Embedded chimeric peptide nucleic acids and uses thereof

Abstract

The present invention relates to an ecPNA having the general structure: H 2 N—X—B—Y—COOH and uses thereof, wherein X is A or C and Y is A or C with the proviso that when X is A, Y is C, and when X is C, Y is A; A represents an oligopeptide structure, the sequence of which comprises a sequence which renders the compound able to enter the nucleus of a cell; B represents a peptide nucleic acid (PNA) structure at least 12 nucleotides in length, the sequence of which is capable of hybridizing with a DNA within the nucleus of the cell, which DNA is within a promoter region of a gene; C represents an oligopeptide structure; and each — represents a chemical linkage between the structures at each side thereof, which may be the same as or different from each other such linkage. The ecPNAs provided herein can upregulate or repress gene transcription and are useful for treating diseases requiring changes in transcription and for induction of inducible pluripotent stem (iPS) cells.

1 . An embedded chimeric peptide nucleic acid (ecPNA) having the general structure:
   H 2 N—X—B—Y—COOH
   wherein X is A or C and Y is A or C with the proviso that when X is A, Y is C, and when X is C, Y is A;   wherein A represents an oligopeptide, the sequence of which comprises a sequence which renders the ecPNA able to enter the nucleus of a cell;   wherein B represents a peptide nucleic acid (PNA) structure at least 12 nucleotides in length, the sequence of which is capable of hybridizing with a DNA within the nucleus of the cell, which DNA is within a promoter region of a gene;   wherein C represents an oligopeptide; and   wherein each — represents a chemical linkage between the structures at each side thereof, which may be the same as or different from each other such linkage.   
     
     
         2 . The ecPNA of  claim 1 , wherein X is A and Y is C. 
     
     
         3 . The ecPNA of  claim 1 , wherein X is C and Y is A. 
     
     
         4 . (canceled) 
     
     
         5 . The ecPNA of  claim 1 , wherein the oligopeptide C is detectable when the ecPNA is bound to the DNA. 
     
     
         6 . The ecPNA of  claim 1 , wherein the sequence of C comprises a sequence which renders the ecPNA able to regulate the transcription and expression of the gene. 
     
     
         7 . The ecPNA of  claim 1 , wherein each — may be a chemical bond or a chemical linker. 
     
     
         8 . The ecPNA of  claim 7 , wherein at least one — is a chemical bond. 
     
     
         9 . The ecPNA of  claim 8 , wherein the chemical bond is a covalent bond, an amide bond, or a peptide bond. 
     
     
         10 . The ecPNA of  claim 7 , wherein at least one — is a chemical linker. 
     
     
         11 . The ecPNA of  claim 10 , wherein the chemical linker comprises an amino acid, biotin, an ether (O), a stable polyether (OO), AEEA (2-aminoethoxy-2-ethoxyacetic acid), or a cleavable disulfide linkage. 
     
     
         12 - 15 . (canceled) 
     
     
         16 . The ecPNA of  claim 1 , wherein A comprises a sequence selected from the group consisting of the following sequences: YGRKKRRQRRR (SEQ ID NO: 6), GRKERRQRRRPPQ (SEQ ID NO: 7), YARKARRQARR (SEQ ID NO: 8), YARAAARARA (SEQ ID NO: 9), YARAARRAARR (SEQ ID NO: 10), YARAARRAARA (SEQ ID NO: 11), PKKKRKV (SEQ ID NO: 12), RQIKIWFQNRRMKWKK (SEQ ID NO: 13), KKWKMRRNQFWIKIQR (SEQ ID NO: 14), RQIKIWFQNRRMKNKK (SEQ ID NO: 15), RQIKIWFPNRRMKWKK (SEQ ID NO: 16), RQPKIWFPNRRMPWKK (SEQ ID NO: 17), RQIKIWFQNMRRKWKK (SEQ ID NO: 18), RQIRIWFQNRRNRWRR (SEQ ID NO 19), RRWRRWWRRWWRRWR (SEQ ID NO: 20), RQILIWFQNRRMKWKK (SEQ ID NO: 22), LLIILRRRIRKQAHAHSK (SEQ ID NO: 23), KLALKLALKALKAALKLA (SEQ ID NO: 24), and AGYLLGKINLKALAALAKKIL (SEQ ID NO: 25). 
     
     
         17 . The ecPNA of  claim 1 , wherein C comprises a sequence selected from the group consisting of the following sequences: DFDLDMLGDFDLDMLG (SEQ ID NO: 26), MLGDFDLDMLGDFDLD (SEQ ID NO: 30), CGSDALDDFDLDML (SEQ ID NO: 27), PEFPGIELLQELQALLQQ (SEQ ID NO: 28), and RHGEKWFLDDFTNNQMDQDY (SEQ ID NO: 29). 
     
