Regulated transcription of targeted genes and other biological events

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.

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 10  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 1  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. 
     
     
         17 . A chimeric protein encoded by a DNA construct of  claim 1 . 
     
     
         18 . A cell containing and capable of expressing at least one DNA construct of  claim 1 . 
     
     
         19 . A cell of  claim 18  which is a mammalian cell. 
     
     
         20 . A cell of  claim 18  which contains
 (a) a first DNA construct encoding a chimeric protein comprising (i) at least one receptor domain capable of binding to a selected ligand and (ii) another protein domain, heterologous with respect to the receptor domain, but capable, upon oligomerization with one or more other like domains, of triggering the activation of transcription of a target gene under the transcriptional control of a transcriptional control element responsive to said oligomerization; and   (b) a target gene under the expression control of a transcriptional control element responsive to said oligomerization;   and which following exposure to the selected ligand expresses the target gene.   
     
     
         21 . A cell of  claim 18  which contains a first set of DNA constructs encoding
 (a) a first chimeric protein containing a DNA-binding domain and at least one receptor domain capable of binding to a first selected ligand moiety; and   (b) a second chimeric protein containing a transcriptional activating domain and at least one receptor domain capable of binding to a second selected ligand (which may be the same or different from the first selected ligand moiety); and   and a second DNA construct encoding a target gene under the transcriptional control of a heterologous transcriptional control sequence which binds with the DNA-binding domain and is responsive to the transcriptional activating domain;   which cell expresses the target gene following exposure to a substance containing the selected ligand moiety(ies).   
     
     
         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 . (canceled) 
     
     
         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.   
     
     
         31 . A method of  claim 30  wherein the cells are grown in a culture medium and the exposing is effected by adding the ligand to the culture medium. 
     
     
         32 . A method of  claim 30  wherein the cells are present within a host organism and the exposing is effected by administering the ligand to the host organism. 
     
     
         33 . A method of  claim 32  wherein the host organism is a mammal and the ligand is administered by oral, bucal, sublingual, transdermal, subcutaneous, intramuscular, intravenous, intra joint or inhalation administration in an appropriate vehicle therfor. 
     
     
         34 - 35 . (canceled) 
     
     
         36 . A kit which comprises at least one DNA construct of  claim 1 . 
     
     
         37 . A kit of  claim 36  which further comprises a ligand to which one or more of the chimeric proteins encoded by the DNA construct(s) bind. 
     
     
         38 . A kit of  claim 37  which further comprises a monomeric ligand reagent as an antagonist for ligand-chimeric protein binding. 
     
     
         39 . A kit of  claim 36  which further comprises at least one DNA construct of  claim 10 . 
     
     
         40 . A kit containing a first DNA construct encoding a chimeric protein containing at least one receptor domain (capable of binding to a selected ligand), fused to a transcriptional activator domain; a second DNA construct encoding a second chimeric protein containing at least one one receptor domain (capable of binding to a selected ligand), fused to a DNA binding domain; and a third DNA construct encoding a target gene under the control of a transcriptional control element containing a DNA sequence to which the DNA binding domain binds and which is transcriptionally activated by exposure to the ligand in the presence of the first and second chimeric proteins. 
     
     
         41 . A kit of  claim 37  in which each DNA construct is linked to a selection marker, which may be the same or different for each different DNA construct, permitting the selection of cells which contain said DNA construct(s). 
     
     
         42 . A kit of  claim 37  in which one or more of the DNA constructs contains a cloning site in place of an action domain or target gene. 
     
     
         43 . A kit of  claim 39  which further comprises positive control cells stably transformed with the DNA construct(s) which are provided in the kit. 
     
     
         44 - 47 . (canceled)