WO1998039418A1 - New applications of gene therapy technology - Google Patents

Abstract

This invention provides new applications of regulated gene therapy relating to treatment of cancer and other disorders.

Description

New Applications of Gene Therapy Technology

Introduction Regulatable transcription of introduced genes in response to administered pharmaceutical agents represent a significant new development in gene therapy. Notable among those systems are the dimerization-controlled system of Schreiber et al (see e.g. PCT/US93/01617) and modifications thereof (see e.g. PCT/US95/ 10559 (filed August 18, 1995), Gilman USSN 08/292,599 (filed August 18, 1994), Gilman USSN 08/373,351 (filed January 17, 1995) and Holt USSN 08/598,776 (filed February 9, 1996).

The development of regulated transcriptional systems now permits new applications of gene therapy.

Summary of the invention

This invention provides, among other aspects, engineered cells which, following exposure to a selected ligand, express a target gene encoding a protein selected from the group consisting of thrombospondin, angiostatin, a soluble receptor for VEGF, a dominant negative form of VEGF, a tumor-specific antigen such as the carcinoembryonic antigen (CEA), a cytokine or (in the case of non- cutaneous cells) beta interferon.

These cells contain a first DNA construct (or pair of such constructs) encoding chimeric responder protein molecules comprising (i) at least one receptor domain capable of binding to a selected ligand and (ii) another protein domain, heterologous with respect to the receptor domain, referred to as the "action" domain. The action domain is capable, upon multimerization of the responder protein molecules, of triggering the activation of transcription of a target gene under the transcriptional control of a transcriptional control element responsive to said multimerLzation. These cells further contain a target gene under the expresssion control of a transcriptional control element responsive to the ligand-mediated multimerization of the chimeric responder protein molecules. In most cases, the target gene preferably encodes a peptide sequence of human origin.

This invention thus provides a method for rendering cells capable of regulatable expression of one of the target genes disclosed herein following exposure of the cells to a selected ligand. In this method one introduces into the cells DNA constructs encoding the chimeric responder protein molecules and a target DNA construct. These constructs may be introduced into cells removed from a human or non-human animal or other organism and maintained in vitro. Alternatively, the constructs may be mtroduced into cells in situ within a host organism by administration of the DNA directly to the organism

Introducing constructs of this mvention, or cells containing them, into a human or non-human organism, combined with administration of a multimerrzing ligand capable of effecting expression of the target gene provides a means for treating diseases responsive to the target gene product Said differently, a human or non-human host organism containing cells engineered in accordance with this invention may be treated for diseases responsive to or affected by the expression of the mtroduced target gene Thus, a human or non-human host organism containing such cells, where the mtroduced target gene is a DNA sequence encodmg thrombospondm, angiostatin, a soluble receptor for VEGF, a dominant negative form of VEGF, a tumor-specific antigen or a cytokme (such as IL-2, IL-4, IL-7, IL- 12, GM-CSF, gamma mterferon, etc), may be treated for various cancers by administration of a ligand capable of mediating the multimerization of the chimeric responder protem molecules Where the introduced target gene encodes a beta mterferon protein such as mterferon β-lB, administration of the ligand constitutes a method for treating or preventing MS episodes Where the mtroduced target gene encodes a nbozyme or antisense sequence directed to an RNA or DNA encodmg a component required for the persistence or spread of HIV, administration of the ligand constitutes a method for treating or preventing HIV infection Preferably the ligand is administered in a pharmaceutical composition which further contains pharmaceutically acceptable excipients Administration may be by any of the various acceptable routes and will comprise an effective amount of the ligand to effect observable expression of the target gene, whether observed directly, e g by observation of the production of the target gene product, or indirectly, e g by observation of the effects of production of the target gene product

With respect to applications to cancer treatment, we note the following A promising new avenue for cancer therapy is "vaccinating" a patient agamst his own tumor Two general strategies are being explored for gene therapy-directed cancer vaccmes One is to augment the lmmunogeniαty of tumor cells by transferrmg mto the cells an engineered gene that directs the production of cytokme that can recruit and locally activate cells of the immune system Among the cytokines that have been used for this purpose are mterleukιn-2, ιnterleukιn-4, rnterleukιn-12, mterferon-ga ma, and granulocyte/macrophage colony-stimulating factor It is contemplated that effectiveness of this approach is limited by the need to produce cytokines withm an optimal therapeutic wmdow Enhanced cytokme prod ichon more effectively recruits and activates the immune system, but systemic circulation of these potent molecules can be toxic. Therefore, the ability to adjust cytokine production precisely and to terminate production if toxic side effects develop will be critical to the practical implementation of tumor vaccination.

In the second strategy, patients are irr-mimized against molecularly-defined tumor-specific antigens. One example is the carcinoembryonic antigen (CEA), which is expressed by many colorectal tumors. Vaccination against molecularly-defined antigens can be achieved using simple techniques known in the art, such as intramuscular injection and microprojectile bombardment, to deliver DNA molecules directing the production and display of the antigen. Animal experiments indicate that this vaccination strategy generates both cellular and humoral immunity. However, the present inventor contemplates that the effectiveness of DNA-mediated vaccination in humans will be limited by the level of antigen expression. For DNA vaccines delivered by intramuscular injection, current technology may require that patients be injected with large amounts of DNA. The subject invention provides materials and methods with which one may reduce the amount of DNA required, preferably by tenfold or more. Furthermore, there may be an optimal level of antigen expression for eliciting an immune response, and more complex i-r-munization schemes, such as "pulsing" the immune system with antigen, may be significantly more effective. These approaches are only possible in conjunction with regulated gene therapy. Finally, regulated antigen expression from a stably-delivered gene cassette provides an opportunity for periodic administration of "boosters." Thus, for example, the anti-tumor activities of the immune system could be redeployed as needed to fight off recurrences of disease. We contemplate two different preferred routes for antigen presentation. The simplest is intramuscular injection of DNA or viral vectors containing the constructs described herein. In addition, however, the use of ex vivo stem, progenitor, and /or peripheral cell gene therapy is also contemplated for delivering antigen-encoding DNA into professional antigen-presenting cells. This strategy has three significant advantages over intramuscular injection. First, these cells are specialized for antigen presentation and they circulate in the body, suggesting that the potency of antigen presentation and of the resulting immune response will be greater than with intramuscular delivery. Second, by directing expression to different types of antigen-presenting cells, the balance between cellular and humoral immunity can be controlled. Third, because transduction is stable, i-r-munization can be life-long and modulated as needed by the administration of dimerizer drugs. In various embodiments of this invention, and particularly in the case of "vaccination" via gene therapy, the chimeric responder constructs are preferably under the expression control of cell-type- or tissue-specific regulatory elements, e.g. promoters and/or enhancers. See e.g. USSN 08/292,596 (filed August 18, 1994) (especially page 24 et seq thereof). Thus where the engineered cells are hematopoietic stem cells or progenitors cells, use of regulatory elements from immunoglobulin heavy or light chain genes (e.g. the Ig kappa light chain promoter/enhancer) provides for B-cell-specific expression of the chimeric responder proteins, and thus B-cell-spe-cific expression of the target gene. See e.g., Borelli, E. et al. (1988) Targeting of an inducible toxic phenotype in animal cells. Proc. Natl. Acad. Sci. USA 85:7572-7576; and, Heyman et al (1989) Thymidine kinase obliteration: creation of transgenic mice with controlled immunodeficiencies. Proc. Natl. Acad. Sci. USA 86:2698-2702. One can provide for macrophage- specific expression of the responder constructs by using the gp91-phox promoter in the responder constructs. See e.g., Targeting of transgene expression to monocyte/macrophages by the gp91-phox promoter and consequent histiocytic malignancies. Proc Natl Acad Sci U S A 88: 8505-9 (1991). See also L. C. Burkly et al, J Immunol 142: 2081-2088 (1989) regarding B cell and macrophage-specific expression using expression control elements from the class II E alpha d gene.

