WO1996006111A1 - Regulatable elimination of gene expression, gene product function and engineered host cells - Google Patents

Regulatable elimination of gene expression, gene product function and engineered host cells Download PDF

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Publication number
WO1996006111A1
WO1996006111A1 PCT/US1995/010591 US9510591W WO9606111A1 WO 1996006111 A1 WO1996006111 A1 WO 1996006111A1 US 9510591 W US9510591 W US 9510591W WO 9606111 A1 WO9606111 A1 WO 9606111A1
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gene
cells
protein
ligand
domain
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PCT/US1995/010591
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English (en)
French (fr)
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Joan S. Brugge
Gerald R. Crabtree
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Ariad Gene Therapeutics, Inc.
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Priority claimed from US08/292,597 external-priority patent/US5834266A/en
Application filed by Ariad Gene Therapeutics, Inc. filed Critical Ariad Gene Therapeutics, Inc.
Priority to AU34092/95A priority Critical patent/AU3409295A/en
Priority to EP95930868A priority patent/EP0776335A4/en
Priority to JP8508238A priority patent/JPH10507624A/ja
Publication of WO1996006111A1 publication Critical patent/WO1996006111A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • C07K7/645—Cyclosporins; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C07K2319/715—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16 containing a domain for ligand dependent transcriptional activation, e.g. containing a steroid receptor domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification

Definitions

  • This invention concerns materials, methods and applications relating to the multimerizing of chimeric proteins with a dimeric or multimeric, preferably non-peptidic, organic compound. Aspects of the invention are exemplified by recombinant modifications of host cells and their use in vitro and in vivo for the regulatable blockade of expression of a target gene, for interference with the function or effect of a target gene product or for the regulatable elimination of a target gene.
  • the materials, methods and applications of the invention provide a means for regulating expression of genes introduced by recombinant modifications of host cells, including completely eliminating a population of genetically engineered cells, thus providing a fail-safe mechanism for controlling genetically engineered cells used in gene therapy.
  • One approach to studying the role of a gene in a host organism is to eliminate it or replace it with a dysfunctional counterpart. This can be accomplished, for example, by genetic engineering of cultured embryonic stem ( ⁇ S") cells followed by introduction of the engineered cells into embryos which develop into whole organisms lacking a functional copy of the gene. Typically such experiments are conducted in mice. However, where the gene product is required for normal development of the host organism, simply eliminating, or “knocking out", the gene may yield severely dysfunctional animals, which may not be useful, or no animals at all.
  • ⁇ S cultured embryonic stem
  • conditional knock-outs i.e., cells or organisms in which a gene can be ablated when desired, i.e., at or after any desired stage of development and for creating cell- type specific (non-conditional) knock-outs, i.e., organisms in which a gene is ablated in a specific type of cell. See for example, Watson et al., Recombinant DNA, 2d ed.
  • This invention provides materials and methods for the genetic engineering of host cells to render the cells, their progeny and organisms containing them, susceptible to blockade of expression of a selected gene, to interference with the functioning of the gene product or to elimination or inactivation of a selected gene, in a regulated fashion.
  • the invention further provides materials and methods for genetic engineering of host cells to render the cells and their progeny susceptible to regulated, programmed cell death, otherwise known as apoptosis.
  • Such cells, as well as organisms containing them are useful as biological reagents for a variety of research purposes, as described infra, including the study of diseases characterized by the dysfunction or absence of a gene or gene product of interest.
  • Table 1 below provides a non-exclusive, illustrative list of diseases linked to the dysfunction or absence of a gene or gene product.
  • the invention involves the adaptation of a method and materials for using homo- and hetero-multimerization of chimeric "responder" proteins to trigger gene transcription in living cells.
  • the terms multimer, multimerize and multime ⁇ zation encompass dimers, trimers and higher order multimers and their formation.
  • the chimeric responder proteins are intracellularly expressed fusion proteins which contain a specific receptor domain and are responsive to the presence of a corresponding multimerizing agent.
  • the multimerizing agent is a multivalent ligand which is capable of binding to more than one of the chimeric protein molecules to yield dimers or higher order multimers of the chimeras.
  • the chimeric proteins are designed such that the ligand-mediated multimerization triggers transcription of a gene under the transcriptional control of an element which is responsive to such multimerization.
  • This gene may be a "blocking" gene, which encodes a blocking factor such as an anti-sense message complementary to, and capable of interfering with transcription of, a target gene or a ribozyme. which is capable of preventing the expression of a target gene.
  • the blocking gene may encode an antibody moiety, such as a single-chain antibody, which is capable of binding to and preferably neutralizing or blocking a biological effect of the gene product of the target gene. Table 1
  • Gaucher's disease glucocerebrosidase glycogen storage diseases several types/genes phenylketonuria phenylalanine hydroxylase severe combined ADA, purine nucleoside phosphorylase, immunodeficiency disease
  • the blocking gene also may encode a dominant negative gene product capable of blocking the effect of a target gene product, or it may encode a protein whose action will lead to the elimination of the selected target gene entirely.
  • An example of a blocking gene whose action will lead to the elimination of a target gene is the gene encoding the protein Cre, which produces Cre recombinase. Expression of the Cre recombinase leads to elimination of a target gene appropriately flanked by "loxP" sequences in the host cells.
  • the gene may be a gene which provides a means for eliminating a population of engineered cells, either growing in culture or in vivo, providing a mechanism for regulating and controlling engineered cells.
  • this invention involves one or more chimeric responder proteins, DNA constructs ("responder” constructs) encoding them, and multi-valent ligand molecules capable of multimerizing the chimeric proteins.
  • the chimeric proteins contain at least one ligand-binding (or "receptor") domain and an action domain capable, upon multimerization of the chimeric protein molecules, of initiating transcription of the blocking gene or the elimination effector gene within a cell.
  • the chimeric proteins may further contain additional domains.
  • These chimeric responder proteins and the responder constructs which encode them are recombinant in the sense that their various components are derived from different sources, and as such, are not found together in nature (i.e., are mutually heterologous).
  • recombinant "blocking" constructs containing a blocking gene under the transcriptional regulation of a control element responsive to the presence of multimerized responder proteins described above.
  • the transcriptional control element is responsive in the sense that transcription of the blocking gene is activated by the presence of the multimerized responder chimeras in cells containing these constructs. Exposure of the cells to the multimerizing ligand results in expression of the blocking gene.
  • the constructs of this invention may contain one or more selectable markers such as a neomycin resistance gene (neo r ) and herpes simples virus-thymidine kinase (HSV-tk). When cells which have been genetically engineered to contain and express the responder and the blocking gene are exposed to the multimerizing ligand, expression of the blocking gene is activated and expression of the target gene or functioning of the target gene product is impaired.
  • neomycin resistance gene neo r
  • HSV-tk herpes simples virus-thymidine kin
  • Cre constructs encoding Cre under the transcriptional regulation of a control element responsive to the presence of multimerized responder proteins described above are also provided.
  • the transcriptional control element is responsive in that transcription of Cre is activated by the presence of the multimerized responder chimeras in cells containing these constru ⁇ s. That is, exposure of the cells to the multimerizing ligand results in expression of Cre.
  • Cells may also contain a "target" gene, preferably, but not necessarily, an endogenous gene to be eliminated, which is flanked by loxP sequences ("floxed").
  • the floxed target gene is introduced into the cell, for example, by homologous recombination using a recombinant "target" construct containing part or all of a copy of the endogenous target gene and/or endogenous flanking DNA sequence thereof, together with loxP DNA elements.
  • the floxed target gene construct may contain one or more selectable markers such as the neomycin resistance gene (neo r ) or herpes simples virus-thymidine kinase gene (HSV-tk).
  • recombinant constructs encoding primary chimeric proteins of this invention permit ligand-regulated apoptosis.
  • These chimeric constructs contain at least the cytoplasmic domain of the fas antigen or Apo-1 antigen, which when cross-linked, induces apoptosis in most cell types.
  • Modified cells are produced by introducing the desired construct ⁇ ) into selected host cells. This may be accomplished using conventional vectors and techniques, many of which are commercially available. If desired, the modified cells into which one or more constructs have been successfully introduced may then be selected, separated from other cells and cultured, again by conventional methods.
  • incorporation of a target gene construct into a host cell may be effected by homologous recombination, by known methods such as that of Gu et al., 1994, Science 265:103-106.
  • the recombinant target gene construct may be introduced by any desired means. Incorporation of the responder constructs and Cre constructs may also be effected using conventional transfection vectors and techniques.
  • Genetically engineered cells which can be grown together with other cells and which can be selectively regulated in, or ablated from, the mixture of cells by addition of an effective amount of an oligomerization ligand which is capable of binding to the primary chimeric protein, are an important aspect of this invention.
  • Contacting the engineered cells with the oligomerization ligand can trigger the regulatable obstruction of the expression of a specific gene through the expression of a target gene which can block or interfere with the function of a target gene.
  • the regulated gene can be one whose expression leads to specific elimination of a target gene, or cell death of the engineered cells.
  • such cells may be permitted to produce an endogenous or heterologous product for some desired period, and may then be deleted by addition of the ligand. In such cases, the cells are engineered to produce a primary chimera in accordance with this invention.
  • the cells which may be further engineered to express a desired gene under ligand-induced regulation, may be grown in culture by conventional means. In that case, addition to the culture medium of the ligand for the optional chimera leads to expression of the desired gene and production of the desired protein. Expression of the gene and production of the protein can then be turned off by adding to the medium an oligomerization antagonist reagent, as is described in detail below. In other cases, production of the protein is constitutive. In any event, the engineered cells can then be eliminated from the cell culture after they have served their intended purpose (e.g.
  • Engineered cells of this invention can also be used in vivo, to modify whole organisms, preferably animals, including humans, such that the cells produce a desired protein or other result within the animal containing such cells. Such uses include gene therapy. Alternatively, the chimeric proteins and oligomerizing molecules can be used extracellularly to bring together proteins which act in concert to initiate a physiological action. To create transgenic animals containing modified cells of this invention the desired constructs are transfected into appropriate cell lines, for example, ES cells.
  • the desired constructs may be directly microinjected into early embryos. See for example, Watson et al., Recombinant DNA, 2d ed., 1992, especially Chapters 14 and 24.
  • tissue-specific expression control sequence such as a promoter or enhancer sequence
  • use of a tissue-specific expression control sequence, such as a promoter or enhancer sequence, in the responder construct permits tissue-specific expression of the chimeric responder protein(s), which in turn permits tissue-specific, regulatable expression of the blocking gene, and thus tissue-specific blockade of the target gene or its gene product.
  • animals may be so produced which comprise cells containing and capable of expressing the responder.
  • Other animals may be produced which comprise cells containing a blocking gene construct. Breeding the two types of engineered animals yields offspring which contain cells containing both of the foregoing constructs.
  • a tissue-specific expression control sequence (promoter/enhancer) in the responder construct also permits tissue-specific expression of the chimeric responder protein(s), which in turn permits tissue-specific, regulatable expression of Cre, and thus tissue-specific elimination of the target gene.
  • animals may be so produced which comprise cells containing and capable of expressing the responder and Cre constru ⁇ s.
  • Other animals may be produced which comprise cells containing a desired target gene constru ⁇ . Breeding the two types of engineered animals yields offspring which contain cells containing all of the foregoing constru ⁇ s. Animals and their progeny may be conveniently chara ⁇ erized by conventional genetic analysis.
  • the constructs may also be introduced by administration, e.g. in suitable vehicles or ve ⁇ ors, dire ⁇ ly into the desired tissue of the whole organisms.
  • the multimerizing ligands useful for triggering the expression of the blocking gene including in the pra ⁇ ice of this invention are capable of binding to two or more chimeric responder proteins containing such receptor domains.
  • the multimerizing ligand may bind to the chimeras in either order or simultaneously, preferably with a Kd value below about 10"*, more preferably below about 10 '7 , even more preferably below about 10 "8 , and in some embodiments below about 10 '9 M.
  • the ligand preferably is a non-protein and has a molecular weight of less than about 5 kDa. Even more preferably, the multimerizing ligand has a molecular weight of less than about 2 Kda, and even more preferably, less than 1500 Da.
  • the receptor domains of the chimeric proteins so multimerized may be the same or different.
  • the chimeric proteins are capable of initiating expression of the blocking gene in the host cell upon exposure to the ligand, following multimerization of the chimeras.
  • transcription of the blocking gene or the elimination effe ⁇ or gene is a ⁇ ivated in genetically engineered cells of this invention following exposure of the cells to a ligand capable of multimerizing the chimeras.
  • genetically engineered cells of this invention contain chimeric proteins as described above and are responsive to the presence of a ligand which is capable of multimerizing those chimera. That responsiveness is manifested by the initiation of expression of the blocking gene and by blockade of expression of the target gene or blocking a biological effe ⁇ of the target gene produ ⁇ .
  • the encoded chimeric responder protein may further comprise an intracellular targeting domain capable of dire ⁇ ing the chimeric protein to a desired cellular compartment.
  • the targeting domain can be a secretory leader sequence, a membrane spanning domain, a membrane binding domain or a sequence dire ⁇ ing the protein to associate with vesicles or with the nucleus, for instance.
  • the a ⁇ ion domains of the chimeric proteins may be sele ⁇ ed from any of the proteins or protein domains (preferably of the species of the desired host cells or organism) which upon multimerization are capable of a ⁇ ivating transcription of a gene, the blocking gene or the regulatable elimination effe ⁇ or gene, in our system, under the transcriptional control of a cognate control element.
  • the a ⁇ ion domain of the chimeric responder protein molecules may comprise a protein domain such as a CD3f (zeta subunit) which is capable, upon exposure to the ligand and subsequent multimerization, of initiating a dete ⁇ able intracellular signal leading to transcriptional a ⁇ ivation via the IL-2 promoter.
  • responder proteins there may be a series of responder proteins, in which one responder protein contains as its a ⁇ ion domain, a DNA-binding protein such as GAL4, while another contains as its a ⁇ ion domain a transcriptional a ⁇ ivation domain such as VP16.
  • Heterodimerization of such responder proteins to form a GAL4-VP16 dimer a ⁇ ivates the transcription of the blocking genes or the elimination effe ⁇ or gene under the transcriptional control of elements to which the heterodimerized responder proteins can bind.
  • multimerization a ⁇ ivates transcription of a blocking gene or an elimination effe ⁇ or gene under the transcriptional control of a transcriptional control element enhancer and/or promoter elements and the like, which is responsive to the multimerization event.
  • This invention further encompasses DNA ve ⁇ ors containing the various constru ⁇ s described herein, whether for introdu ⁇ ion into host cells in tissue culture, for introduttion into embryos or for administration to whole organisms for the introdu ⁇ ion of the constru ⁇ s into cells in vivo.
  • the constru ⁇ may be introduced episomally or for chromosomal integration.
  • the ve ⁇ or may be a viral ve ⁇ or, including for example, an adeno-, adeno associated or retroviral ve ⁇ or.
  • the constru ⁇ s or ve ⁇ ors containing them may also contain sele ⁇ able markers permitting sele ⁇ ion of transfe ⁇ ants containing the constru ⁇ .
  • This invention further encompasses a chimeric protein encoded by any of our DNA constru ⁇ s, as well as cells containing and/or expressing them, including prokaryotic and eucaryotic cells and in particular, yeast, worm, inse ⁇ , mouse or other rodent, and other mammalian cells, including human cells, of various types and lineages, whether frozen or in a ⁇ ive growth, whether in culture or in a whole organism containing them.
  • This invention provides cells, preferably mammalian cells, which contain one or more recombinant DNA constru ⁇ s encoding a responder protein, or series of responder proteins, and a blocking gene constru ⁇ capable of expressing a blocking gene in response to multimerization of the responder protein (s). Additionally, it provides cells which contain one or more recombinant DNA constru ⁇ s encoding a responder protein, or series of responder proteins, a Cre constru ⁇ capable of expressing a Cre gene in response to multimerization of the responder protein(s) and optionally, a target gene constru ⁇ comprising a target gene flanked by loxP sequences which is susceptible to deletion in the presence of Cre.
  • the invention also provides cells which contain recombinant DNA constru ⁇ s encoding proteins whose expression is capable of leading to regulated apoptosis of the cells containing the constru ⁇ .
  • the multimerizing ligands are molecules capable of binding to two or more chimeric responder protein molecules of this invention to form a multimer thereof, and have the formula:
  • n is an integer from 2 to about 5
  • rbm (1) .rbm (n) are receptor binding moieties which may be the same or different and which are capable of binding to the chimeric protein (s).
  • the rbm moieties are covalently attached to a linker moiety which is a bi- or multi-fun ⁇ ional molecule capable of being covalently linked ("— ") to two or more rbm moieties.
  • the ligand has a molecular weight of less than about 5 Kda and is not a protein.
  • ligands examples include those in which the rbm moieties are the same or different and comprise an FK506-type moiety, a cyclosporin-type moiety, a steroid or tetracycline.
  • Cyclosporin-type moieties include cyclosporin and derivatives thereof which are capable of binding to a cyclophilin, naturally occurring or modified, preferably with a Kd value below about 10"* M. In some embodiments it is preferred that the ligand bind to a naturally occurring receptor with a Kd value greater than about 10 "6 M and more preferably greater than about 10 "5 M.
  • Illustrative ligands of this invention are those in which at least one rbm comprises a molecule of FK506, FK520, rapamycin or a derivative thereof modified at C9, CIO or both, which ligands bind to a modified receptor or chimeric molecule containing a modified receptor domain with a Kd value at least one, and preferably 2, and more preferably 3 and even more preferably 4 or 5 or more orders of magnitude less than their Kd values with respe ⁇ to a naturally occurring receptor protein.
