1. At least one DNA cassette with ‘PDGF’ and ‘VEGF’ under any promoter or produced and transfected or transduced by any system (electroporation, viral transduction, chemical transfection) ensuring constitutive or temporary expression of the gene (s), wherein the cassette has the appropriate selection marker like Bleomycin/ Zeocin but not exclusively the mentioned selection markers. The gene(s) in the DNA cassette can be tagged with the expression of Green fluorescent protein or any small DNA tag (His/FLAG tag etc.) so as to confirm the functional expression of the construct visually by Fluorescent Microscopy or Mass Spectrometry, ELISA and or Western Blot for the gene or tagged gene. The invention and application for patent is about the said use of ‘genes’ in one DNA cassette and does not exclude use of any system or cells for the purpose of wound healing and regeneration.

2. A method of production of transgenic carrier cells where the carrier cells can be transgenic mesenchymal stem cells and or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette consisting of ‘PDGF’ and ‘VEGF’ genes along with luciferase gene to provide a biological assay to monitor the expression of said genes.

3. A method of production of transgenic carrier cells where the carrier cells can be transgenic mesenchymal stem cells and or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette consisting of ‘PDGF’ and ‘VEGF’ genes along with CXCR4 gene to intensify the homing mechanism so that the transgenic cells will migrate to the desired site of disease.

4. A method of treating the patients of ischemic diseases including cerebral ischemia, cardiac ischemia, chronic limb ischemia, and other diseases requiring restoring the circulation for example, wound healing, traumatic injury, diabetic wounds, peripheral arterial disease, myocardial infarction and venous thrombosis by infusing the mesenchymal stem cells and somatic cells transfected with the DNA cassette expressing ‘PDGF’ and ‘VEGF’ genes.

5. A method of treating the patients post-surgery for rapid recovery in wound healing and or in healing of the ischemic conditions by infusing the mesenchymal stem cells and somatic cells transfected with the DNA cassette expressing ‘PDGF’ and ‘VEGF’ genes.

6. A method of treating the patients of chronic wound healing and diabetes related conditions by infusing the mesenchymal stem cells and somatic cells transfected with the DNA cassette expressing ‘VEGF’ and ‘PDGF' genes.

7. A method to treat the patients by beneficial/ therapeutic application of genetically modified cells using the plasmid with ‘VEGF’ and ‘PDGF' genes for disorders of burns, non-healing ulcers, and or infections.

8. A method to treat various diseases as well as conditions related to healing and surgery, with or without manifestations in nervous system, including cancer, eye disorders, organ transplant, and expression of apoptotic gene(s) in any other carrier cells where the ‘VEGF’ and ‘PDGF' genes are introduced via viral or non-viral methods of transgenesis.

9. At least one DNA cassette for the treatment of diseases in humans as well as animals i.e. human clinical use as well as veterinary clinical use by virtue of introduction of genetically modified mesenchymal stem cells expressing the ‘VEGF’ and ‘PDGF' genes individually and or in combination.

10. A method of using the temporary, non-genome-integrative gene expression in the mesenchymal stem cells by virtue of adenoviral vectors, or adeno-associated viral vectors or lipofection or electroporation whereas the transgenic cells express the factors only for a limited amount of time and thus provide a natural self-limiting mechanisms to exposure of regenerative factors.

11. A method of using the transgenic mesenchymal stem cells locally in a matrix or spray or fibrin or collagen based gel to cause angiogenesis by virtue of expression of ‘VEGF’ and ‘PDGF' with or without the expression of luciferase gene and CXCR4 gene.

12. A method of using the transgenic mesenchymal stem cells locally in a matrix or spray or fibrin or collagen based gel to cause angiogenesis by virtue of expression of ‘VEGF’ and ‘PDGF' with or without the expression of GFP gene and CXCR4 gene.

13. A method of use of above mentioned transgenic cells in treatment of any ischemic disease in humans or animals.

14. A method of use of above mentioned transgenic cells in treatment of healing of scars and wound healing so as to increase the circulation and healing process.

