Polynucleotides having bioreversible groups

Abstract

The disclosure provides methods and compositions for delivering polynucleotides into cells. The disclosure provides transiently protected polynucleotides comprising an anionic charge-neutralizing moiety/group, which may also confer additional functionality. These compounds can enter the cytosol of cells by endocytic or macropinocytic mechanisms. The transient protecting group is bioreversible, i.e., once inside a cell, it is designed to be removed by enzymatic activity or by passive intracellular methods (e.g., changes in pH or reductive environment).

1 - 93 . (canceled) 
     
     
         94 . A nucleotide construct comprising a structure of Formula (I): 
       
         
           
           
               
               
           
         
       
       or a salt thereof 
       wherein,
 B 1  is a nucleobase; 
 X is an O, S, or NR 5 ; 
 Y is a halo, optionally substituted C 1-6  alkoxy, or a protected hydroxyl group; 
 R 1  is hydroxyl, optionally substituted C 1-6  alkoxy, a protected hydroxyl group, a monophosphate, a disphosphate, a triphosphate, a tetraphosphate, or a pentaphosphate; 
 R 2  is a bioreversible group that comprises an auxiliary moiety selected from the group consisting of peptide, a polypeptide, a carbohydrate, a neutral organic polymer, a positively charged polymer, a therapeutic agent, a targeting moiety, an endosomal escape moiety, and any combination thereof, wherein the auxiliary moiety is linked to the bioreversible group by one or more covalent bonds; 
 R 3  is an O, S, or an optionally substituted amino; 
 R 4  is an H, hydroxyl, optionally substituted C 1-6  alkoxy, a protected hydroxyl group, a monophosphate, a disphosphate, a triphosphate, a tetraphosphate, a pentaphosphate, or absent when R 3  is an optionally substituted amino; and 
 R 5  is H, an optionally substituted C 1-6  alkyl, S-pivaloyl thioethanol, a hydroxyl, an optionally substituted C 1-6  alkoxy, an optionally substituted C 3-8  cycloalkyl, an optionally substituted C 6-12  aryl, or an optionally substituted C 2-9  heterocyclyl. 
 
     
     
         95 . The nucleotide construct of  claim 94 , comprising a structure of Formula I(a): 
       
         
           
           
               
               
           
         
       
       wherein R1 is a 4,4′-dimethoxytrityl group (DMT) protected hydroxyl group. 
     
     
         96 . (canceled) 
     
     
         97 . The nucleotide construct of  claim 94 , wherein R 2  comprises structural Formula V 
       
         
           
           
               
               
           
         
       
       wherein,
 G 1  is the peptide, the polypeptide, the carbohydrate, the neutral organic polymer, the positively charged polymer, the therapeutic agent, the targeting moiety, the endosomal escape moiety, or any combination thereof; 
 L 1  is an optionally substituted C 2-10  alkylene, optionally substituted C 2-10  alkenylene, or optionally substituted C 2-10  alkynylene, wherein each of alkylene, alkenylene, or alkynylene is optionally interrupted with 1-10 heteroatoms selected from O, N, and S; and 
 L 2  is a covalent bond, or is selected from optionally substituted C 1-10  alkylene; optionally substituted C 2-10  alkenylene; optionally substituted C 2-10  alkynylene; and optionally substituted C 6-12  arylene, wherein each of alkylene, alkenylene, or alkynylene is optionally interrupted with 1-10 heteroatoms selected from O, N, and S. 
 
     
     
         98 . The nucleotide construct of  claim 97 , wherein L 1  is optionally substituted C 2-10  alkylene. 
     
     
         99 . (canceled) 
     
     
         100 . The nucleotide construct of  claim 97 , wherein L 2  is a covalent bond. 
     
     
         101 . The nucleotide construct of  claim 97 , wherein L 2  is optionally substituted C 1-10  alkylene or optionally substituted C 6-12  arylene. 
     
     
         102 . The nucleotide construct of  claim 97 , wherein L 2  is not a bond, and G 1  is bound to L 2  via a bond formed by a reaction selected from the group consisting of a pericyclic reaction; an alkylation or arylation of a hydroxyl, thiol, or amino moiety; and a reaction of a hydroxyl, thiol, or amino nucleophile with an electrophile. 
     