  • Linker moieties are also described in detail later, but for the sake of illustration, include such moieties as a C2-C20 alk lene, C4-C18 azalkylene, C6-C24 N-alkylene azalkylene, C6-C18 arylene, C8-C24 ardialkylene or C8-C36 bis-carboxamido alkylene moiety. See also U.S. Patent Application Serial No. 08/481,941 entitled “Rapamycin Regulation of Biological Events", filed on June 7, 1995 and U.S. Patent Application Serial No. 08/292,598, entitled “New Multimerizing Agents," filed 18 August 1994, the contents of which are hereby incorporated by reference.
  • the monomeric rbm's of this invention as well as compounds containing sole copies of an rbm, which are capable of binding to our chimeric proteins but not effe ⁇ ing dimerization or higher order multimerization thereof (in view of the monomeric nature of the individual rbm) are multimerization antagonists.
  • This invention thus provides materials and methods for sele ⁇ ively obstru ⁇ ing the expression or effe ⁇ s of a target gene in engineered cells in response to the presence of a multimerizing ligand which is added to the culture medium or administered to the whole organism.
  • the invention further provides materials and methods for sele ⁇ ively eliminating a target gene in engineered cells in response to the presence of a multimerizing ligand which is added to the culture medium or administered to the whole organism, as the case may be.
  • the invention further provides materials and methods for sele ⁇ ively ablating cells in response to the addition of an oligomerizing ligand.
  • the methods involve providing cells of this invention, or an organism containing such cells, which contain and are capable of expressing (a) one or more DNA constru ⁇ s encoding one or more chimeric proteins capable, following multimerization, of a ⁇ ivating transcription of a blocking gene; (b) a blocking gene under the transcriptional regulation of an element responsive to multimers of the chimeric proteins.
  • the method thus involves exposing the cells to a multimerization ligand capable of binding to the chimeric protein in an amount effe ⁇ ive to result in dete ⁇ able expression of the blocking gene.
  • the method also involves providing cells of this invention, or an organism containing such cells, which contain and are capable of expressing (a) one or more DNA constru ⁇ s encoding one or more chimeric proteins capable, following multimerization, of a ⁇ ivating transcription of a gene encoding Cre; (b) a gene encoding Cre which is under the transcriptional regulation of an element responsive to multimers of the chimeric proteins; and (c) a floxed target gene which is susceptible to recombination and elimination in the presence of Cre.
  • the method thus involves exposing the cells to a multimerization ligand capable of binding to the chimeric protein in an amount effe ⁇ ive to result in dete ⁇ able expression of the Cre gene.
  • the ligand is administered to the host animal by oral, buccal, sublingual, transdermal, subcutaneous, intramuscular, intravenous, intra-joint or inhalation administration in an appropriate vehicle therefor.
  • the target gene is essential for normal fun ⁇ ioning in the healthy cell or animal, obstru ⁇ ing the expression of the gene, the effe ⁇ s of the gene produ ⁇ or eliminating the gene entirely, using the materials and methods of this invention, provides cellular and animal models for corresponding disease states.
  • the target gene is the gene for erythropoietin
  • its obstru ⁇ ion or elimination provides a model for the study of anemia and potential treatments for it.
  • Targeting genes for nerve growth fa ⁇ ors or myelinating proteins provides models of neural degenerative diseases; for bone morphogenic fa ⁇ ors, of osteoporosis; for thrombopoietin, of thrombocytopenia; for antigen receptors or immune cell signaling proteins, of immunodeficiencies; for tyrosine hydroxylase, of Parkinson's disease; etc. See Table 1.
  • This invention further encompasses pharmaceutical or v ⁇ erinary compositions for obstru ⁇ ing the expression or effe ⁇ s of a gene or eliminating a gene from genetically engineered cells of this invention, including eliminating the engineered cells themselves from animal tissue or from a subje ⁇ containing such engineered cells.
  • Such pharmaceutical or veterinary compositions comprise a multimerization ligand of this invention in admixture with a pharmaceutically or veterinarily acceptable carrier and optionally with one or more acceptable excipients.
  • the multimerization ligand can be a homo- multimerization reagent or a hetero-multimerization reagent so long as it is capable of binding to a chimeric responder protein(s) of this invention, triggering expression of the blocking gene or triggering Cre expression in engineered cells of this invention.
  • this invention further encompasses a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising a multimerization antagonist of this invention in admixture with a pharmaceutically acceptable carrier and optionally with one or more pharmaceutically or veterinarily acceptable excipients for preventing or reducing, in whole or part, the level of multimerization of chimeric responder proteins in engineered cells of this invention, in cell culture or in a subje ⁇ , and thus for preventing or reversing the a ⁇ ivation of transcription of the blocking gene in the relevant cells.
  • the use of the multimerization reagents and of the multimerization antagonist reagents to prepare pharmaceutical or veterinary compositions is encompassed by this invention.
  • This invention also offers a method for providing a host organism, preferably an animal, and in many cases a mammal, susceptible to regulatable obstru ⁇ ion of a target gene in response to a multimerization ligand of this invention.
  • the method involves introducing into the organism cells which have been engineered ex vivo in accordance with this invention, i.e. containing a DNA constru ⁇ encoding a chimeric protein hereof, and so forth.
  • DNA constru ⁇ s of this invention into a host organism, e.g. mammal or embryo thereof, under conditions permitting transfe ⁇ ion of one or more cells of the host mammal in vivo.
  • kits for producing cells susceptible to ligand- regulated obstru ⁇ ion of a target gene contains at least one DNA constru ⁇ encoding at least one of our chimeric responder proteins, comprising at least one receptor domain and at least one a ⁇ ion domain (as described elsewhere).
  • the DNA constru ⁇ contains a conventional polylinker to provide the pra ⁇ itioner a site for the incorporation of cell-type specific expression control element (s), such as promoter and/or enhancer elements, to provide for cell-type or tissue-specific expression of one or more of the chimeras.
  • s cell-type specific expression control element
  • the kit may contain a quantity of a ligand of this invention capable of multimerizing the chimeric protein molecules encoded by the DNA constru ⁇ s of the kit, and may contain in addition a quantity of a multimerization antagonist, e.g. monomeric ligand reagent. Where a sole chimeric protein is encoded by the constru ⁇ (s), the multimerization ligand is a homo-multimerization ligand. Where more than one such chimeric protein is encoded, a hetero-multimerization ligand may be included.
  • the kit may further contain a blocking gene constru ⁇ linked to a transcription control element responsive to multimerization of the chimeric responder protein molecules.
  • the kit may contain a Cre constru ⁇ linked to a transcription control element responsive to multimerization of the chimeric responder protein molecules and/or a target gene constru ⁇ containing the target gene flanked by loxP sequence.
  • the kit may also contain at least one DNA constru ⁇ encoding a primary chimeric protein containing at least one receptor domain and one a ⁇ ion domain where one a ⁇ ion domain may be the cytoplasmic domain of Fas or of a TNF receptor as described below.
  • the DNA constru ⁇ s will preferably be associated with one or more sele ⁇ able markers for convenient sele ⁇ ion of transfe ⁇ ants, as well as other conventional ve ⁇ or elements useful for replication in prokaryotes, for expression in eukaryotes, and the like.
  • the sele ⁇ ion markers may be the same or different for each different DNA constru ⁇ , permitting the sele ⁇ ion of cells which contain various combinations of such DNA constru ⁇ (s).
  • one kit of this invention contains ve ⁇ ors comprising a first DNA constru ⁇ encoding a first chimeric responder protein containing at least one receptor domain (capable of binding to a sele ⁇ ed ligand) fused to a transcriptional a ⁇ ivator domain; a second DNA construtt encoding a second chimeric responder protein containing at least one receptor domain fused to a DNA binding domain; and optionally, a third DNA constru ⁇ encoding a blocking gene under the transcriptional control of an element responsive to the multimerization of the first and second chimeric responder proteins.
  • the blocking gene construtt may contain a cloning site, for example, a polylinker sequence, in place of a pre-seletted blocking gene to permit the pra ⁇ itioner to insert any desired blocking gene.
  • the kit may also contain a DNA constru ⁇ encoding Cre under the transcriptional control of an element responsive to the multimerization of the first and second chimeric responder proteins and optionally, a DNA constru ⁇ encoding a target gene flanked by the loxP sequence, permitting deletion of the target gene in the presence of Cre.
  • the fifth DNA constru ⁇ may contain a cloning site in place of a target gene to permit the pra ⁇ itioner to insert any desired target gene.
  • kits of this invention may contain one, two, or more DNA constru ⁇ s encoding chimeric proteins in which one or more of the constru ⁇ s contain a cloning site in place of an a ⁇ ion domain, such as the transcriptional initiation signal generator, transcriptional a ⁇ ivator, the DNA binding protein, or other domains, permitting the user to insert whichever a ⁇ ion domain s/he wishes.
  • a kit may optionally include other elements as described above, such as a DNA constru ⁇ for a target gene under responsive expression control, multimerization ligand, antagonist, etc.
  • kits may also contain positive control cells which were stably transformed with constru ⁇ s of this invention such that they express a reporter gene such CAT (chloramphenicol transferase), beta-gala ⁇ osidase or any other conveniently dete ⁇ able gene produ ⁇ , in response to exposure of the cells to ligand.
  • CAT chloramphenicol transferase
  • beta-gala ⁇ osidase or any other conveniently dete ⁇ able gene produ ⁇
  • Figure 1 is a diagram of the plasmid pSXNeo/IL2 (IL2-SX).
  • IL2-SX the Hindffl-Clal DNA fragment from IL2-SX containing the IL2 enhancer/promoter, is replaced by a minimal IL-2 promoter, which confers basal transcription, and an inducible element containing three tandem NFAT-binding sites.
  • Figure 2 is a flow diagram illustrating the preparation of the intracellular signaling chimera plasmids p#MXFn and p#MFnZ, where n indicates the number of binding domains.
  • Figs. 3A and 3B are a flow diagram of the preparation of the extracellular signalling chimera plasmid p#lFK3/pBJ5.
  • Figs. 4A, 4B and 4C are sequences of the primers used in the constru ⁇ ions of the plasmids employed in the subje ⁇ invention.
  • Figure 5 is a chart of the response of reporter constru ⁇ s having different enhancer groups to rea ⁇ ion of the receptor TAC/CD3f with a ligand.
  • Figure 6 is a chart of the a ⁇ ivity of various ligands with the TAg Jurkat cells described in Example 1.
  • Figure 7 is a chart of the a ⁇ ivity of the ligand FK1012A (8, Figure 9B) with the extracellular receptor 1FK3 (FKBPx3/CD3$.
  • Figure 8 is a chart of the a ⁇ ivation of an NFAT repo ⁇ er via signalling through a myristoylated CD3f/FKBPl2 chimera.
  • Figs. 9A and 9B are the chemical stru ⁇ ures of the allyl-linked FK506 variants and the cyclohexyl-linked FK506 variants, respectively.
  • Figure 10 is a flow diagram of the synthesis of derivatives of FK520.
  • Figs. 11 A and B are a flow diagram of a synthesis of derivatives of FK520 and chemical structures of FK520, where the bottom structures are designed to bind to mutant FKBP12.
  • Figure 12 is a diagrammatic depittion of mutant FKBP with a modified FK520 in the putative cleft.
  • Figure 13 is a flow diagram of the synthesis of heterodimers of FK520 and cyclosporin.
  • Figure 14 is a schematic representation of the oligomerization of chimeric proteins, illustrated by chimeric proteins containing an immunophilin moiety as the receptor domain.
  • Figure 15 depitts ligand-mediated oligomerization of chimeric proteins, showing schematically the triggering of a transcriptional initiation signal.
  • Figure 16 depicts synthetic schemes for HED and HOD reagents based on FK506-type moieties.
  • Figure 17 depitts the synthesis of (CsA)2 beginning with CsA.
  • Figure 18 is an overview of the fusion cDNA construct and protein
  • Figure 19 shows FK1012-induced cell death of the Jurkat T-cell line transfected with a myristoylated Fas-FKBP12 fusion protein (MFF3E), as indicated by the decreased transcriptional activity of the cells.
  • Figure 20A is an analysis of cyclophilin-Fas (and Fas-cyclophilin) fusion constructs in the transient transfection assay. MC3FE was shown to be the most effective in this series.
  • Figure 20B depicts Immunophilin-Fas antigen chimeras and results of transient expression experiments in Jurkat T cells stably transformed with large T- antigen.
  • Myr the myristylation sequence taken from pp60 c src encoding residues 1-
  • FKBP human FKBP12
  • CypC murine cyclophilin C sequence encoding residues 36-212
  • Fas intracellular domain of human Fas antigen encoding residues 179-319 (Oehm et al., /. Biol. Chem. 267 15 (1992): 10709-15).
  • Cells were electroporated with a plasmid encoding a secreted alkaline phosphatase reporter gene under the control of 3 tandem API promoters along with a six fold molar excess of the immunophilin fusion construct. After 24 h (hours) the cells were stimulated with PMA (50ng/mL), which stimulates the synthesis of the reporter gene, and (CsA)2. At 48 hours the cells were assayed for reporter gene activity. Western blots were performed at 24 hours using anti-HA epitope antibodies.
  • Figure 21 depicts the synthesis of modified FK506 type compounds.
  • This invention provides chimeric proteins, organic molecules for multimerizing the chimeric proteins and a system for using them.
  • the fused chimeric proteins have a binding domain for binding to the multimerizing molecule and an action domain, which can effectuate a physiological action or cellular process.
  • the multimerizing molecule is a small organic molecule.
  • the physiological action or cellular process effectuated by multimerization of the chimeric proteins is generally transcription of a gene encoding a regulatable blocking gene.
  • This gene may be a "blocking" gene, which encodes a blocking factor such as an anti-sense message complementary to, and capable of interfering with transcription of a target gene or a ribozyme, which is capable of preventing the expression of a target gene.
  • the blocking gene may encode an antibody moiety, such as a single-chain antibody, which is capable of binding to and preferably neutralizing or blocking a biological effect of the gene product of the target gene.
  • the blocking gene also may encode a dominant negative gene product capable of blocking the effect of a target gene product, or it may encode a protein whose action will lead to the elimination of the target gene entirely.
  • An example of the latter type would be the gene encoding the protein Cre, which produces Cre recombinase and whose expression leads to elimination of a target gene appropriately flanked by "loxP" sequences in the host cells.
  • the constructs will encode gene products such as the cytoplasmic domain of the Fas antigen or TNF receptor, which allow the cells containing such constructs to be readily eliminated through apoptosis.
  • HED heterodimerization, or hetero- multimerization
  • HOD homodimerization, or homo- multimerization
  • homodimerization and “homo-multimerization” refer to the association of like components to form dimers or multimers, which may be linked, as shown in figure 14, by the multivalent ligands of this invention.
  • heterodimerization and “hetero-multimerization” refer to the association of dissimilar components to form dimers or higher order multimers. Homo- multimers thus comprise an association of multiple copies of a particular component, while hetero-multimers comprise an association of copies of different components.
  • Multimerization”, “multimerize” and “multimer”, as the terms are used herein, with or without prefixes, are intended to encompass “dimerization”, “dimerize” and “dimer”, absent an explicit indication to the contrary.
  • fusion, or chimeric, protein molecules containing an action domain and one or more receptor domains that can bind to the multimerization ligands.
  • Intracellular chimeric proteins i.e., proteins which are intended to be located within the cells in which they are produced, will in some embodiments preferably, contain a cellular targeting sequence (e.g. including organelle targeting amino acid sequences). Binding of the ligand to the receptor domains leads to multimerization of the fusion proteins. Multimerization brings the action domains into close proximity with one another thus triggering activation of transcription of a gene which is under the transcriptional control of an element responsive to the multimerization.
  • Cellular processes which can be triggered by receptor multimerization include a change in state, such as a physical state, e.g. conformational change, change in binding partner, cell death, initiation of transcription, channel opening, ion release, e.g. Ca +2 etc. or a chemical state, such as an enzymatically catalyzed chemical reaction, e.g. acylation, methylation, hydrolysis, phosphorylation or dephosphorylation, change in redox state, rearrangement, or the like.
  • a change in state such as a physical state, e.g. conformational change, change in binding partner, cell death, initiation of transcription, channel opening, ion release, e.g. Ca +2 etc.
  • a chemical state such as an enzymatically catalyzed chemical reaction, e.g. acylation, methylation, hydrolysis, phosphorylation or dephosphorylation, change in redox state, rearrangement, or the like.
  • any such process which can be triggered by ligand-mediated multimerization is included within the scope of this technology, although the primary focus here is a ⁇ ivation of transcription, directly or indirettly, of a blocking gene, including a gene whose gene product can function to block expression of a selected target gene or the eliminate the target gene from the cell.
  • cells are modified so as to be responsive to ligand molecules which are capable of binding to, and thus multimerizing, the primary chimeras disclosed herein.
  • Such engineered cells respond to the presence of the multimerizing ligand by a ⁇ ivating transcription of a blocking gene via a transcriptional control element responsive to the multimerized chimeric protein molecules.
  • the modified cells are chara ⁇ erized by a genome containing (a) a genetic constru ⁇ (or series thereof) encoding a primary chimeric protein (or series of primary chimeric proteins) of this invention, which permits ligand- regulated transcriptional a ⁇ ivation of a blocking gene via a corresponding transcriptional control element, and (b) a blocking gene constru ⁇ comprising a recombinant DNA sequence containing a blocking gene under the transcriptional control of a transcriptional control sequence which is a ⁇ ivated by multimerization of the chimeric proteins.
  • the regulated blocking gene encodes the protein Cre
  • the cells additionally contain (c) a target gene flanked by loxP DNA sequences, permitting recombination and elimination of the target gene in the presence of Cre protein.