15. A method of use of above mentioned transgenic cells in treatment of healing of surgical scars. , Description:THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED FIELD OF INVENTION: The present invention relates an engineered DNA cassette using angiogenic and other regenerative factor genes for treatment of chronic wound healing and poor circulation. More specifically, it relates to gene-based strategy used for treatment of chronic wound healing and poor circulation regions/organs using transgenic mesenchymal stem cells or other somatic cells using engineered DNA cassette comprising of specifically angiogenic and other regenerative factor genes alone or in combination. More particularly, the DNA cassette provides 100% patient welfare, safety and assured efficacy of the therapy for the diseases under consideration. BACKGROUND OF THE INVENTION: The ischemic disorders like chronic limb ischemia, myocardial ischemia or cerebral ischemia and others are many times deadly for their consequences when left untreated or remain uncontrolled. With dire consequences like necrosis of affected tissues and limb infections and amputations or long term disability caused due to myocardial and cerebral infarctions, the ischemic disorders need a therapy that will not only alleviate the symptoms but also cure the disease pathology by creating more blood vessels in the affected tissues. Similarly, in diabetic or Berger’s disease patients, the ischemia leads to chronic wounds. Such wounds are hard to heal due to lack of sufficient blood supply or poor circulation. The process of ischemia or wound formation is associated with injury at the locate site associated with inflammatory process which is eventually followed by episodes of intense cellular proliferation, extracellular matrix (ECM) deposition and remodeling, and ultimately leading to the scar formation. During this process, variety of growth factors, cytokines, and lipid mediators produced in response to injury can stimulate angiogenesis. It has been known that vascular endothelial growth factor (VEGF or VEGF-A), and sufficient VEGF levels are believed to be essential for proper wound healing. Similarly, PDGF-B functions as an important factor for cell proliferation, survival and chemotaxis. In addition, it plays a role in platelet activation, secretion of agonists from platelet granules, and in thrombin-induced platelet aggregation. However, during the cardiac, brain or organ specific ischemia, such compromised tissue is unable to cope up with the repair process in the natural way in an effective manner. Similarly, during diabetic condition, such repair capability is also compromised. In order to boost up the repair process, it is mandatory to under support the compromised tissues with regenerative/angiogenic factors under such circumstances. In this regards, the carrier cells such as MSCs or any somatic cells harboring the angiogenic and regenerative factors perform essential function to support the angiogenesis and other necessary steps towards wound healing. Especially, the transgenic MSCs specifically armored to express angiogenic and or other regenerative factors influences wound closure and epidermal repair, granulation tissue formation, and the quality of repair in a beneficial manner. Our invention, the concoction of mesenchymal stem cells or other carrier cells developed from the patient’s own bone marrow or mesenchymal stem cells from various sources like umbilical cord tissue or lymphocytes or fibroblasts expressing the therapeutic genes, enhances healing of tissues by increasing or creating blood vessels by virtue of the co-expression of ‘VEGF’ and ‘PDGF' and other angiogenic and regenerative factors to guarantee the maximum therapeutic effects by virtue of the DNA cassette used for the creation of unique transgenic cells. OBJECTIVES OF THE INVENTION: The primary objective of the present invention is to provide a DNA cassette for producing the transgenic mesenchymal stem cells or somatic cells for treatment of ischemia or directly modifying the body tissues by means of incorporation of transgenically expressed proteins and factors. Another objective of the present invention is to transfect mesenchymal stem cells or carrier somatic cells with the said DNA cassette that contains the VEGF and PDGF genes along with reporter proteins like luciferase or GFP, and one or more suicide genes. Yet another objective of the present invention is to provide a therapeutic agent alone or in combination with other medications to improve the health of patients with chronic non-healing wounds. Yet another objective of the present invention is to ameliorate one or more problems of the prior art and provide a useful alternative. SUMMARY OF THE INVENTION: The embodiments of the present invention described herein below are illustrative only and should not be construed to limit the scope of the present invention in any manner. Unless stated to the contrary, any use of the words such as "including”, "containing”, "comprising", "having" and the like, means "including without limitation" and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive, but can be implemented in various combinations. “Carrier Cells” means transfected cells. The DNA Cassette comprises of individual genes or a combination of DNA sequences for VEGF, PDGF, HGF and other protein factors and luciferase, cytosine deaminase and herpes simplex thymidine kinase along with any other therapeutic gene or peptide. The various genes are expressed at once with the help of IRES sequence separating them or Viral 2A peptide sequences separating the sequences. The map of sequences are shown below, separated by IRES sequence/ Viral 2A sequence. The DNA cassette can be incorporated into the carrier cells of choice by use of an established plasmid. The DNA cassette can be inserted into a bacterial plasmid or viral plasmid during the process of propagation and transgenesis. Bacterial culture can be used for multiplication of Plasmid number. For therapeutic use, the DNA Cassette will be incorporated into mammalian and preferably human carrier mesenchymal stem cells. The selection of cells incorporated with the construct is done by any of the established methods. The gene(s) in the DNA cassette; can be tagged with the expression of Green Fluorescent Protein or any small DNA tag (His/FLAG tag etc.) so as to confirm the functional expression of the construct visually by Fluorescent Microscopy or Mass Spectrometry, ELISA and or Western Blot for the gene or tagged gene. Inclusion of luciferase gene allows a biological assay for monitoring the expression activity of the cells. A linear arrangement of a set of genes from the above said group of genes is shown in figure 1. The transgenesis is conducted with electroporation or lipofection or by use of adenoviral system or adeno-associated virus for a temporary expression of genes and by use of lentiviral systems are used for a permanent expression of therapeutic genes. BRIEF DESCRIPTION OF THE DRAWINGS: A complete understanding of the present invention may be made by reference to the following detailed description which taken in conjugation with the accompanying drawing. The accompanying drawing, which is incorporated into and constitutes a part of the specification, illustrates one or more embodiments of the present invention and, together with the detailed description, it serves to explain the principles and implementations of the invention. Figure 1a: illustrates a linear arrangement of the DNA cassette; Figure 2: illustrates another linear arrangement of the DNA cassette; Figure 3: illustrates a circular arrangement of the DNA cassette; Figure 4: illustrates the expression analysis of construct IschemoHomeBrite in 293-T cells; Figure 5: illustrates the luciferase activity in each flash assay reagent as supernatant and cell lysate in IschemoHomeBrite transfected and un-transfected MSCs; Figure 6 (A) and (B): illustrates the beneficial effect of IschemoHomeBrite against in vitro model for would healing; Figure 7: illustrates the percentage expression of PDGF-B and VEGF proteins in different number of transfected cells as assessed via ELISA assay; Figure 8: illustrates the RT-PCR showing the expression of PDGF-B and VEGF in untreated/un-transfected and transfected (with IschemoHomeBrite) MSCs. Figure 9 (A) and (B): illustrates the increase in the total number of cells in the well supported by IschemoHomeBrite transfected MSCs. (B): illustrates the significant upregulation in the wound healing markers, TIMP1 and CDH1 markers in the wells supported by IschemoHomeBrite transfected MSCs after inducing scratch model in bottom wells of the transwells. DETAILED DESCRIPTION OF THE INVENTION: Before the present invention is described, it is to be understood that this invention is not limited to particular methodologies described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. The present invention describes a DNA cassette for the constitutive or inducible expression of the therapeutic genes within the selected carrier cells. The said DNA cassette comprises of therapeutic VEGF, PDGF, and HGF sequence along with herpes simplex viral thymidine kinase (HSV-tk) and cytosine deaminase (CD) as suicidal switch genes along with Guassia luciferase or any other kind of luciferase gene. Secreted angiogenic proteins are therapeutic to the ischemic tissues and wound beds. The novelty of the present invention lies in the therapeutic mechanism offered by ingenuously allowing the carrier cells to express the secreted angiogenic factors VEGF and PDGF. The carrier cells themselves have a synergistic effect to the proteins delivered at the site of injury. The carrier cells are used to deliver ‘VEGF’ and ‘PDGF' either individually or as they are co-expressed in the carrier cells. The strategy described in the invention has direct applicability for the treatment of diseases of ischemic etiology and wound healing. Our novel invention makes therapy feasible and makes the proteins available in steady physiological rate and manner. The therapy is clinically safe as safety of mesenchymal stem cells is well established and the expression of transgenes does not involve permanent genetic integration. In one embodiment of the invention, an adenoviral or adeno-associated or other viral vector is used for transfection of the protein factors into the carrier cells which does not cause change in the genome of the cells, there is no chance of carcinogenic mutations. The growth factors and angiogenic cytokines will be available for extended but certain period of time at the site of injury providing optimal clinical benefit. To achieve the therapeutic effect, populations of mesenchymal stem cells expressing individual genes mentioned above can also be infused to the patient. Amount of each type of mesenchymal stem cells provides an excellent tool to manipulate the relative ratio of various factors to each other. In another embodiment of the invention, the DNA cassette can be used for gene therapy involving direct delivery of genes to natural tissues. By this delivery, the wound bed can be directly modified to express the angiogenic factors. This will increase the speed of wound healing by many folds. In another embodiment of the invention, the angiogenic genes (VEGF, PDGF and any other angiogenic genes), luciferase gene and CXCR4 homing gene are expressed together into the carrier cells. Expression of CXCR4 gene intensifies the action of mesenchymal stem cells to find the place of injury when administered by intravenous or other remote route. Expression of luciferase provides the capability to monitor the cells by non-invasive or minimally invasive means by use of luciferase assay. In another embodiment of the invention, the DNA cassette additionally incorporates ‘FGF-1’, ‘HGF’, ‘EGF’, ‘FGF-7’, ‘IGF-1’, ‘CXCR4’, ‘luciferase’, ‘IL-10’, ‘IL-4’ genes along with VEGF and PDGF such that they are flanking IRES or 2A sequence. The genes are co-expressed under a promoter that is suitable for the carrier. Once the carrier cells like mesenchymal stem cells are appropriately transfected or transduced with the invented DNA cassette, the cells will be intravenously administered to patient. In yet another embodiment of the invention, the angiogenic and regenerative genes are expressed along with suicidal genes such that the carrier cells can be eliminated at any point of time by the treating team. The transgenic cells should be administered after diagnosis of ischemia and be administered repetitively via systemic and local routes. The cells can be administered by catheter or injection. The promoters used for the expression can be specifically chosen for tissue specific expression such that the genes are expressed only in certain tissues. Our invention manages to get the continuous and safe therapeutic effect while adding the therapeutic genes to off-the-shelf available mesenchymal stem cells or other somatic cells. Mesenchymal stem cells can be produced on industrial scale by using tissue from umbilical cord tissue. Mesenchymal stem cells have hypo-immunogenic properties as well as ability to migrate towards the injured region and thus can provide beneficial effect in cases of surgeries involving nervous system. ILLUSTRATIVE WORKING MECHANISM

