Circular nucleic acid vectors, and methods for making and using the same
View Patent ↗Circular nucleic acid vectors that provide for persistently high levels of protein expression are provided. The circular vectors of the subject invention are characterized by being devoid of expression-silencing bacterial sequences, where in many embodiments the subject vectors include a unidirectional site-specific recombination product hybrid sequence in addition to an expression cassette. Also provided are methods of using the subject vectors for introduction of a nucleic acid, e.g., an expression cassette, into a target cell, as well as preparations for use in practicing such methods. The subject methods and compositions find use in a variety of different applications, including both research and therapeutic applications. Also provided is a highly efficient and readily scalable method for producing the vectors employed in the subject methods, as well as reagents and kits/systems for practicing the same.
1. A circular vector devoid of bacterial transcriptional silencing sequence, said vector comprising:
a product hybrid sequence of a resolvase integrase and an expression cassette,
wherein said vector provides for persistent and high level expression when present in a cell.
2. The circular vector according to claim 1 , wherein said resolvase integrase is a unidirectional site-specific integrase.
3. The circular vector according to claim 2 , wherein said integrase is ΦC31.
4. The circular vector according to claim 1 , wherein said vector ranges in length from about 0.3 to about 10 kb.
5. The circular vector according to claim 1 , wherein said vector is double stranded.
6. The circular vector according to claim 1 , wherein said vector is a deoxyribonucleic acid vector.
7. A kit for introducing an expression cassette into a target cell, said kit comprising:
(a) a vector according to claim 1 ; and
(b) instructions for using said vector for introducing an expression cassette into a target cell.
8. The vector according to claim 1 , wherein said resolvase integrase is selected from the group consisting of ΦC31, R4, TP901-1, A118, and ΦFC1.
9. The vector according to claim 1 , wherein said vector provides for persistent and high level expression for at least 3 weeks when present in a cell.
10. The vector according to claim 1 , wherein said vector provides for persistent and high level expression for at least 28 days when present in a cell.
11. A circular vector devoid of bacterial transcriptional silencing sequence, said vector comprising:
a product hybrid sequence of a resolvase integrase and an expression cassette,
wherein said vector provides for persistent and high level expression for at least 3 weeks when present in a cell.
12. The circular vector according to claim 11 , wherein said resolvase integrase is a unidirectional site-specific integrase.
13. The vector according to claim 12 , wherein said resolvase integrase is selected from the group consisting of ΦC31, R4, TP901-1, A118, and ΦFC1.
14. The circular vector according to claim 13 , wherein said integrase is ΦC31.
15. The circular vector according to claim 11 , wherein said vector ranges in length from about 0.3 to about 10 kb.
16. The circular vector according to claim 11 , wherein said vector is double stranded.
17. The circular vector according to claim 11 , wherein said vector is a deoxyribonucleic acid vector.
18. A circular vector devoid of bacterial transcriptional silencing sequence, said vector comprising:
a product hybrid sequence of a ΦC31 integrase and an expression cassette,
wherein said vector provides for persistent and high level expression for at least 3 weeks when present in a cell.
19. The circular vector according to claim 18 , wherein said vector ranges in length from about 0.3 to about 10 kb.
20. The circular vector according to claim 18 , wherein said vector is double stranded.
21. The circular vector according to claim 18 , wherein said vector is a deoxyribonucleic acid vector.