Novel peptide-conjugated oligomeric compounds

Abstract

The present invention relates to novel amphipathic peptide-conjugated oligomeric compounds and to methods of making and using such compounds. The invention further relates to methods of enhancing the cellular uptake of oligomeric compounds comprising conjugating the compounds to amphipathic moieties such as amphipathic peptides.

1 . An oligomeric compound having at least one amphipathic peptide covalently bound thereto, wherein: 
 each of said amphipathic peptides independently has formula II:      Q 1 -X 1 —(X 2 ) 2 —(X 3 ) 2 —X 2 —X 4 —X 3 —X 4 —X 2 —X 4 —X 3 —(X 2 ) 2 -Q 2   (II)    wherein    one of Q 1  and Q 2  is H and the other of Q 1  and Q 2  is a covalent attachment to the oligomeric compound either directly or through a linking moiety;    each X 1  is, independently, a naturally occurring or non-naturally occurring amino acid;    each X 2  is, independently, a D or L amino acid selected from lysine, histidine, homolysine, diaminobutyric acid, arginine, omithine or homoarginine;    each X 3  is, independently, a D or L amino acid selected from alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, cysteine, or methionine; and    each X 4  is, independently, a D or L amino acid selected from lysine, histidine, homolysine, diaminobutyric acid, arginine, ornithine, homoarginine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, glycine, serine, threonine, aspartate, glutamate, asparagine, or glutamine; and    said oligomeric compound being capable hybridizing a region of a nucleic acid molecule; and    said amphipathic peptide being capable of forming an α-helical structure having at least one hydrophobic face and at least one hydrophilic face.    
     
     
         2 . The oligomeric compound of  claim 1  wherein the oligomeric compound is an oligonucleotide, an oligonucleotide analog, a peptide nucleic acid, a morpholino nucleic acid, a cyclohexenyl nucleic acid. an anhydrohexitol nucleic acid, a locked nucleic acid, a bicyclic nucleic acid, a tricyclic nucleic acid, a phosphonomonoester nucleic acid or a cyclobutyl nucleic acid.  
     
     
         3 . The compound of  claim 2  wherein at least one amphipatic peptide is linked to the oligomeric compound via a linking moiety that has formula III:  
       
         
           
           
               
               
           
         
       
     
     
         4 . The oligomeric compound of  claim 1  where in: 
 each X 1  is, independently glycine or a D or L amino acid selected from alanine, leucine, phenylalanine or tryptophan;    each X 2  is, independently, a D or L amino acid selected from lysine, histidine, homolysine, diaminobutyric acid, arginine, omithine or homoarginine;    each X 3  is, independently, a D or L amino acid selected from alanine, leucine, phenylalanine or tryptophan; and    each X 4  is, independently, a D or L amino acid selected from lysine, arginine, ornithine, homoarginine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, cysteine, methionine or glycine.    
     
     
         5 . The oligomeric compound of  claim 4  comprising at least one amphipathic peptide that is:  
       
         
           
                 
                 
                 
               
                     
                 
                   GKKAFKGAGKGFKK; 
                   (SEQ ID NO: 1) 
                     
                 
                     
                 
                   GGKAFKGAGKGFKG; 
                   (SEQ ID NO: 3) 
                 
                     
                 
                   GKKAFKGAGKGFKG; 
                   (SEQ ID NO: 4) 
                 
                     
                 
                   GRRAFRGAGRGFRG; 
                   (SEQ ID NO: 5) 
                 
                     
                 
                   GKKAFKGAGKKFKK; 
                   (SEQ ID NO: 6) 
                 
                     
                 
                   GKKAFKKAKKKFKK; 
                   (SEQ ID NO: 7) 
                 
                     
                 
                   GRRAFRRARRRFRR; 
                   (SEQ ID NO: 8) 
                 
                     
                 
                   GKKAWKGAGKGWKK; 
                   (SEQ ID NO: 9) 
                 
                     
                 
                   GKKAWKAWAKAWKK; 
                   (SEQ ID NO: 10) 
                 
                     
                 
                   GKKLFKGLGKGFKK; 
                   (SEQ ID NO: 11) 
                 