Design, production and use of DNA constructs and dimerizing agents in the practice of this invention may take advantage of principles, materials and methods disclosed in any of the following: PCT/US94/01667 and PCT/US94/08008 (Crabtree and Schreiber et al), PCT/US95/16982 (Pomerantz et al), PCT/US95/10559 and PCT/US97/03137 (Holt et al), PCT/US96/09948 (Clackson et al), PCT/US95/10591 (Brugge et al) and PCT/US97/22454 (Cerasoli), as well as US priority applications identified in any of the foregoing.

Detailed Description

This invention provides for the delivery of pharmacologically active agents for the treatment and /or prevention of HIV, cancer and MS via gene therapy, preferably via regulatable gene therapy. The invention involves recombinant DNA constructs ("target gene constructs") containing a first DNA sequence encoding a target gene and a second DNA sequence comprising a transcriptional regulatory element, such as a promoter or enhancer sequence, which is responsive to the multimerization of chimeric responder proteins, as is discussed in detail below. Target genes of this invention which are relevant to the treatment of cancer are DNA sequences encoding a cytokine such as IL-2, IL-4, IL-7, IL-12 or GM-CSF; an angiogenesis inhibitory factor such as thrombospondin, angiostatin, a dominant negative VEGF molecule or a soluble receptor for VEGF; and a tumor specific antigen such as CEA. A target gene relevant to MS encodes a beta mterferon protem such as IFN beta-IB Preferably, the foregomg target genes encode a protem of human origin or sequence to avoid undue risks of antigenicity. A target gene relevant to the treatment or prevention of HIV infection is a DNA sequence comprismg a ribozyme or antisense sequence directed to an RNA or DNA sequence, respectively, for an HIV component required for the reproduction or spread of the virus. See e.g. Dropuhc and Jeang, 1994, Human Gene Therapy 5:927-939 (and references cited therem).

DNA sequences for target genes may be readily obtamed by conventional means. For instance, primers may be designed based on the published sequence of a desired target cDNA, synthesized by conventional procedures and used m obtaining target gene DNA through standard PCR techniques. DNA sequence information and other information relevant to the cloning and use of target gene sequences are readily available, as illustrated m the following table:

This invention involves the use of one or more chimeric responder proteins, DNA constructs ("responder" constructs) encoding them, and multi-valent ligand molecules capable of multimerizing the chimeric proteins. These are described in detail in the cited patent documents above. Briefly, the chimeric proteins contain at least one ligand-binding (or "receptor") domain and an action domain capable, upon multimerization of the chimeric responder molecules, of initiating transcription of the target gene wit-hin 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). Also provided are recombinant target constructs containing a target gene under the transcriptional regulation of a transcriptional control element responsive to the presence of the multimerizing agent, i.e, to multimerized responder proteins described above. The transcriptional control element is responsive in the sense that transcription of the target gene is activated by the presence of the multimerized responder chimeras in cells containing these constructs. Said differently, exposure of the cells expressing the chimeric responder constructs and containing a target gene construct responsive to the m timerizing ligand results in expression of the target 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 genetically engineered cells of this invention which contain and express the responder constructs, and contain the target gene construct, are exposed to the multimerizing ligand, expression of the target gene is activated.

To render the mammal responsive to the ligand, certain of the mammal's cells must first be genetically engineered by introdticing into them heterologous DNA constructs, typically in vivo. A variety of systems have been developed which permit the genetic engineering of cells to permit ligand-mediated regulatable expression of a target gene. See e.g. Clackson, "Controlling mammalian gene expression with small molecules", Current Opinion in Chemical Biology 1:210-218 (1997). Materials and methods for implementing those systems are known in the art and may be adapted to the practice of the subject invention. Typically, at least two different heterologous DNA constructs are introduced into the cells, including (a) at least one target DNA construct which comprises a target gene, here a DNA sequence encoding a target protein, i.e., thrombospondin, angiostatin, a soluble receptor for VEGF, a dominant negative form of VEGF, a tumor-specific antigen or a cytokine (such as IL-2, IL-4, IL-7, IL-12, GM-CSF, gamma interferon, etc) operably linked to a transcription control element permitting ligand-mediated expression of the target gene; and (b) one or more DNA constructs encoding and capable of directing the expression of chimeric proteins capable of binding to the ligand and activating expression of the target gene(s) in a ligand-dependent manner.

Preferred regulated expression systems are based on ligand-mediated dimerization of chimeric proteins. In such systems each of the chimeric proteins contains at least one ligand-binding (i.e., receptor) domain and at least one effector domain for activating gene transcription directly or indirectly. The phrase "ligand- binding domain" encompasses protein domains which are capable of binding to the ligand, as in the case of an FKBP domain and the ligand, FK506, discussed below, and further encompasses protein domains which are capable of binding to a complex of the ligand with another binding protein, as in the case of the FRB domain which binds to the rapamycin:FKBP complex. Examples of pairs of receptor domains and ligands which are known in the art and have been demonstrated to be effective in such regulated transcription systems, and which may be used in the practice of the subject invention, include FKBP/FK1012 , FKBP/synthetic divalent FKBP ligands (see WO 96/0609 and WO 97/31898), FRB/rapamycin:FKBP (see e.g., WO 96/41865 and Rivera et al, "A humanized system for pharmacolgic control of gene expression", Nature Medicine 2(9):1028- 1032 (1997)) , cyclophilin/cyclosporin (see e.g. WO 94/18317), DHFR/methotrexate (see e.g. Licitra et al, 1996, Proc. Natl. Acad. Sci. USA 93 12817-12821) and DNA gyrase/coumermycin (see e g Farrar et al, 1996, Nature 383 178-181)

In the case of direct activation of transcription, two chimeric proteins are typically used Each, as mentioned above, contams at least one ligand-binding domain One of the chimeras also contains at least one DNA-binding domain such as GAL4 or ZFHDl, the other contams at least one transcription activation domain such as VP16 or the p65 domam from NF-kappaB The presence of a ligand to which the two chimeric protems can bind, and through which the chimeric proteins can complex with one another to form protem dimers or mulhmers, activates transcription of a target gene linked to a transcription control element containing a DNA sequence which is recognized by, I e , bmds to, the DNA-bmdmg domam Typically the transcription control element also mcludes a ixunimal promoter sequence DNA binding domains and transcription activation domains for use m treatmg human subjects preferably comprise human peptide sequence, as represented by ZFHDl and p65 The transcription control element of a target gene construct to be directly activated by ligand-mediated dimerizahon will typically contain multiple copies of a recognition sequence for the DNA-bmdmg domam and a minimal promoter In embodiments involving a DNA bmdmg domam (DBD), composite DBDs may be used with target gene constructs contammg a correspondmg bmdmg site for the composite DBD, as described m the previously cited Gilman patent documents In all such embodiments, multimerization activates transcription of a target gene under the transcriptional contiol of a transcriptional contiol element (e g enhancer and /or promoter elements and the like) which is responsive to the multimerization event In the case of systems for the indirect activation of transcription, at least one of the chimeric proteins also contams at least one ligand-binding domam and at least one effector domam However, m these embodiments the effector domam comprises a cellular signaling domam such as the cytoplasmic domam of a growth factor receptor, which upon association with one or more like domains triggers transcription of a gene lmked to a responsive promoter Said differently, mutual association of such effector domams is considered to transmit an rntracellular signal, which results in the activation of a responsive promoter For example, clustering of the cytoplasmic portion of the zeta cham of the T Cell receptor triggers transcription of a gene lmked to an IL-2 promoter Numerous promoters responsive to the mutual association of various signaling domams are well known See e g pages 23-26 of PCT/US94/01617 (WO 94/18317) The foregomg may be adapted to the subject invention to provide effector domains for the chimeric proteins and responsive promoters for target DNA constructs.

Alternatively, there are several ligand-mediated regulated transcription systems which are based on mechanisms other than ligand-mediated dimerization which, while not preferred, may be adapted to the practice of the subject invention. In these systems, binding of ligand to a chimeric protein activates transcription of a target gene linked to a responsive tianscription control sequence.

