Methods for Reverting Methylation by Targeting Methyltransferase and Compositions Useful Therefor

Abstract

Methods for restoring a desired pattern of DNA methylation, inducing re-expression of methylation-silenced tumor suppressor genes (TSGs), and/or inhibiting tumorigenicity both in vitro and in vivo in a subject in need thereof by administering an effective amount of one or more miR-29s sufficient to target one or more of DNMT3A and DNMT3B are disclosed.

What is claimed is: 
     
         1 . A pharmaceutical composition for treating a lung cancer-related disease, comprising:
 at least one miR gene product selected from the group consisting of miR-29a, miR-29b, miR-29c and combinations thereof; and,   a pharmaceutically-acceptable carrier.   
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein the at least one miR gene product corresponds to a miR gene product that is down-regulated in cancer cells relative to suitable control cells. 
     
     
         3 . The pharmaceutical composition of  claim 1 , wherein the lung cancer-related disease is non-small cell lung cancer (NSCLC). 
     
     
         4 . A composition used to treat cancer in a patient, the composition comprising an inhibitor of one or more of DNMT1, DNMT3A and/or DNMT3B, wherein the inhibitor comprises one or more miR-29 gene products, and wherein the cancer is characterized as exhibiting an undesirable DNA methylation pattern. 
     
     
         5 . The composition of  claim 4 , wherein the miR-29 gene product comprises to a miRNA that has less than 100% identity to a corresponding wild-type miR gene product and possesses one or more biological activities of the corresponding wild-type miR gene product. 
     
     
         6 . The composition of  claim 4 , wherein the miR-29 gene product includes species variants and variants that are the consequence of one or more mutations in a miR gene. 
     
     
         7 . The composition of  claim 4 , wherein the miR-29 gene product is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to a corresponding wild-type miR gene product. 
     
     
         8 . The composition of  claim 4 , wherein the miR-29 gene product is an oligonucleotide that binds to miR-29. 
     
     
         9 . The composition of  claim 4 , wherein the miR-29 gene product physically interacts with miR-29. 
     
     
         10 . A pharmaceutical composition comprising a nucleic acid that encodes a miRNA having an inhibitory effect on expression of one or more of DNMT1, DNMT3a and/or DNMT3B in an amount effective to treat a cancer. 
     
     
         11 . The pharmaceutical composition of  claim 10 , wherein the miRNA is a mature miRNA, a precursor of the miRNA, or variants and analogs thereof. 
     
     
         12 . The pharmaceutical composition of  claim 10 , wherein the miRNA is a substance comprising a seed sequence of SEQ ID NO: 1 (hsa-miR-29a), SEQ ID NO: 2 (hsa-miR-29b) or SEQ ID NO: 3 (hsa-miR-29c), precursors thereof, or variants and analogs thereof. 
     
     
         13 . The pharmaceutical composition of  claim 10 , wherein the miRNA is comprised within an expression vector for introducing and expressing in cancer cells. 
     
     
         14 . A method of diagnosing whether a subject has a decreases survival prognosis for a lung cancer-related disease, comprising:
 measuring the level of DNMT3A in a test sample from the subject,   wherein an increase in the level of DNMT3A in the test sample, relative to the level of a corresponding DNMT3A in a control sample, is indicative of the subject either having an increased risk for a poor survival prognosis.   
     
     
         15 . A method for restoring a desired pattern of DNA methylation in a subject in need thereof, wherein the method comprises:
 administering to the subject an effective amount of one or more miR-29s sufficient to target at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   restoring the desired pattern of DNA methylation in the subject,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the subject has an undesirable pattern of DNA methylation.   
     
     
         16 . A method for inducing re-expression of at least one methylation-silenced tumor suppressor gene (TSG) in a subject in need thereof, wherein the method comprises:
 administering to the subject an effective amount of one or more miR-29s sufficient to target at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   inducing re-expression of the at least one methylation-silenced TSG in the subject,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the subject has an undesirable decreased expression of at least one TSG.   
     
     
         17 . A method for inducing re-expression of at least one methylation-silenced tumor suppressor gene (TSG) in a cell in need thereof, wherein the method comprises:
 transfecting the cell with an effective amount of one or more miR-29s sufficient to target at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   inducing re-expression of the at least one methylation-silenced TSG in the cell,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the cell is a cancer cell having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         18 . A method for inhibiting tumorigenicity in a subject in need thereof, wherein the method comprises:
 administering to the subject an effective amount of one or more miR-29s sufficient to target at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   inhibiting tumorigenicity in the subject,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the subject has an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         19 . A method for inhibiting tumorigenicity in a cell in need thereof, wherein the method comprises:
 transfecting the cell with an effective amount of one or more miR-29s sufficient to target at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   inhibiting tumorigenicity in the cell,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the cell is a cancer cell having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         20 . A method for reducing global DNA methylation in a subject in need thereof, wherein the method comprises:
 administering to the subject an effective amount of one or more miR-29s sufficient to target at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   reducing global DNA methylation in the subject,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the subject has a cancer having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         21 . The method of  claim 20 , wherein the reducing global DNA methylation includes DNA epigenetic regulation of the cancer by up-regulating expression of the one or more miR-29s in the subject. 
     