  • a homomultimer including homodimers
  • two or more constru ⁇ s are required where a heteromultimer is involved.
  • the chimeric proteins encoded by the first series of constructs will be associated with actuation of gene transcription and will normally be directed to the surface membrane or the nucleus.
  • the encoded chimeric proteins of this invention contain at least one a ⁇ ion domain and at least one ligand binding domain.
  • the two classes are described as follows: (1) constru ⁇ s wherein a ligand- binding domain of the encoded chimera is either extracellular or intracellular, but an a ⁇ ion domain is intracellular, such that ligand-mediated, homo- or hetero-multimerization of the chimeric protein molecules induces a signal which results in a series of events resulting in transcriptional a ⁇ ivation of the seletted gene; (2) constructs wherein a ligand-binding domain and an a ⁇ ion domain of the encoded chimera is intranuclear, such that ligand-mediated, homo- or hetero-multimerization of the protein molecules induces initiation of transcription directly via complexation of multimers with the transcriptional initiation region of the selected blocking gene.
  • Group (1) chimeras activate transcription indirettly and can have somewhat pleiotropic effects; that is, they may attivate a number of wild-type genes in addition to the introduced blocking gene.
  • the transcriptional a ⁇ ivation following multimerization of group (1) chimeric proteins may be slower in onset than in the case of Group (2) chimeras.
  • Group (2) constru ⁇ s attivate transcription more directly and in a manner more narrowly limited to the selected gene. The transcriptional activation in response to multimerization of Group (2) chimeric proteins is typically very rapid.
  • the chimeric protein contains a "binding" or "receptor” domain which is capable of binding to at least one ligand molecule. Since the multimerizing ligand is multivalent, in the sense that it contains more than one receptor- binding site, it can form dimers or higher order homo- or hetero-multimers with the chimeric proteins of this invention.
  • the chimeric protein can have one or a plurality of binding sites, so that, for example, homomultimers can be formed with a divalent ligand.
  • the chimeric protein also contains an "action" domain capable, upon ligand-mediated multimerization of the chimeric protein molecules, of initiating transcription, whether directly or indirettly.
  • the chimeric proteins whether of Group (1) or (2) will typically also contain an intracellular targeting domain comprising a sequence or component which directs the chimeric protein to the desired compartment, for example to the surface membrane in the case of Group (1), or the nucleus in the case of Group (2).
  • a Group (1) chimeric protein may contain a myristate moiety as an intracellular targeting domain, three FKBP 12 moieties as receptor domains; and the a ⁇ ion domain comprising a T cell receptor f subunit.
  • Group (2) chimeric proteins generally comprise a series of at least two chimeras, where the a ⁇ ion domains of one comprise DNA-binding domains, while the a ⁇ ion domains of another comprise transcriptional a ⁇ ivating domains. Multimerization of the Group (2) chimeras brings the DNA-binding domains and transcriptional a ⁇ ivation domains in close proximity. Subsequent interaction of resulting multimers with the DNA sequence to which the DNA-binding domains bind results in the initiation of transcription of the gene associated with the responsive DNA element.
  • the blocking gene is linked to a transcriptional regulatory sequence activated upon multimerization of the chimera's action domain. For example, where the a ⁇ ion domain is a T-cell
  • the blocking gene may be linked to NFAT sequence.
  • the desired regulatable gene encodes the protein Cre
  • a recombinant constru ⁇ comprising a floxed target gene is provided to serve as the object of the Cre- mediated recombination to complete the overall system.
  • Group (1) chimeric proteins of this invention are typically associated with the surface membrane of the engineered cells. Addition of the multimerizing agent to cells containing such proteins results in the generation of a signal leading to transcription of one or more genes. The process involves a number of auxiliary proteins in a series of interactions culminating in the binding of transcription factors to promoter regions associated with the seletted gene(s) In cases in which the transcription fa ⁇ ors bind to promoter regions associated with other genes, transcription is initiated there as well.
  • a constru ⁇ encoding a chimeric protein of this embodiment can encode a signal sequence which can be subject to processing and therefore may not be present in the mature chimeric protein.
  • the chimeric protein will in any event comprise (a) a binding domain capable of binding a pre-determined ligand, (b) an optional (although in many embodiments, preferred) membrane binding element or domain which includes a transmembrane domain or an attached lipid for translocating the fused protein to the cell surface/membrane and retaining the protein bound to the cell surface membrane, and, (c) as the attion domain, a cytoplasmic signal initiation domain.
  • the cytoplasmic signal initiation domain is capable of initiating a signal which results in transcription of a gene having a recognition sequence for the initiated signal in the transcriptional initiation region.
  • the molecular portion of the chimeric protein which provides for binding to a membrane is also referred to as the "retention domain".
  • Suitable retention domains include a moiety which binds directly to the lipid layer of the membrane, such as through lipid participation in the membrane or extending through the membrane, or the like. In such cases the protein becomes translocated to and bound to the membrane, particularly the cellular membrane, as depicted in figure 15. Also see figure 8 of Spencer et al.
  • Group (2) chimeric proteins may contain a cellular targeting sequence which provides for the protein to be translocated to the nucleus.
  • This "signal consensus" sequence has a plurality of basic amino acids, referred to as a bipartite basic repeat as reviewed in Garcia-Bustos et al., Biochimica et Biophysica Atta (1991) 1071, 83-101. This sequence can appear in any portion of the molecule internal or proximal to the N- or C-terminus and results in the chimeric protein being inside the nucleus.
  • One embodiment of this invention involves at least two such Group (2) chimeric proteins: (1) one having an action domain which binds to the DNA of the transcription initiation region associated with the blocking gene in the blocking gene construct and (2) a different chimeric protein containing as an action domain, a transcriptional a ⁇ ivation domain capable, in association with the DNA binding domain of the first chimeric protein, of initiating transcription of the blocking gene constru ⁇ .
  • the two a ⁇ ion domains or transcription fa ⁇ ors can be derived from the same or different protein molecules.
  • the transcription fa ⁇ ors can be endogenous or exogenous to the cellular host. If the transcription fa ⁇ ors are exogenous, but functional within the host and can cooperate with the endogenous RNA polymerase, rather than requiring an exogenous RNA polymerase, for which a gene could be introduced, then an exogenous promoter element functional with the fused transcription factors can be provided within the blocking gene construct for regulating transcription of the blocking gene. By this means the initiation of transcription can be restricted to the blocking gene associated with the heterologous promoter region.
  • transcription factors which require two subunits for attivity.
  • a single transcription factor can be divided into two separate functional domains (e.g. a transcriptional attivator domain and a DNA-binding domain), so that each domain is inactive by itself, but when brought together in close proximity, transcriptional attivity is restored.
  • Transcription factors which can be used include yeast GAL4, which can be divided into two domains as described by Fields and Song, supra. The authors use a fusion of GAL4(1-147)-SNF1 and SNF4- GAL4 (768-881), where the SNF1 and -4 may be replaced by the subjett binding proteins as binding domains. Combinations of GAL4 and VP16 or
  • HNF-1 can be employed.
  • Other transcription factors are members of the Jun, Fos, and ATF/CreB families, Ottl, Spl, HNF-3, the steroid receptor superfamily, and the like.
  • the activation domain may be replaced by one of the binding proteins associated with bridging between a transcriptional activation domain and an RNA polymerase, including, but not limited to RNA polymerase II.
  • these proteins include the proteins referred to as TAF's (transcriptional activation factors), the TFII proteins, particularly B and D, or the like.
  • TAF's transcriptional activation factors
  • TFII proteins transcriptional activation factors
  • B and D or the like.
  • the protein closest to the RNA polymerase will be employed in conjunction with the DNA binding domain to provide for initiation of transcription.
  • the subjett constructs can provide for three or more, usually not more than about 4, proteins to be brought together to provide the transcription initiation complex.
  • an inartivation domain such as ssn-6/TUP-l or Kruppel-family suppressor domain
  • regulation results in turning off the transcription of a gene which is constitutively expressed.
  • a hormone such as growth hormone, blood proteins, immunoglobulins, etc.
  • chimeric proteins of this invention There is a considerable amount of flexibility in the sele ⁇ ion of component domains and their incorporation into the design of chimeric proteins of this invention.
  • the chimeric protein can be designed to contain an extracellular domain sele ⁇ ed from a variety of surface membrane proteins.
  • various cytoplasmic or intracellular domains of the surface membrane proteins which are able to transduce a signal can be employed, depending on which endogenous genes are regulated by the cytoplasmic portion.
  • the ligand- binding domain protein will preferably be one which can bind molecules able to cross the surface membrane or other membrane, as appropriate. These binding domains will generally bind to multivalent ligands comprising naturally occurring or synthetic ligand moieties, which are preferably not nucleic acids or peptidic.
  • a chimeric protein of Group (1) can contain as an action domain, a cytoplasmic domain from one of the various cell surface membrane receptors or variants thereof for which a corresponding recognition sequence is known or available and which is capable of initiating transcription in response to multimerization of the chimeric protein.
  • recognition sequences include those associated with a gene responsive to transcriptional activation triggered by such a receptor.
  • Mutant receptors of interest will dissociate transcriptional activation of a selected gene from activation of genes which can be associated with harmful side effects, such as deregulated cell growth or inappropriate release of cytokines.
  • the receptor-associated cytoplasmic domains of particular interest will have the following characteristics: receptor activation leads to initiation of transcription for relatively few (desirably fewer than 100) and generally innocuous genes in the cellular host; the other factors necessary for transcription initiated by receptor activation are present in the cellular host; genes which are activated other than the selected gene, will not affe ⁇ the intended purpose for which these cells are to be used; multimerization of the cytoplasmic domain or other available mechanism results in signal initiation; and joining of the cytoplasmic domain to a desired ligand-binding domain will not interfere with signalling.
  • a number of different cytoplasmic domains are known.
  • Tyrosine kinase receptors which are attivated by cross-linking, or dimerization include subclass I: EGF-R, ATR2/neu, HER2/neu, HER3/c-erbB-3, Xmrk; subclass II: insulin-R, IGF-1- R (insulin-like growth factor receptor), IRR; subclass III: PDGF-R-A, PDGF- R-B, CSF-l-R (M-CSF/c-Fms), c-kit, STK-l/Flk-2; and subclass IV: FGF-R, fig [acidic FGF], bek [basic FGF]); neurotrophic tyrosine kinases: Trk family
  • Receptors which associate with tyrosine kinases upon cross-linking include the CD3 f-family: CD3 f and CD3 ⁇ , which are found primarily in T cells, and associate with Fyn; ⁇ and ⁇ chains of FceRI, which are found primarily in mast cells and basophils; ⁇ chain of FC7RHI/CDI6, which is found primarily in macrophages, neutrophils and natural killer cells; CD3 ⁇ , ⁇ , and e, which are found primarily in T cells; Ig- ⁇ /MB-1 and Ig-/3/B29, which are found primarily in B cells.
  • cytokine and growth factor receptors associate with common ⁇ subunits which interact with tyrosine kinases and/or other signalling molecules and which can be used as cytoplasmic domains in chimeric proteins of this invention. These include (1) the common ⁇ subunit shared by the GM-CSF,
  • IL-3 and IL-5 receptors (2) the ⁇ chain gpl30 associated with the IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M, and IL-11 receptors; (3) the IL-2 receptor ⁇ subunit associated also with receptors for IL-4, IL-7 and IL-13, and possibly IL-9; and (4) the ⁇ chain of the IL-2 receptor which is homologous to the cytoplasmic domain of the G-CSF receptor.
  • LIF leukemia inhibitory factor
  • CNTF ciliary neurotrophic factor
  • oncostatin M IL-11 receptors
  • IL-2 receptor ⁇ subunit associated also with receptors for IL-4, IL-7 and IL-13, and possibly IL-9
  • (4) the ⁇ chain of the IL-2 receptor which is homologous to the cytoplasmic domain of the G-CSF receptor.
  • the interferon family of receptors which include interferons a/ ⁇ and ⁇ (which can activate one or more members of the JAK, Tyk family of tyrosine kinases) as well as the receptors for growth hormone, erythropoietin and prolattin (which also can attivate JAK2) can also be used as sources for cytoplasmic domains.
  • Other sources of cytoplasmic domains include the TGF-3 family of cell surface receptors. (Reviewed by Kingsley, D., Genes and Development 1994 8 133.)
  • This family of receptors contains serine/threonine kinase a ⁇ ivity in their cytoplasmic domains, which are believed to be attivated by crosslinking.
  • the tyrosine kinases associated with activation and inactivation of transcription factors are of particular interest in providing specific pathways which can be controlled and can be used to initiate or inhibit expression of an exogenous gene.
  • the following table provides a number of receptors and characteristics associated with the receptor and their nuclear response elements that attivate transcription of genes.
  • the list is intended to provide exemplary systems (rather than an exhaustive list) for use in the subjett invention.
  • mutated cytoplasmic domains can be obtained where the signal which is transduced may vary from the wild type, resulting in a restricted or different pathway as compared to the wild-type path way (s).
  • path way for example, in the case of growth fattors, such as EGF and FGF, mutations have been reported where the signal is uncoupled from cell growth but is still maintained with c-fos (Peters, et al., Nature (1992) 358, 678).
  • the tyrosine kinase receptors can be found on a wide variety of cells throughout the body.
  • the CD3f-family, the Ig family and the lymphokine 3-chain receptor family are found primarily on hematopoietic cells, particularly T-cells, B-cells, mast cells, basophils, macrophages, neutrophils, and natural killer cells.
  • the signals required for NF-AT transcription come primarily from the f (zeta) chain of the antigen receptor and to a lesser extent CD3 ⁇ , ⁇ , e. Table 2
  • the cytoplasmic domain as it exists naturally or as it may be truncated, modified or mutated, will be at least about 10, usually at least about 30 amino acids, more usually at least about 50 amino acids, and generally not more than about 400 amino acids, usually not more than about 200 amino acids. (See Romeo, et al., Cell (1992) 68, 889-893.) While any species can be employed, the species endogenous to the host cell is usually preferred. However, in many cases, the cytoplasmic domain from a different species can be used effectively. Any of the above indicated cytoplasmic domains may be used, as well as others which are presently known or may subsequently be discovered.
  • the other chimeric proteins associated with transcription fattors will differ primarily in having a cellular targeting sequence which directs the chimeric protein to the internal side of the nuclear membrane and having transcription factors or portions thereof as the attion domains.
  • the transcription fattor attion domains can be divided into DNA binding domains and a ⁇ ivation domains.
  • the discussion for the chimeric proteins associated with the surface membrane for signal transduction is applicable to the chimeric proteins for direct binding to genomic DNA.
  • a signal peptide or sequence provides for transport of a chimeric protein to the cell surface membrane, where the same or other sequences can result in binding of the chimeric protein to the cell surface membrane. While there is a general motif of signal sequences, two or three N-terminal polar amino acids followed by about 15-20 primarily hydrophobic amino acids, the individual amino acids can be widely varied. Therefore, substantially any signal peptide can be employed which is functional in the host and may or may not be associated with one of the other domains of the chimeric protein. Normally, the signal peptide is processed and will not be retained in the mature chimeric protein.
  • the sequence encoding the signal peptide is at the 5'- end of the coding sequence and will include the initiation methionine codon.
  • the choice of membrane retention domain is not critical to this invention, since it is found that such membrane retention domains are substantially fungible and there is no critical amino acid required for binding or bonding to another membrane region for activation.
  • the membrane retention domain can be isolated from any convenient surface membrane or cytoplasmic protein, whether endogenous to the host cell or not.
  • the transmembrane domain of a cytoplasmic domain or a receptor domain can be employed, which may tend to simplify the construction of the fused protein.
  • the processing signal will usually be added at the 5' end of the coding sequence for N-terminal binding to the membrane and, proximal to the 3' end for C-terminal binding.
  • the lipid membrane retention domain will have a lipid of from about 12 to 24 carbon atoms, particularly 14 carbon atoms, more particularly myristoyl, joined to glycine.
  • the signal sequence for the lipid binding domain is an N-terminal sequence and can be varied widely, usually having glycine at residue 2 and lysine or arginine at residue 7 (Kaplan, et al, Mol. Cell. Biol. (1988) 8, 2435).
  • Peptide sequences involving post-translational processing to provide for lipid membrane binding are described by Carr, et al, PNAS USA (1988) 79, 6128; Aitken, et al, FEBS Lett.
  • An amino acid sequence of interest includes the sequence M-G-S-S-K-S-K-P-K-D-P-S-Q-R.
  • Various DNA sequences can be used to encode such sequence in the fused receptor protein.
  • the transmembrane domain will have from about 18-30 amino acids, more usually about 20-30 amino acids, where the central portion will be primarily neutral., non-polar amino acids, and the termini of the domain will be polar amino acids, frequently charged amino acids, generally having about 1-2 charged, primarily basic amino acids at the termini of the transmembrane domain followed by a helical break residue, e.g. pro- or gly-.
  • the target gene be regulatably eliminated in a cell-specific or tissue-specific manner.
  • Such specificity of expression may be achieved by linking one ore more of the DNA sequences encoding the chimeric protein(s) to a cell-type specific transcriptional regulatory sequence (e.g. promoter/enhancer). Numerous cell-type specific transcriptional regulatory sequences are known. Others may be obtained from genes which are expressed in a cell-specific manner.
  • constructs for expressing the chimeric proteins may contain regulatory sequences derived from known genes for specific expression in sele ⁇ ed tissues. Representative examples are tabulated below:
  • Elastase - acinar RX. Swift, G.H., MacDonald, R.J., Specific expression cell specific of an elastase-human growth fusion in pancreatic acinar cells of transgenic mice, Nature 131 (1985) 600-603.
  • T cells lck promoter Chaffin K.E., Beals, C.R., Wilkie, T.M., Forbush, K-A.
