Expression elements
View Patent ↗The invention relates to improved genetic elements providing high levels of expression of operably-linked genes in a variety of tissues. In particular, fragments of unmethylated, CpG islands of less than 2 kb are shown to provide enhanced transgene expression and have advantages in terms of vector construction and cloning capacity.
1. An isolated polynucleotide comprising:
a) an extended methylation-free CpG island;
b) an expressible open reading frame, operably linked to said extended methylation-free CpG island;
c) a promoter, operably-linked to said open reading frame, wherein said promoter is not naturally linked to said CpG island;
wherein said CpG island is not more than 2 kb in size and comprises the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, and wherein reproducible expression of said open reading frame is obtained in the presence of the CpG island.
2. A polynucleotide according to claim 1 , wherein SEQ ID NO:2 comprises a 987 bp BspE1-Esp3I restriction fragment.
3. A vector comprising the polynucleotide of any of claim 1 or 2 .
4. A vector according to claim 3 wherein the vector is an integrating vector.
5. A vector according to claim 3 wherein the vector is a plasmid.
6. A vector according to claim 3 wherein the operably linked open reading frame is a therapeutic nucleic acid.
7. A vector according to claim 3 , comprising any of SEQ ID NOs 1 or 2, a CMV promoter, a multiple cloning site, a polyadenylation sequence and genes encoding selectable markers under suitable control elements.
8. A host cell comprising the polynucleotide of any of claim 1 or 2 .
9. A pharmaceutical composition comprising the polynucleotide according to any of claim 1 or 2 in combination with a pharmaceutically acceptable excipient.
10. A method for obtaining a desired gene product comprising expressing the polynucleotide according to any of claim 1 or 2 in a cell culture system in order to obtain a desired gene product, wherein the open reading frame encodes the desired gene product.
11. A method for increasing the expression of an endogenous gene comprising inserting the polynucleotide comprising SEQ ID NO 1 or 2 into the genome of a cell in a position operably associated with the endogenous gene thereby increasing the level of expression of the gene.
12. An isolated polynucleotide comprising:
a) an extended methylation-free CpG island;
b) an expressible open reading frame, operably linked to said extended methylation-free CpG island;
c) a promoter, operably-linked to said open reading frame, wherein said promoter
is not naturally linked to said CpG island;
wherein said CpG island is not more than 2 kb in size and comprises the sequence set forth in SEQ ID NO:2, and wherein reproducible expression of said open reading frame is obtained in the presence of the CpG island.
13. An isolated polynucleotide comprising:
a) an extended methylation-free CpG island;
b) an expressible open reading frame, operably linked to said extended methylation-free CpG island;
c) a promoter, operably-linked to said open reading frame, wherein said promoter is not naturally linked to said CpG island;
wherein said CpG island is not more than 2 kb in size and comprises the sequence set forth in SEQ ID NO:1, and wherein reproducible expression of said open reading frame is obtained in the presence of the CpG island.