Polynucleotide
View Patent ↗The present invention relates to a polynucleotide comprising a ubiquitous chromatin opening element (UCOE) which is not derived from 13. The polynucleotide of any one of claims 1 to 7 , wherein the UCOE comprises the sequence of FIG. 20 between nucleotides 1 to 7627 or a functional homologue or fragment thereof an LCR. The present invention also relates to a vector comprising the polynucleotide sequence, a host cell comprising the vector, use of the polynucleoticle, vector or host cell in therapy and in an assay, and a method of identifying UCOEs. The UCOE opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types.
1. A method of obtaining a desired gene product comprising culturing a host cell, wherein the host cell comprises a vector, and the vector comprises a polynucleotide, the polynucleotide comprising:
(i) at least one extended methylation-free CpG-island comprising dual or bi-directional promoters that are divergently transcribed;
(ii) an expressible gene encoding a desired gene product, wherein the expressible gene is operably-linked to the at least one CpG-island; and
(iii) a promoter, operably-linked to the expressible gene, wherein said promoter is not naturally operably-linked to said CpG-island;
wherein the at least one extended methylation-free CpG island facilitates reproducible activation of transcription of the expressible gene.
2. The method of claim 1 , wherein the at least one extended methylation-free CpG island comprises at least one endogenous promoter.
3. The method of claim 1 , wherein the at least one extended methylation-free CpG island comprises dual promoters that transcribe divergently.
4. The method of claim 1 , wherein the at least one extended methylation-free CpG island extends for more than 300 bp or more than 500 bp.
5. The method of claim 1 , wherein the at least one extended methylation-free CpG island comprises a 44 kb DNA fragment spanning the human TATA binding protein gene and 12 kb from each of the 5′ and 3′ flanking sequence.
6. The method of claim 1 , wherein the at least one extended methylation-free CpG island comprises a 25 kb DNA fragment spanning the human TATA binding protein gene with 1 kb 5′ and 5 kb 3′ flanking sequence.
7. The method of claim 1 , wherein the at least one extended methylation-free CpG island comprises the sequence according to FIG. 21 or a functional fragment thereof.
8. The method of claim 1 , wherein the at least one extended methylation-free CpG island comprises a 60 kb DNA fragment spanning the human heterogeneous nuclear ribonucleoprotein A2 gene with 30 kb 5′ and 20 kb 3′ flanking sequence.
9. The method of claim 1 , wherein the at least one extended methylation-free CpG island comprises a 16 kb DNA fragment spanning the human heterogeneous nuclear ribonucleoprotein A2 gene with 5 kb 5′ and 1.5 kb 3′ flanking sequence.
10. The method of claim 1 , wherein the vector comprises a heterologous promoter which is a tissue specific promoter or a substantially ubiquitous promoter.
11. The method of claim 10 , wherein the promoter is the CMV promoter.
12. The method of claim 1 , wherein the expressible gene encodes a therapeutic polypeptide.
13. The method of claim 12 , wherein the expressible gene encodes a therapeutic antibody or fragment thereof.
14. The method of claim 1 , wherein the at least one extended methylation-free CpG-island increases the expression of the operably linked expressible gene.
15. A method for increasing the expression of an endogenous gene, said method comprising inserting an isolated polynucleotide comprising an extended methylation free CpG island into the genome of a cell in a position operably associated with the endogenous gene thereby increasing the level of expression of the gene, wherein said extended methylation-free CpG island comprises dual or bi-directional promoters that are divergently transcribed.