One such sytem relies upon a chimeric protein comprising a GAL4 DNA binding domain, a ligand-binding domain derived from the human progesterone receptor hPRB891 and the VP16 activation domain. The target gene construct comprises a target gene linked to a transcription control sequence comprising GAL4 binding sites. Administration of the progesterone antagonist RU 486 activates expression of the target gene. See e.g. Wang et al, 1994, Proc. Natl. Acad. Sci. USA 91:8180-8184. If used in the practice of the subject invention, it would be preferred to use DNA binding and activation domains of human origin, such as ZFHDl and p65, in place of GAL4 and VP16.

Another such system relies upon a chimeric protein comprising a DNA binding domain and a ligand-binding domain derived from an ecdysone receptor VpEcR or VgEcR. The target gene construct comprises a target gene linked to a transcription control sequence comprising an ecdysone-responsive promoter.

Administration of ecdysone or muristerone A as the ligands activates expression of the target gene. See e.g. No et al, 1996, Proc. Natl. Acad.. Sci. USA 93:3346-3351. Still another such system relies upon a chimeric protem, rtTA, comprising a modified Tet repressor domain and the VP16 tianscription activation domain which in the presence of tetracycline or an analog thereof such as doxycycline activates transcription of a target gene linked to the bacterial tet operon. If used in the practice of the subject invention, it would be preferred to use a transcription activation domain of human origin in place of VP16. See e.g. Gossen et al, 1995, Science 268:1766-1769. Depending on design preferences of the practitioner, a wide variety of ligands may be used. In general, ligands for use in this invention are preferably non- proteinaceous and preferably have a molecular weight below about 5 kD. 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 multimerizing ligand may bind to the chimeras in either order or simultaneously, preferably with a Kd value below about 10^"6, more preferably below about lO^"'⁷, even more preferably below about 10^"°, and in some embodiments below about 10^"° M. FK1012, cyclosporin- based divalent ligands, fujisporin and related types of semisynthetic ligands are disclosed in WO 94/18317 and PCT/US94/08008 (WO 95/02684). Ligands based on synthetic FKBP ligand monomers are disclosed in WO 96/06097 and WO 97/31898, and ligands based on rapamycin and derivatives are disclosed in WO 96/41865. Ligands for the ecdysone receptor, tet system and other proteins are disclosed in various cited references, including those cited and discussed above. All of the foregoing components may be used in the practice of this invention. Those documents also provide guidance in the design of constructs encoding such chimeras, expression vectors containing them, design and use of suitable target gene constructs and their use in engineering host cells.

FKBP, FRB, cyclophilin and other ligand binding domains comprising naturally occurring peptide sequence may be used in the design of chimeric proteins for use in practicing this invention. Alternatively, domains derived from naturally occurring sequences but containing one or more mutations in peptide sequence, generally at up to 10 amino acid positions, and preferably at 1-5 positions, more preferably at 1-3 positions and in some cases at a single amino acid residue, may be used in place of the naturally occurring counterpart sequence and can confer a number of important features. This is described at length in the previously cited patent documents, together with numerous examples of such mutations and corresponding ligands.

This invention further involves DNA vectors containing the various constructs described herein, whether for introduction into host cells in tissue culture, for introduction into embryos or for administration to whole organisms for the introduction of the constructs into cells in vivo. In either case the construct may be introduced episomally or for chromosomal integration. The vector may be a viral vector, including for example an adeno-, adeno associated- or retroviral vector. The constructs or vectors containing them may also contain selectable markers permitting selection of transfectants containing the construct.

This invention further encompasses the genetically engineered cells containing and/or expressing the constiucts described herein, including prokaryotic and eucaryotic cells and in particular, yeast, worm, insect, mouse or other rodent, and other mammalian cells, including human cells, of various types and lineages, whether frozen or in active growth, whether in culture or in a whole organism containing them. To recap, this invention provides materials and methods for regulatably expressing 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 method involves providing cells of this invention (or an organism containing such cells) which contain and are capable of expressing (a) one or more DNA constructs encoding one or more chimeric proteins capable, following multimerization, of activating transcription of a target gene; and, (b) a target gene tmder 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 effective to result in detectable expression of the target gene. In cases in which the cells are growing in culture, exposure to the ligand is effected by adding the ligand to the culture medium. In cases in which the cells are present within a host organism, exposing them to the ligand is effected by administering the ligand to the host organism. For instance, in cases in which the host organism is an animal, in particular, a mammal the ligand is administered to the host animal by oral, bucal, sublingual, transdermal, subcutaneous, intramuscular, intravenous, intra-joint or inhalation administration in an appropriate vehicle therefor.

This invention further encompasses pharmaceutical or veterinary compositions for expressing a target gene in genetically engineered cells of this invention, including from animal tissue or from a subject 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-m timerization reagent or a hetero- multimerization reagent as described in detail elsewhere so long as it is capable of binding to a chimeric responder protein(s) of this invention or triggering expression of the target gene in engineered cells of this invention. Likewise, this invention further encompasses 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 subject, and thus for preventing or reversing the activation of transcription of the target gene in the relevant cells. Thus, the use of the multimerization reagents and of the rniiltimerization 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 expression 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 construct encoding a chimeric protein hereof, and so forth. Alternatively, one can introduce the DNA constructs of this invention into a host organism, e.g. mammal or embryo thereof, under conditions permitting transfection of one or more cells of the host mammal in vivo.

Design and assembly of the constructs

Constructs may be designed in accordance with the principles, illustrative examples and materials and methods disclosed in the patent documents and scientific literature cited herein, with modifications and further exemplification as described herein. Components of the constructs can be prepared in conventional ways, where the coding sequences 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. In the case of DNA constructs encoding fusion proteins, DNA sequences encoding individual domains and sub domains are joined such that they constitute a single open reading frame encoding a fusion protein capable of being translated in cells or cell lysates into a single polypeptide harboring all component domains. The DNA construct encoding the fusion protein may then be placed into a vector that directs the expression of the protein in the appropriate cell type(s). For biochemical analysis of the encoded chimera, it may be desirable to construct plasmids that direct the expression of the protein in bacteria or in reticulocyte-lysate systems. For use in the production of proteins in mammalian cells, the protein-encoding sequence is introduced into an expression vector that directs expression in these cells. Expression vectors suitable for such uses are well known in the art. Various sorts of such vectors are commercially available.

Antisense messages and ribozymes for blocking HIV gene expression

Any gene sequence of an infectious agent such as HIV may be targeted to prevent its expression using ligand-regulated expression of antisense message or ribozyme. 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. For a recent review containing useful background information and guidance, see Altinan, 1993, RNA enzyme-directed gene therapy, Proc. Natl. Acad. Sci. USA 90, 10898-10900 and papers cited therein, including Yu et al., 1993, A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type l.,Proc. Natl. Acad. Sci. USA 90, 6340-6344. See also Efrat et al., Ribozyme-mediated attenuation of pancreatic β-cell glucokinase expression in transgenic mice results in impaired glucose-induced insulin secretion.,Proc. Natl. Acad. Sci. USA 91, 2051-2055.

Interference by a dominant negative gene product 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. For example, raf-1 is a serine/threonine protein kinase that functions 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 factors. 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.

Other examples of dominant negative gene products include certain variants of steroid receptors, growth factor receptors having an inactive protein kinase or lacking the protein kinase domain altogether, cell surface receptors having a nonfunctional extracellular ligand binding domain or intracellular cytoplasmic domain, transcription factor variants that lack a DNA binding domain and /or a transactivation domain. Dominant negative proteins typically disrupt the normal function of a target protein by sequestering it away from its normal partner. Constructs encoding dominant negative variants of VEGF for use in cancer applications can be constructed by random mutagenesis, by selective deletion of gene segments, or by a rational protein engineering. One important requirement is that the dominant negative protein be overexpressed relative to its normal counterpart. The increased expression afforded by the ligand-regulated transcriptional activation of our invention makes this a particularly useful application of the technology.

