Compositions and methods for enhanced gene expression

Abstract

The present disclosure provides polynucleotide cassettes, expression vectors and methods for the expression of a gene in mammalian cells.

What is claimed is: 
     
         1 . A non-naturally occurring polynucleotide cassette for enhanced expression of a transgene in a mammalian cell, comprising in 5′ to 3′ order:
 (a) a first enhancer region; 
 (b) a promoter region; 
 (c) a coding sequence encoding a secretory polypeptide; 
 (d) a second enhancer region; and 
 (e) a polyadenylation site, 
 wherein the coding sequence is operably linked to the promoter region, 
 wherein the expression of the secretory polypeptide from the polynucleotide cassette in mammalian cells is at least 5× higher than the expression of the secretory polypeptide from a reference cassette in the mammalian cells, and 
 wherein the reference cassette comprises, in 5′ to 3′ order, a CMV enhancer sequence (SEQ ID NO:2), a CMV promoter (SEQ ID NO:21), a chimeric intron (SEQ ID NO:22), a 5′UTR (SEQ ID NO:23), a coding sequence encoding the secretory polypeptide, a 3′UTR (SEQ ID NO:25), and an SV40 polyA sequence (SEQ ID NO:26). 
 
     
     
         2 . The polynucleotide cassette of  claim 1 , wherein the secretory polypeptide is an anti-angiogenic polypeptide. 
     
     
         3 . The polynucleotide cassette according to  claim 1 , comprising in 5′ to 3′:
 (a) a first enhancer region comprising a CMV sequence consisting of SEQ ID NO:1 or a sequence having at least 85% identity thereto; 
 (b) a promoter region comprising a CMV sequence consisting of SEQ ID NO:4 or a sequence having at least 85% identity thereto; 
 (c) a 5′UTR region comprising, in 5′ to 3′ order, a TPL sequence consisting of SEQ ID NO:11 or a sequence having at least 85% identity thereto, and an eMLP sequence consisting of SEQ ID NO:12 or a sequence having at least 85% identity thereto; 
 (d) a coding sequence encoding said secretory polypeptide, wherein the coding sequence is operably linked to the promoter region; 
 (e) a second enhancer region comprising a full EES sequence consisting of SEQ ID NO:13 or a sequence having at least 85% identity thereto; and 
 (f) a HGH polyadenylation site consisting of SEQ ID NO:14 or a sequence having at least 85% identity thereto, and 
 optionally wherein the cassette does not comprise an RNA export signal. 
 
     
     
         4 . The polynucleotide cassette of  claim 3 , further comprising each of SEQ ID NO: 76-80. 
     
     
         5 . The polynucleotide cassette according to  claim 1 , comprising in 5′ to 3′:
 (a) a first enhancer region comprising a CMV sequence consisting of SEQ ID NO:1 or a sequence having at least 85% identity thereto; 
 (b) a promoter region, comprising an EF1c sequence consisting of SEQ ID NO:3 or a sequence having at least 85% identity thereto; 
 (c) an intron region comprising an EF1c sequence consisting of SEQ ID NO:5 or a sequence having at least 85% identity thereto; 
 (d) a 5′UTR region comprising an UTR2 sequence consisting of SEQ ID NO:6 or a sequence having at least 85% identity thereto; 
 (e) a coding sequence encoding said secretory polypeptide, wherein the coding sequence is operably linked to the promoter region; 
 (f) a second enhancer region comprising a 511-810 EES sequence consisting of SEQ ID NO:7 or a sequence having at least 85% identity thereto; 
 (g) an WPRE RNA export sequence consisting of SEQ ID NO:8 or a sequence having at least 85% identity thereto; and 
 (h) a BGH polyadenylation site consisting of SEQ ID NO:9 or a sequence having at least 85% identity thereto. 
 
     
     
         6 . The polynucleotide cassette of  claim 5 , further comprising each of SEQ ID NO: 70-75. 
     
     
         7 . The polynucleotide cassette according to  claim 1 , comprising in 5′ to 3′:
 (a) a first enhancer region comprising a CMV sequence consisting of SEQ ID NO:1 or a sequence having at least 85% identity thereto; 
 (b) a promoter region, comprising a CMV sequence consisting of SEQ ID NO:4 or a sequence having at least 85% identity thereto; 
 (c) a 5′UTR region comprising, in 5′ to 3′ order, TPL and eMLP sequences consisting of SEQ ID NO:11 and SEQ ID NO:12, respectively or a sequence having at least 85% identity thereto; 
 (e) a coding sequence encoding a peptide or polypeptide; 
 (f) a second enhancer region comprising a 410-564 EES sequence consisting of SEQ ID NO:16 or a sequence having at least 85% identity thereto; 
 (g) an HPRE RNA export sequence consisting of SEQ ID NO:17 or a sequence having at least 85% identity thereto; and 
 (h) a BGH polyadenylation site consisting of SEQ ID NO:9 or a sequence having at least 85% identity thereto. 
 
