Targeting cells with altered microrna expression

Abstract

The present invention relates to a method of modulating development of a cell. The method includes the step of introducing into the cell a nucleic acid with the capacity to modulate development of the cell, the nucleic acid including a target site for binding of a microRNA, wherein the activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to modulate development of the cell.

1 . A method of modulating development of a cell, the method including the step of introducing into the cell a nucleic acid with the capacity to modulate development of the cell, the nucleic acid including a target site for binding of a microRNA, wherein the activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to modulate development of the cell. 
     
     
         2 . A method according to  claim 1 , wherein the nucleic acid has the capacity to inhibit development of the cell. 
     
     
         3 . A method according to  claim 2 , wherein the nucleic acid has cytotoxic or cytostatic activity. 
     
     
         4 . A method according to  claim 3 , wherein the nucleic acid encodes a gene selected from the group consisting of herpes simplex thymidine kinase,  E. coli  cytosine deaminase,  E. coli nitroreductase, P. aeruginosa  carboxypeptidase, horseradish peroxidase, and  E. coli  purine nucleoside phosphorylase, or an active fragment or variant of any of these genes. 
     
     
         5 . A method according to any one of  claims 1  to  4 , wherein the cell is selected from the group consisting of a cancerous cell, a pre-cancerous cell, an embryonic stem cell, an adult stem cell, a haemopoietic cell including a haemopoietic precursor cell, an adipocyte, a neuronal cell, a sperm cell or a sperm producing cell, a pancreatic islet cell, and a virally infected cell. 
     
     
         6 . A method according to  claim 5 , wherein the cancerous cell is a colorectal cancer cell, a lung cancer cell, a thymus cancer cell, a bladder cancer cell, a breast cancer cell, a prostate cancer cell or a cancerous B cell. 
     
     
         7 . A method according to any one of  claims 2  to  7 , wherein the method is used to inhibit development of a cell with a reduced activity and/or concentration of the microRNA. 
     
     
         8 . A method according to  claim 7 , wherein the method is used to ablate the cell. 
     
     
         9 . A method according to  claim 8 , wherein the microRNA is selected from the group consisting of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-181b-1, hsa-miR-155, hsa-miR-124a, hsa-miR-9 and the corresponding orthologues of the aforementioned microRNAs. 
     
     
         10 . A method according to  claim 1 , wherein the nucleic acid has the capacity to promote development of the cell. 
     
     
         11 . A method according to  claim 2 , wherein the nucleic acid is a nucleic acid encoding a cytokine, a therapeutic protein, or an active fragment or variant of the aforementioned. 
     
     
         12 . A method according to  claims 10  or  11 , wherein the method is used to promote development of a cell with a reduced activity and/or concentration of a microRNA. 
     
     
         13 . A method according to any one of  claims 1  to  12 , wherein the nucleic acid includes two or more target sites for binding of the same or different microRNAs. 
     
     
         14 . A method according to any one of  claims 1  to  13 , wherein the cell is a cell in an animal or human subject. 
     
     
         15 . A method according to any one of  claims 1  to  14 , wherein the method is used to prevent and/or treat a disease, condition or state in an animal or human subject. 
     
     
         16 . A nucleic acid with the capacity to modulate development of a cell, the nucleic acid including a binding site for a microRNA. 
     
     
         17 . A nucleic acid according to  claim 16 , wherein the nucleic acid has the capacity to inhibit development of a cell. 
     
     
         18 . A nucleic acid according to  claim 17 , wherein the nucleic acid has cytotoxic or cytostatic activity. 
     
     
         19 . A nucleic acid according to  claim 18 , wherein the nucleic acid encodes a gene selected from the group consisting of herpes simplex thymidine kinase,  E. coli  cytosine deaminase,  E. coli nitroreductase, P. aeruginosa  carboxypeptidase, horseradish peroxidase, and  E. coli  purine nucleoside phosphorylase, or an active fragment or variant of any of these genes. 
     
     
         20 . A nucleic acid according to any one of  claims 16  to  19 , wherein the binding site is a binding site for a microRNA selected from the group consisting of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-181b-1, hsa-miR-155, hsa-miR-124a, hsa-miR-9 and the corresponding orthologues of the aforementioned microRNAs. 
     
     
         21 . A nucleic acid according to any one of  claims 16  to  20 , wherein the nucleic acid includes two or more binding sites for binding of the same or different microRNAs. 
     
     
         22 . A nucleic acid according to  claim 16 , wherein the nucleic acid has the capacity to promote development of the cell. 
     
     
         23 . A nucleic acid according to  claim 22 , wherein the nucleic acid is a nucleic acid encoding a cytokine, a therapeutic protein, or an active fragment or variant of the aforementioned. 
     
     
         24 . A nucleic acid according to any one of  claims 16  to  23 , wherein the binding site is a binding for a microRNA that is differentially expressed and/or differentially active. 
     
     
         25 . A vector including the nucleic acid according to any one of  claims 16  to  24 . 
     
     
         26 . A vector according to  claim 25 , wherein the vector is a viral vector. 
     
     
         27 . A composition for administration to an animal or human subject, the composition including a nucleic acid according to any one of  claims 16  to  26 . 
     