Applications of target genes encoding a dominant negative variant, ribozyme or antisense message directed against an HIV target gene Intracellular irr-rnirnization is the process of transfering a gene into a cell that protects that cell from a harmful agent, which can be either physical (i.e., irradiation), chemical (i.e., chemotherapeutic drugs), or biological (i.e., infectious agents such as viruses). Intracellular irnmiinization is a particular relevant for treatment or prevention of AIDS, which is caused by the spread of HIV virus in cells of the blood. To treat or prevent HIV infection by intracellular immunization, a patient's blood cells may be transduced with a suitably engineered gene. This gene may be introduced into peripheral blood cells, preferably into progenitor cells of the hematopoietic system, more preferably into totipotent hematopoietic stem cells, through the use of physical DNA transfer methods or viral vectors, such that all offspring of the cells carry the engineered gene and express the encoded gene product. The gene product is a protein or RNA that either blocks establishment of an HIV infection or prevents production of infectious virus from previously-infected cells. Thus, intracellular immunization is expected to reduce virus load, halt the death of CD4 lymphocytes, and prevent the degeneration of imrnune system function that is the basis for morbidity and mortality in AIDS.

Examples of candidate agents for inducing intracellular immunization against a virus such as HIV include antisense RNA, ribozymes that cleave viral RNA, dominant-negative viral proteins (e.g., dominant-negative Tat or Rev proteins for HIV), intracellular antibodies directed against viral proteins, and capsid- nuclease fusion proteins. A common feature of these agents is that they act at least in part stoichiometrically, either by competition, hybridization, or incorporation into multi-component complexes. Therefore, high intracellular concentrations of these agents is essential to their efficacy. Regulated gene therapy permits the controlled high-level expression of intracellular proteins of this type and therefore will augment the efficacy of intracellular immunization agents in actual practice. An additional advantage of regulated gene therapy for this application is enhanced safety. The potential cellular toxicities of these agents are not yet known; regulated gene therapy permits production to be kept below levels associated with toxicity. Finally, regulated gene therapy permits treatment with the intracellular immunization agent to be terminated when the patient is free of danger and restored at a later time, if needed.

The use of regulated gene therapy for producing intracellular immunization may be essential for the successful implementation of stem cell-based gene therapy, because, once administered to the patient, engineered stem cells and their progeny cannot be recovered. The only mechanism for reversing therapy is termination of production of the therapeutic agent. Therefore, regulated gene therapy greatly improves the prospects for intracellular iinmunization.

Soluble VEGF receptor A construct encoding a soluble VEGF receptor may be prepared using conventional methods such as were used in other soluble receptor examples. For instance, the extracellular ligand binding domain of the VEGF receptor may be expressed and purified using the cloned receptor cDNA. Identification of the receptor extracellular domain can be done by performing a Kyte-Doolittle analysis on the coding sequence. In the case of cytokine and growth factor receptors, the extracellular domain is N-terminal of the transmembrane sp-mning (TM) domain. The TM domain marks the end of the ligand binding domain and in the Kyte- Doolittle profile is demarked by a high hydrophobicity index over a span of between 20-30 amino acids. For an example of the Kyte-Doolittle analysis of the EPO- receptor see US Patent No. 5,278,065. To produce the ligand binding domain of a receptor, i.e., a soluble receptor, the cDNA encoding the extracellular domain is cloned into an appropriate expression vector such as pETlla (Invitrogen) for E. coli, pVL1393 (Invitrogen) for insect cells, or pcDNA (Invitrogen) for mammalian cells. A stop codon is introduced at/before the first amino acid of the TM domain. When this so-called soluble receptor is expressed in yeast, insect cells or mammalian cells, the protein is secreted into the cell culture medium (see Kikuchi et al J. Immunol. Methods 167:289 1994). Alternatively, when the ligand binding domain is expressed in E. coli, the soluble receptor collects in the periplasmic space (see Cunningham et al Science 254: 821 1991). To facilitate purification and binding assays the extracellular domain may be expressed fused to an epitope tag such as the epitope for the anti-myc antibody 9E10 or the "Flag" epitope (IBI) (see Kolodziej and Young, Methods Enzymol 194: 508 (1991)). Alternatively, the ligand binding domain may be expressed fused to the heavy chain of an immunoglobulin as described in (Ashkenazi et al PNAS 88:10535 1991). The ligand binding domain can be expressed in E. coli, yeast, insect cells, mammalian cells or produced using an in vitro transcription/ translation system (Promega). Expression in mammalian cells can be accomplished using transient expression or by stable selection of clones using a selectable drug such as G418. For details of expression systems see Goeddel (ed.) Methods Enzymol vol 185 1990). Purification of the expressed protein can be accomplished by standard chromatographic methods, by ligand affinity chromatography or by means of the fusion partner such as an antibody epitope or i-rmαvmoglobulin heavy chain. Tissue-specific or cell-type specific expression

It will be preferred in certain embodiments, that the chimeric proteins be expressed in a cell-specific or tissue-specific manner. Such specificity of expression may be achieved by operably linking one or 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. See e.g. PCT/US95/10591, especially pp. 36-37.

For example, constructs for expressing the chimeric proteins may contain regulatory sequences derived from known genes for specific expression in selected tissues. Representative examples are tabulated below:

Myelin basic Miskimins, R. Knapp, L., Dewey,MJ, Zhang, X. protein Cell and tissue-specific expression of a heterologous gene under control of the myelin basic protein gene promoter in trangenic mice. Brain Res Dev Brain Res 1992 Vol 65: 217-21 spermatids protamine Breitman, M.L., Rombola, FI., Maxwell, I.H., Klintworth, G.K., Bernstein, A. (1990) Genetic ablation in transgenic mice with attenuated diphtheria toxin A gene. Mol. Cell. Biol. 10: 474-479 adipocyte P2 Ross, S.R, Braves, RA, Spiegelman, BM Targeted expression of a toxin gene to adipose tissue: transgenic mice resistant to obesity Genes and Dev 7: 1318-24 1993 muscle myosin light chain Lee, KJ, Ross, RS, Rockman, HA, Harris, AN, O'Brien, TX, van-Bilsen, M., Shubeita, HE, Kandolf, R., Brem, G., Prices et alj. Biol. Chem. 1992 Aug 5, 267: 15875-85

Muscat, GE., Perry, S. , Prentice, H. Kedes, L. The human skeletal alpha-actin gene is

Alpha actin regulated by a muscle-specific enhancer that binds three nuclear factors. Gene Expression 2, 111-26, 1992 liver tyrosine aminotransferase, albumin, apolipoproteins neurofilament Reeben, M. Halmekyto, M. Alhonen, L. neurons proteins Sinervirta, R. Saarma, M. Janne . Tissue specific expression of rat light neurofilament promoter-driven reporter gene in transgenic mice. BBRC 1993: 192: 465-70 lung Lung surfacant Ornitz, D.M., Palmiter, R.D., Hammer, R.E., gene Brinster, R.L., Swift, G.H., MacDonald, R.J. (1985) Specific expression of an elastase human growth fusion in pancreatic acinar cells of transgeneic mice. Nature 131: 600 603

Introduction of Constructs into Cells

Constructs encoding the chimeric responder protein molecules and constructs directing the expression of target genes, all as described herein, can be introduced into cells as one or more DNA molecules or constructs, in many cases in association with one or more markers to allow tor selection of host cells which contam the construct(s) The constructs can be prepared m conventional ways, where the codmg sequences and regulatory regions may be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencmg, or other convenient means Particularly, using PCR, individual fragments mcludmg all or portions of a functional unit may be isolated, where one or more mutations may be mtroduced using primer repair ', kgation, in vitro mutagenesis, etc as appropriate The construct(s) once completed and demonstrated to have the appropriate sequences may then be introduced mto a host cell by any convement means The constructs may be mtegrated and packaged mto non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral vectors, for infection or transduction into cells The constructs may include viral sequences for transfection, if desired Alternatively, the construct may be introduced by fusion, electioporahon, biolistics, transfection, lipofection, or the like The host cells will in some cases be grown and expanded in culture before introduction of the constiuct(s), followed by the appropriate treatment for introduction of the construct(s) and integration of the construct(s) The cells will then be expanded and screened by virtue of a marker present in the construct Various markers which may be used successfully mclude hprt, neomycm resistance, thymidine kmase, hygromycin resistance, etc