1. Construction of the transgenic MSCs

1. The construct/DNA cassette under consideration consists of CMV promoter, which drives the expression of the DNA cassette comprising of PDGF-B, VEGF along with Luciferase and CXCR4B genes and could be used for both bacterial and mammalian cells.

2. The transgenic MSCs with “PDGF-B and VEGF” are created either by viral or non-viral transfection method.

3. The transgenic MSCs are generated using chemical method and a stable line is achieved using the selectivity marker, ‘zeocin’ in our case which is present in the integrin or plasmid used to inert the DNA cassette. However, any appropriate plasmid could be used to carry the DNA cassette and thus could be transfected to MSCs or somatic cells using transfection protocol. Thus, the chief advantage of the DNA cassette with the insert comprising of PDGF-B and VEGF with vacant multiple cloning site is used for industrial scale use of such produced cells to create various combinations of therapeutic genes with luciferase to monitor the expression of construct. The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not as restrictive. The scope of the invention is, therefore, indicated by the appended claims and their combination in whole or in part rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope. EXAMPLES: The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only, and the invention is not limited to these examples, but rather encompasses all variations, which are evident as a result of the teaching provided herein. The figures and specific sequences provided in the description are to illustrate the present invention in which a single DNA cassette with ‘Platelet derived growth factor-B’ with Vascular endothelial growth factor gene is introduced at the multiple cloning site. First the expression level of both PDGF-B and VEGF have been verified as shown in Example 1 while example 2 verifies the beneficial effects of MSCs transfected with DNA cassette in in vitro co-culture environment of transfected MSCs with wound injury model.