     
         28 . A cell including a nucleic acid according to any one of  claims 16  to  26 . 
     
     
         29 . An animal including a cell according to  claim 28 . 
     
     
         30 . A nucleic acid according to any one of  claims 16  to  26 , wherein the nucleic acid is used to inhibit the development of a cell in an animal or human subject. 
     
     
         31 . A nucleic acid according to  claim 30 , wherein the nucleic acid is used to ablate cells in an animal or human subject. 
     
     
         32 . A nucleic acid including a non-naturally occurring binding site for a microRNA that is differentially expressed and/or has differential activity. 
     
     
         33 . A nucleic acid according to  claim 32 , wherein the binding site is a binding site for a microRNA that is differentially expressed and/or differentially active in a cancerous cell as compared to a similar non-cancerous cell. 
     
     
         34 . A nucleic acid according to  claim 32  or  33 , wherein the binding site is a binding site for a microRNA that is downregulated in the cancerous cell as compared to the non-cancerous cell. 
     
     
         35 . A nucleic acid according to any one of  claims 32  to  34 , wherein the binding site is a binding site for a microRNA selected from the group consisting of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-181b-1, hsa-miR-155, hsa-miR-124a, hsa-miR-9 and the corresponding orthologues of the aforementioned microRNAs. 
     
     
         36 . A nucleic acid according to any one of  claims 32  to  35 , wherein the nucleic acid includes two or more binding sites for binding of the same or different microRNAs that are differentially expressed and/or differentially active. 
     
     
         37 . A nucleic acid according to any one of  claims 32  to  36 , wherein the nucleic acid has the capacity to inhibit development of a cell. 
     
     
         38 . A nucleic acid according to  claim 37 , wherein the nucleic acid has cytotoxic or cytostatic activity. 
     
     
         39 . A nucleic acid according to  claim 37  or  38 , wherein the nucleic acid encodes a gene selected from the group consisting of herpes simplex thymidine kinase,  E. coli  cytosine deaminase,  E. coli nitroreductase, P. aeruginosa  carboxypeptidase, horseradish peroxidase, and  E. coli  purine nucleoside phosphorylase, or an active fragment or variant of any of these genes. 
     
     
         40 . A nucleic acid according to  claim 32 , wherein the nucleic acid has the capacity to promote development of the cell. 
     
     
         41 . A nucleic acid according to  claim 40 , wherein the nucleic acid is a nucleic acid encoding a cytokine, a therapeutic protein, or an active fragment or variant of the aforementioned. 
     
     
         42 . A vector including the nucleic acid according to any one of  claims 32  to  41 . 
     
     
         43 . A vector according to  claim 42 , wherein the vector is a viral vector. 
     
     
         44 . A composition for administration to an animal or human subject, the composition including a nucleic acid according to any one of  claims 32  to  43 . 
     
     
         45 . A cell including a nucleic acid according to any one of  claims 32  to  43 . 
     
     
         46 . An animal including a cell according to  claim 45 . 
     
     
         47 . A nucleic acid according to any one of  claims 32  to  43 , wherein the nucleic acid is used to modulate the development of cells in an animal or human subject. 
     
     
         48 . A nucleic acid according to  claim 47 , wherein the nucleic acid is used to inhibit the development of cells in an animal or human subject. 
     
     
         49 . A nucleic acid according to  claim 47 , wherein the nucleic acid is used to ablate cells in an animal or human subject. 
     
     
         50 . A cancerous cell including an exogenous nucleic acid including a binding site for a microRNA, wherein the cancerous cell has a reduced activity and/or concentration of the microRNA as compared to a similar non-cancerous cell. 
     
     
         51 . A cancerous cell according to  claim 50 , wherein the cancerous cell is a colorectal cancer cell, a lung cancer cell, a thymus cancer cell, a bladder cancer cell, a breast cancer cell, a prostate cancer cell or a cancerous B cell. 
     
     
         52 . A cancerous cell according to  claim 50  or  51 , wherein the microRNA is selected from the group consisting of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-181b-1, hsa-miR-155, hsa-miR-124a, hsa-miR-9 and the corresponding orthologues of the aforementioned microRNAs. 
     
     
         53 . A cancerous cell according to any one of  claims 50  to  52 , wherein the exogenous nucleic acid includes two or more binding sites for binding of the same or different microRNAs that are differentially expressed and/or differentially active in the cancerous cell. 
     
     
         54 . A cancerous cell according to any one of  claims 50  to  53 , wherein the exogenous nucleic acid has the capacity to inhibit development of the cell. 
     
     
         55 . A cancerous cell according to  claim 54 , wherein the nucleic acid has cytotoxic or cytostatic activity. 
     
     
         56 . A cancerous cell according to  claim 55 , wherein the exogenous nucleic acid encodes a gene selected from the group consisting of herpes simplex thymidine kinase,  E. coli  cytosine deaminase,  E. coli nitroreductase, P. aeruginosa  carboxypeptidase, horseradish peroxidase, and  E. coli  purine nucleoside phosphorylase, or an active fragment or variant of any of these genes. 
     
