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1. WO2020141106 - ENGINEERED EXTERNALLY REGULATED ARTIFICIAL TRANSCRIPTION REGULATORY SYSTEM BASED ON ENGINEERED NFAT

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CLAIMS

1. An engineered externally regulated artificial transcription regulatory system comprising two components:

a) a first component comprising an NFAT protein lacking its own transactivation domain, fused with at least one first protein domain of a chemical signal-inducible heterodimerization protein complex, and

b) a second component comprising a gene transcription activation or repressor domain, fused to a second protein domain that binds to the at least one first protein domain of the chemical signal-inducible heterodimerization protein complex.

2. An engineered NFAT transcription factor comprising the a) first component and the b) second component of the engineered externally regulated artificial transcription regulatory system of claim 1.

3. The engineered externally regulated artificial transcription regulatory system of claim 1 , or the engineered NFAT transcription factor of claim 2, wherein the at least one first protein domain of the chemical signal-inducible heterodimerization protein complex of the first component is selected from the group consisting of the pairs:

i) FKBP and FRB;

ii) FKBP and calcineurin catalytic subunit A (CnA);

iii) FKBP and cyclophilin;

iv) Homodimer gyrase B (GyrB);

v) DmrA and DmrC;

vi) ABI and PYL1 ; and

vii) GAI and GID1 , and

wherein the second protein domain of the second component is selected from the group consisting of the pairs:

i) FKBP and FRB;

ii) FKBP and calcineurin catalytic subunit A (CnA);

iii) FKBP and cyclophilin;

iv) Homodimer gyrase B (GyrB);

v) DmrA and DmrC;

vi) ABI and PYL1 ; and

vii) GAI and GID1.

4. The regulatory system of claims 1 and 3 or the NFAT transcription factor of claims 2 and 3, wherein the gene transcription activation domain comprises the VP16, VP64, VPR, p65, or a NFAT transactivation domain or other gene transcription activation domains that recruit components of an RNA polymerase complex to effect transcription of NFAT-targeted genes.

5. The regulatory system of claims 1 and 3-4 or the NFAT transcription factor of claims 2-4, wherein the regulatory system or the NFAT transcription factor further comprises a chemical inductor of heterodimerization capable of inducing functional reconstitution of the regulatory system or of the engineered NFAT transcription factor.

6. The regulatory system of claims 1 and 3-5 or the NFAT transcription factor of claims 2-5, wherein the at least one first protein domain and the second protein domain are from two different proteins and have orthologous properties.

7. The regulatory system or the NFAT transcription factor of claims 5 or 6, wherein the regulatory system or the NFAT transcription factor comprises two different chemical inductors of heterodimerization resulting in two different orthologous NFAT transcription factors.

8. The regulatory system of claims 1 and 3-7 or the NFAT transcription factor of claims 2-7, wherein the first and second component form a heterodimer in the presence of a chemical inductor of heterodimerization to form the chemical signal-inducible heterodimerization protein complex, wherein the chemical inductor preferably is a small molecule.

9. The regulatory system of claims 1 and 3-8 or the NFAT transcription factor of claims 2-8, wherein the second component comprises a gene transcription activation domain, which enhances gene transcription of NFAT-dependent genes.

10. The regulatory system of claims 1 and 3-8 or the NFAT transcription factor of claims 2-8, wherein the second component comprises a gene transcription repressor domain, which downregulates gene transcription of NFAT-dependent genes, wherein the regulatory system or the NFAT transcription factor preferably functions ex vivo in T cells, T cell lines and/or CAR T cells.

1 1 . The regulatory system of claims 1 and 3-10 or the NFAT transcription factor of claims 2-10, wherein the regulatory system or the NFAT transcription factor does not comprise the second component, and wherein the first component of the regulatory system or the NFAT transcription factor binds to NFAT DNA binding sites and competes with endogenous NFAT protein, thereby resulting in decreased endogenous NFAT signaling.

12. The regulatory system of claims 1 and 3-10 or the NFAT transcription factor of claims 2-10, wherein the second component comprises a repressor domain, and wherein the binding of the regulatory system or the NFAT transcription factor decreases gene transcription of NFAT-dependent genes, thereby resulting in decreased activation and proliferation of cells.

13. The regulatory system of claims 1 and 3-12 or the NFAT transcription factor of claims 2-12, wherein the regulatory system or the NFAT transcription factor comprises FKBP-FRB, p65 and KRAB.

14. An isolated nucleic acid comprising a nucleic acid encoding a) the first component and/or b) the second component of the regulatory system of claims 1 and 3-13 or of the NFAT transcription factor of claims 2-13.

15. A vector encoding the isolated nucleic acid(s) of claim 14.

16. A cell comprising the regulatory system of claims 1 and 3-13, or the NFAT transcription factor of claims 2-13, or the isolated nucleic acid of claim 14, or the vector of claim 15.

17. The cell of claim 16, wherein the cell is a T cell, wherein the T cell preferably is a CAR T cell.

18. The regulatory system of claims 1 and 3-12, or the NFAT transcription factor of claims 2-12, or the isolated nucleic acid of claim 14, or the vector of claim 15, or the cells of claims 16 or 17 for use as a medicament.

19. The regulatory system of claims 1 and 3-12, or the NFAT transcription factor of claims 2-12, or the isolated nucleic acid of claim 14, or the vector of claim 15, or the cells of claims 16 or 17 for use in a method of treating cancer.

20. The regulatory system of claims 1 and 3-12, or the NFAT transcription factor of claims 2-12, or the isolated nucleic acid of claim 14, or the vector of claim 15, or the cells for use according to claims 18 or 19, wherein the use comprises the use of a chemical inductor of heterodimerization, and wherein the cells preferably are CAR T cells.

21 . The cells for use according to claims 18-20, wherein the cells are CAR T cells, and wherein the CAR T cells are negatively regulated by the regulatory system or by the NFAT transcription factor.

22. The cells for use according to claims 18-21 , wherein the cells are CAR T cells, and wherein the CAR T cells are positively regulated by the regulatory system or by the NFAT transcription factor.

23. A method for upregulating NFAT-dependent genes comprising the following steps:

a) transforming a cell with a nucleic acid encoding the first component and a nucleic acid encoding the second component of the regulatory system of claims 1 and 3-12, or the NFAT transcription factor of claims 2-12, and

b) inducing heterodimerization by adding a suitable chemical inductor.

24. A method for downregulating NFAT-dependent genes comprising the following steps:

a) transforming a cell with a nucleic acid encoding the first component and a nucleic acid encoding the second component of the regulatory system of claims 1 and 3-12, or the NFAT transcription factor of claims 2-12, and

b) inducing heterodimerization by adding a suitable chemical inductor.