1. Expression of IschemoHomeBrite construct. The gene synthesis of PDGF-B:VEGF:Luciferase:CXCR4B construct was carried out to perform molecular and functional study for PDGF-B and VEGF and to evaluate the beneficial role of IschemoHomeBrite construct and their utility in wound healing processes (Figure 1, and 3). a. Detection of genes via WB technique The gene synthesis of PDGF-B:VEGF:Luciferase:CXCR4B gene construct was then used to perform expression studies for IschemoHomeBrite contruct. As illustrated in Figure 4 of the accompanying drawings the expression analysis of construct IschemoHomeBrite in 293-T cells indicates that PDGF-B, VEGF and Luciferase are expressed at once in supernatant and cell lysates, respectively using PDGF-B, VEGF and Luciferase antibodies. b. Detection of genes via RT-PCR technique In order to assess the expression of Guassia luciferase, Luciferase Reporter Assay was performed using protocol known in the prior art. The inclusion of luciferase reporter gene allows monitoring of activity of construct in the body via simple urine based assay in animals/human population as illustrated in Figure 5 of the accompanying drawings. At 48 hours, post-transfection, 20µL of sample was used to measure the luciferase activity in each flash assay reagent as supernatant and cell lysate in IschemoHomeBrite transfected and un-transfected MSCs. * shows the significant difference for the luciferase activity in the cell lysates between the transfected and un-transfected cells where **=P<0.001. # shows the significant difference for the luciferase activity in the supernatant between the transfected and un-transfected cells where ##=P<0.001. Example 2: Transfection of IschemoHomeBrite to umbilical cord derived mesenchymal stem cells Mesenchymal stem cells were produced using umbilical cord tissue using the methods known in the art and have been published. The MSCs were transfected to IschemoHomeBrite via Lipofectamine Plus using protocol known in the prior art. The cells were tested for the expression of PDGF-B and VEGF. Example 3: Quantification of the level of IschemoHomeBrite expression: PDGF-B and VEGF ELISA kits and level of expression of PDGF-B and VEGF were quantified in the IschemoHomeBrite transfected MSCs using a conventional method. Different number of transfected cells was used to quantify the expression of PDGF-B and VEGF separately. The results suggest that transfection of IschemoHomeBrite indeed can be quantified based on the number of transfected MSCs against un-transfected MSCs. The results as shown in Figure 6 (A) of the accompanying drawings suggest that transfection of PDGF-B secreted in the supernatant indeed can be quantified based on the number of transfected MSCs against un-transfected MSCs. The percentage expression of PDGF-B protein in different number of transfected cells as assessed via ELISA assay. The significant difference was found between the protein expression at 1 million and 5 or 10 million cells. There was statistical difference in the cell viability among these three groups, where */** =P<0.05/P=0.005 denote the statistically significant difference for expression of PDGF-B in 1 million transfected cells vs 5 million and 10 million cells. Similarly, the results as shown in Figure 6 (B) of the accompanying drawings suggest that transfection of VEGF secreted in the supernatant indeed can be quantified based on the number of transfected MSCs against un-transfected MSCs. The percentage expression of VEGF protein in different number of transfected cells as assessed via ELISA assay. The significant difference was found between the protein expression at 1 million and 5 or 10 million cells. There was statistical difference in the cell viability among these three groups, where */** =P<0.05/P=0.005 denote the statistically significant difference for expression of VEGF in 1 million transfected cells vs 5 million and 10 million cells. Example 4: In vitro studies to demonstrate the efficacy of IschemoHomeBrite We generated an in vitro model for wound healing by scrapping the uniform monolayer of the MSCs. The interrupted layer developed upon scratch assay is commonly studied for wound healing models Figure 8 (A). We used transwells for performing co-culture of untransfected and IschemoHomeBrite transfected cells with the monolayer wit scratch assay. We first performed the assessment of the total number of cells in the transfected and untranfected co-cultured MSCs. In addition, wound healing markers, TIMP1 (Extracellular Matrix (ECM) remodeling enzymes) and CDH1 (E-Cadherin) (cell adhesion molecule) were used to assess the beneficial role of IschemoHomeBrite in in vitro wound healing model (Figure 9 A and B) using ELISA assay. As illustrated in Figure 9 (A), there was a significant increase in the total number of cells in the well supported with IschemoHomeBrite transfected MSCs cells as compared to those supported by untransfected MSCs. There was statistical difference in the cell numbers between these two groups, where ** =P<0.005 denote the statistically significant difference between the cells supported by untransfected and transfected cells. In addition, the ELISA results indicate upregulation of TIMP1 and CDH1 in the wells supported by IschemoHomeBrite transfected cells after induction of wound via scratch assay. As illustrated in Figure 9 (B), there was a significant increase in the TIMP1 and CDH1 in the well supported with IschemoHomeBrite transfected MSCs cells as compared to those supported by untransfected MSCs. There was statistical difference in the levels of TIMP1 and CDH1, where ** =P<0.005 denote the statistically significant difference between the cells supported by untransfected and transfected cells. While considerable emphasis has been placed herein on the specific elements of the preferred embodiment, it will be appreciated that many alterations can be made and that many modifications can be made in preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiments of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

Claims:1. At least one DNA cassette with ‘PDGF’ and ‘VEGF’ under any promoter or produced and transfected or transduced by any system (electroporation, viral transduction, chemical transfection) ensuring constitutive or temporary expression of the gene (s), wherein the cassette has the appropriate selection marker like Bleomycin/ Zeocin but not exclusively the mentioned selection markers. The gene(s) in the DNA cassette can be tagged with the expression of Green fluorescent protein or any small DNA tag (His/FLAG tag etc.) so as to confirm the functional expression of the construct visually by Fluorescent Microscopy or Mass Spectrometry, ELISA and or Western Blot for the gene or tagged gene. The invention and application for patent is about the said use of ‘genes’ in one DNA cassette and does not exclude use of any system or cells for the purpose of wound healing and regeneration.