                     
                 
                   GKKLFKLFLKLFKK; 
                   (SEQ ID NO: 12) 
                 
                     
                 
                   GKKLWKGLGKGWKK; 
                   (SEQ ID NO: 13) 
                 
                     
                 
                   GKKLWKLWLKLWKK; 
                   (SEQ ID NO: 14) 
                 
                     
                 
                   GKKWFKGWGKGFKK; 
                   (SEQ ID NO: 15) 
                 
                     
                 
                   GKKWFKWFWKFFKK; 
                   (SEQ ID NO: 16) 
                 
                     
                 
                   GKKALKGAGKGLKK; 
                   (SEQ ID NO: 17) 
                 
                     
                 
                   GKKAFKQAQKQFKK; 
                   (SEQ ID NO: 19) 
                 
                     
                 
                   GKKAFKGAEKGFKK; 
                   (SEQ ID NO: 20) 
                 
                     
                 
                   GKKAFKEAGKGFKK; 
                   (SEQ ID NO: 21) 
                 
                     
                 
                   GKKAFKEAEKGFKK; 
                   (SEQ ID NO: 22) 
                 
                     
                 
                   GdKdKdAdFdKdKdAdKdKdKdFdKdK; 
                   (SEQ ID NO: 30) 
                 
                     
                 
                   GdRdRdAdFdRdRdAdRdRdRdFdRdR; 
                   (SEQ ID NO: 31) 
                 
                     
                 
                   GdKdKdLdFdKdLdFdLdKdLdFdKdK; 
                   (SEQ ID NO: 32) 
                 
                     
                 
                   dKdKdFdKdKdKdAdKdKdFdAdKdKG; 
                   (SEQ ID NO: 33) 
                 
                     
                 
                   dKdKdLdLdKdAdAdLdKdLdAdKdKG; or 
                   (SEQ ID NO: 34) 
                 
                     
                 
                   dRdRdFdRdRdRdAdRdRdFdAdRdRG. 
                   (SEQ ID NO: 35) 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         6 . The oligomeric compound of  claim 5  comprising at least one amphipathic peptide that is:  
       
         
           
                 
                 
                 
               
                     
                 
                   GRRAFRRARRRFRR; 
                   (SEQ ID NO: 8) 
                     
                 
                     
                 
                   GKKLFKLFLKLFKK; 
                   (SEQ ID NO: 12) 
                 
                     
                 
                   GKKLWKLWLKLWKK; 
                   (SEQ ID NO: 14) 
                 
                     
                 
                   GKKWFKWFWKFFKK; 
                   (SEQ ID NO: 16) 
                 
                     
                 
                   GdKdKdAdFdKdKdAdKdKdKdFdKdK; 
                   (SEQ ID NO: 30) 
                 
                     
                 
                   GdRdRdAdFdRdRdAdRdRdRdFdRdR; 
                   (SEQ ID NO: 31) 
                 
                     
                 
                   GdKdKdLdFdKdLdFdLdKdLdFdKdK; 
                   (SEQ ID NO: 32) 
                 
                     
                 
                   dKdKdFdKdKdKdAdKdKdFdAdKdKG; 
                   (SEQ ID NO: 33) 
                 
                     
                 
                   dKdKdLdLdKdAdAdLdKdLdAdKdKG; or 
                   (SEQ ID NO: 34) 
                 
                     
                 
                   dRdRdFdRdRdRdAdRdRdFdAdRdRG. 
                   (SEQ ID NO: 35) 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         7 . The oligomeric compound of  claim 1  comprising at least two of said amphipathic peptides.  
     
     
         8 . The oligomeric compound of  claim 1  wherein at least one ampipathic peptide is attached to a terminal position of the oligomeric compound.  
     
     
         9 . The oligomeric compound of  claim 7  where an amphipathic peptide is attached to two terminal ends of the oligomeric compound.  
     
     
         10 . The oligomeric compound of  claim 1  further comprising a targeting moiety.  
     
     
         11 . The oligomeric compound of  claim 10  wherein the targeting moiety is covalently attached to an amphipathic peptide.  
     
