Regulated transcription of targeted genes and other biological events

• US 6,972,193 B1
• Filed: 12/17/1999
• Issued: 12/06/2005
• Est. Priority Date: 02/12/1993
• Status: Expired due to Fees

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Spencer, David M., Ho, Steffan N., Crabtree, Gerald R., Schreiber, Stuart L., Belshaw, Peter, Wandless, Thomas J.
• Primary Examiner(s): MCKELVEY, TERRY ALAN
• Application Number:
  US09/466,568
• Time in Patent Office: 2,181 Days
• Field of Search: 435/320.1, 435/325, 435/419, 435/455, 435/69.1, 536/23.4, 536/23.5, 514/44
• US Class Current: 435/325
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/6425 the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07D 498/18 Bridged systems
• CPC Class Codes: C07F 5/025 Boronic and borinic acid compounds
• CPC Class Codes: C07H 19/01 sharing oxygen
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/035 containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C07K 2319/32 fusions with soluble part of a cell surface receptor, "decoy receptors"
• CPC Class Codes: C07K 2319/42 containing a HA(hemagglutinin)-tag
• CPC Class Codes: C07K 2319/43 containing a FLAG-tag
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C07K 2319/71 : containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/715 : containing a domain for ligand dependent transcriptional activation, e.g. containing a steroid receptor domain
• CPC Class Codes: C07K 2319/81 : containing a Zn-finger domain for DNA binding
• CPC Class Codes: C07K 2319/90 : containing a motif for post-translational modification
• CPC Class Codes: C07K 7/645 : Cyclosporins; Related peptides
• CPC Class Codes: C12N 15/1055 : Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/62 : DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/63 : Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/67 : General methods for enhancing the expression
• CPC Class Codes: C12P 15/00 : Preparation of compounds containing at least three condensed carbocyclic rings gibbanes C12P27/00; naphthacenes C12P29/00

Abstract

Dimerization and oligomerization of proteins are general biological control mechanisms that contribute to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. We have developed a general procedure for the regulated (inducible) dimerization or oligomerization of intracellular proteins.

Claims

1. A nucleic acid composition comprising at least two genetic constructs, each encoding a chimeric protein,

(a) a first construct encoding a first chimeric protein comprising at least one ligand-binding domain and a transcriptional activation domain which is heterologous thereto, and,

(b) a second construct encoding a second chimeric protein comprising at least one ligand-binding domain which may be the same or different from a ligand binding domain of the first chimeric protein, and a DNA binding domain which is heterologous thereto,

wherein the first and second of said chimeric proteins together (i) bind to a ligand to form a ligand cross-linked protein complex, and (ii) in a ligand dependent manner, activate transcription of a gene having a transcriptional regulatory element to which the DNA binding domain binds, and wherein the ligand-binding domain of at least one of the chimeric proteins is an FKBP domain.

4. The composition of claim 1, wherein each of said constructs is provided in a vector including a selectable marker permitting transfection of the vector into host cells and selection of transfectants containing the vector.

5. A mammalian cell which contains and expresses the nucleic acid composition of claim 1, 2, or 3.

15. The composition of claim 1 in which the ligand-binding domain of at least one of the chimeric proteins is an FKBP domain, wherein said FKBP domain comprises FKBP 12 or a variant thereof, and wherein said variant comprises substitution of one or more of Tyr26, Phe36, Asp37, Tyr82 and Phe99 with another amino acid residue.

16. The composition of claim 1 in which the ligand binding domain of at least one of the chimeric proteins specifically binds to FK506, FK520, rapamycin, or a derivative of FK506, FK520, or rapamycin.

17. A eukaryotic cell containing and capable of expressing the nucleic acid composition of claim 1, 2, or 3.

2. A nucleic acid composition comprising at least two genetic constructs, each encoding a chimeric protein,

(a) a first construct encoding a first chimeric protein comprising at least one ligand-binding domain and a signal initiation domain which is heterologous thereto;

and,

(b) a second construct encoding a second chimeric protein comprising at least one ligand-binding domain which may be the same or different from the ligand binding domain of the first chimeric protein, and an intra-cellular localization domain which is heterologous thereto,

wherein the first and second of said chimeric proteins together (i) bind to a ligand to form a ligand cross-linked protein complex, and (ii) in a ligand dependent manner, activate an intra-cellular signaling pathway.

6. The composition of claim 2, wherein the intra-cellular localization domain is a nuclear localization domain.

7. The composition of claim 2, wherein the intra-cellular localization domain is a cytoplasmic localization domain.

8. The composition of claim 2, wherein the intra-cellular localization domain comprises a secretory leader sequence, a membrane retention domain, a nuclear localization domain, or a vesicle targeting domain.

9. The composition of claim 8, wherein the membrane retention domain comprises a plasma membrane targeting sequence for attachment of a myristoyl moiety or a prenyl moiety.

18. The composition of claim 2 in which the ligand binding domain of at least one of the chimeric proteins is an FKBP domain.

19. The composition of claim 2 in which the ligand-binding domain of at least one of the chimeric proteins is an FKBP domain, wherein said FKBP domain comprises FKBP12 or a variant thereof, and wherein said variant comprises substitution of one or more of Tyr26, Phe36, Asp37, Tyr82 and Phe99 with another amino acid residue.

20. The composition of claim 2 in which the ligand binding domain of the at least one chimeric proteins specifically binds to FK506, FK520, rapamycin, or a derivative of FK506, FK520, or rapamycin.

21. The composition of claim 2 or 3 in which the activation of a cellular signaling pathway regulates, in a ligand dependent manner, at least one of cell proliferation, differentiation, or death.

3. A nucleic acid composition comprising at least two genetic constructs, each encoding a chimeric protein,

(a) a first construct encoding a first chimeric protein comprising at least one ligand-binding domain, a signal initiation domain which is heterologous thereto, and a cytoplasmic domain of a cell surface receptor;

and,

(b) a second construct encoding a second chimeric protein comprising at least one ligand-binding domain which may be the same or different from the ligand binding domain of the first chimeric protein, a signal initiation domain which is heterologous thereto and which may be the same or different from the signal initiation domain of the first chimeric protein, and a cytoplasmic domain of a cell surface receptor which may be the same or different from the cytoplasmic domain of a cell surface receptor of the first chimeric protein,

wherein the first and second of said chimeric proteins together (i) bind to a ligand to form a ligand cross-linked protein complex, and (ii) in a ligand dependent manner, activate a cellular signaling pathway.

10. The composition of claim 3, in which the ligand-binding domain of at least one of the chimeric proteins is an FKBP domain.

11. The composition of claim 3 in which the ligand-binding domain of at least one of the chimeric proteins is an FKBP domain, wherein said FKBP domain comprises FKBP12 or a variant thereof, and wherein said variant comprises substitution of one or more of Tyr26, Phe36, Asp37, Tyr82 and Phe99 with another amino acid residue.

12. The composition of claim 3 in which the ligand binding domain of at least one of the chimeric proteins specifically binds to FK506, FK520, rapamycin, or a derivative of FK506, FK520, or rapamycin.

13. The composition of claim 3 in which the cytoplasmic domain of a cell surface receptor is selected from the group consisting of a tyrosine kinase receptor, a cytokine receptor and a growth factor receptor.

14. The composition of claim 3 in which the cytoplasmic domain of a cell surface receptor is selected from the group consisting of a Fas receptor and a TNF receptor.