2. A method of production of transgenic carrier cells where the carrier cells can be transgenic mesenchymal stem cells and or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette consisting of ‘PDGF’ and ‘VEGF’ genes along with luciferase gene to provide a biological assay to monitor the expression of said genes.

3. A method of production of transgenic carrier cells where the carrier cells can be transgenic mesenchymal stem cells and or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette consisting of ‘PDGF’ and ‘VEGF’ genes along with CXCR4 gene to intensify the homing mechanism so that the transgenic cells will migrate to the desired site of disease.

4. A method of treating the patients of ischemic diseases including cerebral ischemia, cardiac ischemia, chronic limb ischemia, and other diseases requiring restoring the circulation for example, wound healing, traumatic injury, diabetic wounds, peripheral arterial disease, myocardial infarction and venous thrombosis by infusing the mesenchymal stem cells and somatic cells transfected with the DNA cassette expressing ‘PDGF’ and ‘VEGF’ genes.

5. A method of treating the patients post-surgery for rapid recovery in wound healing and or in healing of the ischemic conditions by infusing the mesenchymal stem cells and somatic cells transfected with the DNA cassette expressing ‘PDGF’ and ‘VEGF’ genes.

6. A method of treating the patients of chronic wound healing and diabetes related conditions by infusing the mesenchymal stem cells and somatic cells transfected with the DNA cassette expressing ‘VEGF’ and ‘PDGF' genes.

7. A method to treat the patients by beneficial/ therapeutic application of genetically modified cells using the plasmid with ‘VEGF’ and ‘PDGF' genes for disorders of burns, non-healing ulcers, and or infections.

8. A method to treat various diseases as well as conditions related to healing and surgery, with or without manifestations in nervous system, including cancer, eye disorders, organ transplant, and expression of apoptotic gene(s) in any other carrier cells where the ‘VEGF’ and ‘PDGF' genes are introduced via viral or non-viral methods of transgenesis.

9. At least one DNA cassette for the treatment of diseases in humans as well as animals i.e. human clinical use as well as veterinary clinical use by virtue of introduction of genetically modified mesenchymal stem cells expressing the ‘VEGF’ and ‘PDGF' genes individually and or in combination.

10. A method of using the temporary, non-genome-integrative gene expression in the mesenchymal stem cells by virtue of adenoviral vectors, or adeno-associated viral vectors or lipofection or electroporation whereas the transgenic cells express the factors only for a limited amount of time and thus provide a natural self-limiting mechanisms to exposure of regenerative factors.

11. A method of using the transgenic mesenchymal stem cells locally in a matrix or spray or fibrin or collagen based gel to cause angiogenesis by virtue of expression of ‘VEGF’ and ‘PDGF' with or without the expression of luciferase gene and CXCR4 gene.

12. A method of using the transgenic mesenchymal stem cells locally in a matrix or spray or fibrin or collagen based gel to cause angiogenesis by virtue of expression of ‘VEGF’ and ‘PDGF' with or without the expression of GFP gene and CXCR4 gene.

13. A method of use of above mentioned transgenic cells in treatment of any ischemic disease in humans or animals.

14. A method of use of above mentioned transgenic cells in treatment of healing of scars and wound healing so as to increase the circulation and healing process.

