Compositions and methods for expression of multiple biologically active polypeptides from a single vector for treatment of cardiac conditions and other pathologies

Abstract

The present invention provides compositions and methods useful for treating disorders amenable to therapy via introduction of multigenic expression vectors. More particularly, the invention provides vectors and polynucleotides encoding polypeptides for treatment of cardiac disorders wherein said polypeptides may comprise a cytokine, a chemokine, and/or an angiogenic polypeptide, or functional derivatives thereof. Also provided, as compositions of the invention, are linkers useful for connecting and expressing functional (biologically active) polypeptides from single, multigenic-expression constructs.

1 - 164 . (canceled) 
     
     
         165 . A method of treating a cardiac condition in a subject comprising: providing to said subject a pharmaceutical composition comprising a polynucleotide, wherein said polynucleotide encodes a S100 calcium binding protein (S100) polypeptide, a stromal cell derived factor (SDF) polypeptide, and a vascular endothelial growth factor (VEGF) polypeptide; or fragments and variants thereof. 
     
     
         166 . The method of  claim 165 , wherein said S100 polypeptide, said SDF polypeptide, and said VEGF polypeptide are separated from each other by one or more linkers. 
     
     
         167 . The method of  claim 166 , wherein said one or more linkers are selected from the group consisting of a rigid linker, a flexible linker, a peptide linker, a cleavable linker, a self-cleavable linker, a linker comprising a sequence that mediates a ribosome skipping translational effect, and any combination thereof. 
     
     
         168 . The method of  claim 167 , wherein said ribosome skipping translational effect linker comprises a 2A peptide. 
     
     
         169 . The method of  claim 166 , wherein said one or more linkers are selected from the group consisting of a RAKR linker, a fp2a linker, a p2a linker, a GSG-p2a linker, and a fmdv linker, and any combination thereof. 
     
     
         170 . The method of  claim 166 , wherein said one or more linkers comprise a sequence as shown in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. 
     
     
         171 . The method of  claim 166 , wherein said one or more linkers comprise a sequence selected from the group consisting of APVKQ (SEQ ID NO: 42), GPVKQ (SEQ ID NO: 43), VPVKQ (SEQ ID NO: 44), IPVKQ (SEQ ID NO: 45), MPVKQ (SEQ ID NO: 46), APIKQ (SEQ ID NO: 47), GPIKQ (SEQ ID NO: 48), VPIKQ (SEQ ID NO: 49), IPIKQ (SEQ ID NO: 50), MPIKQ (SEQ ID NO: 51), APAKQ (SEQ ID NO: 52), GPAKQ (SEQ ID NO: 53), VPAKQ (SEQ ID NO: 54), IPAKQ (SEQ ID NO: 55), MPAKQ (SEQ ID NO: 56), APVRQ (SEQ ID NO: 57), GPVRQ (SEQ ID NO: 58), VPVRQ (SEQ ID NO: 59), IPVRQ (SEQ ID NO: 60), MPVRQ (SEQ ID NO: 61), APIRQ (SEQ ID NO: 62), GPIRQ (SEQ ID NO: 63), VPIRQ (SEQ ID NO: 64), IPIRQ (SEQ ID NO: 65), MPIRQ (SEQ ID NO: 66), APARQ (SEQ ID NO: 67), GPARQ (SEQ ID NO: 68), VPARQ (SEQ ID NO: 69), IPARQ (SEQ ID NO: 70), MPARQ (SEQ ID NO: 71), APVKN (SEQ ID NO: 72), GPVKN (SEQ ID NO: 73), VPVKN (SEQ ID NO: 74), IPVKN (SEQ ID NO: 75), MPVKN (SEQ ID NO: 76), APIKN (SEQ ID NO: 77), GPIKN (SEQ ID NO: 78), VPIKN (SEQ ID NO: 79), IPIKN (SEQ ID NO: 80), MPIKN (SEQ ID NO: 81), APAKN (SEQ ID NO: 82), GPAKN (SEQ ID NO: 83), VPAKN (SEQ ID NO: 84), IPAKN (SEQ ID NO: 85), IVIPAKN (SEQ ID NO: 86), APVRN (SEQ ID NO: 87), GPVRN (SEQ ID NO: 88), VPVRN (SEQ ID NO: 89), IPVRN (SEQ ID NO: 90), IVIPVRN (SEQ ID NO: 91), APIRN (SEQ ID NO: 92), GPIRN (SEQ ID NO: 93), VPIRN (SEQ ID NO: 94), IPIRN (SEQ ID NO: 95), MPIRN (SEQ ID NO: 96), APARN (SEQ ID NO: 97), GPARN (SEQ ID NO: 98), VPARN (SEQ ID NO: 99), IPARN (SEQ ID NO: 100) or MPARN (SEQ ID NO: 101). 
     
     
         172 . The method of  claim 165 , wherein said S100 polypeptide is a S100A1 polypeptide or fragment or variant thereof. 
     
     
         173 . The method of  claim 165 , wherein said SDF polypeptide is a SDF1, a SDF-1α, a SDF-1β; or fragments or variants thereof. 
     
     
         174 . The method of  claim 165 , wherein said VEGF polypeptide is a VEGF121, a VEGF121b, a VEGF145, a VEGF165, a VEGF165b, a VEGF189, a VEGF191, a VEGF206, or fragments or variants thereof. 
     
     
         175 . The method of  claim 165 , wherein said subject is a human subject. 
     
     
         176 . A method comprising: contacting at least one cardiac cell with a polynucleotide that encodes a S100 calcium binding protein (S100) polypeptide, a stromal cell derived factor (SDF) polypeptide, and a vascular endothelial growth factor (VEGF) polypeptide; or fragments and variants thereof, wherein said S100 polypeptide, said SDF polypeptide, and said VEGF polypeptide are separated from each other by one or more cleavable linkers. 
     
     
         177 . The method of  claim 176 , wherein said polynucleotide is introduced in vivo. 
     
     
         178 . The method of  claim 177 , wherein said introduction in vivo is selected from a group consisting of percutaneous coronary artery catheterization, coronary venous blockade, cardiac recirculation, antegrade coronary artery infusion, retrograde perfusion, direct injection, ultrasound targeted microbubble destruction, and any combination thereof. 
     
     
         179 . The method of  claim 176 , wherein said cardiac cell is a myocardial cell. 
     
     
         180 . The method of  claim 176 , wherein said one or more cleavable linkers are cleaved to release said S100 polypeptide, said SDF polypeptide, and said VEGF polypeptide. 
     
     
         181 . A method of treating a cardiac disease or disorder in a subject comprising:
 administering to said subject an amount of a composition comprising at least one microbubble, wherein said microbubble comprises a lipid, a gas, and a plasmid comprising a polynucleotide; and   contacting said subject with an ultrasonic energy sufficient to result in ultrasound disruption of said at least one microbubble and delivering said plasmid to the heart of said subject,   
       wherein said polynucleotide encodes a S100 calcium binding protein (S100) polypeptide, a stromal cell derived factor (SDF) polypeptide, and a vascular endothelial growth factor (VEGF) polypeptide; or fragments and variants thereof. 
     
     
         182 . The method of  claim 181 , wherein said lipid forms a shell enclosing said gas and said plasmid. 
     
     
         183 . The method of  claim 181 , wherein said gas is a perfluorocarbon gas, or a perfluoropropane gas. 
     
     
         184 . The method of  claim 181 , wherein said microbubble comprises at least one of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine glycerol.