Systems and methods for nucleic acid expression in vivo

Abstract

The present invention provides compositions, systems, kits, and methods for expression of one or more biomolecules in a subject, human or non-human mammal, (e.g., at therapeutic levels for the extended periods of time required to produce therapeutic effects). In certain embodiments, compositions, systems, kits, and methods are provided that comprise a first composition comprising polycationic structures (e.g., empty cationic liposomes, cationic micelles, cationic emulsions, or cationic polymers) and a second composition comprising expression vectors (e.g., non-viral expression vectors not associated with liposomes or other carriers) encoding one or more biomolecules of interest.

1 . A system comprising:
 a) a first composition comprising a first amount of polycationic structures, wherein said first composition is free, or essentially free, of nucleic acid molecules; and   b) a second composition comprising a therapeutically effective amount of expression vectors,   wherein said expression vectors comprise nucleic acid sequences encoding one or more therapeutic biomolecules; and   at least one of the following:   i) wherein the ratio of said first amount of said polycationic structures to said therapeutically effective amount of expression vectors is 5:1 to 25:1;   ii) wherein 2.0% to 15.0% of said first composition comprises dexamethasone palmitate and/or dexamethasone;   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 expression vectors comprise circularized synthetically amplified nucleic acid, plasmid-based vector, or minicircle DNA. 
     
     
         3 . The system of  claim 1 , wherein said one or more therapeutic biomolecules comprise one or more monoclonal antibodies (mAb), or antigen-binding portion thereof. 
     
     
         4 . The system of  claim 3 , wherein said antigen-binding portion of said mAb is selected from a Fab, F(ab)2, and/or scFv. 
     
     
         5 . The system of  claim 3 , wherein said mAb or antigen-binding portion thereof specifically binds to a pathogen or pathogen component, a tumor antigen, or a cytokine. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The system of  claim 3 , wherein said one or more mAb or antigen-binding portion thereof comprise a first mAb or antigen-binding portion thereof that specifically binds to a first target molecule and a second mAb or antigen-binding portion thereof that specifically binds to a second, different, target molecule. 
     
     
         9 . The system of  claim 8 , wherein said one or more mAb or antigen-binding portion thereof comprise a first mAb or antigen-binding portion thereof that specifically binds to a first target molecule, a second mAb or antigen-binding portion thereof that specifically binds to a second target molecule, and a third mAb or antigen-binding portion thereof that specifically binds to a third target molecule, wherein said first, second, and third target molecules are different molecules. 
     
     
         10 . The system of  claim 1 , wherein said one or more therapeutic biomolecules comprise one or more CRISPR/Cas9 components in one or more expression cassettes in said expression vectors. 
     
     
         11 . The system of  claim 1 , wherein said one or more therapeutic biomolecules comprise a nucleic acid, optionally wherein said nucleic acid is an antisense oligonucleotide, ribozyme, an shRNA, miRNA, siRNA, piRNA, snoRNA, tsRNA, or srRNA. 
     
     
         12 . (canceled) 
     
     
         13 . The system of  claim 1 , wherein said expression vectors
 (a) encode a first therapeutic biomolecule and a second therapeutic biomolecule, wherein said first and second therapeutic biomolecules: i) express for different lengths of time than one another, and/or ii) are the same;   (b) comprise at least one of the following: an R6K origin of replication, an hr3 enhancer, a BV3 signal sequence, a Syn21 sequence, a delta-p10 sequence, or an MITD (MHC class I trafficking signal) sequence;   (c) are CpG-free or CpG-reduced; or   (d) contain a plurality of CpG motifs, and/or are not CpG-free or CpG-reduced.   
     
     
         14 - 16 . (canceled) 
     
     
         17 . A method of expressing one or more therapeutic biomolecules in a subject, comprising: a) administering a first composition of a system of  claim 1  into a subject; and b) administering a second composition of said system into said subject. 
     
     
         18 . A method of expressing a monoclonal antibody (mAb), Fab, F(ab)2, and/or scFv in a subject comprising:
 a) administering a first composition to a subject,
 wherein said first composition comprises a first amount of polycationic structures, and 
   wherein said first composition is free, or essentially free, of nucleic acid molecules; and   b) administering a second composition to said subject within about 300 minutes of administering said first composition,   wherein said second composition comprises a therapeutically effective amount of expression vectors encoding said mAb, said Fab, said F(ab)2, and/or scFv, and   wherein, as a result of said administering said first composition and said administering said second composition, said first therapeutic protein is expressed in said subject.   
     
     
         19 . The method of  claim 18 , wherein said subject has at least one symptom of a disease or condition, or has at least physiological trait to be altered, and wherein said first therapeutic protein is expressed in said subject at a therapeutic level with respect to said disease or condition, or at an effective level sufficient to alter said physiological or disease trait. 
     
     
         20 . The method of  claim 18 , wherein said first therapeutic protein is expressed in said subject at a prophylactic level with respect sufficient to prevent the subject from acquiring one or more infectious diseases. 
     
     
         21 . An aqueous composition comprising or consisting essentially of:
 a) polycationic structures or neutral lipid present in said composition at a concentration of between 500 nM and 500 mM;   b) dexamethasone and/or dexamethasone palmitate present in said composition at a concentration between 1-10% of said composition; and   c) a physiologically tolerable buffer, and   wherein said composition is free, or essentially free, of nucleic acid molecules.   
     
     
         22 . The composition of  claim 21 , wherein
 (a) said polycationic structure are cationic lipids that are present as small unilamellar vesicles;   (b) said polycationic structures comprise DOTAP; or   (c) said polycationic structures are present in said composition at a concentration of between 800 nM and 1500 nM, or between 10 mM and 100 mM.   
     
     
         23 . The composition of  claim 21 , wherein said physiologically tolerable buffer is selected from the group consisting of: saline buffer, 5% dextrose in water, lactated ringers buffer, and any combination thereof. 
     
     
         24 - 26 . (canceled) 
     
     
         27 . The composition of  claim 21 , wherein said neutral lipids
 (a) are present as multi-lamellar vesicles;   (b) comprise DMPC, or   (c) are present in said composition at a concentration of between 800 nM and 1500 nM, or between 10 mM and 100 mM.   
     
     
         28 - 41 . (canceled) 
     
     
         42 . A system comprising:
 a) a composition of  claim 18 , and   b) a syringe, wherein at least part of said composition is located inside said syringe.   
     
     
         43 . The system of  claim 42 , wherein said composition located inside said syringe contains a therapeutic and/or prophylactic dose of said polycationic structures. 
     
     
         44 - 47 . (canceled)