15. A method of use of above mentioned transgenic cells in treatment of healing of surgical scars.
, Description:THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
FIELD OF INVENTION:
The present invention relates an engineered DNA cassette using angiogenic and other regenerative factor genes for treatment of chronic wound healing and poor circulation. More specifically, it relates to gene-based strategy used for treatment of chronic wound healing and poor circulation regions/organs using transgenic mesenchymal stem cells or other somatic cells using engineered DNA cassette comprising of specifically angiogenic and other regenerative factor genes alone or in combination. More particularly, the DNA cassette provides 100% patient welfare, safety and assured efficacy of the therapy for the diseases under consideration.
BACKGROUND OF THE INVENTION:
The ischemic disorders like chronic limb ischemia, myocardial ischemia or cerebral ischemia and others are many times deadly for their consequences when left untreated or remain uncontrolled. With dire consequences like necrosis of affected tissues and limb infections and amputations or long term disability caused due to myocardial and cerebral infarctions, the ischemic disorders need a therapy that will not only alleviate the symptoms but also cure the disease pathology by creating more blood vessels in the affected tissues. Similarly, in diabetic or Berger’s disease patients, the ischemia leads to chronic wounds. Such wounds are hard to heal due to lack of sufficient blood supply or poor circulation. The process of ischemia or wound formation is associated with injury at the locate site associated with inflammatory process which is eventually followed by episodes of intense cellular proliferation, extracellular matrix (ECM) deposition and remodeling, and ultimately leading to the scar formation. During this process, variety of growth factors, cytokines, and lipid mediators produced in response to injury can stimulate angiogenesis. It has been known that vascular endothelial growth factor (VEGF or VEGF-A), and sufficient VEGF levels are believed to be essential for proper wound healing. Similarly, PDGF-B functions as an important factor for cell proliferation, survival and chemotaxis. In addition, it plays a role in platelet activation, secretion of agonists from platelet granules, and in thrombin-induced platelet aggregation. However, during the cardiac, brain or organ specific ischemia, such compromised tissue is unable to cope up with the repair process in the natural way in an effective manner. Similarly, during diabetic condition, such repair capability is also compromised. In order to boost up the repair process, it is mandatory to under support the compromised tissues with regenerative/angiogenic factors under such circumstances. In this regards, the carrier cells such as MSCs or any somatic cells harboring the angiogenic and regenerative factors perform essential function to support the angiogenesis and other necessary steps towards wound healing. Especially, the transgenic MSCs specifically armored to express angiogenic and or other regenerative factors influences wound closure and epidermal repair, granulation tissue formation, and the quality of repair in a beneficial manner. Our invention, the concoction of mesenchymal stem cells or other carrier cells developed from the patient’s own bone marrow or mesenchymal stem cells from various sources like umbilical cord tissue or lymphocytes or fibroblasts expressing the therapeutic genes, enhances healing of tissues by increasing or creating blood vessels by virtue of the co-expression of ‘VEGF’ and ‘PDGF' and other angiogenic and regenerative factors to guarantee the maximum therapeutic effects by virtue of the DNA cassette used for the creation of unique transgenic cells.
OBJECTIVES OF THE INVENTION:
The primary objective of the present invention is to provide a DNA cassette for producing the transgenic mesenchymal stem cells or somatic cells for treatment of ischemia or directly modifying the body tissues by means of incorporation of transgenically expressed proteins and factors.
Another objective of the present invention is to transfect mesenchymal stem cells or carrier somatic cells with the said DNA cassette that contains the VEGF and PDGF genes along with reporter proteins like luciferase or GFP, and one or more suicide genes.
Yet another objective of the present invention is to provide a therapeutic agent alone or in combination with other medications to improve the health of patients with chronic non-healing wounds.
Yet another objective of the present invention is to ameliorate one or more problems of the prior art and provide a useful alternative.
SUMMARY OF THE INVENTION:
The embodiments of the present invention described herein below are illustrative only and should not be construed to limit the scope of the present invention in any manner. Unless stated to the contrary, any use of the words such as "including”, "containing”, "comprising", "having" and the like, means "including without limitation" and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive, but can be implemented in various combinations.
“Carrier Cells” means transfected cells. The DNA Cassette comprises of individual genes or a combination of DNA sequences for VEGF, PDGF, HGF and other protein factors and luciferase, cytosine deaminase and herpes simplex thymidine kinase along with any other therapeutic gene or peptide. The various genes are expressed at once with the help of IRES sequence separating them or Viral 2A peptide sequences separating the sequences. The map of sequences are shown below, separated by IRES sequence/ Viral 2A sequence. The DNA cassette can be incorporated into the carrier cells of choice by use of an established plasmid. The DNA cassette can be inserted into a bacterial plasmid or viral plasmid during the process of propagation and transgenesis.
Bacterial culture can be used for multiplication of Plasmid number. For therapeutic use, the DNA Cassette will be incorporated into mammalian and preferably human carrier mesenchymal stem cells. The selection of cells incorporated with the construct is done by any of the established methods. The gene(s) in the DNA cassette; can be tagged with the expression of Green Fluorescent Protein or any small DNA tag (His/FLAG tag etc.) so as to confirm the functional expression of the construct visually by Fluorescent Microscopy or Mass Spectrometry, ELISA and or Western Blot for the gene or tagged gene. Inclusion of luciferase gene allows a biological assay for monitoring the expression activity of the cells. A linear arrangement of a set of genes from the above said group of genes is shown in figure 1. The transgenesis is conducted with electroporation or lipofection or by use of adenoviral system or adeno-associated virus for a temporary expression of genes and by use of lentiviral systems are used for a permanent expression of therapeutic genes.
BRIEF DESCRIPTION OF THE DRAWINGS:
A complete understanding of the present invention may be made by reference to the following detailed description which taken in conjugation with the accompanying drawing. The accompanying drawing, which is incorporated into and constitutes a part of the specification, illustrates one or more embodiments of the present invention and, together with the detailed description, it serves to explain the principles and implementations of the invention.
Figure 1a: illustrates a linear arrangement of the DNA cassette;
Figure 2: illustrates another linear arrangement of the DNA cassette;
Figure 3: illustrates a circular arrangement of the DNA cassette;
Figure 4: illustrates the expression analysis of construct IschemoHomeBrite in 293-T cells;
Figure 5: illustrates the luciferase activity in each flash assay reagent as supernatant and cell lysate in IschemoHomeBrite transfected and un-transfected MSCs;

Figure 6 (A) and (B): illustrates the beneficial effect of IschemoHomeBrite against in vitro model for would healing;
Figure 7: illustrates the percentage expression of PDGF-B and VEGF proteins in different number of transfected cells as assessed via ELISA assay;
Figure 8: illustrates the RT-PCR showing the expression of PDGF-B and VEGF in untreated/un-transfected and transfected (with IschemoHomeBrite) MSCs.
Figure 9 (A) and (B): illustrates the increase in the total number of cells in the well supported by IschemoHomeBrite transfected MSCs. (B): illustrates the significant upregulation in the wound healing markers, TIMP1 and CDH1 markers in the wells supported by IschemoHomeBrite transfected MSCs after inducing scratch model in bottom wells of the transwells.