In some instances, one may have a target site for homologous recombination, where it is desired that a construct be mtegrated at a particular locus For example, one can delete and/or replace an endogenous gene (at the same locus or elsewhere) with a recombinant target construct of this invention For homologous recombmahon, one may generally use either Ω or O-vectors See, for example, Thomas and Capecchi, Cell (1987) 51, 503-512, Mansour, et al , Nature (1988) 336, 348-352, and Joyner, et al , Nature (1989) 338, 153-156

The constructs may be mtroduced as a smgle DNA molecule encodmg all of the genes, or different DNA molecules having one or more genes The constructs may be mtroduced simultaneously or consecutively, each with the same or different markers

Vectors contammg useful elements such as bacterial or yeast origins of replication, selectable and/or amplifiable markers, promoter/enhancer elements for expression m procaryotes or eucaryotes, etc which may be used to prepare stocks of construct DNAs and for carrymg out transfections are well known m the art, and many are commercially available Introduction of Constructs into host Organisms

Cells which have been modified ex vivo with the DNA constructs may be grown in culture under selective conditions and cells which are selected as havmg the desired construct(s) may then be expanded and further analyzed, using, for example, the polymerase cham reaction for determining the presence of the construct m the host cells. Once modified host cells have been identified, they may then be used as planned, e.g. grown m culture or mtroduced into a host organism. Dependmg upon the nature of the cells, the cells may be mtroduced mto a host organism, e.g. a mammal, m a wide variety of ways. Hematopoietic cells may be administered by in_jection mto the vascular system, there bemg usually at least about 104 cells and generally not more than about 10l0, more usually not more than about 108 cells. The number of cells which are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be 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. Alternatively, with skin cells which may be used as a graft, the number of cells 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 104 and not more than about 108 and may be applied as a dispersion, generally being injected at or near the site of interest. The cells will usually be in a physiologically-acceptable medium.

Cells engmeered m accordance with this invention may also be encapsulated, e.g. using conventional materials and methods. See e.g. Uludag and Sefton, 1993, J Biomed. Mater. Res. 27(10):1213-24; Chang et al, 1993, Hum Gene Ther 4(4):433-40; Reddy et al, 1993, J Infect Dis 168(4):1082-3, Tai and Sun, 1993, FASEB J 7(ll):1061-9; Emerich et al, 1993, Exp Neurol 122(l):37-47; Emerich et al, 1994, Exp Neurol 130:141-150; Sagen et al, 1993, J Neurosci 13(6):2415-23; Aebischer et al, 1994, Exp Neurol 126(2):151-8; Savelkoul et al, 1994, J Immunol Methods 170(2):185-96; Winn et al, 1994, PNAS USA 91(6):2324-8; Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry 18(5):935-46; Emerich et al, J Comparative Neurology 349:148-164 (1994); Joseph, 1994, Cell Transplanation 3(5):355-364 and Kordower et al, 1994, PNAS USA 91(23):10898-902. The cells may then be mtroduced m encapsulated form mto an animal host, preferably a mammal and more preferably a human subject m need thereof. Preferably the encapsulating material is semipermeable, permitting release mto the host of secreted protems produced by the encapsulated cells. In many embodiments the semipermable encapsulation renders the encapsulated cells immunologically isolated from the host organism in which the encapsulated cells are introduced. In those embodiments the cells to be encapsulated may express one or more chimeric proteins containing component domains derived from viral proteins or proteins from other species (and no longer preferably contain a composite DNA binding domain as described in detail in the Gilman references, supra). For example in those cases the chimeras may well contain elements derived from GAL4 and VP16. In such cases, the cells may be engineered as disclosed in International Patent Applications PCT/US94/01617 or PCT/US94/08008 or in WO96/06111. Instead of ex vivo modification of the cells, in many situations one may wish to modify cells in vivo. For this purpose, various techniques have been developed for modification of target tissue and cells in vivo. A number of virus vectors have been developed, such as adenovirus, adeno-associated virus, and retioviruses, which allow for transfection and random integration of the virus into the host, as described below.

Adenoviral Vectors:

A viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors. Knowledge of the genetic organization of adenovirus, a 36 kB, linear and double-stranded DNA virus, allows substitution of a large piece of adenoviral DNA with foreign sequences up to 8 kB. In contrast to retrovirus, the infection of adenoviral DNA into host cells does not result in chromosomal integration because adenoviral DNA replicates in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in the human.

Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair (bp) inverted terminal repeats (ITR), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. T e expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan (1990) Radiotherap. Oncol. 19:197). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5' tripartite leader (TL) sequence which makes them preferred mRNAs for translation.

The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al., (1988) BioTechniques 6:616; Rosenfeld et al, (1991) Science 252:431-434; and Rosenfeld et al., (1992) Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al., (1992) cited supra), endothelial cells (Lemarchand et al., (1992) PNAS USA 89:6482-6486), hepatocytes (Herz and Gerard, (1993) PNAS USA 90:2812-2816) and muscle cells (Quantin et al., (1992) PNAS USA 89:2581-2584). Adenovirus vectors have also been used in vaccine development (Grunhaus and Horwitz (1992) Siminar in Virology 3:237; Graham and Prevec (1992) Biotechnology 20:363). Experiments in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al. (1991) ; Rosenfeld et al. (1992) Cell 68:143), muscle injection (Ragot et al. (1993) Nature 361:647), peripheral intravenous injection (Herz and Gerard (1993) Proc. Natl. Acad. Sci. U.S.A. 90:2812), and stereotactic inoculation into the brain (Le Gal La Salle et al. (1993) Science 254:988).

Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors. Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral El and E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones et al., (1979) Cell 16:683; Berkner et al., supra; and Graham et al., in Methods in Molecular Biology, E.J. Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109- 127). Expression of the inserted chimeric gene can be under control of, for example, the E1A promoter, the major late promoter (MLP) and associated leader sequences, the viral E3 promoter, or exogenously added promoter sequences.

Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the method of the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector. As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to intioduce the nucleic acid of interest at the position from which the El coding sequences have been removed. However, the position of insertion of the nucleic acid of interest in a region within the adenovirus sequences is not critical to the present invention. For example, the nucleic acid of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described previously by Karlsson et. al. (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.

A preferred helper cell line is 293 (ATCC Accession No. CRL1573). This helper cell line, also termed a "packaging cell line" was developed by Frank Graham (Graham et al. (1987) J. Gen. Virol. 36:59-72 and Graham (1977) J.General Virology 68:937-940) and provides E1A and E1B in trans. However, helper cell lines may also be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or eoithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.

Various adenovirus vectors have been shown to be of use in the transfer of genes to mammals, including humans. Replication-deficient adenovirus vectors have been used to express marker proteins and CFTR in the pulmonary epithelium. Because of their ability to efficiently infect dividing cells, their tropism for the lung, and the relative ease of generation of high titer stocks, adenoviral vectors have been the subject of much research in the last few years, and various vectors have been used to deliver genes to the lungs of human subjects (Zabner et al., Cell 75:207-216, 1993; Crystal, et al., Nat Genet. 8:42-51, 1994; Boucher, et al., Hum Gene Ther 5:615-639, 1994). The first generation Ela deleted adenovirus vectors have been improved upon with a second generation that includes a temperature-sensitive E2a viral protein, designed to express less viral protein and thereby make the virally infected cell less of a target for the immune system (Goldman et al., Human Gene Therapy 6:839 851,1995). More recently, a viral vector deleted of all viral open reading frames has been reported (Fisher et al., Virology 217:11-22, 1996). Moreover, it has been shown that expression of viral IL-10 inhibits the immune response to adenoviral antigen (Qin et al., Human Gene Therapy 8:1365-1374, 1997).

