1. At least one DNA cassette with one or more of the genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’ 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), under the regulation of glucose responsive regulatory elements wherein the plasmid bearing 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 or more DNA cassettes and does not exclude use of any system or cells for the purpose of treating diabetes, insulin deficiencies and metabolic syndromes.

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 genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’ along with luciferase gene to provide a biological assay to monitor the expression of said genes.

3. A method of treating the patients of diabetes and complications due to diabetes and muscle injuries like traumatic injury and diabetic wounds by infusing the DNA cassette for the expression of urocortin-3, IGF-1, Glucokinase and Furin Cleavable Insulin.

4. 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 ‘FGF-1’, ‘HGF’, ‘EGF’, ‘VEGF’, ‘FGF-7’, ‘IGF-1’, ‘CXCR4’, ‘luciferase’, ‘IL-10’, ‘IL-4’ and ‘PDGF' genes along with genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’.

5. A method to treat the patients by consecutive injections of circular DNA molecules expressing ‘glucokinase’ and ‘glucose inducible insulin gene expression’ in that order in skeletal muscle tissue or liver tissue.

6. A method to treat various diseases involving insulin deficiencies with genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’ where these therapeutic genes are introduced via viral or non-viral methods of transgenesis by use of consecutive injections of circular DNA molecules expressing ‘glucokinase’ and ‘glucose inducible insulin gene expression’ in that order in skeletal muscle tissue or liver tissue by use of tissue specific regulatory elements viz. creatinine Kinase promoter for muscles and albumin promoter for liver specific expression.

7. 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 with genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’.

8. 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 or DNA minicircles 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.

9. A method of using the transgenic mesenchymal stem cells locally in a matrix or spray or fibrin or collagen based gel to cause wound healing by virtue of expression of therapeutic genes including but not exclusively with genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’ with or without the expression of luciferase gene and CXCR4 gene.

10. A method of using the transgenic mesenchymal stem cells locally in a matrix or spray or fibrin or collagen based gel to cause muscle strengthening by virtue of expression of genes including but not excluding ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’.

11. A method of use of above mentioned DNA cassette or transgenic host cells in treatment of any injury or chronic disease in humans or animals

12. A method of use of above mentioned DNA cassette specifically in combination of a safety switch to control the life of transgene expressing cells so that there is a control over the therapy after administration.

13. A method of use of above mentioned DAN cassette and transgenic cells in treatment of healing of surgical or non-surgical injuries especially in muscles.

14. A method of treating single gene deficiencies or multiple gene deficiencies where insulin gene in the cassette can be replaced by the therapeutic gene for the respective gene deficiency.

15. A method of using the therapeutic mechanism of the DNA cassette to treat cystic fibrosis by inserting the gene CFTR and surfactant proteins in the DNA cassette.