DETAILED DESCRIPTION OF THE INVENTION:
Before the present invention is described, it is to be understood that this invention is not limited to particular methodologies described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results.
The present invention describes a DNA cassette for the constitutive or inducible expression of the therapeutic genes within the selected carrier cells. The said DNA cassette comprises of therapeutic VEGF, PDGF, and HGF sequence along with herpes simplex viral thymidine kinase (HSV-tk) and cytosine deaminase (CD) as suicidal switch genes along with Guassia luciferase or any other kind of luciferase gene. Secreted angiogenic proteins are therapeutic to the ischemic tissues and wound beds.
The novelty of the present invention lies in the therapeutic mechanism offered by ingenuously allowing the carrier cells to express the secreted angiogenic factors VEGF and PDGF. The carrier cells themselves have a synergistic effect to the proteins delivered at the site of injury. The carrier cells are used to deliver ‘VEGF’ and ‘PDGF' either individually or as they are co-expressed in the carrier cells. The strategy described in the invention has direct applicability for the treatment of diseases of ischemic etiology and wound healing.
Our novel invention makes therapy feasible and makes the proteins available in steady physiological rate and manner. The therapy is clinically safe as safety of mesenchymal stem cells is well established and the expression of transgenes does not involve permanent genetic integration.
In one embodiment of the invention, an adenoviral or adeno-associated or other viral vector is used for transfection of the protein factors into the carrier cells which does not cause change in the genome of the cells, there is no chance of carcinogenic mutations. The growth factors and angiogenic cytokines will be available for extended but certain period of time at the site of injury providing optimal clinical benefit. To achieve the therapeutic effect, populations of mesenchymal stem cells expressing individual genes mentioned above can also be infused to the patient. Amount of each type of mesenchymal stem cells provides an excellent tool to manipulate the relative ratio of various factors to each other.
In another embodiment of the invention, the DNA cassette can be used for gene therapy involving direct delivery of genes to natural tissues. By this delivery, the wound bed can be directly modified to express the angiogenic factors. This will increase the speed of wound healing by many folds.
In another embodiment of the invention, the angiogenic genes (VEGF, PDGF and any other angiogenic genes), luciferase gene and CXCR4 homing gene are expressed together into the carrier cells. Expression of CXCR4 gene intensifies the action of mesenchymal stem cells to find the place of injury when administered by intravenous or other remote route. Expression of luciferase provides the capability to monitor the cells by non-invasive or minimally invasive means by use of luciferase assay.
In another embodiment of the invention, the DNA cassette additionally incorporates ‘FGF-1’, ‘HGF’, ‘EGF’, ‘FGF-7’, ‘IGF-1’, ‘CXCR4’, ‘luciferase’, ‘IL-10’, ‘IL-4’ genes along with VEGF and PDGF such that they are flanking IRES or 2A sequence. The genes are co-expressed under a promoter that is suitable for the carrier. Once the carrier cells like mesenchymal stem cells are appropriately transfected or transduced with the invented DNA cassette, the cells will be intravenously administered to patient.
In yet another embodiment of the invention, the angiogenic and regenerative genes are expressed along with suicidal genes such that the carrier cells can be eliminated at any point of time by the treating team.
The transgenic cells should be administered after diagnosis of ischemia and be administered repetitively via systemic and local routes. The cells can be administered by catheter or injection. The promoters used for the expression can be specifically chosen for tissue specific expression such that the genes are expressed only in certain tissues.
Our invention manages to get the continuous and safe therapeutic effect while adding the therapeutic genes to off-the-shelf available mesenchymal stem cells or other somatic cells. Mesenchymal stem cells can be produced on industrial scale by using tissue from umbilical cord tissue. Mesenchymal stem cells have hypo-immunogenic properties as well as ability to migrate towards the injured region and thus can provide beneficial effect in cases of surgeries involving nervous system.

ILLUSTRATIVE WORKING MECHANISM
1. Construction of the transgenic MSCs
1. The construct/DNA cassette under consideration consists of CMV promoter, which drives the expression of the DNA cassette comprising of PDGF-B, VEGF along with Luciferase and CXCR4B genes and could be used for both bacterial and mammalian cells.
2. The transgenic MSCs with “PDGF-B and VEGF” are created either by viral or non-viral transfection method.
3. The transgenic MSCs are generated using chemical method and a stable line is achieved using the selectivity marker, ‘zeocin’ in our case which is present in the integrin or plasmid used to inert the DNA cassette. However, any appropriate plasmid could be used to carry the DNA cassette and thus could be transfected to MSCs or somatic cells using transfection protocol.
Thus, the chief advantage of the DNA cassette with the insert comprising of PDGF-B and VEGF with vacant multiple cloning site is used for industrial scale use of such produced cells to create various combinations of therapeutic genes with luciferase to monitor the expression of construct.
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not as restrictive. The scope of the invention is, therefore, indicated by the appended claims and their combination in whole or in part rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.
EXAMPLES:
The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only, and the invention is not limited to these examples, but rather encompasses all variations, which are evident as a result of the teaching provided herein. The figures and specific sequences provided in the description are to illustrate the present invention in which a single DNA cassette with ‘Platelet derived growth factor-B’ with Vascular endothelial growth factor gene is introduced at the multiple cloning site. First the expression level of both PDGF-B and VEGF have been verified as shown in Example 1 while example 2 verifies the beneficial effects of MSCs transfected with DNA cassette in in vitro co-culture environment of transfected
MSCs with wound injury model.