     
         103 . The nucleotide construct of  claim 97 , wherein L 2  is not a bond, and G 1  is bound to L 2  via an amide bond, a sulfonamide bond, a carboxylic ester, a thioester, an optionally substituted C 6-12  aryl or C 2-9  heteroaryl, an imine, a hydrazone, an oxime or a succinimide. 
     
     
         104 . The nucleotide construct of  claim 97 , wherein G 1  is the peptide, the polypeptide, the neutral organic polymer, or any combination thereof;
 L 1  is an optionally substituted C 2-6  alkylene, optionally substituted C 2-6  alkenylene, or optionally substituted C 2-6  alkynylene, wherein each of alkylene, alkenylene, or alkynylene is optionally interrupted with 1 or 2 oxygen atoms; and   L 2  is a covalent bond, or is selected from optionally substituted C 1-6  alkylene; optionally substituted C 2-6  alkenylene; optionally substituted C 2-6  alkynylene; and optionally substituted C 6-10  arylene.   
     
     
         105 . The nucleotide construct of  claim 104 , wherein G 1  comprises a delivery domain. 
     
     
         106 . The nucleotide construct of  claim 105 , wherein the delivery domain comprises one or more a peptide transduction domains (PTDs). 
     
     
         107 . The nucleotide construct of  claim 104 , wherein the neutral organic polymer of G 1  is poly(ethyleneglycol). 
     
     
         108 . The nucleotide construct of  claim 97 , wherein L 1  is 
       
         
           
           
               
               
           
         
       
       wherein L 2′  is a covalent bond or is selected from optionally substituted C 1-10  alkylene; optionally substituted C 2-10  alkenylene; optionally substituted C 2-10  alkynylene; and optionally substituted C 6-12  arylene, wherein each of alkylene, alkenylene, or alkynylene is optionally interrupted with 1-10 heteroatoms selected from O, N, and S; 
       G 2  is a conjugating moiety, a hydrophilic functional group, a small molecule, a peptide, a polypeptide, a carbohydrate, a neutral organic polymer, a positively charged polymer, a therapeutic agent, a targeting moiety, an endosomal escape moiety, or combination thereof. 
     
     
         109 . The nucleotide construct of  claim 94 , wherein X is O. 
     
     
         110 . The nucleotide construct of  claim 94 , wherein the peptide, the polypeptide, the protein, the carbohydrate, the neutral organic polymer, the positively charged polymer, the therapeutic agent, the targeting moiety, the endosomal escape moiety, or combination thereof is linked to the bioreversible group through a condensation reaction with the aldehyde group to form an imine, enamine, or hydrazone bond. 
     
     
         111 . The nucleotide construct of  claim 94 , wherein the peptide, the polypeptide, the protein, the carbohydrate, the neutral organic polymer, the positively charged polymer, the therapeutic agent, the targeting moiety, the endosomal escape moiety, or combination thereof is linked to the bioreversible group by one or more nitrogen containing complementary conjugating moieties having the structure of Formula III: 
       
         
           
           
               
               
           
         
       
       wherein,
 A 1 , A 2 , A 4 , and A 5  are each individually a N or CR 8 ; 
 A 3  and A 6  are C; 
 R 6 -R 7  are each individually a H, optionally substituted C 1-6  alkyl, optionally substituted C 2-6  alkenyl, optionally substituted imine, and optionally substituted enamine; and 
 each R 8  is individually a H, optionally substituted C 1-6  alkyl, optionally substituted C 1-6  alkenyl, optionally substituted C 2-6  alkynyl, halide, hydroxyl, —CHO, optionally substituted C 1-6  acyl, carboxylic acid, cyano, nitro, optionally substituted amino, thiol, optionally substituted C 2-9  heterocyclyl, optionally substituted C 6-12  aryl, optionally substituted C 3-8  cycloalkyl, and optionally substituted C 4-8  cycloalkenyl. 
 
     
     
         112 . The nucleotide construct of  claim 94 , wherein R 2  comprises a delivery domain. 
     
     
         113 . The nucleotide construct of  claim 105 , wherein the delivery domain comprises one or more peptide transduction domains (PTDs). 
     