Adenoviruses can also be cell type specific, i.e., infect only restricted types of cells and /or express a transgene only in restricted types of cells. For example, the viruses comprise a gene under the transcriptional control of a transcription initiation region specifically regulated by target host cells, as described e.g., in U.S. Patent No. 5,698,443, by Henderson and Schuur, issued December 16, 1997. Thus, replication competent adenoviruses can be restricted to certain cells by, e.g., inserting a cell specific response element to regulate a synthesis of a protein necessary for replication, e.g., E1A or E1B.

DNA sequences of a number of adenovirus types are available from Genbank. For example, human adenovirus type 5 has GenBank Accession

No.M73260. The adenovirus DNA sequences may be obtained from any of the 42 human adenovirus types currently identified. Various adenovirus strains are available from the American Type Culture Collection, Rockville, Maryland, or by request from a number of commercial and academic sources. A transgene as described herein may be incorporated into any adenoviral vector and delivery protocol, by the same methods (restriction digest, linker ligation or filling in of ends, and ligation) used to insert the CFTR or other genes into the vectors.

Adenovirus producer cell lines can include one or more of the adenoviral genes El, E2a, and E4 DNA sequence, for packaging adenovirus vectors in which one or more of these genes have been mutated or deleted are described, e.g., in PCT/US95/15947 (WO 96/18418) by Kadan et al.; PCT/US95/07341 (WO 95/346671) by Kovesdi et al; PCT /FR94/ 00624 (W094/28152) by Imler et al.;PCT/FR94/00851 (WO 95/02697) by Perrocaudet et al, PCT/US95/ 14793 (WO96/14061) by Wang et al

AAV vectors: Yet another viral vector system useful for delivery of the sub_ject chimeric genes is the adeno-associated virus (AAV) Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (For a review, see Muzyczka et al., Curr Topics m Micro and Immunol. (1992) 158 97-129)

AAV has not been associated with the cause of any disease AAV is not a tiansforming or oncogemc virus AAV integration mto chromosomes of human cell lmes does not cause any significant alteration in the growth properties or morphological characteristics of the cells These properties of AAV also recommend it as a potentially useful human gene therapy vector.

AAV is also one of the few viruses that may integrate its DNA into non- dividing cells, e.g., pulmonary epithelial cells, and exhibits a high frequency of stable integration (see for example Flotte et al., (1992) Am. J. Respir. Cell Mol. Biol 7-349-356; Samulski et al., (1989) J Virol. 63:3822-3828; and McLaughlin et al , (1989) J Virol. 62:1963-1973) Vectors contammg as little as 300 base pairs of AAV can be packaged and can mtegrate Space for exogenous DNA is limited to about 4.5 kb An AAV vector such as that described m Tratschm et al., (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA mto cells. A variety of nucleic acids have been mtroduced into different cell types using AAV vectors (see for example Hermonat et al , (1984) PNAS USA 81.6466-6470, Tratschm et al , (1985) Mol Cell. Biol 4:2072-2081, Wondisford et al, (1988) Mol Endocrmol 2.32-39; Tratschm et al., (1984) J Virol. 51.611 619, and Flotte et al., (1993) J. Biol Chem. 268:3781-3790)

The AAV-based expression vector to be used typically includes the 145 nucleotide AAV inverted terminal repeats (ITRs) flankmg a restriction site that can be used for subcloning of the transgene, either directly using the restriction site available, or by excision of the transgene with restriction enzymes followed by blunting of the ends, ligation of appropriate DNA linkers, restriction digestion, and ligation mto the site between the ITRs The following protems have been expressed using various AAV-based vectors: neomycm phosphotransferase, chloramphemcol acetyl transferase, Fanconi's anemia gene, cystic fibrosis transmembrane conductance regulator, and granulocyte macrophage colony-stimulating factor (Kotin, R.M., Human Gene Therapy 5:793-801, 1994, Table I). A transgene incorporating the various DNA constructs of this invention can similarly be included in an AAV-based vector. As an alternative to inclusion of a constitutive promoter such as CMV to drive expression of the recombinant DNA encoding the chimeric protein(s), e.g. chimeric proteins comprising an activation domain or DNA-binding domain, an AAV promoter can be used (ITR itself or AAV p5 (Flotte, et al. J. Biol.Chem. 268:3781-3790, 1993)).

Such a vector can be packaged into AAV virions by reported methods. For example, a human cell line such as 293 can be co transfected with the AAV-based expression vector and another plasmid containing open reading frames encoding AAV rep and cap (which are obligatory for replication and packaging of the recombinant viral construct) tmder the control of endogenous AAV promoters or a heterologous promoter. In the absence of helper virus, the rep proteins Rep68 and Rep 78 prevent accumulation of the replicative form, but upon superinfection with adenovirus or herpes virus, these proteins permit replication from the ITRs (present only in the construct containing the transgene) and expression of the viral capsid proteins. This system results in packaging of the transgene DNA into AAV virions (Carter, B.J., Current Opinion in Biotechnology 3:533-539, 1992; Kotin, R.M, Human Gene Therapy 5:793-801, 1994)). Typically, three days after transfection, recombinant AAV is harvested from the cells along with adenoviurs and the contaminating adenovirus is then inactivated by heat treatment.

Methods to improve the titer of AAV can also be used to express the transgene in an AAV virion. Such strategies include, but are not limited to: stable expression of the ITR-flanked transgene in a cell line followed by transfection with a second plasmid to direct viral packaging; use of a cell line that expresses AAV proteins inducibly, such as temperature-sensitive indticible expression or pharmacologically indticible expression. Alternatively, a cell can be transformed with a first AAV vector including a 5' ITR, a 3' ITR flanking a heterologous gene, and a second AAV vector which includes an inducible origin of replication, e.g., SV40 origin of replication, which is capable of being induced by an agent, such as the SV40 T antigen and which includes DNA sequences encoding the AAV rep and cap proteins. Upon induction by an agent, the second AAV vector may replicate to a high copy number, and thereby increased numbers of infectious AAV particles may be generated (see, e.g, U.S. Patent No. 5,693,531 by Chiorini et al., issued December 2, 1997. In yet another method for producing large amounts of recombinant AAV, a chimeric plasmid is used which incorporate the Epstein Barr Nuclear Antigen (EBNA) gene , the latent origin of replication of Epstein Barr virus (oriP) and an AAV genome. These plasmids are maintained as a multicopy extra- chromosomal elements in cells, such as in 293 cells. Upon addition of wild-type helper ftmctions, these cells will produce high amotmts of recombinant AAV (U.S. Patent 5,691,176 by Lebkowski et al., issued Nov. 25, 1997). In another system, an AAV packaging plasmid is provided that allows expression of the rep gene, wherein the p5 promoter, which normally controls rep expression, is replaced with a heterologous promoter (U.S. Patent 5,658,776, by Flotte et al., issued Aug. 19, 1997). Additionally, one may increase the efficiency of AAV transduction by treating the cells with an agent that facilitates the conversion of the single stranded form to the double stranded form, as described in Wilson et al., WO96/39530. AAV stocks can be produced as described in Hermonat and Muzyczka (1984) PNAS 81:6466, modified by using the pAAV/Ad described by Samulski et al. (1989) J. Virol. 63:3822. Concentration and purification of the virus can be achieved by reported methods such as banding in cesium chloride gradients, as was used for the initial report of AAV vector expression in vivo (Flotte, et al. J.Biol. Chem. 268:3781-3790, 1993) or chromatographic purification, as described in O'Riordan et al., WO97/08298.

Methods for in vitro packaging AAV vectors are also available and have the advantage that there is no size limitation of the DNA packaged into the particles (see, U.S. Patent No. 5,688,676, by Zhou et al., issued Nov. 18, 1997). This procedure involves the preparation of cell free packaging extracts.