16. A method of changing the eye color of a person by adding an iris specific promoter driving the iris specific expression of blue eye color. , Description: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 novelty of the present invention lies in the therapeutic mechanism offered by ingenuously allowing the host cells to treat the diabetes mellitus developed from various etiologies including that developed from a complete pancreatectomy. The host cells are used to express ‘insulin’, ‘IGF’, ‘urocortin-3’, ‘glucokinase’, ‘luciferase’, and “cytosine deaminase” or “herpes simplex thymidine kinase” either individually or in combination. The strategy described in the invention has direct applicability for the treatment of diseases of diabetes mellitus and its complications. Our novel invention makes therapy easily feasible and makes the proteins available in steady physiological rate and manner and is clinically safe. The insulin can also be delivered by use of delivery vehicles by use of mesenchymal stem cells as safety of mesenchymal stem cells is well established. The present invention describes a concoction comprising of therapeutic genes including insulin, insulin like growth factor, glucokinase, urocortin-3 and luciferase, cytosine deaminase, herpes simplex thymidine kinase and EGFR domains. The safety genes or suicide genes are thymidine kinase or cytosine deaminase which convert a nontoxic drug to a toxic compound in gene modified cells. Unlike the mammalian thymidine kinase, HSV-TK is characterized by 1000 fold higher affinity to specific nucleoside analogs including ganciclovir. HSV-TK phosphorylates nucleoside analogs, including acyclovir and GCV, and their resulting triphosphate form incorporates into DNA via the action of DNA polymerase, leading to chain termination and cell death. HSV-TK/GCV also induces apoptosis through CD95-L independent CD95 aggregation, leading to the formation of a Fas-associated death domain protein (FADD) and caspase-8-containing death-inducing signaling complex. In another approach, truncated EGFR domain and anti-EGFR monoclonal antibody can also be used for the elimination of gene therapy cells. HSV-TK expressing cells die after a three days of exposure to ganciclovir. The co-expression of insulin and glucokinase genes intramuscularly along with luciferase gene and a truncated EGFR domain enables body to create the insulin and glucokinase under the regulatory control resulting in muscle expression and ability to monitor and withdraw the therapy at any time. The said invention is useful against type 1 diabetes mellitus, type 2 diabetes mellitus, post-surgical insulin requirement, cases of muscle injury, improving metabolic syndromes, diabetic cardiomyopathy, and diabetic neuropathy. In addition to muscles, the insulin can also be therapeutically expressed in the liver along with luciferase and suicide genes. The glucokinase is naturally expressed in the hepatocytes and because of liver is actively involved in the glycogen metabolism where glycogen synthesis and glycogenolysis take place by use of enzyme glucose-6-phophate resulting into production and utilization of glucose, the hepatocytes also make an excellent host cells for the diabetes therapy with the DNA cassette. The said therapeutic genes will be expressed together by virtue of connecting P2A sequences such that the sequences get dissociated automatically upon translation and formation of protein.

