Regulated transcription of targeted genes and other biological events
First Claim
1. A eukaryotic cell comprising at least one genetic construct encoding a chimeric protein, which chimeric protein comprises:
- (a) at least one ligand-binding domain which binds to a selected ligand to form a ligand cross-linked protein complex including the chimeric protein, the ligand having 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
(b) an action domain, which is heterologous with respect to at least one of the ligand-binding domains, and which induces a biological process in the cell as a result of formation of the ligand cross-linked protein complex.
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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. Using gene transfer techniques to introduce our artificial receptors, one can turn on or off the signaling pathways that lead to the overexpression of therapeutic proteins by administering orally active "dimerizers" or "de-dimerizers", respectively. Since cells from different recipients can be configured to have the pathway overexpress different therapeutic proteins for use in a variety of disorders, the dimerizers have the potential to serve as "universal drugs". They can also be viewed as cell permeable, organic replacements for therapeutic antisense agents or for proteins that would otherwise require intravenous injection or intracellular expression (e.g., the LDL receptor or the CFTR protein).
75 Citations
129 Claims
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1. A eukaryotic cell comprising at least one genetic construct encoding a chimeric protein, which chimeric protein comprises:
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(a) at least one ligand-binding domain which binds to a selected ligand to form a ligand cross-linked protein complex including the chimeric protein, the ligand having 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 (b) an action domain, which is heterologous with respect to at least one of the ligand-binding domains, and which induces a biological process in the cell as a result of formation of the ligand cross-linked protein complex. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 86, 104, 105, 106, 107)
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62. A eukaryotic cell comprising two or more genetic constructs, each encoding a different chimeric protein, each of which chimeric proteins independently comprises
(a) at least one ligand-binding domain which binds to a selected ligand to form a ligand cross-linked protein complex containing at least the two different chimeric proteins, the ligand having 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 (b) at least one of (i) an action domain, which is heterologous with respect to at least one of the ligand-binding domains, and which induces a biological process as a result of formation of the ligand cross-linked protein complex, and (ii) an intracellular targeting domain, which is heterologous with respect to at least one of the ligand-binding domains, and which induces cellular localization of the ligand cross-linked protein complex, with the proviso that at least one of the chimeric proteins includes an action domain. - View Dependent Claims (63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129)
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Specification