  • adipocyte P2 Ross, S.R, Braves, RA, Spiegelman, B.M. Targeted expression of a toxin gene to adipose tissue: transgenic mice resistant to obesity, Genes and Dev. 7 (1993) 1318- 24.
  • the human skeletal alpha-actin gene is regulated by a muscle- specific enhancer that binds three nuclear fa ⁇ ors, Gene Expression 2 (1992) 111-26. neurons neurofilament Reeben, M. Halmekyto, M. Alhonen, L. Sinervirta, R. proteins Saarma, M. Janne, J., Tissue-specific expression of rat light neurofilament promoter-driven reporter gene in transgenic mice, BBRC 192 (1993) 465-70. liver tyrosme aminotransf er ⁇ ase, albumin, apolipoproteins
  • upstream sequences are then usually fused to an easily detectable reporter gene like beta-gala ⁇ osidase, in order to be able to follow the expression of the gene under the control of upstream regulatory sequences.
  • an easily detectable reporter gene like beta-gala ⁇ osidase
  • the complete upstream sequences are introduced into the cells of interest to determine whether the initial clone contains the control sequences. Reporter gene expressor is monitored as evidence of expression.
  • deletions may be made in the 5' flanking sequences to determine which sequences are minimally required for cell-type specific expression. This can be done by making transgenic mice with each construtt and monitoring beta-gal expression, or by first examining the expression in specific culture cells, 5 with comparison to expression in non-specific cultured cells.
  • the ligand binding ("dimerization" or "receptor”) domain of any of the chimeric proteins of this invention can be any convenient domain which binds to a natural or preferably, to a synthetic ligand.
  • the location of the binding domain in the chimeric protein, as expressed and located within a cell, can be 5 internal or external to the cellular membrane, depending upon the nature of the chimeric protein and the choice of ligand.
  • binding proteins are known, including receptors and binding proteins associated with the cytoplasmic regions indicated above. Binding proteins for which ligands are known or may be readily produced are of particular interest. These ligands are 0 preferably small organic molecules.
  • the receptors or ligand binding domains include the FKBPs and cyclophilin receptors, the steroid receptors, the t ⁇ racycline receptor, the other receptors indicated above, and the like, as well as "unnatural" receptors, which can be obtained from antibodies, particularly the heavy or light chain subunit, mutated sequences thereof, random amino acid sequences obtained by stochastic procedures, combinatorial syntheses, and the like.
  • the receptor domains will be at least about 50 amino acids, and fewer than about 350 amino acids, usually fewer than 200 amino acids, either as the natural domain or truncated active portion thereof.
  • the binding domain will be small, usually less than 25 kDa, to allow efficient transfection in viral vectors, and will be monomeric, nonimmunogenic. Additionally, it should bind synthetically accessible, cell permeable, nontoxic ligands that can be configured for dimerization.
  • the receptor domain of the chimeric proteins expressed by a host cell can be intracellular or extracellular, depending upon the design of the construtt encoding the chimeric protein and the availability of an appropriate ligand.
  • the binding domain can be on either side of the membrane, but for hydrophilic ligands, particularly protein ligands, the binding domain will usually be external to the cell membrane, unless there is a transport system for internalizing the ligand in a form in which it is available for binding.
  • the construtt can encode a signal peptide and transmembrane domain 5' or 3' of the receptor domain sequence or by having a lipid attachment signal sequence 5' or 3' of the receptor domain sequence. Where the receptor domain is between the signal peptide and the transmembrane domain, the receptor domain will be extracellular.
  • the portion of the construtt encoding the receptor can be subjected to mutagenesis for a variety of reasons.
  • the mutagenized protein can provide for higher binding affinity, allow for discrimination by the ligand of the naturally occurring receptor and the mutagenized receptor, provide opportunities to design a receptor-ligand pair, or the like.
  • the change in the receptor can involve changes in amino acids known to be at the binding site, random mutagenesis using combinatorial techniques, where the codons for the amino acids associated with the binding site or other amino acids associated with conformational changes can be subjett to mutagenesis by changing the codon(s) for the particular amino acid, either with known changes or randomly, expressing the resulting proteins in an appropriate prokaryotic host and then screening the resulting proteins for binding.
  • FKBPl2's Phe36 to Ala and/or Asp37 to Gly or Ala to accommodate a substituent at positions 9 or 10 of FK506 or FK520 or related ligands.
  • mutant FKBP 12 moieties which contain Val., Ala, Gly, Met or other small amino acids in place of one or more of Tyr26, Phe36, Asp37, Tyr82 and Phe99 are of particular interest as receptor domains for FK506-type and FK520-type ligands containing modifications at C9 and/or CIO.
  • Antibody subunits e.g.
  • heavy or light chain particularly fragments, more particularly all or part of the variable region, or fusions of heavy and light chain to create high-affinity binding
  • Antibodies can be prepared against haptenic molecules which are physiologically acceptable and the individual antibody subunits screened for binding affinity.
  • the cDNA encoding the subunits can be isolated and modified by deletion of the constant region, portions of the variable region, mutagenesis of the variable region, or the like, to obtain a binding protein domain that has the appropriate affinity for the ligand.
  • haptenic compound can be employed as the ligand or to provide an epitope for the ligand.
  • natural receptors can be employed, where the binding domain is known and there is a useful ligand for binding.
  • the ability to employ in vitro mutagenesis or combinatorial modifications of sequences encoding proteins allows for the production of libraries of proteins which can be screened for binding affinity for different ligands. For example, one can totally randomize a sequence of 1 to 5, 10 or more codons, at one or more sites in a DNA sequence encoding a binding protein, make an expression construtt and introduce the expression construtt into a unicellular microorganism, and develop a library. One can then screen the library for binding affinity to one or desirably a plurality of ligands. The best affinity sequences which are compatible with the cells into which they would be introduced can then be used as the binding domain.
  • the ligand would be screened with the host cells to be used to determine the level of binding of the ligand to endogenous proteins.
  • a binding profile could be defined by weighting the ratio of binding affinity to the mutagenized binding domain with the binding affinity to endogenous proteins. Those ligands which have the best binding profile could then be used as the ligand.
  • Phage display techniques as a non-limiting example, can be used in carrying out the foregoing.
  • the transduced signal will normally result from ligand-mediated multimerization of the chimeric protein molecules, i.e. as a result of multimerization following ligand binding, although other binding events, for example allosteric activation, can be employed to initiate a signal.
  • the construtt of the chimeric protein will vary as to the order of the various domains and the number of repeats of an individual domain. For the extracellular receptor domain in the 5 '-3' direction of transcription, the construtt will encode a protein comprising the signal peptide, the receptor domain, the transmembrane domain and the signal initiation domain, which last domain will be intracellular, either nuclear or cytoplasmic.
  • the receptor domain is intracellular
  • different orders may be employed, where the signal peptide can be followed by either the receptor or signal initiation domain, followed by the remaining domain, or with a plurality of receptor domains, the signal initiation domain can be sandwiched between receptor domains.
  • the active site of the signal initiation domain will be internal to the sequence and not require a free carboxyl terminus.
  • Either of the domains can be present in multiple copies, particularly the receptor domain, usually having not more than about 5 repeats, more usually not more than about 3 repeats.
  • the ligand for the receptor domains of the chimeric surface membrane proteins will usually be multimeric in the sense that it will have at least two binding sites, each of which being capable of binding to a receptor domain.
  • the ligand will be a dimer or higher order multimer, usually not greater than about tetrameric, of small synthetic organic molecules, the individual molecules typically being at least about 150 D and fewer than about 5 kD, usually fewer than about 3 kD.
  • a variety of pairs of synthetic ligands and receptors can be employed.
  • dimeric FK506 can be used with an FKBP receptor
  • dimerized cyclosporin A can be used with the cyclophilin receptor
  • dimerized estrogen with an estrogen receptor
  • dimerized glucocorticoids with a glucocorticoid receptor
  • dimerized tetracycline with the tetracycline receptor
  • dimerized vitamin D with the vitamin D receptor
  • higher orders of the ligands e.g. trimeric can be used.
  • unnatural receptors such as antibody subunits, modified antibody subunits or modified receptors and the like
  • any of a large variety of compounds can be used.
  • a significant characteristic of these ligand units is that they bind the receptor with high affinity (preferably with a K d ⁇ 10 "8 M) and they are able to be dimerized chemically.
  • the ligand can have different receptor binding molecules with different epitopes which are referred to as "HED" reagents, since they can mediate hetero-dimerization or hetero-multimerization of chimeric proteins having the same or different binding domains.
  • HED Hexathiophene
  • the ligand may comprise FK506 or an FK506-type moiety and a CsA or a cyclosporin type moiety. Both moieties are covalently attached to a common linker moiety.
  • Such a ligand would be useful for mediating the multimerization of a first and second chimeric protein where the first chimeric protein contains a receptor domain such as an FKBP 12 which is capable of binding to the FK506-type moiety and the second chimeric protein contains a receptor domain such as cyclophilin which is capable of binding to the cyclosporin A-type moiety.
  • a receptor domain such as an FKBP 12 which is capable of binding to the FK506-type moiety
  • the second chimeric protein contains a receptor domain such as cyclophilin which is capable of binding to the cyclosporin A-type moiety.
  • the cells may be procaryotic, but are preferably eucaryotic, including plant, yeast, worm, insect and mammalian. Those cells may be mammalian cells, from any mammal of interest, particularly domesticated animals, such as horses, cows, pigs, dogs, cats, rats, mice and so forth.
  • various types of cells can be involved, such as osteoclasts, osteoblasts, neuronal, hematopoietic, neural, mesenchymal, cutaneous, mucosal, stromal, muscle, spleen, reticuloendothelial, epithelial, endothelial, hepatic, kidney, pancreatic, gastrointestinal, pulmonary, etc.
  • Hematopoietic cells which include any of the nucleated cells which may be involved with the lymphoid or myelomonocytic lineages and members of the T- and B-cell lineages, macrophages and monocytes, myoblasts and fibroblasts are of particular interest.
  • stem and progenitor cells such as hematopoietic, neural, stromal, muscle, hepatic, pulmonary and gastrointestinal progenitor cells.
  • the cells can be autologous cells, syngeneic cells, allogenic cells and even in some cases, xenogeneic cells.
  • the cells may be modified by changing the major histocompatibility complex ("MHC") profile, by inactivating ⁇ 2 - microglobulin to prevent the formation of functional Class I MHC molecules, by inactivating Class II molecules, by providing for expression of one or more MHC molecules, by enhancing or inactivating cytotoxic capabilities, by enhancing or inhibiting the expression of genes associated with the cytotoxic attivity, or the like.
  • MHC major histocompatibility complex
  • specific clones or oligoclonal cells may be of interest, where the cells have a particular specificity, such as T cells and B cells having a specific antigen specificity or homing target site specificity.
  • Ligands A wide variety of ligands, including both naturally occurring and synthetic substances, can be used in this invention to effect multimerization of the chimeric protein molecules. Applicable and readily observable or measurable criteria for selecting a ligand are: (A) the ligand is physiologically acceptable (i.e., lacks undue toxicity towards the cell or animal for which it is to be used), (B) it has a reasonable therapeutic dosage range, (C) desirably (for applications in whole animals, including gene therapy applications), it can be taken orally (is stable in the gastrointestinal system and absorbed into the vascular system), (D) it can cross the cellular and other membranes, as necessary, and (E) binds to the receptor domain with reasonable affinity for the desired application.
  • A the ligand is physiologically acceptable (i.e., lacks undue toxicity towards the cell or animal for which it is to be used), (B) it has a reasonable therapeutic dosage range, (C) desirably (for applications in whole animals, including gene therapy applications
  • a first desirable criterion is that the compound is relatively physiologically inert, but for its a ⁇ ivating capability with the receptors.
  • the ligand should not have a strong biological effe ⁇ on native proteins.
  • the ligands will be non-peptide and non-nucleic acid.
  • the subjett compounds will for the most part have two or more units, where the units can be the same or different, joined together through a central linking group.
  • the "units” will be individual moieties (e.g., FK506, FK520, cyclosporin A, a steroid, etc.) capable of binding the receptor domain.
  • Each of the units will usually be joined to the linking group through the same reactive moieties, at least in homodimers or higher order homo-multimers.
  • binding affinities will be reflected in Kd values well below 10 "4 , preferably below 10 "6 , more preferably below about 10 '7 , although binding affinities below 10 '9 or 10 "10 are possible, and in some cases will be most desirable.
  • ligands comprise multimers, usually dimers, of compounds capable of binding to an FKBP protein and/or to a cyclophilin protein.
  • Such ligands includes homo- and heteromultimers (usually 2-4, more usually 2-3 units) of cyclosporin A, FK506, FK520, and rapamycin, and derivatives thereof, which retain their binding capability to the natural or mutagenized binding domain.
  • Many derivatives of such compounds are already known, including synthetic high affinity FKBP ligands, which can be used in the pra ⁇ ice of this invention. See Holt et al., /. Am. Chem. Soc. 1993, 115, 9925 ⁇ 9935.
  • Sites of interest for linking of FK506 and analogs thereof include positions involving annular carbon atoms from about 17 to 24 and substituent positions bound to those annular atoms, e.g. 21 (allyl), 22, 37, 38, 39 and 40, or 32 (cyclohexyl), while the same positions except for 21 are of interest for FK520.
  • sites of interest include MeBmt, position 3 and position 8.
  • Various functionalities which can be conveniently introduced at those sites are alkyl groups to form ethers, acylamido groups, N-alkylated amines, where a 2- hydroxyethylimine can also form a 1,3-oxazoline, or the like.
  • the substituents will be from about 1 to 6, usually 1 to 4, and more usually 1 to 3 carbon atoms, with from 1 to 3, usually 1 to 2 heteroatoms, which will usually be oxygen, sulfur, nitrogen, or the like.
  • By using different derivatives of the basic strutture one can create different ligands with different conformational requirements for binding.
  • mutagenizing receptors one can have different receptors of substantially the same sequence having different affinities for modified ligands not differing significantly in strutture.
  • ligands which can be used are steroids.
  • the steroids can be multimerized, so that their natural biological attivity is substantially diminished without loss of their binding capability with respect to a chimeric protein containing one or more steroid receptor domains.
  • glucocorticoids and estrogens can be so used.
  • Various drugs can also be used, where the drug is known to bind to a particular receptor with high affinity. This is particularly so where the binding domain of the receptor is known, thus permitting the use in chimeric proteins of this invention of only the binding domain, rather than the entire native receptor protein.
  • enzymes and enzyme inhibitors can be used.
  • amide groups including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, or the like.
  • the particular monomer can be modified by oxidation, hydroxylation, substitution, reduction, etc., to provide a site for coupling. Depending on the monomer, various sites can be selected as the site of coupling.
  • the multimeric ligands can be synthesized by any convenient means, where the linking group will be at a site which does not interfere with the binding of the binding site of a ligand to the receptor. Where the active site for physiological activity and binding site of a ligand to the receptor domain are different, it will usually be desirable to link at the active site to inactivate the ligand.
  • Various linking groups can be employed, usually of from 1-30, more usually from about 1-20 atoms in the chain between the two molecules
  • linking groups will be primarily composed of carbon, hydrogen, nitrogen, oxygen, sulphur and phosphorous.
  • the linking groups can involve a wide variety of fun ⁇ ionalities, such as amides and esters, both organic and inorganic, amines, ethers, thioethers, disulfides, quaternary ammonium salts, hydrazines, etc.
  • the chain can include aliphatic, alicyclic, aromatic or heterocyclic groups. The chain will be selected based on ease of synthesis and the stability of the multimeric ligand. Thus, if one wishes to maintain long-term a ⁇ ivity, a relatively inert chain will be used, so that the multimeric ligand link will not be cleaved.
  • esters and amides particularly peptides
  • circulating and/or intracellular proteases can cleave the linking group.
  • ligands such as alkylene, usually of from 2 to 20 carbon atoms, azalkylene (where the nitrogen will usually be bttween two carbon atoms), usually of from 4 to 18 carbon atoms), N-alkylene azalkylene (see above), usually of from 6 to 24 carbon atoms, arylene, usually of from 6 to 18 carbon atoms, ardialkylene, usually of from 8 to 24 carbon atoms, bis-carboxamido alkylene of from about 8 to 36 carbon atoms, etc.
  • alkylene usually of from 2 to 20 carbon atoms
  • azalkylene where the nitrogen will usually be bttween two carbon atoms
  • N-alkylene azalkylene usually of from 6 to 24 carbon atoms
  • arylene usually of from 6 to 18 carbon atoms
  • ardialkylene usually of from 8 to 24 carbon atoms
  • Illustrative groups include decylene, octadecylene, 3- azapentylene, 5-azadecylene, N-butylene 5-azanonylene, phenylene, xylylene, j>- dipropylenebenzene, bis-benzoyl 1,8-diaminoottane and the like.
  • Multivalent or other ligand molecules containing linker moieties as described above can be evaluated with chimeric proteins of this invention bearing corresponding receptor domains using materials and methods described in the examples which follow.
  • the ligand will be selected to be able to be transferred across the membrane in a bioattive form, that is, it will be membrane permeable.
  • Various ligands are hydrophobic or can be made so by appropriate modification with lipophilic groups.
  • the linking bridge can serve to enhance the lipophilicity of the ligand by providing aliphatic side chains of from about 12 to 24 carbon atoms.
  • one or more groups can be provided which will enhance transport across the membrane, desirably without endosome formation.
  • multimeric ligands need not be employed.
  • molecules can be employed where two different binding sites provide for dimerization of the receptor.
  • binding of the ligand can result in a conformational change of the receptor domain, resulting in attivation, e.g. multimerization, of the receptor.
