Methods for intracellular delivery and enhanced gene targeting

Abstract

Disclosed herein are methods and compositions for enhancing gene targeting. The method entails co-administrating to a cell a targeting molecule and a means of enhancing the function of the targeting molecule upon delivery to the cell. The means of enhancing the function of the targeting molecule including one or more of a stressor that induces cellular stress, a proton sponge molecule, and an endosome or lysosome inhibitor. Compositions disclosed include a targeting molecule and one or more of a stressor that induces cellular stress, a proton sponge molecule, and an endosome or lysosome inhibitor.

1 . A composition for enhancing gene targeting in a cell, comprising:
 a targeting molecule, wherein the targeting molecule binds a target molecule in the cell;   (A) a stressor, wherein the stressor induces a cellular stress response; and   (B) a proton sponge molecule,   wherein the composition enhances the function of the targeting molecule when administered to the cell,   wherein the stressor comprises arsenic trioxide, H 2 O 2 , glutathione, lipofectamine, or heat shock, and   wherein the proton sponge molecule comprises inorganic ammonium or an inorganic ammonium salt.   
     
     
         2 . The composition of  claim 1 , wherein the targeting molecule comprises an antisense oligonucleotide (ASO), a splicing switch oligonucleotide (SSO), an siRNA, an miRNA, or an shRNA. 
     
     
         3 . The composition of  claim 1 , wherein the composition is administered to the cell in vitro, in vivo, or ex vivo. 
     
     
         4 . The composition of  claim 1 , wherein the cell is a cultured cell or part of an argan or tissue. 
     
     
         5 . The composition of  claim 1 , wherein the target molecule comprises a nucleotide sequence. 
     
     
         6 . The composition of  claim 5 , wherein the nucleotide sequence is found in the cell nucleus or the cell nucleolus, or in the cell cytoplasm. 
     
     
         7 . The composition of  claim 6 , wherein the nucleotide sequence comprises an mRNA, an ncRNA, a piRNA, an miRNA, a viral RNA, or a promoter sequence. 
     
     
         8 . The composition of  claim 1 , wherein the composition is administered as a liquid, solid, vapor or any other formulation, or is administered orally, via injection, absorption, or inhalation. 
     
     
         9 . The composition of  claim 1 , wherein the cell is a cancer cell. 
     
     
         10 . The composition of  claim 9 , wherein the target molecule is selected from the group consisting of (i) a mutant β-globin, (ii) BCL-2, (iii) β-catenin, and (iv) androgen receptor. 
     
     
         11 . The composition of  claim 10 , wherein the targeting molecule is an ASO having a sequence comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. 
     
     
         12 . The composition of  claim 10 , further comprising a second targeting molecule, wherein the second targeting molecule binds a second target molecule in the cell. 
     
     
         13 . The composition of  claim 12 , wherein the second targeting molecule comprises an ASO, an SSO, an siRNA, an miRNA, or an shRNA. 
     
     
         14 . The composition of  claim 12 , wherein the second targeting molecule is a second ASO that binds a target molecule selected from the group consisting of (i) a mutant β-globin, (ii) BCL-2, (iii) β-catenin, and (iv) androgen receptor, and wherein the second targeting molecule binds a different target molecule from the first target molecule. 
     
     
         15 . The composition of  claim 14 , wherein the second ASO has a sequence comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.