Codon optimized synthetic plasmids

Abstract

One aspect of the current invention is an optimized synthetic mammalian expression plasmid (e.g. pAV0201). This new plasmid comprise a therapeutic element, and a replication element. The therapeutic element of the new plasmid comprises a eukaryotic promoter; a 5′ untranslated region (“UTR”); a codon-optimized-eukaryotic therapeutic gene sequence; and a poly adenylation signal. The therapeutic elements of this plasmid are operatively linked and located in a first operatively-linked arrangement. Additionally, the optimized synthetic mammalian expression plasmid comprises replication elements, wherein the replication elements are operatively linked and located in a second operatively-linked arrangement. The replication elements comprise a selectable marker gene promoter, a ribosomal binding site, and an origin of replication. The first-operatively-linked arrangement and the second-operatively-linked arrangement comprise a circular structure of the codon optimized synthetic mammalian expression plasmid.

1 .- 31 . (canceled) 
     
     
         32 . A method for plasmid mediated gene supplementation comprising:
 delivering into a subject a codon optimized synthetic mammalian expression plasmid; wherein the codon optimized synthetic mammalian expression plasmid encodes a growth hormone releasing hormone (“GHRH”) or functional biological equivalent in the subject.   
     
     
         33 . The method of  claim 32 , wherein delivering into the cells of the subject the codon optimized synthetic mammalian expression plasmid is via electroporation. 
     
     
         34 . The method of  claim 32 , wherein the cells of the subject are somatic cells, stem cells, or germ cells. 
     
     
         35 . The method of  claim 32 , wherein the codon optimized synthetic mammalian expression plasmid is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21. 
     
     
         36 . The method of  claim 32 , wherein the encoded GHRH is a biologically active polypeptide; and the encoded functional biological equivalent of GHRH is a polypeptide that has been engineered to contain a distinct amino acid sequence while simultaneously having similar or improved biologically activity when compared to the GHRH polypeptide. 
     
     
         37 . The method of  claim 32 , wherein the encoded GHRH or functional biological equivalent thereof facilitates growth hormone (“GH”) secretion in the subject. 
     
     
         38 . The method of  claim 32 , wherein the codon optimized synthetic mammalian expression plasmid encodes a protein sequence chosen from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. 
     
     
         39 . The method of  claim 32 , wherein the codon optimized synthetic mammalian expression plasmid comprises a 5′ untranslated region (UTR). 
     
     
         40 . The method of  claim 39 , wherein the 5′ UTR comprises a portion of a human growth hormone 5′ UTR. 
     
     
         41 . The method of  claim 32 , wherein the codon optimized synthetic mammalian expression plasmid comprises an origin of replication comprising SEQ ID NO:12. 
     
     
         42 . The method of  claim 32 , wherein the codon optimized synthetic mammalian expression plasmid comprises a prokaryotic ribosomal binding site (“RBS”). 
     
     
         43 . The method of  claim 32 , wherein the codon optimized synthetic mammalian expression plasmid comprises a eukaryotic poly A signal. 
     
     
         44 . The method of  claim 39 , wherein the 5′ UTR comprises a portion of a eukaryotic 5′ UTR. 
     
     
         45 . The method of  claim 32 , wherein the codon optimized synthetic mammalian expression plasmid comprises a prokaryotic promoter that comprises PNEO and a transposon fragment (“Tn5”). 
     
     
         46 . The method of  claim 32 , wherein the codon optimized synthetic mammalian expression plasmid comprises a selectable marker gene. 
     
     
         47 . The method of  claim 32 , wherein the codon optimized synthetic mammalian expression plasmid comprises SEQ ID NO: 2. 
     
     
         48 . The method of  claim 32 , wherein the delivering into a subject is accomplished by administration selected from the group consisting of: electroporation, a gene gun, and applying large volumes of liquid.