  • Other mechanisms may also be operative for inducing the signal., such as binding a single receptor with a change in conformation resulting in attivation of the cytoplasmic domain.
  • Monomeric ligands can be used for reversing the effect of the multimeric ligand, i.e., for preventing, inhibiting or disrupting multimer formation or maintenance.
  • a monomeric ligand can be used.
  • the parent ligand moitty can be modified at the same site as the multimer, using the same procedure, except substituting a monofunctional compound for the polyfunctional compound.
  • monoamines particularly of from 2 to 20 (although they can be longer), and usually 2 to 12, carbon atoms can be used, such as ethylamine, hexylamine, benzylamine, etc.
  • the monovalent parent compound can be used, in cases, or at dosage levels, in which the parent compound does not have undue undesirable physiological attivity such as immunosuppression, mitogenesis, toxicity, and so forth.
  • HED/HOD reagents that will fail to bind appreciably to their wild type receptors (e.g., FKBP 12) due to the presence of substituents ("bumps") on the reagents that sterically clash with sidechain residues in the receptor's binding pocket.
  • substituents e.g., FKBP 12
  • Using "bumped" ligand moieties and receptor domains bearing compensatory mutations should enhance the specificity and thus the potency of these reagents.
  • Bumped reagents should not bind to the endogenous, wild type receptors, which can otherwise act as a "buffer" toward dimerizers based on natural ligand moieties.
  • the generation of novel receptor-ligand pairs should simultaneously yield the HED reagents that will be used when heterodimerization is required, example, regulated vesicle fusion may be achieved by inducing the heterodimerization of syntaxin (a plasma membrane fusion protein) and synaptobrevin (a vesicle membrane fusion protein) using a HED reagent. This would not only provide a research tool, but could also serve as the basis of a gene therapy treatment for diabetes, using appropriately modified secretory cells.
  • spiro-epoxide (depicted in Figure 16C), which has been prepared by adaptation of known procedures. See e.g. Fisher et al., / Org Chem 56 8(1991): 2900-7 and Edmunds et al., Jet. Lett. 32 48 (1991):819-820.
  • a particularly interesting series of C9 derivatives are characterized by their sp 3 hybridization and reduced oxidation state at C9. Several such compounds have been synthesized according to the reactions shown in Figure 16C.
  • This intermediate can now be used in the coupling with any activated FK506 (or bumped-FK506) molecule.
  • Deprotection with catalytic tetrakis-triphenylphosphine palladium in the presence of dimedone at room temperature in THF removes the amine protecting group.
  • Immediate treatment with an activated FK506 derivative, followed by desilylation leads to a dimeric product. This technique has been used to synthesize the illustrated HOD and HED reagents.
  • Cyclosporin A is a cyclic undecapeptide that binds with high affinity (6nM) to its intracellular receptor cyclophilin, an 18 kDa monomeric protein.
  • the resulting complex like the FKBP12-FK506 complex, binds to and inactivates the protein phosphatase calcineurin resulting in the immunosuppressive properties of the drug.
  • CsA has been dimerized via its MeBmtl sidechain in 6 steps and 35% overall yield to give (CsA)2 ( Figure 17, steps 1-4 were conducted as reported in Eberle et al., / Org Chem 57 9 (1992): 2689-91).
  • Blocking gene constructs encode gene produtts which are capable of blocking expression of target genes, the interfering with the function of the gene products encoded by target gene or eliminating the gene from the host cell entirely.
  • Blocking genes of this latter type may genes which encode gene produtts such as the Cre recombinase, whose expression leads to elimination of a target gene appropriately flanked by loxP sequences in host cells.
  • Constructs encoding the blocking gene products of the present invention have a responsive element in the 5' region, which responds to ligand-mediated multimerization of the chimeric receptor protein, presumably via the generation and transduttion of a transcription initiation signal as discussed infra. Therefore, it will be necessary to select at least one transcription initiation system, e.g.
  • transcription factor which is activated either directly or indirettly, by the cytoplasmic domain or can be activated by association of two domains. It will also be necessary to select at least one promoter region which is responsive to the resulting transcription initiation system. Either the promoter region or the gene under its transcriptional control need be selected. In other words, an attion domain can be selected for the chimeric proteins, which is encoded by a "first" series construtt, based on the role of that attion domain in initiating transcription via a given promoter or responsive element. See e.g. Section 111(A) "Cytoplasmic domains", above.
  • the responsive element can be included in the blocking gene construtt to provide an expression cassette for integration into the genome either as an episome or by chromosomal integration. It is not necessary to have isolated the particular sequence of the responsive element, so long as a gene is known, which is transcriptionally a ⁇ ivated by the cytoplasmic domain upon natural ligand binding to the protein comprising the cytoplasmic domain. Homologous recombination is then used for insertion of the gene of interest downstream from the promoter region such that the inserted gene is under the transcriptional regulation of the endogenous promoter region.
  • sequence of a specific responsive element can be used in conjun ⁇ ion with a different transcription initiation region, which has other advantageous or desired properties, such as a high or low rate of transcription, binding by particular transcription factors, including tissue specific binding fattors, and the like.
  • the expression construtt will therefore have at its 5' end in the direction of transcription, the responsive element and the promoter sequence which allows induced transcription initiation of a target gene of interest, usually a therapeutic gene.
  • the transcriptional termination region is not as important, and can be used to enhance the lifetime of or make short half-lived mRNA by inserting AU sequences which serve to reduce the stability of the mRNA and, as a consequence, limit the period of a ⁇ ivity of the protein. Any region can be employed which provides for the necessary transcriptional termination, and as appropriate, translational termination.
  • the responsive element can be a single sequence or it can be a repeated sequence, but it would usually not be repeated more than about 5 times, often not more than about 3 times.
  • Homologous recombination can be used to remove or ina ⁇ ivate endogenous transcriptional control sequences, including promoter, enhancer and other responsive elements, which are responsive to the multimerization of Group (1) and Group (2) chimeric proteins as discussed above. Additionally, homologous recombination can be used to insert responsive transcriptional control sequences upstream of a desired endogenous gene.
  • the target gene can be any sequence of interest, the absence of which provides a desired phenotype.
  • the target gene can encode a surface membrane protein, a secreted protein, a cytoplasmic protein, or there can be a plurality of target genes which can express different types of produtts.
  • the encoded proteins can be involved in homing, cytotoxicity, proliferation, immune response, inflammatory response, clotting or dissolving of clots, hormonal regulation, or the like (See Table 1).
  • the target gene may alternatively encode a product of unknown function, in which case the modified cells or organisms in which the target gene is regulatably obstructed can be used to identify or study that function.
  • the target gene need not be endogenous to the engineered cell — in some embodiments it is a gene of an infective agent e.g. batteria or viruses. In such embodiments, it is a viral or bacterial gene or gene product which is regulatably obstructed.
  • the gene encoding the blocking agent is expressed under the transcriptional regulation of an element responsive to the multimerization of chimeric proteins, as described in detail elsewhere.
  • the blocking gene may encode an anti-sense message or a ribozyme, an antibody or related form thereof, or a dominant negative form of the target gene product.
  • the blocking gene can be a gene that is capable of eliminating the target gene from the host genome completely, such as the gene encoding the protein Cre, which produces Cre recombinase. Expression of this recombinase leads to elimination of a target gene appropriately flanked by "loxP" sequences in the host cells.
  • an antisense message or a ribozyme contains sufficient sequence complementary to the target gene such that it specifically recognizes the target message and blocks its expression.
  • Altman "RNA enzyme-directed gene therapy," Proc. Natl. Acad. Sci. USA 90 (1993) 10898-10900 and papers cited therein, including Yu et al., "A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1," Proc. Natl Acad. Sci. USA 90 (1993) 6340- 6344.
  • the fun ⁇ ion of a target gene product can be blocked by ligand- regulated expression of an antibody, antibody fragment or other antibody like moiety, that specifically recognizes the encoded target protein and blocks its cellular function. This is done by expressing a gene encoding a neutralizing or otherwise blocking antibody against the target gene product. By regulatably expressing such an antibody gene, one regulatably obstructs the functioning of the target protein. As in other embodiments, this may be effected in a cell-type specific manner by expressing the chimeric proteins using a cell-type specific promoter.
  • intracellularly expressed antibody moieties blocking a gene produ ⁇ function examples include an antibody to HTV I gpl20 protein expressed in a mammalian cell (Marasco et al. PNAS 90:7889 1993) and an anti-p21ras antibody expressed in Xenopus oocytes (Biocca et al. BBRC 197: 422 1993).
  • Intracellular expression of the anti-gpl20 antibody blocked processing of the envelop precursor and reduced the infettivity of HIV-1 particles.
  • the anti-ras antibody blocked insulin-mediated meiotic maturation of Xenopus oocytes.
  • the antibody should be selected such that it binds to the target gene or gene product and blocks its cellular function.
  • a preferred form of the antibody is the so-called single chain form, since this ensures that the heavy and light chains will associate within the cell.
  • Other configurations, such as two chain antibodies, particularly two chain F. b s, or F v fragments, or antibody chains fused to other proteins are also possible.
  • the antibody may need to be targeted to the same compartment as the desired gene product to be disrupted. This can be accomplished by fusing a localization sequence to the antibody coding sequence. This could be at the N-terminus, C-terminus, or embedded within the antibody moiety amino acid sequence.
  • Protein-protein interactions that are critical for a cellular process can be selectively blocked by expression of a non-functional variant of one of the protein partners.
  • raf-1 is a serine/threonine protein kinase that funttions in growth factor-stimulated proliferation pathway (Schaap et al. J.
  • Biol. Chem. 268: 20232 1993 It is composed to two domains, an N-terminal regulatory domain and C-terminal kinase domain. Constitutive overexpression of the N-terminal domain of p74raf-l in cultured cells blocked mitogenesis induced by growth fattors. This domain also interfered with an oncogenic variant of p21ras. Such a system could be useful for models of cancer or the role of growth factors on cellular proliferation.
  • dominant negative gene produtts include certain variants of steroid receptors, growth fattor receptors having an inactive protein kinase or lacking the protein kinase domain altogether, cell surface receptors having a non-functional extracellular ligand binding domain or intracellular cytoplasmic domain, transcription fattor variants that lack a DNA binding domain and/or a transattivation domain.
  • Dominant negative proteins typically disrupt the normal function of a target protein by sequestering it away from its normal partner.
  • Dominant negative proteins can be constructed by random mutagenesis, by selettive deletion of gene segments, or by a rational protein engineering where the domain structure and function of the protein is understood. Often the dominant negative protein is an inactive version of a protein with enzymatic attivity.
  • the Cre constructs and floxed target gene constructs for use in this embodiment of the invention will generally be as described by Barinaga (1994) and by Gu et al. (1994), cited above, and references cited therein, with the proviso that the Cre-encoding DNA sequence will be linked to a promoter/enhancer sequence responsive to multimerization of Group (1) chimeras or to a DNA sequence to which multimerized Group (2) chimeras are capable of binding and initiating Cre transcription.
  • modified cells or organisms in which the target gene is regulatably eliminated can be used to identify or study the function of the gene that is eliminated either at the cellular level or at the level of the organism.
  • Cre constructs will have a responsive element in the 5' region, which responds to ligand-mediated multimerization of the chimeric receptor protein, presumably via the generation and transduttion of a transcription initiation signal as discussed infra. Therefore, it will be necessary to select at least one transcription initiation system, which utilizes at least one transcription factor, which is activated either directly or indirettly, by the cytoplasmic domain or can be activated by association of two domains. It will also be necessary to select at least one promoter region which is responsive to the resulting transcription initiation system. Either the promoter region or the gene under its transcriptional control need be selected.
  • an attion domain can be selected for the chimeric proteins (encoded by a "first" series construtt) based on the role of that attion domain in initiating transcription via a given promoter or responsive element. See e.g. Settion 111(A) "Cytoplasmic domains", above.
  • the responsive element can be included in the Cre gene construct to provide an expression cassette for integration into the genome whether as an episome or by incorporation into the chromosome. It is not necessary to have isolated the particular sequence of the responsive element, so long as a gene is known which is transcriptionally activated by the cytoplasmic domain upon natural ligand binding to the protein comprising the cytoplasmic domain. Homologous recombination could then be used for insertion of the gene of interest downstream from the promoter region to be under the transcriptional regulation of the endogenous promoter region.
  • the specific responsive element sequence that can be used in conjunction with a different transcription initiation region, which can have other aspects, such as a high or low attivity as to the rate of transcription, binding of particular transcription factors and the like.
  • the expression construtt will therefore have at its 5' end in the direction of transcription, the responsive element and the promoter sequence which allows for induced transcription initiation of a target gene of interest, usually a therapeutic gene.
  • the transcriptional termination region is not as important, and can be used to enhance the lifetime of or make short half-lived mRNA by inserting AU sequences which serve to reduce the stability of the mRNA and, therefore, limit the period of attion of the protein. Any region can be employed which provides for the necessary transcriptional termination, and as appropriate, translational termination.
  • the responsive element can be a single sequence or can be multimerized, usually having not more than about 5 repeats, usually having about 3 repeats. Homologous recombination can also be used to remove or inactivate endogenous transcriptional control sequences, including promoter and/or responsive elements, which are responsive to the multimerization event, and/or to insert such responsive transcriptional control sequences upstream of a desired endogenous gene.
  • endogenous transcriptional control sequences including promoter and/or responsive elements, which are responsive to the multimerization event, and/or to insert such responsive transcriptional control sequences upstream of a desired endogenous gene.
  • a wide variety of genes can be employed as the target gene to be eliminated from selected host cells.
  • the target gene can be any sequence of interest, the absence of which provides a desired phenotype.
  • the target gene can encode a surface membrane protein, a secreted protein, a cytoplasmic protein, or there can be a plurality of target genes which can express different types of produtts.
  • the encoded proteins can be involved in homing, cytotoxicity, proliferation, immune response, inflammatory response, clotting or dissolving of clots, hormonal regulation, or the like (See Table 1).
  • the target gene may alternatively encode a product of unknown function, in which case the modified cells or organisms in which the target gene is regulatably eliminated can be used to identify or study that function. v. Regulated Apoptosis
  • the genetically modified recombinant cells such as where one wishes to terminate the treatment provided by the modified cells, where the cells have become neoplastic, particularly in a patient, where a genetic therapy has been deleterious rather than beneficial or where the removal of the cells from a subjett, particularly a recombinant animal, after the engineered cells have expressed a desired protein, may be of interest for research.
  • one may provide for the expression of the Fas antigen or TNF receptor fused to a binding domain.
  • Constru ⁇ s encoding the primary chimera may be designed for constitutive expression using conventional materials and methods, so that the modified cells have such proteins on their surface or present in their cytoplasm.
  • constructs described herein can be introduced as one or more DNA molecules or constructs, where there will usually be at least one marker and there may be two or more markers, which will allow for selection of host cells which contain the constructs.
  • the constructs can be prepared in conventional ways, where the genes and regulatory regions may be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing, or other convenient means. Particularly, using PCR, individual fragments including all or portions of a functional unit may be isolated, where one or more mutations may be introduced using "primer repair", ligation, in vitro mutagenesis, etc. as appropriate. Once the constructs are completed and have been demonstrated to contain the appropriate, desired sequences, they may be introduced into the host cell by any convenient means.
  • the constructs may be integrated and packaged into non-replicating, defective viral genomes like adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV) or others, including retroviral vectors, for infection or transduttion into cells.
  • the constructs may include viral sequences for transfection, if desired.
  • the construtt may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like.
  • the host cells will usually be grown and expanded in culture before introduction of the construct ⁇ ), followed by the appropriate treatment for introduction of the construtt(s) and integration of the construct ⁇ ).
  • the cells will then be expanded and screened by virtue of a marker present in the construct.
  • markers which may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.
  • a target site for homologous recombination where it is desired that a construtt be integrated at a particular locus.
  • an endogenous gene at the same locus or elsewhere, can be deleted and/or replaced with a recombinant construtt of this invention, using materials and methods known in the art for homologous recombination.
  • the recombinant constructs of this invention also can be used to introduce the floxed target gene into particular cells, using materials and methods known in this art.
  • homologous recombination one may generally use either ⁇ or O-vectors.
  • the constructs may be introduced as a single DNA molecule encoding all of the genes, or as different DNA molecules having one or more genes.
  • the constructs may be introduced simultaneously or consecutively, each with the same or different markers.
  • one construtt would contain a therapeutic gene under the control of a specific responsive element (e.g. NFAT), another encoding the receptor fusion protein comprising the signaling region fused to the ligand receptor domain (e.g. as in MZF3E).
  • a third DNA molecule encoding a homing receptor or other product that increases the efficiency of delivery of the therapeutic product may also be introduced.
  • Vectors containing useful elements such as bacterial or yeast origins of replication, selettable and/or amplifiable markers, promoter/enhancer elements for expression in prokaryotes or eukaryotes, etc. which may be used to prepare stocks of construct DNAs and for carrying out transfections are well known in the art, and many are commercially available.
  • the cells which have been modified with the DNA constructs may be grown in culture under selective conditions and cells which are seletted as having the constru ⁇ may then be expanded and further analyzed. For example, the polymerase chain reattion may be used for verifying the presence of the construtt in the host cells. Once the modified host cells have been identified, they may then be grown in culture or introduced into a host organism, as appropriate for the purpose for which they were developed.
  • Hematopoietic cells may be administered by injection into the vascular system, there being usually at least about 10 4 cells and generally not more than about 10 10 , more usually not more than about 10 8 cells.
  • the number of cells which are administered will depend upon a number of circumstances, including the purpose of introducing the modified cells, the lifetime of the cells and the administration protocol used. For example, the number of administrations, the ability of the cells to multiply, the stability of the therapeutic agent, the physiologic need for the therapeutic agent, and the like will all be considered in determining the number of modified cells to be administered.
