Fusion protein comprising a Caspase domain and a nuclear hormone receptor binding domain and methods and uses thereof
View Patent ↗The present invention relates to a fusion protein comprising a Caspase domain or a functionally active variant thereof and a ligand binding domain of a nuclear hormone receptor, a nucleic acid coding for the fusion protein, a vector or cell comprising the nucleic acid, a method of producing the fusion protein, a non-human transgenic animal containing the nucleic acid, the use of the fusion protein for ligand-mediated induction of apoptosis of a cell, or for studying the function of a cell, tissue and/or organ or the use of a transgenic organism for studying the function of a cell at various developmental stages or as a disease model, a method for inducing apoptosis of a cell expressing a fusion protein or for identifying a ligand, or a medicament comprising a fusion protein, the nucleic acid, the vector or the cell, particularly for the treatment of cancer or for or after transplantation, particularly as safety mechanism.
1. A fusion protein comprising
(a) a Caspase domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67 and, SEQ ID NO: 68; and
(b) a ligand binding domain of a nuclear hormone receptor or a functionally active variant thereof having the ability to be activated upon binding of a ligand, wherein upon exposure of the ligand binding domain of the nuclear hormone receptor to the ligand, the fusion protein is capable of inducing apoptosis in a cell.
2. The fusion protein of claim 1 , wherein the nuclear hormone receptor is selected from the group consisting of an estrogen receptor, a progesterone receptor, a glucocorticoid receptor and an androgen receptor.
3. The fusion protein of claim 1 , wherein the nuclear hormone receptor is a mammalian nuclear hormone receptor.
4. The fusion protein of claim 1 , wherein the functionally active variant of the nuclear hormone receptor is mutant ER(T2) estrogen receptor.
5. The fusion protein of claim 1 , wherein the binding domain of the nuclear hormone receptor ligand is linked to the N-terminal or C-terminal end of the Caspase domain.
6. The fusion protein of claim 1 , wherein the ligand binding domain of the nuclear hormone receptor is linked to the Caspase domain directly.
7. The fusion protein of claim 1 , wherein the ligand binding domain of the nuclear hormone receptor is linked to the Caspase domain via a linker.
8. The fusion protein of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 32 and SEQ ID NO: 37.
9. A nucleic acid coding for the fusion protein according to claim 1 .
10. A vector comprising the nucleic acid according to claim 9 .
11. An isolated cell comprising the nucleic acid according to claim 9 .
12. A method for producing the fusion protein according to claim 1 comprising
culturing a cell comprising a nucleic acid coding for the fusion protein according to claim 1 under conditions conducive to the production of the fusion protein.
13. An in vitro method for ligand-mediated induction of apoptosis of a cell, the cell comprising a nucleic acid coding for the fusion protein according to claim 1 , the method comprising contacting the fusion protein according to claim 1 on the surface of the cell with a ligand.
14. The method of claim 13 , the method being used for studying the function of a cell, tissue or organ.
15. An in virtro method for identifying a ligand to a ligand binding domain of a nuclear hormone receptor or a functionally active variant thereof, the method comprising
contacting the ligand binding domain of the nuclear hormone receptor of a cell comprising a nucleic acid coding for the fusion protein according to claim 1 with a substance; and
identifying the substance as a ligand, depending on its capability to induce apoptosis of the cell.
16. A composition comprising (i) the fusion protein according to claim 1 or the nucleic acid according to claim 9 and (ii) a pharmaceutically acceptable carrier or excipient.
17. The fusion protein of claim 1 , wherein the Caspase domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 68.
18. The fusion protein of claim 1 , wherein the ligand binding domain comprises the ER(T2) estrogen receptor ligand binding domain.
19. The fusion protein of claim 1 , comprising a deletion of the Caspase recruitment domain (CARD).
20. The fusion protein of claim 1 , further comprising a myristoylation signal sequence.