     
         12 . An oligomeric compound having at least one amphipathic peptide covalently bound thereto, wherein: 
 each of said amphipathic peptides independently has formula II:      Q 1 -X 1 —(X 2 ) 2 —(X 3 ) 2 —X 2 —X 4 —X 3 —X 4 —X 2 —X 4 —X 3 —(X 2 ) 2 -Q 2   (II)    wherein    one of Q 1  and Q 2  is H and the other of Q 1  and Q 2  is a covalent attachment to the oligomeric compound either directly or through a linking moiety;    each X 1  is, independently, a naturally occurring or non-naturally occurring amino acid;    each X 2  is, independently, a D or L amino acid selected from lysine, histidine, homolysine, diaminobutyric acid, arginine, omithine or homoarginine;    each X 3  is, independently, a D or L amino acid selected from alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, cysteine, or methionine; and    each X 4  is, independently, a D or L amino acid selected from lysine, histidine, homolysine, diaminobutyric acid, arginine, ornithine, homoarginine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, glycine, serine, threonine, aspartate, glutamate, asparagine, or glutamine;    said oligomeric compound being capable hybridizing a region of a nucleic acid molecule;    said amphipathic peptide being capable of forming an α-helical structure having at least one hydrophobic face and at least one hydrophilic face; and    said oligomeric compound further comprises at least one targeting moiety attached thereto.    
     
     
         13 . The oligomeric compound of  claim 12  wherein the targeting moiety is covalently linked to the oligomeric compound.  
     
     
         14 . The oligomeric compound of  claim 12  wherein the targeting moiety is covalently linked to the amphipathic peptide.  
     
     
         15 . The oligomeric compound of  claim 12  wherein the targeting moiety is a ligand that binds to a cell-surface protein.  
     
     
         16 . The oligomeric compound of  claim 15  wherein said targeting moiety is a mono- or multivalent ligand carrying one or multiple units of transferrin, folate, methotrexate, epidermal growth factor, nerve growth factor, insulin, alpha-fetoprotein, galactose, galactosamine, N-acetylgalactose, lactose, mannose, glucose, fucose, rhamnose, a polyclonal antibody, or a monoclonal antibody.  
     
     
         17 . The oligomeric compound of  claim 12  wherein the targeting moiety is a mono- or multivalent ligand carrying one or multiple units of Vitamin B 12 , ibuprofen, cholesterol, or low-density lipoprotein.  
     
     
         18 . The oligomeric compound of  claim 12  wherein the targeting moiety is a peptide comprising an arginine-glycine-aspartic acid sequence.  
     
     
         19 . The oligomeric compound of  claim 12  wherein the oligomeric compound is an oligonucleotide, an oligonucleotide analog, a peptide nucleic acid, a morpholino nucleic acid, a cyclohexenyl nucleic acid. an anhydrohexitol nucleic acid, a locked nucleic acid, a bicyclic nucleic acid, a tricyclic nucleic acid, a phosphonomonoester nucleic acid or a cyclobutyl nucleic acid.  
     
     
         20 . The oligomeric compound of  claim 19  wherein the oligomeric compound is an oligonucleotide, an oligonucleotide analog, a peptide nucleic acid, a morpholino nucleic acid or a locked nucleic acid, a bicyclic nucleic acid.  
     
     
         21 . The compound of  claim 12  wherein at least one amphipatic peptide is linked to the oligomeric compound via a linking moiety that has formula III:  
       
         
           
           
               
               
           
         
       
     
     
         22 . The oligomeric compound of  claim 12  where in: 
 each X 1  is, independently glycine or a D or L amino acid selected from alanine, leucine, phenylalanine or tryptophan;    each X 2  is, independently, a D or L amino acid selected from lysine, histidine, homolysine, diaminobutyric acid, arginine, omithine or homoarginine;    each X 3  is, independently, a D or L amino acid selected from alanine, leucine, phenylalanine or tryptophan; and    each X 4  is, independently, a D or L amino acid selected from lysine, arginine, omithine, homoarginine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, cysteine, methionine or glycine.