Stable episomal vectors
View Patent ↗The present invention provides a vector system which can be stably translocated from the cytoplasm of a cell to the nucleus where it is a stable nuclear episomal vector. A basic vector system according to the present invention is generally composed of nucleic acid encoding (a) a promoter functional both in cytoplasm and nucleus, operably linked to (b) a plasmid maintenance/translocation factor and (c) an origin of replication.
1. A vector system capable of facilitated translocation from the cytoplasm to the nucleus of a eukaryotic cell, comprising a nucleic acid comprising:
(a) a promoter element functional both in the cytoplasm and nucleus of a cell which promoter element comprises a T7 promoter or a SP6 promoter, said promoter element being operably linked to:
(b) a gene sequence for a plasmid maintenance factor and a translocation factor, encoding either (i) EBNA-1 or (ii) EBNA-1 and T-antigen of SV40, and
(c) a eukaryotic origin of replication, wherein the promoter element is bound to its cognate T7 or SP6 RNA polymerase.
2. The vector system of claim 1 wherein said eukaryotic origin of replication is oriP.
3. The vector system of claim 1 wherein said promoter element is a hybrid promoter.
4. The vector system of claim 1 wherein said promoter element further comprises at least a portion of an SV 40 promoter.
5. The vector system of claim 1 , wherein the nucleic acid further comprises a multiple cloning site operably linked to a nuclear promoter.
6. The vector system of claim 5 wherein said multiple cloning site further comprises a nucleic acid sequence encoding a target gene.
7. The vector system of claim 1 wherein:
(a) said plasmid maintenance factor and said translocation factor are EBNA-1,
(b) said promoter element comprises a T7 promoter and a SV40 promoter, and
(c) said eukaryotic origin of replication is oriP.
8. A method of expressing a target gene, comprising culturing a host cell containing the vector system of claim 1 , said nucleic acid further comprising a target gene, under conditions that promote expression of said target gene in said host cell.
9. A composition comprising the vector system of claim 1 , further comprising a pharmaceutically acceptable carrier, and wherein the nucleic acid further comprises a target gene.
10. A method for facilitating translocation of a vector from the cytoplasm of a target cell to the nucleus of the target cell in vitro comprising:
(a) incorporating into a vector:
(i) a promoter element functional in both the cytoplasm and the nucleus of a cell, which promoter element comprises a T7 or SP6 promoter, said promoter element being operably linked to:
(ii) a gene sequence for a plasmid maintenance factor and a translocation factor, comprising either (i) EBNA-1 or (ii) EBNA-1 and T antigen of SV40, and
(iii) an eukaryotic origin of replication; and
(b) contacting the target cell with the vector
wherein the vector is delivered to the cytoplasm of the target cell and the target cell expresses the translocation and maintenance factor thereby mediating transfer of the vector to the nucleus of the target cell, and
wherein (i) the translocation factor facilitates translocation of the vector into the nucleus of the cell, and (ii) the promoter is bound to its cognate T7 or SP6 RNA polymerase prior to said step of contacting.
11. The method of claim 10 , wherein the vector is an EBV vector that comprises a T7 promoter and a SV40 promoter, and the maintenance and translocation factors are EBNA-1.
12. The vector system of claim 1 wherein the nucleic acid further comprises a prokaryotic origin of replication.
13. The method of claim 10 , wherein the nucleic acid further comprises a prokaryotic origin of replication.