     
         57 . An animal including a cancerous cell according to any one of  claims 50  to  56 . 
     
     
         58 . An animal according to  claim 57 , wherein the animal is a transgenic animal. 
     
     
         59 . An animal according to  claim 57  or  58 , wherein the animal is used to identify microRNAs that are differentially expressed between cancerous and non-cancerous cells. 
     
     
         60 . A method of preventing and/or treating a disease, condition or state associated with target cells in a subject, the method including the step of introducing into cells in the subject a nucleic acid with the capacity to modulate development of a cell, the nucleic acid including a target site for binding of the microRNA, wherein the activity of the microRNA in the target cells results in a level of activity of the nucleic acid sufficient to modulate development of the target cells in the subject. 
     
     
         61 . A method according to  claim 60 , wherein the disease, condition or state is selected from the group consisting of a cancer, including colorectal cancer, lung cancer, thymus cancer, bladder cancer, breast cancer and prostate cancer; human B cell chronic lymphocytic leukemia; B cell (Burkitt) Lymphoma; a disease or disorder of pancreatic endocrine cells including diabetes; a disease or condition associated with viral infection of cells including EBV, HIV, Hepatitis and Herpes infection of cells; 5q-myelodysplastic syndrome (macrocytic anaemia); a disease or conditions associated with haemopoietic dysfunction, an autoimmune and inflammatory diseases including Crohn's disease; fragile X mental retardation; Di George syndrome; Wilms tumour; a disease or condition associated with neuron dysfunction; a disease or condition associated with adipocyte dysfunction; a disease that can be treated with embryonic or adult stem cells; and a disease or condition associated with sperm producing cells. 
     
     
         62 . A method according to  claim 61 , wherein the disease is a cancer. 
     
     
         63 . A method according to  claim 62 , wherein the target cell is a cancerous cell or a pre-cancerous cell. 
     
     
         64 . A method according to  claim 63 , wherein the cancerous cell or pre-cancerous is a colorectal cancer cell, a lung cancer cell, a thymus cancer cell, a bladder cancer cell, a breast cancer cell, a prostate cancer cell or a cancerous B cell. 
     
     
         65 . A method according to any one of  claims 60  to  64 , wherein the nucleic acid has the capacity to inhibit development of the cell. 
     
     
         66 . A method according to  claim 65 , wherein the nucleic acid has cytotoxic or cytostatic activity. 
     
     
         67 . A method according to  claim 66 , wherein the nucleic acid encodes a gene selected from the group consisting of herpes simplex thymidine kinase,  E. coli  cytosine deaminase,  E. coli nitroreductase, P. aeruginosa  carboxypeptidase, horseradish peroxidase, and  E. coli  purine nucleoside phosphorylase, or an active fragment or variant of any of these genes. 
     
     
         68 . A method according to any one of  claims 60  to  67 , wherein the target cells have a reduced activity and/or expression of the microRNA. 
     
     
         69 . A method according to any one of  claims 60  to  68 , wherein the method is used to ablate the target cells in the subject. 
     
     
         70 . A method according to any one of  claims 60  to  69 , wherein the microRNA is selected from the group consisting of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-181b-1, hsa-miR-155, hsa-miR-124a, hsa-miR-9 and the corresponding orthologues of the aforementioned microRNAs. 
     
     
         71 . A method according to  claim 60 , wherein the nucleic acid has the capacity to promote development of the cell. 
     
     
         72 . A method according to  claim 71 , wherein the nucleic acid is a nucleic acid encoding a cytokine, a therapeutic protein, or an active fragment or variant of the aforementioned. 
     
     
         73 . A method according to  claim 71  or  72 , wherein the method is used for the promotion of development a cell with a reduced activity and/or concentration of a microRNA. 
     
     
         74 . A method of inhibiting development of a cell, the cell having a reduced activity and/or concentration of a microRNA, the method including the step of introducing into the cell a nucleic acid with the capacity to inhibit development of the cell, the nucleic acid including a target site for binding of the microRNA, wherein the reduced activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to inhibit development of the cell. 
     
     
         75 . A method of promoting development of a cell, the cell having a reduced activity and/or concentration of a microRNA, the method including the step of introducing into the cell a nucleic acid with the capacity to promote development of the cell, the nucleic acid including a target site for binding of the microRNA, wherein the reduced activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to promote development of the cell. 
     
     
         76 . A method of detecting altered microRNA activity and/or concentration in a cancerous or pre-cancerous cell, the method including the steps of:
 determining the level of expression of a reporter nucleic acid in the cancerous or pre-cancerous cells and determining the level of expression of a reporter nucleic acid in non-cancerous cells; and   detecting a reduced activity of the microRNA in the cancerous cells by an increase in the expression of the reporter nucleic acid in the cancerous cells as compared to the level of expression of the reporter nucleic acid in the non-cancerous cells.   
     
     
         77 . A method of modulating the concentration of a nucleic acid expressed in a cancerous cell, the cancerous cell having an altered activity and/or concentration of a microRNA as compared to a similar non-cancerous cell, the method including the step of introducing a target site for binding of the microRNA into the nucleic acid to be expressed in the cell.