1. Expression of IschemoHomeBrite construct.
The gene synthesis of PDGF-B:VEGF:Luciferase:CXCR4B construct was carried out to perform molecular and functional study for PDGF-B and VEGF and to evaluate the beneficial role of IschemoHomeBrite construct and their utility in wound healing processes (Figure 1, and 3).

a. Detection of genes via WB technique
The gene synthesis of PDGF-B:VEGF:Luciferase:CXCR4B gene construct was then used to perform expression studies for IschemoHomeBrite contruct. As illustrated in Figure 4 of the accompanying drawings the expression analysis of construct IschemoHomeBrite in 293-T cells indicates that PDGF-B, VEGF and Luciferase are expressed at once in supernatant and cell lysates, respectively using PDGF-B, VEGF and Luciferase antibodies.
b. Detection of genes via RT-PCR technique

In order to assess the expression of Guassia luciferase, Luciferase Reporter Assay was performed using protocol known in the prior art. The inclusion of luciferase reporter gene allows monitoring of activity of construct in the body via simple urine based assay in animals/human population as illustrated in Figure 5 of the accompanying drawings. At 48 hours, post-transfection, 20µL of sample was used to measure the luciferase activity in each flash assay reagent as supernatant and cell lysate in IschemoHomeBrite transfected and un-transfected MSCs. * shows the significant difference for the luciferase activity in the cell lysates between the transfected and un-transfected cells where **=P<0.001. # shows the significant difference for the luciferase activity in the supernatant between the transfected and un-transfected cells where ##=P<0.001.

Example 2: Transfection of IschemoHomeBrite to umbilical cord derived mesenchymal stem cells

Mesenchymal stem cells were produced using umbilical cord tissue using the methods known in the art and have been published. The MSCs were transfected to IschemoHomeBrite via Lipofectamine Plus using protocol known in the prior art. The cells were tested for the expression of PDGF-B and VEGF.

Example 3: Quantification of the level of IschemoHomeBrite expression:

PDGF-B and VEGF ELISA kits and level of expression of PDGF-B and VEGF were quantified in the IschemoHomeBrite transfected MSCs using a conventional method. Different number of transfected cells was used to quantify the expression of PDGF-B and VEGF separately. The results suggest that transfection of IschemoHomeBrite indeed can be quantified based on the number of transfected MSCs against un-transfected MSCs. The results as shown in Figure 6 (A) of the accompanying drawings suggest that transfection of PDGF-B secreted in the supernatant indeed can be quantified based on the number of transfected MSCs against un-transfected MSCs. The percentage expression of PDGF-B protein in different number of transfected cells as assessed via ELISA assay. The significant difference was found between the protein expression at 1 million and 5 or 10 million cells. There was statistical difference in the cell viability among these three groups, where */** =P<0.05/P=0.005 denote the statistically significant difference for expression of PDGF-B in 1 million transfected cells vs 5 million and 10 million cells. Similarly, the results as shown in Figure 6 (B) of the accompanying drawings suggest that transfection of VEGF secreted in the supernatant indeed can be quantified based on the number of transfected MSCs against un-transfected MSCs. The percentage expression of VEGF protein in different number of transfected cells as assessed via ELISA assay. The significant difference was found between the protein expression at 1 million and 5 or 10 million cells. There was statistical difference in the cell viability among these three groups, where */** =P<0.05/P=0.005 denote the statistically significant difference for expression of VEGF in 1 million transfected cells vs 5 million and 10 million cells.

Example 4: In vitro studies to demonstrate the efficacy of IschemoHomeBrite

We generated an in vitro model for wound healing by scrapping the uniform monolayer of the MSCs. The interrupted layer developed upon scratch assay is commonly studied for wound healing models Figure 8 (A). We used transwells for performing co-culture of untransfected and IschemoHomeBrite transfected cells with the monolayer wit scratch assay. We first performed the assessment of the total number of cells in the transfected and untranfected co-cultured MSCs. In addition, wound healing markers, TIMP1 (Extracellular Matrix (ECM) remodeling enzymes) and CDH1 (E-Cadherin) (cell adhesion molecule) were used to assess the beneficial role of IschemoHomeBrite in in vitro wound healing model (Figure 9 A and B) using ELISA assay. As illustrated in Figure 9 (A), there was a significant increase in the total number of cells in the well supported with IschemoHomeBrite transfected MSCs cells as compared to those supported by untransfected MSCs. There was statistical difference in the cell numbers between these two groups, where ** =P<0.005 denote the statistically significant difference between the cells supported by untransfected and transfected cells. In addition, the ELISA results indicate upregulation of TIMP1 and CDH1 in the wells supported by IschemoHomeBrite transfected cells after induction of wound via scratch assay. As illustrated in Figure 9 (B), there was a significant increase in the TIMP1 and CDH1 in the well supported with IschemoHomeBrite transfected MSCs cells as compared to those supported by untransfected MSCs. There was statistical difference in the levels of TIMP1 and CDH1, where ** =P<0.005 denote the statistically significant difference between the cells supported by untransfected and transfected cells.
While considerable emphasis has been placed herein on the specific elements of the preferred embodiment, it will be appreciated that many alterations can be made and that many modifications can be made in preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiments of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.