     
         114 . The nucleotide construct of  claim 113 , comprising a structure of Formula I(c): 
       
         
           
           
               
               
           
         
       
       wherein R 2′  is the residue of the bioreversible group bound to the PTD; and 
       z′ is a number from 1 to 10, wherein when z′ is greater than 1, the PTDs are linked together through poly(C 1-4  alkyleneoxide) groups having 1-10 repeating units. 
     
     
         115 . The nucleotide construct of  claim 113 , wherein the PTD is a cationic peptide sequence having 5-10 arginine and/or lysine residues over 5-15 amino acids. 
     
     
         116 . The nucleotide construct of  claim 113 , wherein the PTD comprises the sequence RKKRRQRRR (SEQ ID NO:1). 
     
     
         117 . (canceled) 
     
     
         118 . The nucleotide construct of  claim 113 , wherein delivery domain comprises a sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   PEG-(PTD); 
                 
                     
                     
                 
                     
                   GG-(PTD)-PEG-(PTD); 
                 
                     
                     
                 
                     
                   PEG-(PTD)-PEG-(PTD); 
                 
                     
                     
                 
                     
                   GG-(PTD)-PEG-PEG-PEG-(PTD); 
                 
                     
                     
                 
                     
                   PEG-(PTD)-PEG-PEG-PEG-(PTD); 
                 
                     
                     
                 
                     
                   GG-(PTD)-PEG-(PTD)-PEG-(PTD); 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   GG-(PTD)-PEG-PEG-PEG-(PTD)-PEG-PEG-PEG-(PTD); 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       wherein PEG is a poly(ethyleneglycol) linker having one to ten repeat units. 
     
     
         119 . The nucleotide construct of  claim 94 , wherein R 2  comprises the targeting moiety. 
     
     
         120 . The nucleotide construct of  claim 119 , wherein the targeting moiety is a ligand, carbohydrate, antibody, FAb, ScFv, or single-domain antibody. 
     
     
         121 . The nucleotide construct of  claim 94 , wherein B 1  is a non-natural nucleotide base. 
     
     
         122 - 124 . (canceled) 
     
     
         125 . A method of manufacturing a polynucleotide construct, comprising
 (1) de-blocking a first nucleoside or nucleotide which comprises a DMT protecting group at the 5′ position, by removing the DMT group under acidic conditions in an aprotic solvent system, wherein the first nucleoside or nucleotide may be fixed to a solid support at the 3′ position or alternatively the 3′ position is protected with non-acid labile hydroxyl protecting group and the first nucleoside or nucleotide is in solution;   (2) coupling the de-blocked first nucleoside or nucleotide with either an activated nucleotide of claim  88 - 114  or activated nucleotide comprising a phosphoramidite at the 3′ position in the presence of an acidic azole catalyst;   (3) oxidizing the coupled nucleotides with an oxidizing agent in a solvent system comprising a protic solvent and a weak base; and   (4) de-blocking the coupled nucleotides by removing a DMT group at the 5′ position of the coupled nucleotides under acidic conditions in an aprotic solvent system;   wherein steps (2)-(4) are repeated from 1 to 149 times, and wherein the polynucleotide construct comprises at least one nucleotide construct from  claim 94 .   
     
     
         126 . The method of manufacture of  claim 125 , wherein the first nucleoside or nucleotide of step (1) has been attached to a solid support, and wherein the resulting polynucleotide construct is cleaved from the solid support after the last de-blocking step. 
     
     
         127 . (canceled) 
     
     
         128 . The method of manufacture of  claim 125 , wherein post synthesis of the polynucleotide construct, if a nucleobase comprises one or more protecting groups, then the protecting groups are removed; and/or for any bioreversible groups which comprise a hydrophilic functional group or conjugating moiety that is protected by a protecting group, then the protecting group is removed. 
     
     
         129 . The method of manufacture of  claim 125  or  128 , wherein post synthesis of the polynucleotide construct, a small molecule, a peptide, a polypeptide, a carbohydrate, a neutral organic polymer, a positively charged polymer, a therapeutic agent, a targeting moiety, and/or an endosomal escape moiety is linked to one or more conjugating moieties of one or more bioreversible groups. 
     
     
         130 . (canceled)