  • the number of modified cells used would depend upon the size of the layer to be applied to the burn or other lesion. Generally, for myoblasts or fibroblasts, the number of cells will be at least about 10' 4 and not more than about 10 8 and may be applied as a dispersion, generally being inje ⁇ ed at or near the site of interest. The cells will usually be in a physiologically-acceptable medium.
  • the vector may be administered by injection, e.g. intravascularly or intramuscularly, inhalation, or other parenteral mode.
  • the manner of the modification will depend on the nature of the tissue, the efficiency of cellular modification required, the number of opportunities to modify the particular cells, the accessibility of the tissue to the DNA composition to be introduced, and the like.
  • the DNA introduction need not result in integration in every case. In some situations, transient maintenance of the DNA introduced may be sufficient. In this way, one could have a short term effect, where cells could be introduced into the host and then turned on after a predetermined time, for example, after the cells have been able to home to a particular site.
  • the ligand providing for attivation of the cytoplasmic domain may then be administered as desired.
  • various protocols may be employed.
  • the ligand may be administered parenterally or orally. The number of administrations will depend upon the fattors described above.
  • the ligand may be taken orally as a pill, powder, or dispersion; bucally; sublingually; injected intravascularly, intraperitoneally, subcutaneously; by inhalation, or the like.
  • the ligand (and monomeric compound) may be formulated using conventional methods and materials well known in the art for the various routes of administration. The precise dose and particular method of administration will depend upon the above fattors and be determined by the attending physician or human or animal healthcare provider. For the most part, the manner of administration will be determined empirically.
  • the monomeric compound may be administered or other single binding site compound which can compete with the ligand.
  • the monomeric binding compound can be administered in any convenient way, particularly intravascularly, if a rapid reversal is desired.
  • one may provide for the presence of an inattivation domain (or transcriptional silencer) with a DNA binding domain.
  • cells may be eliminated through apoptosis via signalling through Fas or TNF receptor as discussed Example 4(B) below.
  • the particular dosage of the ligand for any application may be determined in accordance with the procedures used for therapeutic dosage monitoring, where maintenance of a particular level of expression is desired over an extended period of times, for example, greater than about two weeks, or where there is repetitive therapy, with individual or repeated doses of ligand over short periods of time, with extended intervals, for example, two weeks or more.
  • a dose of the ligand within a predetermined range would be given and monitored for response, so as to obtain a time-expression level relationship, as well as observing therapeutic response. Depending on the levels observed during the time period and the therapeutic response, one could provide a larger or smaller dose the next time, following the response. This process would be iteratively repeated until one obtained a dosage within the therapeutic range.
  • the ligand is chronically administered, once the maintenance dosage of the ligand is determined, one could then do assays at extended intervals to be assured that the cellular system is providing the appropriate response and level of the expression product.
  • the system is subjett to many variables, such as the cellular response to the ligand, the efficiency of expression and, as appropriate, the level of secretion, the attivity of the expression product, the particular need of the patient, which may vary with time and circumstances, the rate of loss of the cellular attivity as a result of loss of cells or expression activity of individual cells, and the like. Therefore, it is expected that individual patient would be monitored for the proper dosage for the individual, even if there were universal cells which could be administered to the population at large.
  • B- and T-cells may be used in the investigation and/or treatment of cancer, infettious diseases, metabolic deficiencies, cardiovascular disease, hereditary coagulation deficiencies, autoimmune diseases, joint degenerative diseases, such as arthritis, pulmonary disease, kidney disease, endocrine abnormalities, etc.
  • Various cells involved with structure, such as fibroblasts and myoblasts may be used in the treatment and/or investigation of genetic deficiencies, such as connective tissue deficiencies, arthritis, hepatic disease, etc
  • IL2-SX The plasmid pSXNeo/IL2 (IL2-SX) ( Figure 1), which contains the placental secreted alkaline phosphatase gene under the control of human IL-2 promoter (- 325 to +47; MCB(86) 6, 3042), and related plasmid variants (i.e. NFAT-SX, NFB-SX, OAP/Octl-SX, and AP-l-SX) in which the reporter gene is under the transcriptional control of the minimal IL-2 promoter (-325 to -294 and -72 to + 47) combined with synthetic oligomers containing various promoter elements ⁇ i.e.
  • NFAT, NKB, OAP/Oct-l, and API were made by three piece ligations of 1) pPL/SEAP (Berger, et al, Gene (1988) 66,1) cut with Sspl and Hind ⁇ i; 2) pSV2/Neo (Southern and Berg, /. Mol. Appl Genet. (1982) 1, 332) cut with Ndel, blunted with Klenow, then cut with Pvul; and 3) various promoter-containing plasmids (i.e.
  • NFAT-CD8 contains 3 copies of the NFAT-binding site (-286 to -257; Genes and Dev.
  • cxl2lacZ-Oct contains 4 copies of the OAP/Oct-l/(ARRE- 1) binding site (MCB, (1988) 8, 1715) from the human IL-2 enhancer; B-CD8 contains 3 copies of the NFB binding site from the murine light chain (EMBO (1990) 9, 4425) and AP1-LUCIF3H contains 5 copies of the AP-1 site (5'-TGA- CTCAGCGC-3') from the metallothionen promoter.
  • pCDL-SR (MCB 8, 466- 72) (Tac-IL2 receptor -chain), encoding the chimeric receptor TAC/TAC/Z (TTZ) (PNAS 88, 8905-8909)
  • TAg Jurkat cells a derivative of the human T-cell leukemia line Jurkat stably transfected with the SV40 large T antigen (Northrup, et al, J. Biol. Chem. [1993]).
  • Each reporter plasmid contains a multimerized oligonucleotide of the binding site for a distinct IL-2 enhancer-binding transcription fattor within the context of the minimal IL-2 promoter or, alternatively, the intact IL-2 enhancer/promoter upstream of the reporter gene.
  • aliquots of cells (approximately 10 5 ) were placed in microtiter wells containing log dilutions of bound anti-TAC (CD25) mAb (33B3.1; AMAC, Westbrook, ME).
  • ionomycin (1 ⁇ m) and PMA 25 ng/ml
  • Example 2 Inhibitory Activity of the Immunosuppressant Drugs FK506 and Cyclosporin A (CsA) or the Dimeric Derivative Compounds FK1012A (8), FK1012B (5), and CsA dimer (PB-1-218).
  • Example 3 Activity of the Dimeric FK506 Derivative, FK1012A, on the Chimeric FKBP12/CD3 (1FK3) Receptor.
  • NFAT-inducible secreted alkaline phosphatase reporter plasmid NFAT-SX.
  • 5 ⁇ g of pBJ5 was used, instead of lFK3/pBJ5, in a parallel transfection. After 24 hours, aliquots of each transfettion containing approximately 10 5 cells were incubated with log dilutions of the drug, FK1012A, as indicated.
  • ionomycin (1 ⁇ m) and PMA 25 ng/ml
  • Example 4A Activity of the Dimeric FK506 Derivative, FK1012B, on the Myristoylated Chimeric CD3/FKBP12 (MZF3E) Receptor.
  • FK1012 FK 1012s were found to achieve high affinity, 2:1 binding stoichiometry (K d (l) - 0.1 nM; K d (2) - 0.8 nM) and do not inhibit calcineurin-mediated TCR signaling.
  • the ligands are neither
  • (CsA)2 cyclosporin A-based homodimerizing agent which binds to the CsA receptor, cyclophilin, with 1:2 stoichiometry, but which does not bind to calcineurin.
  • (CsA)2 does not inhibit signalling pathways and is thus neither immunosuppressive nor toxic.
  • SEAP alkaline phosphatase
  • FK1012-induced signaling can be terminated by a deaggregation process induced by a nontoxic, monomeric version of the ligand called FK506-M.
  • pBJ5 containing a myristoylated chimeric receptor was co-transfected with 4 ⁇ g NFAT-SX.
  • MZE, MZF1E, MZF2E and MZF3E contain 0, 1, 2, or 3 copies of FKBP12, respectively, downstream of a myristoylated CD3 cytoplasmic domain (see Figure 2).
  • 5 ⁇ g of pBJ5 was used in a parallel transfettion. After 24 hours, aliquots of each transfettion containing approximately 10 5 cells were incubated with log dilutions of the drug, FK1012B, as indicated.
  • the intracellular domains of the TCR, CD3 and zeta-chains interact with cytoplasmic protein tyrosine kinases following antigen stimulation.
  • Specific members of the Src family (lck and/or fyn) phosphorylate one or more tyrosine residues of attivation motifs within these intracellular domains (tyrosine a ⁇ ivation motif, TAM).
  • TAM tyrosine a ⁇ ivation motif
  • the tyrosine kinase ZAP-70 is recruited (via its two SH2 domains) to the tyrosine phosphorylated T-cell-receptor, a ⁇ ivated, and is likely to be involved in the further downstream attivation of phospholipase C.
  • the Fas antigen is a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily of cell surface receptors.
  • Fas antigen and antibodies to its extracellular domain activates a poorly understood signaling pathway that results in programmed cell death or apoptosis.
  • the Fas antigen and its associated apoptotic signaling pathway are present in most cells including possibly all tumor cells.
  • the pathway leads to a rapid and unique cell death (2 h) that is characterized by condensed cytoplasm, the absence of an inflammatory response and fragmentation of nucleosomal DNA, none of which are seen in necrotic cell death.
  • Transgenic animals can be designed with "death" responder genes under the control of cell-specific promoters. Target cells may then be chemically ablated in the adult animal by administering a HOD reagent to the animal. In this way, the role of specific brain cells in memory or cognition or immune cells in the indu ⁇ ion and maintenance of autoimmune disorders could be assessed.
  • Death responder genes may also be introduced into tumors using the human gene therapy technique developed by M.
  • FIG. 19 An exemplary chimeric cDNA has been constructed consisting of three FKBP 12 domains fused to the cytoplasmic signaling domain of the Fas antigen (Figure 19).
  • This construtt when expressed in human Jurkat and murine D10 T cells, can be induced to dimerize by an FK1012 reagent and initiate a signaling cascade resulting in FK1012-dependent apoptosis.
  • the LDy, for FK1012A-mediated death of cells transiently transfetted with MFF3E is 15 nM as determined by a loss of reporter gene attivity (Figure 19; for a discussion of the assay, see legend to Figure 20).
  • the stable transfectants represent a homogeneous population of cells, they have been used to ascertain that death is due to apoptosis rather than necrosis as evidenced by membrane blebbing and nucleosomal DNA fragmentation). Because the transient transfection protocol is more convenient, it has been used as an initial assay system, as is described below.
  • the data show that only dimerization and not aggregation is required for initiation of signal transduttion by the Fas cytoplasmic tail.
  • Mutation of the N-terminal glycine of the myristoylation signal to an alanine prevents myristoylation and hence membrane localization.
  • the mutated construct ( ⁇ MFF3E) was equally potent as an inducer of FKl012-dependent apoptosis, indicating that membrane localization is not necessary for Fas-mediated cell death.
  • a 320 bp Xhol-EcoRI fragment was obtained by PCR comprising the transmembrane and cytoplasmic domains of CD3. These two fragments were ligated and inserted into a S ⁇ CII-£CORI digested pBluescript (Stratagene) to provide plasmid SPZ/KS.
  • plasmid rhFKBP (provided by S. Schreiber, Nature (1990) 346, 674) was used with P#6052 and P#6053 to obtain a 340 bp Xhol-Satl fragment containing human FKBP 12. This fragment was inserted into pBluescript digested with Xhol and Sail to provide plasmid FK12/KS, which was the source for the FKBP12 binding domain.
  • SPZ/KS was digested with Xhol, phosphatased (cell intestinal alkaline phosphatase; CIP) to prevent self-annealing, and combined with a 10-fold molar excess of the Xh ⁇ - Sail FKBP12-containing fragment from FK12/KS.
  • Clones were isolated that contained monomers, dimers, and trimers of FKBP 12 in the corrett orientation.
  • the clones 1FK1/KS, 1FK2/KS, and 1FK3/KS are comprised of in the direction of transcription; the signal peptide from the murine MHC class II gene I-E, a monomer, dimer or trimer, respettively, of human FKBP 12, and the transmembrane and cytoplasmic portions of CD3.
  • the construtt MZ/pBJ5 (MZE/pBJ5) is digested with restriction enzymes Xhol and Sail, the TCR ⁇ fragment is removed and the resulting vector is ligated with a 10 fold excess of a monomer, dimer, trimer or higher order multimer of FKBP 12 to make MF1E, MF2E, MF3E or MF n E/pBJ5.
  • Active domains designed to contain compatible flanking restrittion sites i.e. Xhol and Sail
  • Example 7 Construction of Nuclear Chimera A. GAL4 DNA binding domain - FKBP domain(s) - epitope tag. The
  • GAL4 DNA binding domain (amino acids 1-147) was amplified by PCR using a 5' primer (#37) that contains a Sacll site upstream of a Kozak sequence and a translational start site, and a 3' primer (#38) that contains a SaU site.
  • the PCR product was isolated, digested with Sacll and Sail, and ligated into pBluescript II KS (+) at the SacW and Sail Sites, generating the construtt pBS-GAL4. The construtt was verified by sequencing.
  • Sac ⁇ l/Sa ⁇ l fragment from pBS-GAL4 was isolated and ligated into the IFKl/pBJ5 and IFK3/pBJ5 constructs (containing the myristoylation sequence, see Example 6) at the S ⁇ CII and Xhol sites, generating constructs GF1E, GF2E and GF3E.
  • HNF1 Dimerization/DNA Binding Domain - FKBP Domain(s) - Tag The HNFla dimerization/DNA binding domain (amino acids 1-282) was amplified by PCR using a 5' primer (#39) that contains a Sacll site upstream of a Kozak sequence and a translational start site, and a 3' primer (#40) that contains a SaU site. The PCR product was isolated, digested with S ⁇ CII and SaU, and ligated into pBluescript II KS (+) at the Sacll and SaU sites, generating the construtt pBS-HNF. The construtt was verified by sequencing.
  • SacH/Sall fragment from pBS-HNF was isolated and ligated into the IFKl/pBJ5 and IFK3/pBJ5 constructs at the S ⁇ CII and Xhol sites, generating construtts HF1E, HF2E and HF3E.
  • construtts were made in three steps: (i) a construtt was created from IFK3/pBJ5 in which the myristoylation sequence was replaced by a start site immediately upstream of an Xhol site, generating construtt SF3E; (ii) a nuclear localization sequence was inserted into the Xhol site, generating construtt NF3E; (iii) the VP16 attivation domain was cloned into the SaU site of NF3E, generating construtt NF3V1E.
  • Multimers of the FKBP12 domain were obtained by isolating the FKBP12 sequence as an Xhol/ SaU fragment from pBS-FKBP12 and ligating this fragment into NF1E linearized with Xhol. This resulted in the generation of the construtts NF2E and NF3E.
  • Threonine at position 128 results in a defective NLS.
  • the VP16 transcriptional activation domain (amino acids 413-490) was amplified by PCR using a 5' primer (#43) that contains SaU site and a 3' primer (#44) that contains an Xhol site.
  • the PCR product was isolated, digested with SaU and Xhol, and ligated into MF3E at the Xhol and SaU sites, generating the construct MV1E.
  • the construtt was verified by sequencing.
  • Multimerized VP16 domains were created by isolating the single VP16 sequence as a Xhol/Satl fragment from MV1E and ligating this fragment into MV1E linearized with Xhol.
  • Construtts MV2E, MV3E and MV4E were generated in this manner.
  • DNA fragments encoding one or more multiple VP16 domains were isolated as Xh ⁇ /Sa fragments from MV1E or MV2E and ligated into NFIE linearized with SaU, generating the construtts NFIVIE and NF1V3E.
  • Multimers of the FKBP 12 domain were obtained by isolating the FKBP 12 sequence as an Xhol/ SaU fragment from pBS-FKBP12 and ligating this fragment into NFIVIE linearized with Xhol. This resulted in the generation of the construtts NF2V1E and NF3V1E.
  • Jurkat TAg cells were transfetted with the indicated construtts (5 ⁇ g of each construtt) by ele ⁇ roporation (960 ⁇ F, 250 v). After 24 hours, the cells were resuspended in fresh media and aliquoted. Half of each transfettion was incubated with the dimeric FK506 derivative, (Example 14) at a final concentration of 1 ⁇ M. After 12 hours, the cells were washed and cellular extracts were prepared by repeated freeze-thaw. Chloramphenicol acetyltransferase (CAT) activity was measured by standard protocols. Molecular Cloning: A Laboratory Manual., Sambrook et al. eds. (1989) CSH Laboratory, pp.
  • linker is a linker moiety such as described herein which is covalently linked to "n" (an integer from 2 to about 5, usually 2 or 3) receptor binding moieties ("rbm"'s) which may be the same or different.
  • the receptor binding moiety is a ligand (or analog thereof) for a known receptor, such as are enumerated in Settion V(C), and including FK506, FK520, rapamycin and analogs thereof which are capable of binding to an FKBP; as well as cyclosporins, tetracyclines, other antibiotics and macrolides and steroids which are capable of binding to respective receptors.
  • the linker is a bi- or multi-functional molecule capable of being covalently linked (“— ”) to two or more receptor binding moieties.
  • the linker would comprise up to about 40 atoms and may include nitrogen, oxygen and sulfur in addition to carbon and hydrogen.
  • Illustrative linker moieties are disclosed in Section VI(A) and in the various Examples and include among others C1-C30 alkyl, alkylene, or arylalkyl groups which may be substituted or unsubstituted and may be straight-chain, branched or cyclic.