For additional detailed guidance on AAV technology which may be useful in the practice of the subject invention, including methods and materials for the incorporation of a transgene, the propagation and purification of the recombinant AAV vector containing the transgene, and its use in transfecting cells and mammals, see e.g. Carter et al, US Patent No. 4,797,368 (10 Jan 1989); Muzyczka et al, US Patent No. 5,139,941 (18 Aug 1992); Lebkowski et al, US Patent No. 5,173,414 (22 Dec 1992); Srivastava, US Patent No. 5,252,479 (12 Oct 1993); Lebkowski et al, US Patent No. 5,354,678 (11 Oct 1994); Shenk et al, US Patent No. 5,436,146(25 July 1995); Chatterjee et al, US Patent No. 5,454,935 (12 Dec 1995), Carter et al WO 93/24641 (published 9 Dec 1993), and Natsoulis, U.S. Patent No. 5,622,856 (April 22, 1997). Further information regarding AAVs and the adenovirus or herpes helper functions required can be found in the following articles: Berns and Bohensky (1987), "Adeno- Associated Viruses: An Update", Advances in Virus Research, Academic Press, 33:243-306. The genome of AAV is described in Laughlin et al. (1983) "Cloning of infectious adeno-associated virus genomes in bacterial plasmids", Gene, 23: 65-73. Expression of AAV is described in Beaton et al. (1989) "Expression from the Adeno-associated virus p5 and pl9 promoters is negatively regulated in trans by the rep protein", J. Virol., 63:4450- 4454. Construction of rAAV is described in a number of publications: Tratschin et al. (1984) "Adeno-associated virus vector for high frequency integration, expression and rescue of genes in mammalian cells", Mol. Cell. Biol., 4:2072-2081; Hermonat and Muzvczka (1984) "Use of adeno-associated virus as a mammalian DNA cloning vector: Transduction of neomycin resistance into mammalian tissue culture cells", Proc. Natl. Acad. Sci. USA, 81:6466-6470; McLaughlin et al. (1988) "Adeno- associated virus general transduction vectors: Analysis of Proviral Structures", J. Virol., 62:1963-1973; and Samtilski et al. (1989) "Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression", J. Virol., 63:3822-3828. Cell lines that can be transformed by rAAV are those described in Lebkowski et al. (1988) "Adeno-associated virus: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types", Mol. Cell. Biol., 8:3988-3996. "Producer" or "packaging" cell lines used in manufacturing recombinant retroviruses are described in Dougherty et al. (1989) J. Virol., 63:3209-3212; and Markowitz et al. (1988) J. Virol., 62:1120- 1124.

Hybrid Adenovirus-AAV Vectors:

Hybrid Adenovirus-AAV vectors represented by an adenovirus capsid containing a nucleic acid comprising a portion of an adenovirus, and 5' and 3' ITR sequences from an AAV which flank a selected transgene under the control of a promoter. See e.g. Wilson et al, International Patent Application Publication No. WO 96/13598. This hybrid vector is characterized by high titer tiansgene delivery to a host cell and the ability to stably integrate the tiansgene into the host cell chromosome in the presence of the rep gene. This virus is capable of infecting virtually all cell types (conferred by its adenovirus sequences) and stable long term transgene integration into the host cell genome (conferred by its AAV sequences).

The adenovirus nucleic acid sequences employed in the this vector can range from a minimum sequence amount, which requires the use of a helper virus to produce the hybrid virus particle, to only selected deletions of adenovirus genes, which deleted gene products can be supplied in the hybrid viral process by a packaging cell. For example, a hybrid virus can comprise the 5' and 3' inverted terminal repeat (ITR) sequences of an adenovirus (which function as origins of replication). The left terminal sequence (5') sequence of the Ad5 genome that can be used spans bp 1 to about 360 of the conventional adenovirus genome (also referred to as map units 0-1) and includes the 5' ITR and the packaging /enhancer domain. The 3' adenovirus sequences of the hybrid virus include the right terminal 3' ITR sequence which is about 580 nucleotides (about bp 35,353- end of the adenovirus, referred to as about map tmits 98.4-100.

The AAV sequences useful in the hybrid vector are viral sequences from which the rep and cap polypeptide encoding sequences are deleted and are usually the cis acting 5' and 3' ITR sequences. Thus, the AAV ITR sequences are flanked by the selected adenovirus sequences and the AAV ITR sequences themselves flank a selected tiansgene. The preparation of the hybrid vector is further described in detail in published PCT application entitled "Hybrid Adenovirus-AAV Virus and Method of Use Thereof", WO 96/13598 by Wilson et al.

For additional detailed guidance on adenovirus and hybrid adenovirus- AAV technology which may be useful in the practice of the subject invention, including methods and materials for the incorporation of a transgene, the propagation and purification of recombinant virus containing the transgene, and its use in transfecting cells and mammals, see also Wilson et al, WO 94/28938, WO 96/13597 and WO 96/26285, and references cited therein.

Retroviruses:

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin (1990) Retroviridae and their Replication" In Fields, Knipe ed. Virology. New York: Raven Press). The resulting DNA then stably integrates into cellular chromosomes as a provirtis and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsial proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene, termed psi , functions as a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin (1990), supra).

In order to construct a retroviral vector, a nucleic acid of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and psi components is constructed (Mann et al. (1983) Cell 33:153). When a recombinant plasmid containing a human cDNA, together with the retroviral LTR and psi sequences is introduced into this cell line (by calcium phosphate precipitation for example), the psi sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and

Rtibenstein (1988) "Retroviral Vectors", In: Rodriguez and Denhardt ed. Vectors: A Survey of Molecular Cloning Vectors and their Uses. Stoneham:Butterworth; Temin, (1986) "Retrovirus Vectors for Gene Transfer: Efficient Integration into and Exprssion of Exogenous DNA in Vertebrate Cell Genome", In: Kucherlapati ed. Gene Transfer. New York: Plenum Press; Mann et al., 1983, supra). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al. (1975) Virology 67:242). A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene tiansfer for gene therapy purposes (for a review see

Miller, A.D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding a fusion protein of the present invention, e.g., a transcriptional activator, rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. A preferred retroviral vector is a pSR MSVtkNeo (Mttller et al. (1991) Mol. Cell Biol. 11:1785 and pSR MSV(Xbal) (Sawyers et al. (1995) J. Exp. Med. 181:307) and derivatives thereof. For example, the unique BamHI sites in both of these vectors can be removed by digesting the vectors with BamHI, filling in with Klenow and religating to produce pSMTN2 and pSMTX2, respectively, as described in PCT /US96/ 09948 by Clackson et al. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include Crip, Cre, 2 and Am.

Retroviruses have been used to mtroduce a variety of genes mto many different cell types, including neural cells, epithelial cells, endothehal cells, lymphocytes, myob lasts, hepatocytes, bone marrow cells, in vitro and /or in vivo (see for example Eglihs et al., (1985) Science 230.1395-1398; Danos and Mulligan, (1988) PNAS USA 85:6460 6464; Wilson et al., (1988) PNAS USA 85:3014-3018; Armentano et al., (1990) PNAS USA 87:6141-6145; Huber et al., (1991) PNAS USA 88:8039 8043, Ferry et al., (1991) PNAS USA 88:8377-8381; Chowdhury et al., (1991) Science 254:1802-1805; van Beusechem et al, (1992) PNAS USA 89:7640-7644; Kay et al., (1992) Human Gene Therapy 3:641-647; Dai et al., (1992) PNAS USA 89:10892-10895; Hwu et al., (1993) J. Immunol. 150:4104-4115; U.S. Patent No 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications WO93/25234, WO94/06920, and WO94/11524). For mstance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protem (Roux et al., (1989) PNAS USA 86:9079-9083, Julan et al., (1992) J. Gen Virol 73:3251-3255; and Goud et al., (1983) Virology 163:251-254); or couplmg cell surface ligands to the viral env protems (Neda et al., (1991) J. Biol. Chem. 266:14143-14146). Couplmg can be m the form of the chemical cross-linking with a protein or other variety (e.g. lactose to convert the env protein to an asialoglycoprotein), as well as by generatmg fusion protems (e.g. single-chain antibody /env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certam tissue types, and can also be used to convert an ecotropic vector m to an amphotropic vector.