     
     
         8 . The polynucleotide cassette of  claim 7 , further comprising each of SEQ ID NO: 81-86. 
     
     
         9 . The polynucleotide cassette according to  claim 1 , comprising in 5′ to 3′:
 (a) a first enhancer region comprising a CMV sequence consisting of SEQ ID NO:1 or a sequence having at least 85% identity thereto; 
 (b) a promoter region, comprising an actin sequence consisting of SEQ ID NO:96 or a sequence having at least 85% identity thereto; 
 (c) a 5′ UTR comprising an eMLP sequences consisting of SEQ ID NO:12 or a sequence having at least 85% identity thereto; 
 (e) a coding sequence encoding a peptide or polypeptide; 
 (f) a second enhancer region comprising a 511-810 EES sequence consisting of SEQ ID NO:7 or a sequence having at least 85% identity thereto; 
 (g) an HPRE RNA export sequence consisting of SEQ ID NO:17 or a sequence having at least 85% identity thereto; and 
 (h) a rabbit Beta Globin polyadenylation site consisting of SEQ ID NO:20 or a sequence having at least 85% identity thereto. 
 
     
     
         10 . The polynucleotide cassette of  claim 9 , further comprising each of SEQ ID NO: 87-91. 
     
     
         11 . The polynucleotide cassette according to  claim 1 , comprising in 5′ to 3′:
 (a) a first enhancer region comprising a CMV sequence consisting of SEQ ID NO:1 or a sequence having at least 85% identity thereto; 
 (b) a promoter region, comprising a CMV sequence consisting of SEQ ID NO:4 or a sequence having at least 85% identity thereto; 
 (c) an intron region comprising an CMVc sequence consisting of SEQ ID NO:18 or a sequence having at least 85% identity thereto; 
 (d) a 5′UTR region comprising a UTR1 sequence consisting of SEQ ID NO:19 or a sequence having at least 85% identity thereto; 
 (e) a coding sequence encoding a peptide or a polypeptide; 
 (f) a second enhancer region comprising a full EES sequence consisting of SEQ ID NO:13 or a sequence having at least 85% identity thereto; 
 (g) an WPRE RNA export sequence consisting of SEQ ID NO:8 or a sequence having at least 85% identity thereto; and 
 (h) a rabbit Beta Globin polyadenylation site consisting of SEQ ID NO:20 or a sequence having at least 85% identity thereto. 
 
     
     
         12 . The polynucleotide cassette of  claim 11 , further comprising each of SEQ ID NO: 92-95. 
     
     
         13 . A recombinant virus comprising:
 a) a capsid protein, and   b) the polynucleotide cassette according to  claim 1 .   
     
     
         14 . The recombinant virus of  claim 13  wherein the recombinant virus is a recombinant adeno-associated virus. 
     
     
         15 . The recombinant virus of  claim 14  wherein the capsid protein is an AAV variant 7m8 capsid protein or is derived from the AAV variant 7m8 capsid protein. 
     
     
         16 . A pharmaceutical composition comprising the recombinant virus of  claim 13  and a pharmaceutically acceptable excipient. 
     
     
         17 . A method for expressing a transgene in mammalian cells, comprising contacting one or more mammalian cells with an amount of the recombinant virus of claim  13 , wherein the secretory polypeptide is expressed in the one or more mammalian cells at a level that is at least 5× higher than that obtained by contacting the cells with a recombinant virus comprising a reference cassette encoding the secretory polypeptide, wherein the reference cassette comprises, in 5′ to 3′ order, a CMV enhancer sequence (SEQ ID NO:2), a CMV promoter (SEQ ID NO:21), a chimeric intron (SEQ ID NO:22), a 5′UTR (SEQ ID NO:23), a coding sequence encoding the secretory polypeptide, a 3′UTR (SEQ ID NO:25), and an SV40 polyA sequence (SEQ ID NO:26). 
     
     
         18 . A method for the treatment or prophylaxis of a disease in a mammal in need of treatment or prophylaxis for a disease, comprising administering to the mammal an effective amount of the pharmaceutical composition of  claim 16 . 
     
     
         19 . The method of  claim 18 , wherein the disease is an ocular disease and the pharmaceutical composition is administered to the eye of the mammal. 
     
     
         20 . The method of  claim 19 , wherein the ocular disease is selected from the group consisting of choroidal neovascularization and macular degeneration.