Compositions and methods for enhanced gene expression
The present disclosure provides polynucleotide cassettes, expression vectors and methods for the expression of a gene in mammalian cells.
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 comprising a cytomegalovirus (CMV) sequence consisting of SEQ ID NO:1 or a sequence having at least 99% identity to SEQ ID NO:1;
(b) a promoter region, comprising an elongation factor 1 alpha (EF1α) sequence consisting of SEQ ID NO:3 or a sequence having at least 99% identity to SEQ ID NO:3;
(c) an intron region comprising an EF1α sequence consisting of SEQ ID NO:5 or a sequence having at least 99% identity to SEQ ID NO:5;
(d) a 5′untranslated region (UTR) region comprising an UTR2 sequence consisting of SEQ ID NO:6 or a sequence having at least 99% identity to SEQ ID NO:6;
(e) a coding sequence encoding a secretory polypeptide, wherein the coding sequence is operably linked to the promoter region;
(f) a second enhancer region comprising a 511-810 expression enhancer sequence (EES) sequence consisting of SEQ ID NO:7 or a sequence having at least 99% identity to SEQ ID NO:7;
(g) a woodchuck hepatitis virus post-transcriptional element (WPRE) RNA export sequence consisting of SEQ ID NO:8 or a sequence having at least 99% identity to SEQ ID NO:8; and
(h) a bovine growth hormone (BGH) polyadenylation site consisting of SEQ ID NO:9 or a sequence having at least 99% identity to SEQ ID NO:9.
2. The polynucleotide cassette of claim 1 , further comprising each of SEQ ID NO: 70-75.
3. A recombinant adeno-associated virus (AAV) comprising:
a) a capsid protein, wherein the capsid protein is an AAV variant 7m8 capsid protein or is derived from the AAV variant 7m8 capsid protein, and
b) the polynucleotide cassette according to claim 1 .
4. A pharmaceutical composition comprising the recombinant virus of claim 3 and a pharmaceutically acceptable excipient.
5. A method for expressing a transgene in mammalian retinal cells, comprising
contacting one or more mammalian retinal cells with an amount of a recombinant AAV2 virus comprising: (a) a capsid protein, wherein the capsid protein is an AAV variant 7m8 capsid protein or is derived from the AAV variant 7m8 capsid protein, and (b) the polynucleotide cassette according to claim 1 ,
wherein the secretory polypeptide is expressed in the one or more mammalian retinal cells at a level that is 3× higher than that obtained by contacting the cells with a recombinant AAV2 virus comprising a reference cassette encoding the secretory polypeptide, and
wherein the reference cassette comprises, in 5′ to 3′ order, a CMV enhancer sequence as set forth in SEQ ID NO:2, a CMV promoter as set forth in SEQ ID NO:21, a chimeric intron as set forth in SEQ ID NO:22, a 5′UTR as set forth SEQ ID NO:23, a coding sequence encoding the secretory polypeptide, a 3′UTR as set forth in SEQ ID NO:25, and an SV40 polyA sequence as set forth in SEQ ID NO:26.
6. A method for the treatment of a disease in a mammal in need of treatment for a disease, comprising administering to the mammal an effective amount of a pharmaceutical composition comprising a recombinant AAV2 virus comprising: (a) a capsid protein, wherein the capsid protein is an AAV variant 7m8 capsid protein or is derived from the AAV variant 7m8 capsid protein, and (b) the polynucleotide cassette according to claim 1 , wherein the disease is an ocular disease and the pharmaceutical composition is administered by intravitreal injection to an eye of the mammal.
7. The method of claim 6 , wherein the ocular disease is selected from the group consisting of choroidal neovascularization and macular degeneration.
8. The polynucleotide cassette of claim 1 , wherein the secretory polypeptide is selected from the group consisting of soluble fms-like tyrosine kinase-I (sFLT-1), a vascular endothelial growth factor (VEGF)-binding fragment of sFLT-1, and aflibercept.
9. The polynucleotide of cassette of claim 1 , wherein the secretory polypeptide is aflibercept.
10. The recombinant virus of claim 3 , wherein the secretory polypeptide is aflibercept.
11. The pharmaceutical composition of claim 4 , wherein the secretory polypeptide is aflibercept.
12. The method of claim 5 , wherein the secretory polypeptide is aflibercept.
13. The method of claim 7 , wherein the macular degeneration is non-exudative age-related macular degeneration.