Systems and Methods for Nucleic Acid Expression In Vivo

Abstract

The present invention provides compositions, systems, kits, and methods for generating expression of one or more proteins and/or biologically active nucleic acid molecules in a subject (e.g., at therapeutic levels for extended periods required to produce therapeutic effects). In certain embodiments, systems and kits are provided that comprise a first composition comprising a first amount of polycationic structures, and a second composition comprising a therapeutically effective amount of expression vectors (e.g., non-viral expression vectors not associated with liposomes) that are CpG-free or CpG-reduced, where the expression vectors comprise a first nucleic acid sequence encoding: i) a first therapeutic protein or proteins, and/or ii) a first biologically active nucleic acid molecule or molecules.

We claim: 
     
         1 . A system comprising:
 a) a first composition comprising a first amount of polycationic structures liposomes, wherein said first composition is free, or essentially free, of nucleic acid molecules; and   b) a second composition comprises non-viral expression vectors,   wherein said non-viral expression vectors are CpG-free or CpG-reduced,   wherein each of said non-viral expression vectors comprises a first nucleic acid sequence encoding: i) a first therapeutic protein, and/or ii) a first biologically active nucleic acid molecule; and   at least one of the following:   i) wherein the ratio of said first amount of said polycationic structures to said non-viral expression vectors is 5:1 to 25:1;   ii) wherein 2.0% to 6.0% of said first composition comprises dexamethasone or dexamethasone palmitate;   iii) wherein said first composition further comprises neutral lipid; and   iv) wherein said polycationic structures comprise empty liposomes, and wherein said empty liposomes present in said first composition have a z-average diameter of about 20-85 nm.   
     
     
         2 . The system of  claim 1 , wherein said wherein said polycationic structures comprise empty liposomes, and wherein said empty liposomes present in said first composition have a z-average diameter of about 20-85 nm. 
     
     
         3 . The system of  claim 1 , wherein said non-viral expression vectors each further comprise an regulating nucleic acid sequence, wherein said regulating nucleic acid sequence reduces the duration of expression of said first nucleic acid sequence that would occur in the absence of said regulating nucleic acid sequence. 
     
     
         4 . The system of  claim 3 , wherein said regulating nucleic acid sequence is selected from the group consisting of: a promoter, an enhancer, a second nucleic acid sequence encoding a second protein, and/or a second biologically active nucleic acid molecule, 
     
     
         5 . The system of  claim 1 , wherein said first amount of polycationic structures in said first composition comprises a mixture of cationic lipid and neutral lipid that reduces the expression of said first therapeutic protein and/or first biologically active nucleic acid molecule compared to such expression when only said cationic lipid is employed in said method. 
     
     
         6 . The system of  claim 1 , wherein said first nucleic acid sequence encodes said therapeutic protein, and wherein said therapeutic protein is selected from the group consisting of: human G-CSF, Rituximab, and human Factor IX. 
     
     
         7 . The system of  claim 1 , wherein said non-viral expression vectors comprise a first nucleic acid sequence encoding said first therapeutic protein. 
     
     
         8 . The system of  claim 1 , wherein said non-viral expression vectors comprise a first nucleic acid sequence encoding said first therapeutic protein, wherein said first therapeutic protein comprises a monoclonal antibody light chain. 
     
     
         9 . The system of  claim 1 , wherein said non-viral expression vectors comprise a first nucleic acid sequence encoding said first therapeutic protein, wherein said first therapeutic protein comprises a monoclonal antibody heavy chain. 
     
     
         10 . The system of  claim 1 , wherein said non-viral expression vectors comprise: i) a first nucleic acid sequence encoding said first therapeutic protein, wherein said first therapeutic protein comprises a monoclonal antibody heavy chain, and ii) a second nucleic acid sequence encoding a second therapeutic protein, wherein said second therapeutic protein comprises a monoclonal antibody light chain. 
     
     
         11 . The system of  claim 1 , wherein said non-viral expression vectors comprise a first nucleic acid sequence encoding said first biologically active nucleic acid molecule. 
     
     
         12 . The system of  claim 1 , wherein said non-viral expression vectors comprise a first nucleic acid sequence encoding said first biologically active nucleic acid molecule, wherein said first biologically active nucleic acid molecule comprises a shRNA sequence. 
     
     
         13 . The system of  claim 1 , wherein said non-viral expression vectors comprise a first nucleic acid sequence encoding said first biologically active nucleic acid molecule, wherein said first biologically active nucleic acid molecule comprises a miRNA sequence. 
     
     
         14 . The system of  claim 1 , wherein said non-viral expression vectors comprise a first nucleic acid sequence encoding said first biologically active nucleic acid molecule, wherein said first biologically active nucleic acid molecule comprises an antisense sequence or ribozyme. 
     
     
         15 . The system of  claim 1 , wherein said non-viral expression vectors comprise a first nucleic acid sequence encoding said first biologically active nucleic acid molecule, wherein said first biologically active nucleic acid molecule comprises a CRISPR single guide RNA sequence. 
     
     
         16 . The system of  claim 1 , wherein said at least one of the following is i) wherein the ratio of said first amount of said polycationic structures to said non-viral expression vectors is 5:1 to 25:1. 
     
     
         17 . The system of  claim 1 , wherein said at least one of the following is ii) wherein 2.0% to 6.0% of said first composition comprises dexamethasone or dexamethasone palmitate. 
     
     
         18 . The system of  claim 1 , wherein said at least one of the following is iii) wherein said first composition further comprises neutral lipid. 
     
     
         19 . The system of  claim 1 , wherein said at least one of the following is iv) wherein said polycationic structures comprise empty liposomes, and wherein said empty liposomes present in said first composition have a z-average diameter of about 20-85 nm.