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 comprising in 5′ to 3′ order:
(a) a first enhancer region comprising a 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 a CMV sequence consisting of SEQ ID NO:4 or a sequence having at least 99% identity to SEQ ID NO:4;
(c) a 5′UTR region comprising, in 5′ to 3′ order, a tripartite leader (TPL) sequence consisting of SEQ ID NO:11 or a sequence having at least 99% identity to SEQ ID NO:11, an enhancer element from the adenovirus major late promoter (eMLP) sequence consisting of SEQ ID NO:12 or a sequence having at least 99% identity to SEQ ID NO:12, a mammalian intron acceptor, and a Kozak sequence;
(d) a coding sequence encoding a secretory polypeptide, wherein the coding sequence is codon optimized for translation in primates and operably linked to the promoter region;
(e) an enhancer region comprising a full expression enhancer sequence (EES) sequence consisting of SEQ ID NO:13 or a sequence having at least 99% identity to SEQ ID NO:13; and
(f) a human growth hormone (HGH) polyadenylation site consisting of SEQ ID NO:14 or a sequence having at least 99% identity to SEQ ID NO:14;
and wherein:
the first enhancer region (a), the promoter region (b), and the 5′ UTR region (c) define a 5′ arm;
the second enhancer region (e), and the HGH polyadenylation site (f) define a 3′ arm.
2. The polynucleotide cassette according to claim 1 , wherein the cassette does not comprise an RNA export signal.
3. The polynucleotide cassette of claim 1 , further comprising each of SEQ ID NOs: 76-80.
4. The polynucleotide cassette according to claim 1 , wherein the 5′ arm comprises a sequence with at least 99% identity to SEQ ID NO: 47.
5. The polynucleotide cassette according to claim 1 , wherein the 5′ arm consists of SEQ ID NO: 47.
6. The polynucleotide cassette according to claim 1 , wherein the 3′ arm comprises a sequence with at least 99% identity to SEQ ID NO: 48.
7. The polynucleotide cassette according to claim 1 , wherein the 3′ arm consists of SEQ ID NO: 48.
8. The polynucleotide cassette according to claim 4 , wherein the 3′ arm comprises a sequence with at least 99% identity to SEQ ID NO: 48.
9. The polynucleotide cassette according to claim 5 , wherein the 3′ arm consists of SEQ ID NO: 48.
10. The polynucleotide cassette of claim 1 , wherein the secretory polypeptide is selected from the group consisting of soluble fms-like tyrosine kinase-1, a VEGF-binding fragment of sFLT-1, and aflibercept.
11. The polynucleotide cassette of claim 1 , wherein the secretory polypeptide is aflibercept.
12. A recombinant adeno-associated 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) an adeno-associated virus vector genome comprising the polynucleotide cassette according to claim 1 .
13. The recombinant adeno-associated virus of claim 12 , wherein the secretory polypeptide is aflibercept.
14. A pharmaceutical composition comprising the recombinant adeno-associated virus of claim 12 and a pharmaceutically acceptable excipient.
15. The pharmaceutical composition of claim 14 , wherein the secretory polypeptide is aflibercept.
16. The polynucleotide cassette according to claim 1 , wherein the 5′ arm comprises a sequence with at least 99% identity to SEQ ID NO: 47, and wherein the 3′ arm comprises a sequence with at least 99% identity to SEQ ID NO: 48.
17. The polynucleotide cassette according to claim 1 , wherein the 5′ arm comprises SEQ ID NO: 47, and wherein the 3′ arm comprises SEQ ID NO: 48.