Claims:1. At least one DNA cassette with one or more of the genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’ 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), under the regulation of glucose responsive regulatory elements wherein the plasmid bearing 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 or more DNA cassettes and does not exclude use of any system or cells for the purpose of treating diabetes, insulin deficiencies and metabolic syndromes.
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 genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’ along with luciferase gene to provide a biological assay to monitor the expression of said genes.
3. A method of treating the patients of diabetes and complications due to diabetes and muscle injuries like traumatic injury and diabetic wounds by infusing the DNA cassette for the expression of urocortin-3, IGF-1, Glucokinase and Furin Cleavable Insulin.
4. 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 ‘FGF-1’, ‘HGF’, ‘EGF’, ‘VEGF’, ‘FGF-7’, ‘IGF-1’, ‘CXCR4’, ‘luciferase’, ‘IL-10’, ‘IL-4’ and ‘PDGF' genes along with genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’.
5. A method to treat the patients by consecutive injections of circular DNA molecules expressing ‘glucokinase’ and ‘glucose inducible insulin gene expression’ in that order in skeletal muscle tissue or liver tissue.
6. A method to treat various diseases involving insulin deficiencies with genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’ where these therapeutic genes are introduced via viral or non-viral methods of transgenesis by use of consecutive injections of circular DNA molecules expressing ‘glucokinase’ and ‘glucose inducible insulin gene expression’ in that order in skeletal muscle tissue or liver tissue by use of tissue specific regulatory elements viz. creatinine Kinase promoter for muscles and albumin promoter for liver specific expression.
7. 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 with genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’.
8. 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 or DNA minicircles 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.
9. A method of using the transgenic mesenchymal stem cells locally in a matrix or spray or fibrin or collagen based gel to cause wound healing by virtue of expression of therapeutic genes including but not exclusively with genes ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’ with or without the expression of luciferase gene and CXCR4 gene.
10. A method of using the transgenic mesenchymal stem cells locally in a matrix or spray or fibrin or collagen based gel to cause muscle strengthening by virtue of expression of genes including but not excluding ‘furin cleavable insulin’, ‘insulin like growth factor-1, ‘urocortin-3’, ‘urocortin-2’, ‘glucokinase’, ‘luciferase’, ‘cytosine deaminase’, ‘herpes simplex thymidine kinase’, and ‘EGFR truncated domain’.
11. A method of use of above mentioned DNA cassette or transgenic host cells in treatment of any injury or chronic disease in humans or animals
12. A method of use of above mentioned DNA cassette specifically in combination of a safety switch to control the life of transgene expressing cells so that there is a control over the therapy after administration.
13. A method of use of above mentioned DAN cassette and transgenic cells in treatment of healing of surgical or non-surgical injuries especially in muscles.
14. A method of treating single gene deficiencies or multiple gene deficiencies where insulin gene in the cassette can be replaced by the therapeutic gene for the respective gene deficiency.
15. A method of using the therapeutic mechanism of the DNA cassette to treat cystic fibrosis by inserting the gene CFTR and surfactant proteins in the DNA cassette.
16. A method of changing the eye color of a person by adding an iris specific promoter driving the iris specific expression of blue eye color.
, Description: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 novelty of the present invention lies in the therapeutic mechanism offered by ingenuously allowing the host cells to treat the diabetes mellitus developed from various etiologies including that developed from a complete pancreatectomy. The host cells are used to express ‘insulin’, ‘IGF’, ‘urocortin-3’, ‘glucokinase’, ‘luciferase’, and “cytosine deaminase” or “herpes simplex thymidine kinase” either individually or in combination. The strategy described in the invention has direct applicability for the treatment of diseases of diabetes mellitus and its complications. Our novel invention makes therapy easily feasible and makes the proteins available in steady physiological rate and manner and is clinically safe. The insulin can also be delivered by use of delivery vehicles by use of mesenchymal stem cells as safety of mesenchymal stem cells is well established.
The present invention describes a concoction comprising of therapeutic genes including insulin, insulin like growth factor, glucokinase, urocortin-3 and luciferase, cytosine deaminase, herpes simplex thymidine kinase and EGFR domains.
The safety genes or suicide genes are thymidine kinase or cytosine deaminase which convert a nontoxic drug to a toxic compound in gene modified cells. Unlike the mammalian thymidine kinase, HSV-TK is characterized by 1000 fold higher affinity to specific nucleoside analogs including ganciclovir. HSV-TK phosphorylates nucleoside analogs, including acyclovir and GCV, and their resulting triphosphate form incorporates into DNA via the action of DNA polymerase, leading to chain termination and cell death. HSV-TK/GCV also induces apoptosis through CD95-L independent CD95 aggregation, leading to the formation of a Fas-associated death domain protein (FADD) and caspase-8-containing death-inducing signaling complex. In another approach, truncated EGFR domain and anti-EGFR monoclonal antibody can also be used for the elimination of gene therapy cells. HSV-TK expressing cells die after a three days of exposure to ganciclovir.
The co-expression of insulin and glucokinase genes intramuscularly along with luciferase gene and a truncated EGFR domain enables body to create the insulin and glucokinase under the regulatory control resulting in muscle expression and ability to monitor and withdraw the therapy at any time.
The said invention is useful against type 1 diabetes mellitus, type 2 diabetes mellitus, post-surgical insulin requirement, cases of muscle injury, improving metabolic syndromes, diabetic cardiomyopathy, and diabetic neuropathy.
In addition to muscles, the insulin can also be therapeutically expressed in the liver along with luciferase and suicide genes. The glucokinase is naturally expressed in the hepatocytes and because of liver is actively involved in the glycogen metabolism where glycogen synthesis and glycogenolysis take place by use of enzyme glucose-6-phophate resulting into production and utilization of glucose, the hepatocytes also make an excellent host cells for the diabetes therapy with the DNA cassette.
The said therapeutic genes will be expressed together by virtue of connecting P2A sequences such that the sequences get dissociated automatically upon translation and formation of protein.