REGULATED TRANSCRIPTION OF TARGETED GENES AND OTHER BIOLOGICAL EVENTS

• US 20110003385A1
• Filed: 01/08/2010
• Published: 01/06/2011
• Est. Priority Date: 02/12/1993
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: 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.
• Application Number:
  US12/684,784
• Publication Number:
  US 20110003385A1
• Time in Patent Office: Days
• US Class Current: 435/375
• 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. In principle, any two target proteins can be induced to associate by treating the cells or organisms that harbor them with cell permeable, synthetic ligands. To illustrate the practice of this invention, we have induced: (1) the intracellular aggregation of the cytoplasmic tail of the ζ chain of the T cell receptor (TCR)-CD3 complex thereby leading to signaling and transcription of a reporter gene, (2) the homodimerization of the cytoplasmic tail of the Fas receptor thereby leading to cell-specific apoptosis (programmed cell death) and (3) the heterodimerization of a DNA-binding domain (Gal4) and a transcription-activation domain (VP16) thereby leading to direct transcription of a reporter gene.

Regulated intracellular protein association with our cell permeable, synthetic ligands offers new capabilities in biological research and medicine, in particular, in gene therapy.

Claims

1. A DNA construct encoding a chimeric protein comprising (a) at least one receptor domain, capable of binding to a selected ligand, fused to (b) a heterologous additional protein domain capable of initiating a biological process upon exposure to the ligand, said ligand being capable of binding to two or more chimeric protein molecules.

2. A DNA construct of claim 1 wherein the chimeric protein further comprises an intracellular targeting domain capable of directing the chimeric protein to a desired cellular compartment.

3. A DNA construct of claim 2 wherein the intracellular targeting domain comprises a secretory leader sequence, a membrane spanning domain, a membrane binding domain or a sequence directing the protein to associate with vesicles or with the nucleus.

4. A DNA construct of claim 1 wherein the chimeric protein has a K_d value for binding to the selected ligand of less than or equal to about 10⁻

6 M.

5. A DNA construct of claim 1 wherein the selected ligand is less than about 5 kDa in molecular weight.

6. A DNA construct of claim 1 wherein the heterologous additional protein domain comprises:

(a) a protein domain capable, upon exposure to the ligand, of initiating a detectable intracellular signal;

(b) a DNA-binding protein;

or

(c) a transcriptional activation domain.

7. A DNA construct of claim 6 wherein the intracellular signal is capable of activating transcription of a gene under the transcriptional control of transcriptional control element responsive to said oligomerization.

8. A DNA construct of claim 7 wherein the additional protein domain is the zeta subunit of CD3.

9. A DNA construct of claim 1, wherein the chimeric protein is capable of binding to an FK506-type ligand, a cyclosporin A-type ligand, tetracycline or a steroid ligand.

10. A DNA construct encoding a target gene under the transcriptional control of an transcription control element responsive to the oligomerization of a chimeric protein of claim 1.

11. A DNA construct of claim 10 in which the target gene is not naturally under the transcriptional control of the responsive transcriptional control element.

12. A DNA construct containing (a) a transcriptional control element responsive to the oligomerization of a chimeric protein of claim 1 and (b) flanking DNA sequence from a target gene permitting the homologous recombination of the transcriptional control element into a host cell in association with the target gene.

13. A DNA construct of claim 11 wherein the target gene encodes a surface membrane protein, a secreted protein, a cytoplasmic protein or a ribozyme or an antisense sequence.

14. A DNA vector containing a DNA construct of claim 13 and a selectable marker permitting transfection of the DNA construct into host cells and selection of transfectants containing the construct.

15. A DNA vector of claim 14 wherein the vector is a viral vector.

16. A viral vector of claim 15 which is an adeno-, adeno associated- or retroviral vector.

18. A cell containing and capable of expressing at least one DNA construct of claim 12 or 13.

19. A cell of claim 18 which is a mammalian cell.

30. A method for activating the transcription of a target gene in cells which comprises

(a) providing cells containing and capable of expressing (i) at least one DNA construct of claim 7 and (ii) a target gene under the expression control of a transcription control element responsive to the oligomerization of said DNA construct;

and,

(b) exposing the cells to a ligand capable of binding to the chimeric protein encoded by the DNA construct in an amount effective to result in expression of the target gene.

43. A host organism containing a cell of claim 18.

17. (canceled)

20-21. -21. (canceled)

22. A DNA composition comprising

(a) a first DNA construct encoding a chimeric protein comprising (i) at least one receptor domain, capable of binding to a selected ligand, fused to (ii) a heterologous additional protein domain capable of initiating a biological process upon exposure to the ligand;

and

(b) a second DNA construct encoding a target gene under the transcriptional control of an transcription control element responsive to the oligomerization of a chimeric protein.

23. A DNA composition comprising

(a) a DNA construct encoding a first chimeric protein comprising (i) at least one first receptor domain, capable of binding to a selected first ligand moiety, fused to (ii) a heterologous additional protein domain capable of initiating a biological process upon exposure to the ligand in the presence of a second chimeric protein;

and,

(b) a DNA construct encoding the second chimeric protein comprising (i) at least one receptor domain, capable of binding to a selected second ligand moiety, fused to (ii) a heterologous additional protein domain capable of initiating a biological process upon exposure to the ligand in the presence of the first chimeric protein;

wherein the first and second receptor moieties may be the same or different and the first and second selected ligand moieties may be the same or different; and

(c) a target DNA construct encoding a target gene under the transcriptional control of a transcriptional control element responsive to the oligomerization of a chimeric protein.

24-29. -29. (canceled)

31-42. -42. (canceled)

44-46. -46. (canceled)