Compositions and methods for the delivery of molecules into live cells

Abstract

The present disclosure provides methods and compositions related to the cytosolic delivery of proteins and cell-impermeable small molecules into live cells using an endosomolytic dimer of cell-penetrating peptide TAT.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 
     
         1 . A composition having the formula:
   X-Y-Z   wherein   X is a cysteine (C),   Y is an amino acid residue covalently coupled to a hydrophobic moiety,   Z is a cell-penetrating peptide (CPP) moiety, wherein the CPP moiety has a net positive charge and has an amino acid sequence with 50% or more residues having a guanidinium group, and   X and Y, and Y and Z are linked by amide bonds.   
     
     
         2 . The composition of  claim 1 , wherein the CPP moiety comprises an amino acid sequence with at least 85% identity to the sequence set forth in SEQ ID NO:1. 
     
     
         3 . The compound of  claim 2 , wherein the CPP moiety comprises an amino acid sequence with the sequence set forth in SEQ ID NO:1. 
     
     
         4 . The compound of  claim 1 , wherein Y is lysine (K). 
     
     
         5 . The compound of  claim 1 , wherein the hydrophobic moiety comprises a rhodamine group. 
     
     
         6 . The compound of  claim 5 , wherein the rhodamine group is tetramethylrhodamine (TMR). 
     
     
         7 . The compound of  claim 1 , where the compound is capable of forming a homodimer by the formation of a disulfide bond between the N-terminal cysteine (C) residues. 
     
     
         8 . A method of enhancing endosomal permeability in a cell, the method comprising contacting a cell with the compound of  claim 1 . 
     
     
         9 . The method of  claim 8 , wherein the cell is contacted under conditions sufficient to permit endocytosis of the compound. 
     
     
         10 . The method of  claim 8 , wherein the cell is contacted with a concentration of the compound of at least 1 μM. 
     
     
         11 . The method of  claim 10 , wherein the cell is contacted with a concentration of the compound of at least 5 μM. 
     
     
         12 . The method of  claim 8 , wherein the cell is in a culture that lacks albumin. 
     
     
         13 . The method of  claim 8 , further comprising contacting the cell with a cell-impermeable molecule. 
     
     
         14 . The method of  claim 8 , wherein the cell is obtained from a living subject and is contacted with the compound ex vivo. 
     
     
         15 . The method of  claim 8 , wherein the cell is in a living subject and is contacted in vivo. 
     
     
         16 . The method of  claim 15 , wherein the cell is contacted in vivo by administering an amount of the compound to the subject effective to enhance the endosomal permeability of the cell. 
     
     
         17 . The method of  claim 14 , wherein the subject is a mammal. 
     
     
         18 . The method of  claim 15 , wherein the subject is a mammal.