Construction of fully-deleted adenovirus-based gene delivery vectors and uses thereof
The embodiments disclosed herein relate to the construction of fully-deleted Adenovirus-based gene delivery vectors packaged without helper Adenovirus, and more particularly to their use in gene therapy for gene and protein expression, vaccine development, and immunosuppressive therapy for allogeneic transplantation. In an embodiment, a method for propagating an adenoviral vector includes (a) providing an Adenovirus packaging cell line; (b) transfecting a fully-deleted Adenoviral vector construct into the cell line; and optionally (c) transfecting a packaging construct into the cell line, wherein the fully-deleted Adenoviral vector construct and optionally the packaging construct can transfect the Adenovirus packaging cell line resulting in the encapsidation of a fully-deleted Adenoviral vector independent of helper Adenovirus. In an embodiment, a target cell is transduced with the encapsidated fully-deleted Adenoviral vector for treating a condition, disease or a disorder.
1. A method for propagating a fully-deleted adenoviral-based gene transfer vector comprising:
(a) providing an Adenovirus packaging cell line;
(b) transfecting, into the cell line, a fully-deleted Adenoviral vector construct; and
(c) transfecting, into the cell line, a replication defective circular packaging construct having a subset of Adenoviral late genes including L1, L2, L3, L4, L5, E2A, and E4, while being absent of at least one inverted terminal repeat and a packaging signal,
wherein the fully-deleted Adenoviral vector construct and the packaging construct can transfect the Adenovirus packaging cell line resulting in the encapsidation of a fully-deleted adenoviral-based gene transfer vector independent of helper Adenovirus,
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector includes both adenoviral inverted terminal repeats, the packaging signal, and at least one DNA insert which comprises a gene sequence encoding a protein of interest, and
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector is absent of adenoviral structural genes.
2. The method of claim 1 wherein the packaging construct itself is incapable of being packaged.
3. The method of claim 1 wherein the Adenovirus packaging cell line is a human cell line.
4. The method of claim 3 wherein the human cell line is selected from one of A549 cells or primary cells.
5. The method of claim 1 wherein the encapsidated fully-deleted Adenoviral vector is replication deficient.
6. A method for propagating a fully-deleted adenoviral-based gene transfer vector comprising:
(a) providing an Adenovirus packaging cell line;
(b) transfecting, into the cell line, a fully-deleted Adenoviral vector construct; and
(c) transfecting, into the cell line, a replication defective circular packaging construct that is incapable of being packaged, the replication defective circular packaging construct having a subset of Adenoviral late genes including L1, L2, L3, L4, L5, E2A, and E4, while being absent of at least one inverted terminal repeat and a packaging signal,
wherein the fully-deleted Adenoviral vector construct and the packaging construct can transfect the Adenovirus packaging cell line resulting in the encapsidation of a fully-deleted adenoviral-based gene transfer vector independent of helper Adenovirus,
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector includes both adenoviral inverted terminal repeats, the packaging signal, and at least one DNA insert which comprises a gene sequence encoding a protein of interest, and
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector is absent of adenoviral structural genes.
7. The method of claim 6 wherein the Adenovirus packaging cell line is a human cell line.
8. The method of claim 7 wherein the human cell line is selected from one of A549 cells or primary cells.
9. The method of claim 6 wherein the encapsidated fully-deleted Adenoviral vector is replication deficient.
10. A method for propagating a fully-deleted adenoviral-based gene transfer vector comprising:
(a) providing an Adenovirus packaging cell line, wherein the Adenovirus packaging cell line is a human cell line;
(b) transfecting, into the cell line, a fully-deleted Adenoviral vector construct; and
(c) transfecting, into the cell line, a replication defective circular packaging construct having a subset of Adenoviral late genes including L1, L2, L3, L4, L5, E2A, and E4, while being absent of at least one inverted terminal repeat and a packaging signal,
wherein the fully-deleted Adenoviral vector construct and the packaging construct can transfect the Adenovirus packaging cell line resulting in the encapsidation of a fully-deleted adenoviral-based gene transfer vector independent of helper Adenovirus,
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector includes both adenoviral inverted terminal repeats, the packaging signal, and at least one DNA insert which comprises a gene sequence encoding a protein of interest, and
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector is absent of adenoviral structural genes.
11. The method of claim 10 wherein the packaging construct itself is incapable of being packaged.
12. The method of claim 10 wherein the human cell line is selected from one of A549 cells or primary cells.
13. The method of claim 10 wherein the encapsidated fully-deleted Adenoviral vector is replication deficient.
14. A target cell transduced with the encapsidated fully-deleted Adenoviral vector produced by the method of claim 10 .
15. A method for propagating a fully-deleted adenoviral-based gene transfer vector comprising:
(a) providing an Adenovirus packaging cell line, wherein the Adenovirus packaging cell line is a human cell line;
(b) transfecting, into the cell line, a fully-deleted Adenoviral vector construct; and
(c) transfecting, into the cell line, a replication defective circular packaging construct that is incapable of being packaged, the replication defective circular packaging construct having a subset of Adenoviral late genes including L1, L2, L3, L4, L5, E2A, and E4, while being absent of at least one inverted terminal repeat and a packaging signal,
wherein the fully-deleted Adenoviral vector construct and the packaging construct can transfect the Adenovirus packaging cell line resulting in the encapsidation of a fully-deleted adenoviral-based gene transfer vector independent of helper Adenovirus,
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector includes both adenoviral inverted terminal repeats, the packaging signal, and at least one DNA insert which comprises a gene sequence encoding a protein of interest, and
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector is absent of adenoviral structural genes.