     
         18 . The ecPNA of  claim 1 , wherein C comprises a sequence selected from the group consisting of the sequence of an engrailed repression domain, the sequence of the HID (HDAC interaction domain) of the Sin3A protein, and MSRRKQSKPRQIL (SEQ ID NO: 21). 
     
     
         19 . (canceled) 
     
     
         20 . The ecPNA of  claim 1 , wherein the gene is a γ-globin gene. 
     
     
         21 . The ecPNA of  claim 3 , wherein the PNA structure comprises the sequence TACTCTAAGACTATT (PNA78) (SEQ ID NO: 1). 
     
     
         22 - 26 . (canceled) 
     
     
         27 . A method of detecting the presence of a DNA within the nucleus of a cell which comprises contacting the cell with the ecPNA of  claim 1  under conditions such that the ecPNA enters the cell and the PNA structure B hybridizes to the DNA to form a hybridization product, and then detecting the resulting hybridization product. 
     
     
         28 . A method for upregulating transcription of a γ-globin gene in a mammalian bone marrow cell comprising contacting the cell with the ecPNA of  claim 20 . 
     
     
         29 . (canceled) 
     
     
         30 . A method for treating a β-globin disorder in a mammal comprising administering to said mammal a therapeutically effective amount of the ecPNA of  claim 20 . 
     
     
         31 - 35 . (canceled) 
     
     
         36 . A method for generating an inducible pluripotent stem (iPS) cell from a source cell comprising administering to the source cell an amount effective for inducing the generation of the iPS cell of an ecPNA of  claim 1 , wherein the ecPNA upregulates transcription of a gene selected from the group consisting of the following genes: OCT4, SOX2, c-NYC, KLF4, LIN28, NANOG, PRDM14, and NFRKB. 
     
     
         37 - 45 . (canceled) 
     
     
         46 . An ecPNA having the general structure:
   H 2 N—X—B—Y—COOH
   wherein X is D or C and Y is D or C with the proviso that when X is D, Y is C, and when X is C, Y is D;   wherein D represents a compound which renders the ecPNA able to bind to a receptor on a cell;   wherein B represents a peptide nucleic acid (PNA) structure at least 12 nucleotides in length, the sequence of which is capable of hybridizing with a DNA within the nucleus of the cell, which DNA is within a promoter region of a gene;   wherein C represents an structure; and   wherein each — represents a chemical linkage between the structures at each side thereof, which may be the same as or different from each other such linkage.   
     
     
         47 . The ecPNA of  claim 1 ,
 wherein A comprises a sequence selected from the group consisting of the following sequences: YGRKKRRQRRR (SEQ ID NO: 6), GRKKRRORRRPPQ (SEQ ID NO: 7), YARKARRQARR (SEQ ID NO: 8), YARAAARQARA (SEQ ID NO: 9), YARAARRAARR (SEQ ID NO: 10), YARAARRAARA (SEQ ID NO: 11), PKKKRKV (SEQ ID NO: 12), RQIKIWFQNRRMKWKK (SEQ ID NO: 13), KKWKMRRNQFWIKIQR (SEQ ID NO: 14), RQIKIWFQNRRMKWKK (SEQ ID NO: 15), RQIKIWFQNRRMKWKK (SEQ ID NO: 16), RQPKIWFPNRRMPWKK (SEQ ID NO: 17), RQIKINFQNMRRKWKK (SEQ ID NO: 18), RQIRIWFQNRRNRWRR (SEQ ID NO 19), RRWRRWWRRWWRRWR (SEQ ID NO: 20), RQILIWFQNRRMKWKK (SEQ ID NO: 22), LLIILRRRIRKQAHAHSK (SEQ ID NO: 23), KLALKLALKALKAALKLA (SEQ ID NO: 24), and AGYLLGKINLKALAALAKKIL (SEQ ID NO: 25);   wherein C represents an oligopeptide the sequence of which comprising a sequence selected from the group consisting of the following sequences: DFDLDMLGDFDLDMLG (SEQ ID NO: 26), MLGDFDLDMLGDFDLD (SEQ ID NO: 30), CGSDALDDFDLDML (SEQ ID NO: 27), PEFPGIELQELELQELQALLQQ (SEQ ID NO: 28), RHGEKNFLDDFTNNQMDQDY (SEQ ID NO: 29), an engrailed repression domain, the sequence of the HID (HDAC interaction domain) of the Sin3A protein, and MSRRKQSKPRQIL (SEQ ID NO: 21); and   wherein the PNA structure comprises the sequence TACTCTAAGACTATT (SEQ ID NO: 1).