analyzing molecular semaphore before and after islet transplantation.
[00012] In accordance with an embodiment, the method includes developing a panel including pre transplant molecular parameters and post-transplant molecular parameters. Based on the panel, pre transplant molecular parameters are measured using an in-vitro measuring means. Next, based on the panel post transplant molecular parameters is measured using a non-invasive in-vivo measuring means. Then, detecting cell functioning, cell rejection based on the measured pre transplant molecular parameters and post transplant molecular parameters. Finally, providing customized treatments to a patient based on the detected cell functioning, cell rejection.
[00013] In accordance with an embodiment, the pre transplant molecular parameters include glut1 receptor, Ca2+ influx, and glucose kinase.
[00014] In accordance with an embodiment, the post transplant molecular parameters include Cell Free DNA, miRNA375, Insulin levels, CD30 levels, HMGB1.
[00015] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

Brief Description of Figures
[00016] The detailed description is set forth with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The use of the same reference numbers in different figures indicates similar or identical items.
[00017] Fig.1 illustrates a flowchart for a method of analyzing molecular semaphore before and after islet transplantation, according to an embodiment herein; and
[00018] Fig.2 illustrates a flowchart for a method of developing a panel, according to an embodiment herein.

Detailed Description of the Invention
[00019] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[00020] As mentioned above, there is a need to provide a non-invasive procedure to improve functioning and increase longevity of transplanted islet cells. In particular, there is a need to provide a method for defining functionality of islet cells before and after islet transplantation to enable customized treatment. The embodiments herein achieve this by providing a method for analyzing molecular semaphore before and after islet transplantation.
[00021] Referring now to the drawings, and more particularly to Fig.1 to Fig.2, where similar reference characters denote corresponding features consistently throughout the figures, there are shown preferred embodiments.
[00022] Islet transplantation includes islet cells harvested from a cadaver pancreas. Sterility tests and viability tests are performed on the islet cells. In the in-vitro condition of the islet cells and in-vivo condition of the islet cells, functionality tests are performed for assessing quality of the islet cells. For performing the functionality tests, bio signatures are identified and molecular parameters and their signaling pathways are assessed. The molecular parameters and signaling pathways are measured in in-vitro conditions before the process of islet transplantation and in in-vivo conditions after the process is islet transplantation.
[00023] Every molecular parameter has a specific functionality and defines a role for functioning of the islet cells after transplantation. Thereby, measuring the molecular parameters is important for identifying the functioning of islets cells after isolation from a cadaver pancreas and after transplantation of the islet cells to a recipient.
[00024] In an embodiment, a set of molecular parameters of the islet cells before transplantation include glut1 receptor, Ca2+ influx, and glucose kinase. These molecular parameters are tested in the in-vitro conditions, using in-vitro measuring means. They are dependent on signaling pathways involved in diabetes.
[00025] In an embodiment, after the transplantation of the islet cells into a recipient, another set of molecular parameters of the islet cells are tested regularly in in-vivo conditions. The another set of molecular parameters include Cell Free DNA, miRNA375, Insulin levels, CD30 levels, HMGB1. These molecular parameters are tested using an in-vivo measuring means for assessing the graft and cell functions.
[00026] In an embodiment, a panel is developed which includes the molecular parameters for testing in in-vivo conditions and the molecular parameters for testing in in-vitro conditions.
[00027] In an embodiment, a panel is developed based on the biosignatures of the islet cells identified in in-vitro conditions before the islet transplantations and in-vivo conditions after the islet transplantations. The identified biosignatures in the panel are used for predicting or detecting a presence of rejection of transplanted islet cells. The biosignatures of beta cell stress and beta cell (dys) function enable analyzing of pathogenesis before and after transplantation of the islet cells. The process enables timely prevention or treatment strategies for an individual based on the identified bio signatures.
[00028] Fig.1 illustrates a flow chart of the method for analyzing molecular semaphore before and after islet transplantation. The method includes developing 101 a panel including pre transplant molecular parameters and post-transplant molecular parameters.
[00029] Next, the method includes measuring 102 pre transplant molecular parameters using an in-vitro measuring means, wherein the molecular parameters include glut1 receptor, Ca2+ influx, and glucose kinase.
[00030] Then, measuring 103 post-transplant molecular parameters using a noninvasive in-vivo measuring means, wherein the molecular parameters include Cell Free DNA, miRNA375, Insulin levels, CD30 levels, HMGB1.
[00031] Then, based on the measured pre transplant molecular parameters and post-transplant molecular parameters, detecting 104 cell functioning, cell rejection, death of rejected cells, expression of insulin from the cells.
[00032] Finally, providing 105 customized treatments to a patient based on the detected cell functioning, cell rejection, death of rejected cells, expression of insulin from the cells.
[00033] Fig.2 illustrates a method of developing a panel of pre transplant molecular parameters and post-transplant molecular parameters. The method includes the steps of data mining 201 tissue specific pathway analysis using available assays.