16. The method of claim 15 wherein the human cell line is selected from one of A549 cells or primary cells.
17. The method of claim 15 wherein the encapsidated fully-deleted Adenoviral vector is replication deficient.
18. A method for propagating a fully-deleted adenoviral-based gene transfer vector comprising:
(a) providing an Adenovirus packaging cell line;
(b) transfecting, into the cell line, a fully-deleted Adenoviral vector construct; and
(c) transfecting, into the cell line, a replication defective circular packaging construct having a subset of Adenoviral late genes including L1, L2, L3, L4, L5, E2A, and E4, while being absent of at least one inverted terminal repeat and a packaging signal,
wherein the fully-deleted Adenoviral vector construct and the packaging construct can transfect the Adenovirus packaging cell line resulting in the encapsidation of a fully-deleted replication deficient adenoviral-based gene transfer vector independent of helper Adenovirus,
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector includes both adenoviral inverted terminal repeats, the packaging signal, and at least one DNA insert which comprises a gene sequence encoding a protein of interest, and
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector is absent of adenoviral structural genes.
19. The method of claim 18 wherein the packaging construct itself is incapable of being packaged.
20. The method of claim 18 wherein the Adenovirus packaging cell line is a human cell line.
21. The method of claim 20 wherein the human cell line is selected from one of A549 cells or primary cells.
22. A method for propagating a fully-deleted adenoviral-based gene transfer vector comprising:
(a) providing an Adenovirus packaging cell line;
(b) transfecting, into the cell line,. a fully-deleted Adenoviral vector construct; and
(c) transfecting, into the cell line, a replication defective circular packaging construct that is incapable of being packaged, the replication defective circular packaging construct having a subset of Adenoviral late genes including L1, L2, L3, L4, L5, E2A, and E4, while being absent of at least one inverted terminal repeat and a packaging signal,
wherein the fully-deleted Adenoviral vector construct and the packaging construct can transfect the Adenovirus packaging cell line resulting in the encapsidation of a fully-deleted replication deficient adenoviral-based gene transfer vector independent of helper Adenovirus,
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector includes both adenoviral inverted terminal repeats, the packaging signal, and at least one DNA insert which comprises a gene sequence encoding a protein of interest, and
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector is absent of adenoviral structural genes.
23. The method of claim 22 wherein the Adenovirus packaging cell line is a human cell line.
24. The method of claim 23 wherein the human cell line is selected from one of A549 cells or primary cells.
25. A method for propagating a fully-deleted adenoviral-based gene transfer vector comprising:
(a) providing an Adenovirus packaging cell line, wherein the Adenovirus packaging cell line is a human cell line;
(b) transfecting, into the cell line, a fully-deleted Adenoviral vector construct; and
(c) transfecting, into the cell line, a replication defective circular packaging construct having a subset of Adenoviral late genes including L1, L2, L3, L4, L5, E2A, and E4, while being absent of at least one inverted terminal repeat and a packaging signal,
wherein the fully-deleted Adenoviral vector construct and the packaging construct can transfect the Adenovirus packaging cell line resulting in the encapsidation of a fully-deleted replication deficient adenoviral-based gene transfer vector independent of helper Adenovirus,
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector includes both adenoviral inverted terminal repeats, the packaging signal, and at least one DNA insert which comprises a gene sequence encoding a protein of interest, and
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector is absent of adenoviral structural genes.
26. The method of claim 25 wherein the packaging construct itself is incapable of being packaged.
27. The method of claim 25 wherein the human cell line is selected from one of A549 cells or primary cells.
28. A method for propagating a fully-deleted adenoviral-based gene transfer vector comprising:
(a) providing an Adenovirus packaging cell line, wherein the Adenovirus packaging cell line is a human cell line;
(b) transfecting, into the cell line, a fully-deleted Adenoviral vector construct; and
(c) transfecting, into the cell line, a replication defective circular packaging construct that is incapable of being packaged, the replication defective circular packaging construct having a subset of Adenoviral late genes including L1, L2, L3, L4, L5, E2A, and E4, while being absent of at least one inverted terminal repeat and a packaging signal,
wherein the fully-deleted Adenoviral vector construct and the packaging construct can transfect the Adenovirus packaging cell line resulting in the encapsidation of a fully-deleted replication deficient adenoviral-based gene transfer vector independent of helper Adenovirus,
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector includes both adenoviral inverted terminal repeats, the packaging signal, and at least one DNA insert which comprises a gene sequence encoding a protein of interest, and
wherein the encapsidated fully-deleted adenoviral-based gene transfer vector is absent of adenoviral structural genes.
29. The method of claim 28 wherein the human cell line is selected from one of A549 cells or primary cells.