  • alkyl substituents are saturated straight-chain, cyclic or branched hydrocarbon moieties, preferably of one to about twelve carbon atoms, including methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, t-butyl, cyclobutyl, cyclopropylmethylene, pentyl, hexyl, heptyl, octyl and so forth, and may be optionally substituted with one or more substituents such as lower alkoxy, carboxy, amino (substituted or unsubstituted), phenyl, aryl, mercapto, halo (fluoro, chloro, bromo or iodo), azido or cyano.
  • These compounds may be prepared using commercially available materials and/or procedures known in the art. Engineered receptors for these compounds may be obtained as described infra. Compounds of particular interest are those which bind to a receptor with a Kd of less than 10 "6 , preferably less than about 10 7 and even more preferably, less than 10 '8 M.
  • One subclass of oligomerizing agents of interest are those in which one or more of the receptor binding moieties is FK506, an FK506-type compound or a derivative thereof, wherein the receptor binding moieties are covalently attached to the linker moiety through the allyl group at C21 (using FK506 numbering) as per compound 5 or 13 in Fig 9 A, or through the cyclohexyl ring (C29-C34), e.g. through the C32 hydroxyl as per compounds 8, 16, 17 in Fig 9B.
  • Compounds of this class may be prepared by adaptation of methods disclosed herein, including in the examples which follow.
  • oligomerizing agents of interest are those in which at least one of the receptor binding moieties is FK520 or a derivative thereof, wherein the molecules of FK520 or derivatives thereof are covalently attached to the linker moiety as in FK1040A or FK 1040B in Figure 10.
  • Compounds of this class may be prepared by adaptation of Scheme 1 in Figure 10, Scheme 2 in Figs. 11A and 11B or Scheme 3 in Fig 12 and Fig 13.
  • a further subclass of oligomerizing agents of interest are those in which at least one of the receptor binding moieties is cyclosporin A or a derivative.
  • oligomerizing agents of this invention may be homo-oligomerizing reagents (where the rbm's are the same) or hetero-oligomerizing agents (where the rbm's are different). Hetero- oligomerizing agents may be prepared by analogy to the procedures presented herein, including Scheme 3 in Figure 13 and as discussed elsewhere herein.
  • Merck silica gel 60F glass plates (0.25 mm). Components were visualized by illumination with long wave ultraviolet light, exposed to iodine vapor, and/or by dipping in an aqueous eerie ammonium molybdate solution followed by heating. Solvents for chromatography were HPLC grade. Liquid chromatography was performed using forced flow (flash chromatography) of the indicated solvent system on E. Merck silica gel 60 (230- 400 mesh).
  • THF Tetrahydrofuran
  • benzene toluene
  • diethyl ether Triethylamine and acetonitrile were distilled from calcium hydride.
  • Dichloromethane was distilled from phosphorous pentoxide.
  • Dim ⁇ hylformamide (DMF) was distilled from calcium hydride at reduced pressure and stored over 4A molecular sieves.
  • N,N'-disuccinimidyl carbonate (36 mg, 0.14 mmol) was added in one portion and the solution was stirred at 20°C for 46 h.
  • the reaction mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate solution (10 mL). The phases were separated and the aqueous layer was back-extracted with dichloromethane (2 x 10 mL). The organic phases were combined and dried (MgS0 4 ), concentrated, and subjetted to flash chromatography (3:1 to 2:1 to 1:1 hexane:ethyl acetate). The desired mixed carbonate was isolated as a clear, colorless oil (16.8 mg, 0.014 mmol, 51%).
  • Deprotettion of the FK506 Dimer (4 to 5).
  • the protected dimer (3.3 mg, 1.4 ⁇ mol) was placed in a 1.5-mL polypropylene tube fitted with a spin vane.
  • Acetonitrile (0.5 mL, 3 mM final concentration) was added and the solution stirred at 20°C as HF (55 ⁇ L, 48% aqueous solution; Fisher) was added.
  • the solution was stirred 18 h at room temperature.
  • the deprotetted FK506 derivative was then partitioned between dichloromethane and saturated aqueous sodium bicarbonate in a 15-mL test tube. The tube was vortexed extensively to mix the phases and, after separation, the organic phase was removed with a pipet.
  • aqueous phase was back-extratted with dichloromethane (4 x 2 mL), and the combined organic phases were dried (MgS0 4 ), concentrated and subjected to flash chromatography (1:1:1 hexane:THF:tther to 1:1 THF:ether) providing the desired dimer as a clear, colorless oil (1.7 mg, 0.93 ⁇ mol, 65%).
  • Example 10 Reduction of FK506 with L-Selectride (FK506 to 6).
  • Danishefsky and coworkers have shown that the treatment of FK506 with L- Selectride provides 22-dihydro-FK506 with a boronate ester engaging the C24 and C22 hydroxyl groups (Coleman and Danishefsky, Heterocycles (1989) 28, 157- 161; Fisher, et al, J. Org. Chem. (1991) 56, 2900-2907).
  • the solution was diluted with ethyl acetate (20 mL) and washed with saturated aqueous sodium bicarbonate (10 mL) and the phases were separated. The aqueous phase was then back-extratted with ethyl acetate (2 x 10 mL), and the organic phases were combined, dried (MgS0 ), and the resulting oil was subjetted to flash chromatography (1:1 to 1:2 hexane:ethyl acetate) providing the desired mixed carbonate as a clear, colorless oil (89.0 mg, 0.088 mmol, 61%).
  • benzylamine 15
  • octylenediamine decamethylenediamine (16)
  • bis-p-dibenzylamine N-methyl diethyleneamine
  • tris-aminoethylamine tris-aminopropylamine
  • 1,3,5- triaminomethylcyclohexane etc.
  • Example 11 Oxidative Cleavage and Reduction of FK506 (1 to 9).
  • the osmylation was performed according to the procedure of Kelly (VanRheenen, et al, Tetrahedron Lett. (1976) 17, 1973-1976).
  • the cleavage was performed according to the procedure of Danishefsky (Zell, et al, J. Org. Chem. (1986) 51, 5032-5036).
  • the aldehyde reduction was performed according to the procedure of Krishnamurthy (J. Org. Chem., (1981) 46, 4628-4691).
  • the reaction was then diluted with 50% aqueous methanol (1.0 mL) and sodium periodate (175 mg, 0.82 mmol, 10 equiv) was added in one portion.
  • the cloudy mixture was stirred 40 min at room temperature, diluted with ether (10 mL), and washed with saturated aqueous sodium bicarbonate solution (5 mL). The phases were separated and the aqueous layer was back-extratted with ether (2 x 5 mL).
  • the combined organic layers were dried (MgS0 4 ) and treated with solid sodium sulfite (50 mg).
  • the rea ⁇ ion mixture was diluted with ether (10 mL) and washed with saturated aqueous sodium bicarbonate solution (10 mL). The phases were separated and the aqueous layer was back-extratted with ether (2 xlO mL). The organic phases were combined and dried (MgS0 4 ), concentrated, and subjected to flash chromatography (2:1 to 1:1 hexane:ethyl acetate). The desired mixed carbonate was isolated as a clear, colorless oil (32.6 mg, 0.028 mmol, 75%).
  • the protected monomer (6.2 mg, 5.3 ⁇ mol) was placed in a 1.5 mL polypropylene tube fitted with a spin vane. Acetonitrile (0.5 mL, 11 mM final concentration) was added and the solution stirred at room temperature as HF (55 ⁇ L, 48% aqueous solution; Fisher, 3.0 N final concentration) was added. The solution was stirred 18 h at room temperature. The deprotected FK506 derivative was then partitioned between dichloromethane and saturated aqueous sodium bicarbonate in a 15 mL test tube. The tube was vortexed extensively to mix the phases and, after separation, the organic phase was removed with a pipet.
  • aqueous phase was back-extratted with dichloromethane (4 x 2 mL), and the combined organic phases were dried (MgS0 ), concentrated and subjected to flash chromatography (1:1 to 0:1 hexane:ethyl acetate) providing the desired deprotected benzylcarbamate as a clear, colorless oil (3.9 mg, 4.1 ⁇ mol, 78%).
  • dimeric compounds of the subject invention are prepared.
  • Example 12 Preparation of the Mixed Carbonate of FK506 (12).
  • a 10-mL flask was charged with 24, 32-bis [(tert-butyldimethylsilyI)oxy]-FK506 (339.5 mg., 0.329 mmol), 4-methylmorpholine N-oxide (193 mg, 1.64 mmol, 5 equiv), water (0.20 mL) and THF (8.0 mL, 41 mN final concentration).
  • Osmium tetroxide (0.183 mL, 0.033 mmol, 0.1 equiv, 0.18 M solution in water) was added via syringe. The clear, colorless solution was stirred at room temperature for 4.5 h.
  • the reaction was diluted with 50% aqueous methanol (4.0 mL) and sodium periodate (700 mg, 3.29 mmol, 10 equiv) was added in one portion.
  • the cloudy mixture was stirred 25 min at room temperature, diluted with ether (20 mL), and washed with saturated aqueous sodium bicarbonate solution (10 mL). The phases were separated and the aqueous layer was back-extratted with ether (2x10 mL). The combined organic layers were dried over MgS0 4 and solid sodium sulfite (50 mg).
  • the aqueous phase was back-extratted with ether (2x10 mL).
  • the organic phases were combined and dried (MgS0 4 ), concentrated, and subjetted to flash chromatography (3:1 to 2:1 to 1:1 hexane/tthyl acetate).
  • the desired mixed carbonate 12 was isolated as a clear, colorless oil (217 mg, 0.184 mmol, 56% overall for 4 steps).
  • Example 14 Preparation of FK1012-A (p-xylylenediamine bridge) (13).
  • the protected dimer (11.0 mg, 4.9 ⁇ mol) was placed in a 1.5-mL polypropylene tube fitted with a spin vane.
  • Acetonitrile (0.50 mL, 10 mM final concentration) was added, and the solution stirred at 20 °C as HF (55 ⁇ L, 48% aqueous solution; Fisher, 3.0 N final concentration) was added.
  • the solution was stirred 16h at room temperature.
  • the deprotected FK506 derivative was then partitioned between dichloromethane and saturated aqueous sodium bicarbonate in a 15-mL test tube.
  • the tube was vortexed extensively to mix the phases and, after separation, the organic phase was removed with a pipet.
  • the aqueous phase was back-extracted with dichloromethane (4x2 mL), and the combined organic phases were dried (MgS0 4 ), concentrated and subjetted to flash chromatography (1:1:1 hexane/THF/ether to 1:1 THF/ether) providing FK1012-A as a clear, colorless oil (5.5 mg, 3.0 ⁇ mol, 63%).
  • Example 15 Preparation of 24, 24', 32, 32'-tetrakis[(ter- butyldimethylsilyl)oxy]-FK1012-B (diaminodecane bridge).
  • a dry, 1-mL conical glass vial was charged with the mixed carbonate (53.3 mg, 0.0453 mmol) and acetonitrile (2.0 mL, 11 mM final concentration).
  • Triethylamine (16 ⁇ L, 0.11 mmol, 5 equiv) was added followed by diaminodecane (61 ⁇ L, 0.0226 mmol, 0.37 M solution in DMF).
  • Example 16 Preparation of FK1012-B (diaminodecane -1,10 bridge) (14).
  • the protected dimer (18.0 mg, 7.8 ⁇ mol) was placed in a 1.5-mL polypropylene tube fitted with a stirring flea.
  • Acetonitrile (0.45 mL, 16 mM final concentration) was added, and the solution stirred at room temperature as HF (55 ⁇ L, 48% aqueous solution; Fisher, 3.6 N final concentration) was added.
  • the solution was stirred 17 h at 23 °C.
  • the product FK1012-B was then partitioned between dichloromethane and saturated aqueous sodium bicarbonate in a 15-mL test tube.
  • the tube was vortexed extensively to mix the phases and, after separation, the organic phase was removed with a pipet.
  • the aqueous phase was back-extratted with dichloromethane (4x2 mL), and the combined organic phases were dried (MgS0 4 ), concentrated and subjetted to flash chromatography (100% ethyl acetate to 20:1 ethyl acetate/methanol) affording FK1012-B as a clear, colorless oil (5.3 mg, 2.9 ⁇ mol, 37%).
  • Example 17 Preparation of 24, 24', 32, 32'-tetrakis[(tert- butyldimethylsilyl)oxy]-FK1012-C (bis-p-aminomethylbenzoyl diaminodecane bridge). A dry 25-mL tear-shaped flask was charged with the diamine linker (15.1 mg, 0.0344 mmol) and 1.0 mL of DMF.
  • the mixed carbonate and triethylamine (0.100 mL, 0.700 mmol, 20 equiv) were dissolved in 2.0 mL of dichloromethane then added slowly (4x0.50 mL) to the stirring solution of bis-p-aminomethylbenzoyl, diaminodecane -1,10.
  • the flask containing the mixed carbonate 12 was washed with dichloromethane (2x0.50 mL) to ensure complete transfer of the mixed carbonate 12.
  • reaction mixture was purified by reversed phase HPLC using a 5 cm x 25 cm, 12 ⁇ , 100 A, C18 column at 70°C eluting with 70% acetonitrile/H 2 0 containing 0.1% (v/v) Trifluoroacetic acid to give 112 mg (72%) of the desired monoacetate (2).
  • MeBmt(OAc)-OCOIm 1 CsA (3) MeBmt(OAc)-OCOIm 1 CsA (3).
  • MeBmt(OAc)-OH 1 -CsA (2) (57 mg, 45.5 ⁇ mol) and carbonyldiimidazole (15 mg, 2 eq., 91 ⁇ mol.) were transferred into a 50 mL round bottom flask and dissolved in dry THF (6 mL).
  • Diisopropylethylamine 32 ⁇ L, 4 eq., 182 ⁇ mol was added and then the solvent was removed on a rotary evaporator at room temperature. The residue was purified by flash chromatography on silica gel using ethyl acetate as eluent to give 45 mg (73%) of the desired carbamate (3).
  • MeBmt(OAc)- OCOIm ⁇ CsA (3) (7.5 mg, 5.54 ⁇ mol, 3.1 eq.) was dissolved in THF (100 ⁇ L).
  • Diisopropylethylamine (62 ⁇ L, 5 eq., 8.93 ⁇ mol of a solution containing 100 ⁇ L of amine in 4 mL THF) was added followed by tris(2-aminoethyl)amine (26 ⁇ L, 1.79 ⁇ mol, 1 eq. of a solution containing 101 mg of tris-amine in 10 mL THF). This solution was allowed to stir under N 2 atmosphere for 5 days.
  • Diaminodecane CsA Dimer (8) Solid Na metal (200 mg, excess) was reacted with dry methanol (10 mL) at 0°C. Diaminodecane CsA Dimer Diacetate (5) (4.0 mg) was dissolved in MeOH (5 mL). 2.5 mL of the NaOMe solution was added to the solution of (5).
  • the diaminodecane CsA Dimer Diacetate (5) was prepared by replacing the tris(2-aminoethyl)amine with 0.45 eq. of 1,10-diaminodecane.
  • the p-xylene diamine CsA Dimer (4) was prepared by replacing the tris(2-aminoethyl)amino with 0.45 eq. of p-xylylene diamine.
  • Position 8 D-isomer analogues are produced by feeding the producing organism with the D-amino analogue to obtain inco ⁇ oration specifically at that site. See Patchett, et al, ].
  • the position 3 analogues are prepared by poly-lithiation/alkylation of CsA, specifically at the -carbon of Sac3. See
  • MeBmt(OH)- ⁇ -OH'-CsA 38 mg, 31 mmol, 1218.6 g/mol
  • carbonyl- diimidazole 20 mg, 4eq., 124 mmol, 162.15 g/mol
  • Diisopropylethylamine 22 mL, 4 eq., 125 mmol, 129.25 g/mol
  • the residue was purified by flash chromatography on silica gel using 0-20% acetone in ethyl acetate as eluent to give 32mg (78% yield) of a white solid.
  • MeBmt(OH)- ⁇ -OCOIm'-CsA (12.5 mg, 9.52 mmol, 1312.7 g/mol) was dissolved in DCM (200mL). To this solution was added 22ml (0.5eq., 4.75 mmol) of a solution of xylylene diamine (14.7 mg, 136.2g mol) in DMSO (0.5 mL) and the reaction mixture was stirred for 72 hours at room temperature under a nitrogen atmosphere concentrating slowly.
  • reaction was diluted with acetonitrile (2 mL) filtered through glass wool and purified by reverse phase HPLC (Beckman C18, 10m, 100A, 1cm x 25cm, 5mL/min, 50 to 90% ACN/H 2 O(+0.1%TFA) over 30 minutes, 70°C) to give 6.1 mg (49% yield) of a white solid.
  • reverse phase HPLC Beckman C18, 10m, 100A, 1cm x 25cm, 5mL/min, 50 to 90% ACN/H 2 O(+0.1%TFA) over 30 minutes, 70°C
  • MeBm OAcJ- ⁇ -Br'-CsA (26 mg, -80% pure, 15.7 mmol, 1323.57 g/mol) was dissolved in THF (500 mL). This solution was added by syringe pump over 15 hours to a THF solution of the magnesium enolate of ethyl hydrogen malonate
  • N-(6-aminohexyl) FK506 carbamate bis-TBS-N-(6-(Boc-amino)hexyl) FK506 carbamate (5.9 mg, 1278.88 g/mol, 4.61 mmol) was transferred to a polypropylene tube in ACN (700 ml) followed by aqueous HF (49%, lOOmL). The reaction was complete after six hours at room temperature and was quenched by the slow addition of a saturated solution of NaHCO 3 . The mixture was diluted with saturated NaHCO 3 (4mL), H2O (4mL) and extracted with DCM (3 x 10 mL). The combined organic phases were dried with MgSO 4 , filtered and evaporated to give 3.6 mg (82% yield) of crude product.
