Vectors comprising guinea pig CMV regulatory elements
The invention comprises novel polynucleotides, and related vectors, host cells, methods, and compositions, containing transcriptional enhancers providing very high levels of expression of operably-linked expressible nucleic acid sequences in eukaryotic cells. Advantageously the enhancers may be used in combination with their naturally-associated promoters and/or other genetic elements that increase transcription. The invention comprises eukaryotic expression vectors that are capable of providing increased levels of expression in many cell types over that obtainable from human or murine CMV IE enhancer/promoter elements.
1. An isolated polynucleotide comprising:
(a) an element comprising at least 200 contiguous nucleotides of SEQ ID NO:1; and
(b) an element comprising an expressible polynucleotide sequence, wherein said element in (a) is directly operably linked to said expressible polynucleotide sequence in (b) and is not naturally operably linked to said expressible polynucleotide sequence.
2. An isolated polynucleotide according to claim 1 , comprising at least 500 contiguous nucleotides of SEQ ID NO: 1.
3. An isolated polynucleotide according to either of claim 1 or 2 , comprising nucleotides 50 to 550 of SEQ ID NO: 1.
4. An isolated polynucleotide according to either of claim 1 or 2 , comprising nucleotides 275 to 775 of SEQ ID NO: 1.
5. An isolated polynucleotide according to either of claim 1 or 2 , wherein the element in (a) comprises the promoter from the immediate/early regulatory region of guinea pig CMV.
6. An isolated polynucleotide according to claim 5 , comprising nucleotides 679 to 880 of SEQ ID NO: 1.
7. An isolated polynucleotide according to claim 1 or claim 2 , comprising nucleotides 1 to 887 of SEQ ID NO:1
8. An isolated polynucleotide according to claim 1 or claim 2 , further comprising an extended, methylation-free CpG island operably linked to said expressible nucleic acid sequence.
9. A vector comprising the polynucleotide of claim 1 or claim 2 .
10. A eukaryotic expression vector according to claim 9 .
11. A vector according to claim 9 , comprising the polynucleotide sequence of nucleotides 1 to 1003 and 1747 to 5749 of SEQ ID NO: 2.
12. A vector according to claim 9 , comprising nucleotides 1 to 9328 and 10072 to 14119 of SEQ ID NO: 3.
13. A vector according to claim 9 , comprising nucleotides 1 to 2592 and 3336 to 7383 of SEQ ID NO: 4.
14. An isolated host cell comprising an isolated polynucleotide according to any of claims 1 or 2 , or a vector comprising the polynucleotide of claim 1 .
15. A method of expressing a polypeptide comprising inserting an expressible nucleic acid sequence encoding said polypeptide into an expression vector according to claim 9 into an appropriate host cell and culturing said host cell in suitable conditions to allow expression.
16. The method according to claim 15 , wherein said polypeptide is a therapeutically useful polypeptide.
17. The method according to claim 16 , wherein said polypeptide is selected from the group consisting of: an immunoglobulin, a functional epitope-binding fragment of an immunoglobulin, a growth factor, a soluble receptor and a blood clotting factor.
18. A pharmaceutical preparation comprising a polynucleotide according to claim 1 or claim 2 , a vector comprising the polynucleotide of claim 1 , or a host cell according to claim 1 and a pharmaceutically acceptable carrier, excipient, buffer or medium.