     
         22 . The method of  claim 20 , wherein the administering includes combining the one or more miR-29s with an effective amount of one or more nucleoside analogs sufficient to block at least one de novo and maintenance methyltransferase (DNMT) pathway. 
     
     
         23 . The method of  claim 22 , wherein the one or more nucleoside analogs comprise decitabine. 
     
     
         24 . A method for reducing global DNA methylation in a cell in need thereof, wherein the method comprises:
 transfecting the cell with an effective amount of one or more miR-29s sufficient to target at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   reducing global DNA methylation in the cell,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the cell is a cancer cell having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         25 . The method of  claim 24 , wherein the reducing global DNA methylation includes DNA epigenetic modification of the cell by up-regulating expression of the one or more miR-29s in the cancer cell. 
     
     
         26 . The method of  claim 25 , wherein the transfecting includes combining the one or more miR-29s with an effective amount of one or more nucleoside analogs sufficient to block at least one de novo and maintenance methyltransferase (DNMT) pathway. 
     
     
         27 . The method of  claim 26 , wherein the one or more nucleoside analogs comprise decitabine. 
     
     
         28 . A method for increasing expression of at least one tumor suppression gene (TSG) in a subject in need thereof, wherein the method comprises:
 administering to the subject an effective amount of one or more miR-29s sufficient to target at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   increasing expression of the at least one TSG in the subject,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the subject has a cancer having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         29 . The method of  claim 28 , wherein the at least one TSG comprises FHIT and/or WWOX. 
     
     
         30 . A method for increasing expression of at least one tumor suppression gene (TSG) in a cell in need thereof, wherein the method comprises:
 transfecting the cell with an effective amount of one or more miR-29s sufficient to target at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   increasing expression of the at least one TSG in the cell,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the cell is a cancer cell having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         31 . The method of  claim 30 , wherein the at least one TSG comprises FHIT and/or WWOX. 
     
     
         32 . A method for increasing expression of at least one FHIT and/or WWOX enzyme in a subject in need thereof, wherein the method comprises:
 administering to the subject an effective amount of one or more miR-29s sufficient to decrease expression of at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   increasing expression of the at least one FHIT and/or WWOX enzyme in the subject,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the subject has a cancer having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         33 . A method for increasing expression of at least one FHIT and/or WWOX enzyme in a cell in need thereof, wherein the method comprises:
 transfecting the cell with an effective amount of one or more miR-29s sufficient to decrease expression of at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   increasing expression of at least one FHIT and/or WWOX enzyme in the cell,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the cell is a cancer cell an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         34 . A method for reducing global DNA methylation in a subject in need thereof, wherein the method comprises:
 administering to the subject an effective amount of one or more miR-29s sufficient to decrease expression of at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   reducing global DNA methylation in the subject,   wherein the reducing global DNA methylation includes inducing expression of the one or more miR-29s in the subject,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the subject has a cancer having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         35 . A method for reducing global DNA methylation in a cell in need thereof, wherein the method comprises:
 transfecting the cell with an effective amount of one or more miR-29s sufficient to decrease expression of at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   reducing global DNA methylation in the cell,   wherein the reducing global DNA methylation includes inducing expression of the one or more miR-29s in the cell,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the cell is a cancer cell having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         36 . A method for restoring expression of at least one tumor suppressor gene (TSG) in a subject in need thereof, wherein the method comprises:
 administering to the subject an effective amount of one or more miR-29s sufficient to decrease expression of at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   restoring expression of the at least one TSG in the subject,   wherein the restoring expression of the at least one TSG includes inducing expression of the one or more miR-29s in the subject,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the subject has a cancer having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         37 . The method of  claim 36 , wherein the at least one TSG comprises FHIT and/or WWOX. 
     
     
         38 . A method for restoring expression of at least one tumor suppression gene (TSG) in a cell in need thereof, wherein the method comprises:
 transfecting the cell with an effective amount of one or more miR-29s sufficient to decrease expression of at least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and   restoring expression of the at least one TSG in the cell,   wherein the restoring expression of the at least one TSG includes inducing expression of the one or more miR-29s in the cell,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the cell is a cancer cell having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         39 . The method of  claim 38 , wherein the at least one TSG comprises FHIT and/or WWOX. 
     
     
         40 . A method for developing an epigenetic therapy in a subject in need thereof, wherein the method comprises:
 administering to the subject an effective amount of one or more miR-29s sufficient to normalize an aberrant pattern of methylation and reactivate expression of at least one tumor suppressor gene (TSG); and   developing the epigenetic therapy in the subject,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the subject has a cancer having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.   
     
     
         41 . A method for developing an epigenetic therapy in a cell in need thereof, wherein the method comprises:
 transfecting the cell with an effective amount of one or more miR-29s sufficient to normalize an aberrant pattern of methylation and reactivate expression of at least one tumor suppressor gene (TSG); and   developing the epigenetic therapy in the cell,   wherein the one or more miR-29s comprise at least one isolated or synthetic miR-29 selected from the group consisting of miR-29a, miR-29b, and miR-29c, and   wherein the cell is a cancer cell having an increased expression of one or more of DNMT1, DNMT3a and/or DNMT3B.