[00034] Using the data, a plurality of bio markers is identified 202, in pre transplant islet cells and post-transplant islet cells. In an embodiment, more than twenty bio markers are identified.
[00035] From the identified plurality of bio markers, biosignatures having noninvasive procedures are determined 203. The biosignatures increase or decrease and give an early indication about acceptance or rejection or functionality of transplanted cells. Identification and validation of these bio-signatures predict presence of rejection and are important for analyzing pathogenesis before and after transplant.
[00036] Thereby, using the determined noninvasive biosignatures, a measuring means detects 204 a presence of rejection in the transplanted islet cells. The measuring means also detects cell functionality of the islet cells.
[00037] Based on the measured data using the measuring means, the islet cells are accordingly treated and developed 205, such as determining adjustments of immunosuppressive drugs.
[00038] Finally, a panel is developed 206 including these bio signatures of pre transplant molecular parameters and post-transplant molecular parameters. In an embodiment, the panel developed is used in routine clinical implementation on islet transplanted patients. The panel is used for providing customized treatments to the patients, based on detected cell functioning, cell rejection using the panel. As every patient may have different cell functioning, cell rejection, the medications, treatments such as determining adjustments of immunosuppressive drugs without pain, can be personalized based on individual requirements and enhance cell longevity of transplanted islet cells.
[00039] A main advantage of the present disclosure is that the method provides a faster diagnosis of cell functioning, cell rejection, death of rejected cells, expression of insulin from the cells etc., before start of clinical symptoms.
[00040] Another advantage of the present disclosure is that the method provides timely prevention or treatment strategies for islet transplantation depending on the individuals.
[00041] Still another advantage of the present disclosure is that the method provides monitoring of rejection episodes enabling easier immunosuppressant drug adjustments without pain.
[00042] Yet another advantage of the present disclosure is that the method is practical, cost-effective and capable of repetitive usage in islet transplantation recipients to extend cell longevity.
[00043] Another advantage of the present disclosure is that the method provides prediction of graft damaging complications at the earliest stages of islet transplantation.
[00044] Still another advantage of the present disclosure is that the method provides clinically useful assays having high sensitivity, specificity, and diagnostic activity that can assess islet functioning and early rejection properties.
[00045] Yet another advantage of the present disclosure is that the method provides biosignature panel development for early diagnosis, treatment response, and surrogate end point and outcome prediction in islet transplantation, leading to a tailored and individualized treatment
[00046] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
,CLAIMS:We claim:
1. A method of analyzing a molecular biosignature in an islet transplant before and after islet transplantation, comprising the steps of
identifying a biosignature by measuring marker level in the transplant, in-vitro, before the islet transplantation, wherein the marker is selected from the group consisting of a glut1 receptor, Ca2+ influx, and glucose kinase, and a combination thereof,
identifying a biosignature by measuring marker level in the transplant, in-vivo, after the islet transplantation, wherein the marker is selected from the group consisting of Cell free DNA, miRNA375, Insulin level, CD30 level, and HMGB1, and a combination thereof,
comparing the biosignature of step (a) and step (b) to determine the difference in the biosignature level.
2. The method as claimed in claim 1, wherein the islet transplant is isolated from cadaver pancreas.
3. The method as claimed in claim 1, wherein the difference in the biosignature level is used for predicting risk of islet transplant rejection.
4. The method as claimed in claim 1, wherein the different in the biosignature level is used for detecting presence of islet transplant rejection cell.
5. The method as claimed in claim 1, wherein the difference in the biosignature level is used to customized treatment in a patient who has received the islet transplantation.
6. A method of determining the risk of islet transplant rejection in a patient who has received an islet transplantation, comprising
identifying a biosignature by measuring marker level in the sample, in-vitro, before the islet transplantation, wherein the marker is selected from the group consisting of a glut1 receptor, Ca2+ influx, and glucose kinase, and a combination thereof,
identifying a biosignature by measuring marker level in the transplant, in-vivo, after the islet transplantation, wherein the marker is selected from the group consisting of Cell free DNA, miRNA375, Insulin level, CD30 level, and HMGB1, and a combination thereof,
comparing the biosignature identified in-vitro and in-vivo, wherein the difference in the biosignature level determine risk of islet transplant rejection.
7. A method of customizing treatment in a patient who has received an islet transplantation, comprising
identifying a biosignature by measuring marker level in the sample, in-vitro, before the islet transplantation, wherein the marker is selected from the group consisting of a glut1 receptor, Ca2+ influx, and glucose kinase, and a combination thereof,
identifying a biosignature by measuring marker level in the transplant, in-vivo, after the islet transplantation, wherein the marker is selected from the group consisting of Cell free DNA, miRNA375, Insulin level, CD30 level, and HMGB1, and a combination thereof,
comparing the biosignature identified in-vitro and in-vivo, wherein the difference in the biosignature level determine customizing treatment.
8. The method as claimed in claim 7, where customizing treatment include adjustment of an immunosuppressive drug.