Regulated apoptosis
AssigneesPresident and Fellows of Harvard College, The Board of Trustees of the Leland Stanford Jr. University
InventorsGerald R. Crabtree, Stuart L. Schreiber, David M. Spencer, Thomas J. Wandless, Peter Belshaw
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Abstract
We have developed a general procedure for the regulated (inducible) dimerization or oligomerization of intracellular proteins and disclose methods and materials for using that procedure to regulatably initiate cell-specific apoptosis (programmed cell death) in genetically engineered cells.
Claims235
A genetic construct encoding a chimeric protein comprising at least one ligand-binding domain which binds to a selected ligand and an action domain which is heterologous with respect to the ligand-binding domain,
- wherein the selected ligand binds to the ligand-binding domain and to a ligand-binding domain of another protein to form a ligand cross-linked complex with the two protein molecules, and further has one or more of the following characteristics:
- (i) the ligand is not a protein;
- (ii) the ligand has a molecular weight less than 5 kD;
- and
- (iii) the ligand is membrane permeable;
- and
- wherein the action domain induces apoptosis in a cell following formation of the ligand cross-linked complex.
A vector comprising a genetic construct encoding a chimeric protein comprising at least one ligand-binding domain which binds to a selected ligand and an action domain which is heterologous with respect to the ligand-binding domain, wherein the selected ligand binds to the ligand-binding domain and to a ligand-binding domain of another protein to form a ligand cross-linked complex with the two protein molecules, and further has one or more of the following characteristics:
- (i) the ligand is not a protein;
- (ii) the ligand has a molecular weight less than 5 kD;
- and
- (iii) the ligand is membrane permeable;
- and
- wherein the action domain induces apoptosis in a cell following formation of the ligand cross-linked complex;
- and which vector further comprises one or more of a bacterial or yeast origin of replication, a selectable marker, an amplifiable marker, a promoter, and an enhancer element for expression of the chimeric protein in procaryotic or eucaryotic cells.
A cell comprising at least one genetic construct encoding a chimeric protein comprising at least one ligand-binding domain which binds to a selected ligand and an action domain which is heterologous with respect to the ligand-binding domain, wherein the selected ligand binds to the ligand-binding domain and to a ligand-binding domain of another protein to form a ligand cross-linked complex with the two protein molecules, and further has one or more of the following characteristics:
- (i) the ligand is not a protein,
- (ii) the ligand has a molecular weight less than 5 kD, and
- (iii) the ligand is membrane permeable;
- and
- wherein the action domain induces apoptosis in the cell following formation of the ligand cross-linked complex.
A method for producing a cell which is selectively killed, which comprises introducing a genetic construct into a host cell, which genetic construct encodes a chimeric protein comprising at least one ligand-binding domain which binds to a selected ligand and an action domain which is heterologous with respect to the ligand-binding domain, wherein the selected ligand binds to the ligand-binding domain and to a ligand-binding domain of another protein in the host cell to form a ligand cross-linked complex with the two protein molecules, and further has one or more of the following characteristics:
- (i) the ligand is not a protein;
- (ii) the ligand has a molecular weight less than 5 kD;
- and
- (iii) the ligand is membrane permeable;
- and
- wherein the action domain induces apoptosis in the host cell following formation of the ligand cross-linked complex.
A kit which comprises at least one genetic construct encoding a first chimeric protein comprising at least one ligand-binding domain which binds to a selected ligand and an action domain which is heterologous with respect to the ligand-binding domain, wherein the selected ligand binds to the ligand-binding domain and to a ligand-binding domain of another protein to form a ligand cross-linked complex with the two protein molecules, and further has one or more of the following characteristics:
- (i) the ligand is not a protein;
- (ii) the ligand has a molecular weight less than 5 kD;
- and
- (iii) the ligand is membrane permeable;
- and
- wherein the action domain induces apoptosis in a cell following formation of the ligand cross-linked complex.
Dependencies are inferred from the claim text.