  • MeBmt(OH)- ⁇ -CH 2 COOH'-CsA (2.86 mg, 2.27 mmol, 1260.66 g/mol) and N-(6-aminohexyl) FK506 carbamate (crude, 2.16 mg, 2.28 mmol, 949.21 g/mol) were dissolved in DCM (900mL).
  • reaction mixture was diluted with acetonitrile (1 mL) filtered through glass wool and purified by reverse phase HPLC (Beckman C18, 1cm x 25cm, 5mL/min, 50 to 90% ACN/H 2 O over 25 minutes, 50°C) to give 2.4 mg (48% yield) of a white solid.
  • HED and HOD (and antagonist) reagents for chimeric proteins containing corresponding binding domains bearing compensatory mutations.
  • An illustrative HED reagent is depicted in Figure 21 that contains modifications at C9 and CIO'.
  • This invention thus encompasses a class of FK506-type compounds comprising an FK506-type moiety which contains, at one or both of C9 and CIO, a functional group comprising -OR, -R, -(CO)OR, -NH(CO)H or - NH(CO)R, where R is substituted or unsubstituted, alkyl or arylalkyl which may be straightchain, branched or cyclic, including substituted or unsubstituted peroxides, and carbonates.
  • FK506-type moieties include FK506, FK520 and synthetic or naturally occurring variants, analogs and derivatives thereof (including rapamycin) which retain at least the (substituted or unsubstituted) C2 through C15 portion of the ring stru ⁇ ure of FK506 and are capable of binding with a natural or modified FKBP, preferably with a Kd value below about 10 "6 M.
  • This invention further encompasses homo- and hetero-dimers and higher order oligomers containing one or more of such FK506-type compounds covalently linked to a linker moiety of this invention.
  • FK506-type compounds are also of interest, whether or not covalently attached to a linker moiety or otherwise modified without abolishing their binding affinity for the corresponding FKBP.
  • Such monomeric compounds may be used as oligomerization antagonist reagents, i.e., as antagonists for oligomerizing reagents based on a like FK506-type compound.
  • the compounds and oligomers comprising them in accordance with this invention bind to natural., or preferably mutant, FKBPs with an affinity at least 0.1% and preferably at least about 1% and even more preferably at least about 10% as great as the affinity of FK506 for FKBP 12. See e.g. Holt et al., infra.
  • Receptor domains for these and other ligands of this invention may be obtained by strutture-based, site-directed or random mutagenesis methods.
  • FKBP12 moieties which contain Val., Ala, Gly, Met or other small amino acids in place of one or more of Tyr26, Phe36, Asp37, Tyr82 and Phe99 as receptor domains for FK506-type and FK520-type ligands containing modifications at C9 and/or CIO.
  • FKBP's with small replacements such as Gly or Ala for Asp37 in conjunction with FK506-type and FK520-type ligands containing substituents at
  • CIO e.g., -NHCOR, where R is alkyl, preferably lower alkyl such as methyl for example; or -NHCHO
  • FKBP's with small replacements such as Gly or Ala for Phe36, Phe99 and Tyr26 in conjunction with FK506-type and FK520-type ligands containing replacements at C9 (e.g., oxazalines or imines).
  • Site-directed mutagenesis may be conducted using the megaprimer mutagenesis protocol (see e.g., Sakar and Sommer, BioTech iques 8 4 (1990): 404-407).
  • cDNA sequencing is performed with the Sequenase kit.
  • mutant FKBPl2s may be carried out in the plasmid pHNl + in the E. coli strain XA90 since many FKBP 12 mutants have been expressed in this system efficiently. Mutant proteins may be conveniently purified by frattionation over
  • mutant FKBP 12s may be subjected to a binding assay using LH20 resin and radiolabeled 3 H 2 -dihydroFK506 and 3 H,-dihyroCsA that we have used previously with FKBPs and cyclophilins.
  • a binding assay using LH20 resin and radiolabeled 3 H 2 -dihydroFK506 and 3 H,-dihyroCsA that we have used previously with FKBPs and cyclophilins.
  • yeast two-hybrid or "interaction trap” system.
  • the two-hybrid system has been used to detect proteins that interatt with each other.
  • a "bait” fusion protein consisting of a target protein fused to a transcriptional attivation domain is co-expressed with a cDNA library of potential "hooks” fused to a DNA-binding domain.
  • a protein-protein (bait- hook) interaction is detected by the appearance of a reporter gene product whose synthesis requires the joining of the DNA-binding and activation domains.
  • the yeast two-hybrid system mentioned here was originally developed by Elledge and co-workers. Durfee et al., Genes & Development 7 4
  • the first uses the ability of FK506 to bind to FKBP12 and create a composite surface that binds to calcineurin.
  • the sequence-specific transcriptional activator is thus comprised of: DNA-binding domain-mutant FKBP12— bump-FK506— calcineurin A-activation domain (where — refers to a noncovalent binding interaction).
  • the second strategy uses the ability of FKl012s to bind two FKBPs simultaneously.
  • a HED version of an FK1012 may be used to screen for the following ensemble: DNA-binding domain-mutant FKBP 12— bump- FK506-normal FK506— wildtype FKBPl2-a ⁇ ivation domain.
  • Calcineurin-GAL4 activation domain fusion as a bait A derivative of pSE1107 that contains the GAL4 a ⁇ ivation domain and calcineurin A subunit fusion construct has been constructed. Its ability to act as a bait in the proposed manner has been verified by studies using the two-hybrid system to map out calcineurin's FKBP-FK506 binding site.
  • hFKBP 12-GAL4 activation domain fusion as a bait: hFKBP 12 cDNA may be excised as an EcoRI-Hindl ⁇ fragment that covers the entire open reading frame, blunt-ended and ligated to the blunt-ended Xho I site of pSEH07 to generate the full-length hFKBP-GAL4 attivation domain protein fusion.
  • hFKBP 12 may be digested with EcoRI and Hindlll, blunted and cloned into pASl (Durfee et al., supra) that has been cut with Ncol and blunted. This plasmid is further digested with Ndel to eliminate the Ndel fragment between the Ndel site in the polylinker sequence of pASl and the 5' end of hFKBP 12 and religated. This generated the hFKBP 12-GAL4 DNA binding domain protein fusion.
  • hFKBP was reamplified with primers #11206 and #11210, Primer Table:
  • 3S ⁇ *FK37 5*-CTGTC CCG GGA NNN N NNN TTT CTT TCC ATC TTC AAG C-.' R S X X X K G D E l
  • 3SmF 27 5'- ⁇ GTC CCG GGA GGA ATC AAA TTT CTT TCC ATC TTC AAG CA " R S S 0 F K K G 0 E L M NNN NNN NNN GTG CAC CAC GCA GG-3" X X X H V V C
  • Primer Table Primer Table:Primers used in the construction of a regionally localized hFKBPl2 cDNA library for use in screening for compensatory mutations. Mutant hFKBP 12 cDNA fragments were then prepared using the primers listed below that contain randomized mutant sequences of hFKBP at defined positions by the polymerase chain reaction, and were inserted into the GAL4 DNA binding domain-hFKBP(NdeI/BamHI) constru ⁇ .
  • Yeast strain S. cerevisiae Y153 carries two selectable marker genes (his3/0- galactosidase) that are integrated into the genome and are driven by GAL4 promoters. (Durfee, supra.)
  • the FKBP12-FK506 complex binds with high affinity to calcineurin, a type 2B protein phosphatase. Since we use C9- or ClO-bumped ligands to serve as a bridge in the two-hybrid system, only those FKBPs from the cDNA library that contain a compensator)' mutation generate a transcriptional attivator. For convenience, one may prepare at least three distinct libraries (using primers 11207-11209, Primer Table) that will each contain 8,000 mutant FKBPl2s.
  • Randomized sites were chosen by inspecting the FKBP12-FK506 strutture, which suggested clusters of residues whose mutation might allow binding of the offending C9 or CIO substituents on bumped FK506s.
  • the libraries are then individually screened using both C9- and ClO-bumped FK506s.
  • the interaction between a bumped- FK506 and a compensatory hFKBP 12 mutant can be detected by the ability of host yeast to grow on his drop-out medium and by the expression of ⁇ - galactosidase gene. Since this selection is dependent on the presence of the bumped-FK506, false positives can be eliminated by subtra ⁇ ive screening with replica plates that are supplemented with or without the bumped-FK506 ligands.
  • GAL4 activation domain to screen hFKBP12 mutant cDNA libraries is a simple way to identify compensatory mutations on FKBP 12.
  • mutations that allow bumped-FK506s to bind hFKBP 12 may disrupt the interaction between the mutant FKBP12— bumped-FK506 complex and calcineurin. If the initial screening with calcineurin as a bait fails, the wild type hFKBP 12-GAL4 activation domain will instead be used.
  • An FK1012 F ⁇ ED reagent consisting of: native-FK506-bumped-FK506 ( Figure 16) may be synthesized and used as a hook. The FK506 moi ⁇ y of the FK1012 can bind the FKBP12-GAL4 attivation domain.
  • FK1012 and a compensatory mutant of FKBP12 will allow host yeast to grow on his drop-out medium and to express /3-galactosidase.
  • the sele ⁇ ion is based solely on the ability of hFKBP 12 mutant to interatt with the bumped-FK506.
  • the same subtractive screening strategy can be used to eliminate false positives.
  • an in vivo assay may be used to determine the binding affinity of the bumped-FK506s to the compensatory hFKBP 12 mutants.
  • /3-gal activity is determined by the degree of interaction between the "bait” and the "prey”.
  • FKBP 12 mutants can be estimated by the corresponding /3-galactosidase activities produced by host yeasts at different HED (native-FK506-bumped- FK506) concentrations. Using the same strategy, additional randomized mutant FKBP12 cDNA libraries may be created in other bump-contact residues with low-affinity compensatory FKBP12 mutants as templates and may be screened similarly.
  • Some high-affinity hFKBP12 mutants for bump-FK506 may contain several combined point mutations at discrete regions of the protein.
  • the size of the library that contains appropriate combined mutations can be too large for the yeast two-hybrid system's capacity (e.g., > 10 8 mutations).
  • the use of bacteriophage as a vehicle for exposing whole funttional proteins should greatly enhance the capability for screening a large numbers of mutations. See e.g. Bass et al., Proteins: Strutture, Function & Genetics 8 4 (1990): 309-14; McCafferty et al., Nature 348 6301 (1990): 552-4; and Hoogenboom, Nucl. Acids Res. 19
  • mutant hFKBP 12 fusion phages can be screened with bumped-FK506-Sepharose as an affinity matrix, which can be synthesized in analogy to our original FK506-based affinity matrices.
  • One class of modified CsA derivatives of this invention are CsA analogs in which (a) NMeValll is replaced with NMePhe (which may be substituted or unsubstituted) or NMeThr (which may be unsubstituted or substituted on the threonine betahydroxyl group) or (b) the pro-S methyl group of NMeValll is replaced with a bulky group of at least 2 carbon atoms, preferably three or more, which may be straight, branched and/or contain a cyclic moiety, and may be alkyl (ethyl, or preferably propyl, butyl, including t-butyl, and so forth), aryl, or arylalkyl.
  • These compounds include those CsA analogs which contain NMeLeu, NMelle, NMePhe or specifically the unnatural
  • This invention further encompasses homo- and hetero-dimers and higher order oligomers containing one or more such CsA analogs.
  • the compounds and oligomers comprising them in accordance with this invention bind to natural., or preferably mutant, cyclophilin proteins with an affinity at least 0.1% and preferably at least about 1% and even more preferably at least about 10% as great as the affinity of CsA for cyclophilin.
  • a two step strategy may be used to prepare the modified [MeVal n ]CsA derivatives starting from CsA.
  • the residue Me Vail 1 is removed from the macrocycle.
  • a selected amino acid is introduced at the (former) MeValll site and the linear peptide is cyclized.
  • the advantage of this strategy is the ready access to several modified [MeVal ⁇ ]CsA derivatives in comparison with a total synthesis.
  • the synthetic scheme is as follows:
  • the ester MeBmtl-MeValll bond is then reduced selectively in the presence of the N-methyl amide bonds, e.g. using DIBAL-H.
  • the resulting diol is then transformed to the corresponding di-ester with another acid-induced N t O shift.
  • bumped-CsAs to cvclophilins can be evaluated by the same methods described for FK506s and FK1012s. Once cvclophilins are identified with compensatory mutations, bumped (CsA)2 HED and HOD reagents may be synthesized according to the methods discussed previously. Of particular interest are bumped CsA compounds which can form dimers which themselves can bind to a cyclophilin protein with 1:2 stoichiom ⁇ ry.
  • Homo dimers and higher order homo-oligomers, heterodimers and h ⁇ ero-higher order oligomers containing at least one such CsA or modified CsA moiety may be designed and evaluated by the methods developed for FK1012A and (CsA)2, and optimize the linker element in analogy to the FK1012 studies.
  • Cvclophilins that bind our position 11 CsA variants (2) by accommodating the extra bulk on the ligand may be now be prepared. Cvclophilins with these compensatory mutations may be identified through the structure-based site-directed and random mutagenesis/screening protocols described in the FK1012 studies.
  • the subjett method and compositions provide for great versatility in the production of cells for a wide variety of pu ⁇ oses.
  • cells can have a wide variety of lifetimes in a host, there is the opportunity to treat both chronic and acute indications so as to provide short- or long-term protection.
  • Cells can be provided which will produce a wide variety of proteins or other gene products which may serve to correct a deficit or inhibit an undesired result, such as attivation of cytolytic cells, to inactivate a destructive agent, to kill a restricted cell population, or as is the focus here, to provide regulatable obstruction of the expression of a target gene or functioning of the target gene produtt.
  • the cells may be readily activated by administering the multimerizing drug at a dose which can result in a rapid response of the engineered cells.
  • Cells can be provided where the expressed chimeric receptor is intracellular, avoiding immune response due to a foreign protein on the cell surface. Furthermore, the intracellular chimeric receptor protein provides for efficient signal transduttion upon ligand binding, apparently more efficiently than the receptor binding at an extracellular receptor domain.

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WO1996041865A1 (en) * 1995-06-07 1996-12-27 Ariad Gene Therapeutics, Inc. Rapamcycin-based regulation of biological events
EP0785992A1 (en) * 1994-10-25 1997-07-30 The Board Of Trustees Of The Leland Stanford Junior University Conditional transformation of genetically engineered cells
WO1998039418A1 (en) * 1997-03-07 1998-09-11 Ariad Gene Therapeutics, Inc. New applications of gene therapy technology
US5858990A (en) * 1997-03-04 1999-01-12 St. Elizabeth's Medical Center Fas ligand compositions for treatment of proliferative disorders
US5928868A (en) * 1996-04-26 1999-07-27 Massachusetts Institute Of Technology Three hybrid screening assay
WO1999061055A1 (en) 1998-05-22 1999-12-02 The Board Of Trustees Of The Leland Stanford Junior University Bifunctional molecules and therapies based thereon
US6153383A (en) * 1997-12-09 2000-11-28 Verdine; Gregory L. Synthetic transcriptional modulators and uses thereof
US6270998B1 (en) 1991-04-26 2001-08-07 Osaka Bioscience Institute DNA coding for human cell surface antigen
US6303848B1 (en) 1998-01-16 2001-10-16 Large Scale Biology Corporation Method for conferring herbicide, pest, or disease resistance in plant hosts
US6426185B1 (en) 1998-01-16 2002-07-30 Large Scale Biology Corporation Method of compiling a functional gene profile in a plant by transfecting a nucleic acid sequence of a donor plant into a different host plant in an anti-sense orientation
EP1229924A1 (en) * 1999-11-19 2002-08-14 The Board Of Trustees Of The Leland Stanford Junior University Targeted bifunctional molecules and therapies based thereon
US6506379B1 (en) 1995-06-07 2003-01-14 Ariad Gene Therapeutics, Inc. Intramuscular delivery of recombinant AAV
US6887842B1 (en) 1999-11-19 2005-05-03 The Board Of Trustees Of The Leland Stanford Junior University Modulating a pharmacokinetic property of a drug by administering a bifunctional molecule containing the drug
US6984635B1 (en) 1998-02-13 2006-01-10 Board Of Trustees Of The Leland Stanford Jr. University Dimerizing agents, their production and use
US7220552B1 (en) 1999-11-19 2007-05-22 The Board Of Trustees Of The Leland Stanford Junior University Bifunctional molecules and their use in the disruption of protein-protein interactions
WO2008097875A1 (en) * 2007-02-08 2008-08-14 Neurologix, Inc. Novel methods for treating neurodegenerative disorders
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US5858990A (en) * 1997-03-04 1999-01-12 St. Elizabeth's Medical Center Fas ligand compositions for treatment of proliferative disorders
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US6153383A (en) * 1997-12-09 2000-11-28 Verdine; Gregory L. Synthetic transcriptional modulators and uses thereof
US6183965B1 (en) 1997-12-09 2001-02-06 President And Fellows Of Harvard College Synthetic transcriptional modulators and uses thereof
US6426185B1 (en) 1998-01-16 2002-07-30 Large Scale Biology Corporation Method of compiling a functional gene profile in a plant by transfecting a nucleic acid sequence of a donor plant into a different host plant in an anti-sense orientation
US6303848B1 (en) 1998-01-16 2001-10-16 Large Scale Biology Corporation Method for conferring herbicide, pest, or disease resistance in plant hosts
US6984635B1 (en) 1998-02-13 2006-01-10 Board Of Trustees Of The Leland Stanford Jr. University Dimerizing agents, their production and use
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EP0776335A4 (en) 1999-09-15
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JPH10507624A (ja) 1998-07-28
EP0776335A1 (en) 1997-06-04

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