Other Viral Systems:

Other viral vector systems that may have application m gene therapy have been derived from herpes virus, e , Herpes Simplex Virus (U.S Patent No. 5,631,236 by Woo et al , issued May 20, 1997), vaccmia virus (Ridgeway (1988) Ridgeway, "Mammalian expression vectors," In: Rodriguez R L, Denhardt D T, ed. Vectors: A survey of molecular cloning vectors and their uses. Stoneham: Butterworth,; Baichwal and Stigden (1986) "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press; C'oupar et al. (1988) Gene, 68:1-10), and several RNA viruses. Preferred viruses include an alphavirus, a poxivirtis, an arena virus, a vaccinia virus, a polio virus, and the like. In particular, herpes virus vectors may provide a unique strategy for persistence of the recombinant gene in cells of the central nervous system and ocular tissue (Pepose et al., (1994) Invest Ophthalmol Vis Sci 35:2662-2666). They offer several attractive features for various mammalian cells (Friedmann (1989) Science, 244:1275-1281 ; Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra; Coupar et al., 1988; Horwich et al.(1990) J.Virol., 64:642-650).

With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al., 1990, supra). This suggested that large portions of the genome could be replaced with foreign genetic material. The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer. Chang et al. recently introduced the chloramphemcol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre- surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high liters of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al. (1991) Hepatology, 14-.124A).

Administration of Viral Vectors:

Generally the DNA or viral particles are transferred to a biologically compatible solution or pharmaceutically acceptable delivery vehicle, such as sterile saline, or other aqueous or non-aqueous isotonic sterile injection solutions or suspensions, numerous examples of which are well known in the art, including Ringer's, phosphate buffered saline, or other similar vehicles. Delivery of the transgene as naked DNA; as lipid-, liposome-, or otherwise formulated DNA; or as a recombinant viral vector is then preferably carried out via in vivo, lung-directed, gene therapy. This can be accomplished by various means, including nebulization/inhalation or by instillation via bronchoscopy. Recently, recombinant adenovirus encoding CFTR was administered via aerosol to human subjects in a phase I clinical trial. Vector DNA and CFTR expression were clearly detected inthe nose and airway of these patients with no acute toxic effects (Bellonet al., Human Gene Therapy, 8(l):15-25, 1997).

Preferably, the DNA or recombinant virus is administered insufficient amounts to transfect cells within the recipient's airways, including without limitation various airway epithelial cells, leukocytes residing within the airways and accessible airway smooth muscle cells, and provide sufficient levels of transgene expression to provide for observable ligand-responsive transcription of a target gene, preferably at a level providing therapeutic benefit without undue adverse effects.

Optimal dosages of DNA or virus depends on a variety of factors, as discussed previously, and may thus vary somewhat from patient to patient. Again, therapeutically effective doses of viruses are considered to be in the range of about 20 to about 50 ml of saline solution containing concentrations of from about I X 107 to about 1 X 1010 pfti of virus/ml, e.g. from 1 X 108 to 1 X 109 pfu of virus /ml.

In a preferred embodiment, the ratio of viral particle containing a target gene versus viral particles containing nucleic acids encoding the chimeric proteins of the invention is about 1:1. However, other ratios can also be used. For example, in certain instances it may be desirable to administer twice as many particles having the target gene as those encoding the chimeric proteins. Other ratios include 1:3, 1:4, 1:10, 2:1, 3:1, 4:1, 5:1, 10:1. The optimal ratio can be determined by performing in vitro assays using the different ratios of viral particles to determine which ratio results in highest expression and lowest background expression of the target gene. Similarly, in situations in which the chimeric proteins are encoded by two different nucleic acids each encapsidated separately, one can vary the ratio between the three viral particles, according to the result desired.

In accordance with in vivo genetic modification, 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. By employing an attenuated or modified retrovirus carrying a target transcriptional initiation region, if desired, one can activate the virus using one of the subject transcription factor constructs, so that the virus may be produced and transfect adjacent cells. 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 dimerizing ligand may be administered to the patient as desired to activate transcription of the target gene. Depending upon the binding affinity of the ligand, the response desired, the manner of administration, the half-life, the number of cells present, various protocols may be employed. The ligand may be administered parenterally or orally. The number of administrations will depend upon the factors 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 antagonist 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 factors 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. In the event that transcriptional activation by the ligand is to be reversed or terminated, a monomeric compound which can compete with the dimerizing ligand may be administered. Thus, in the case of an adverse reaction or the desire to terminate the therapeutic effect, an antagonist to the dimerizing agent can be administered in any convenient way, particularly intravascularly, if a rapid reversal is desired. Alternatively, one may provide for the presence of an inactivation domain (or transcriptional silencer) with a DNA binding domain. In another approach, cells may be eliminated through apoptosis via signalling through Fas or TNF receptor as described elsewhere. See e.g.,PCT/US94/01617 and PCT/US94/08008. 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. Where 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.

It should be appreciated that the system is subject to many variables, such as the cellular response to the ligand, the efficiency of expression and, as appropriate, the level of secretion, the activity of the expression product, the particular need of the patient, which may vary with time and circumstances, the rate of loss of the cellular activity as a result of loss of cells or expression activity of individual cells, and the like. Therefore, it is expected that for each individual patient, even if there were universal cells which could be administered to the population at large, each patient would be monitored for the proper dosage for the individual.

Biological research

This invention is also applicable to a wide range of biological experiments in which precise contiol over a target gene is desired. These include: (1) expression of a protein or RNA of interest for biochemical purification; (2) regulated expression of a protein or RNA of interest in tissue culture cells for the purposes of evaluating its biological function; (3) regulated expression of a protein or RNA of interest in transgenic animals for the purposes of evaluating its biological function; (4) regulating the expression of another regulatory protein that acts on an endogenous gene for the purposes of evaluating the biological function of that gene.

Claims

Claims:

1. A cell which contains

(a) a first DNA construct or pair of first DNA constructs encoding chimeric responder protein molecules comprising (i) at least one receptor domain capable of binding to a selected ligand and (ii) another protein domain, heterologous with respect to the receptor domain, but capable, upon multimerization of the responder protein molecules, of triggering the activation of transcription of a target gene tmder the transcriptional control of a transcriptional contiol element responsive to said multimerization; and

(b) a target gene tinder the expresssion control of a transcriptional control element responsive to said multimerization;

and which, following exposure to the selected ligand, expresses the target gene,

wherein the target gene encodes thrombospondin, angiostatin, a soluble receptor for VEGF, a dominant negative form of VEGF or a tumor-specific antigen.

2. An engineered cell of claim 1 in which the target gene encodes a peptide sequence of human origin.

3. A method for rendering cells capable of regulatable expression of a target gene following exposure of the cells to a selected ligand, wherein the target gene is selected from the group consisting of DNA sequences encoding thrombospondin, angiostatin, a soluble receptor for VEGF, a dominant negative form of VEGF or a tumor-specific antigen, which method comprises introducing into the cells:

(i) one or more first DNA constructs, each encoding a chimeric responder protein comprising (i) at least one receptor domain capable of binding to a selected ligand and (ii) another protein domain, heterologous with respect to the receptor domain, but capable, upon multimerization of the chimeric responder protein molecules, of triggering the activation of transcription of the target gene tmder the transcriptional control of a transcriptional control element responsive to said multimerization; and (ii) a target DNA construct comprising the target gene linked to and tinder the expresssion control of a transcriptional control element responsive to said multimerization.

4. A method of claim 3 wherein the DNA constructs are introduced into cells maintained in vitro or into cells in situ within a host organism.

5. A method for treating or preventing cancer in a mammalian host organism containing cells which:

(a) contain, and are capable of expressing, a first DNA construct or pair of first DNA constructs encoding chimeric responder protein molecules comprising (i) at least one receptor domain capable of binding to a selected ligand and (ii) another protein domain, heterologous with respect to the receptor domain, but capable, upon multimerization of the responder protein molecules, of triggering the activation of tianscription of a target gene under the transcriptional control of a transcriptional control element responsive to said multimerization; and

(b) contain a target gene under the expresssion contiol of a transcriptional control element responsive to said multimerization;

(c) express the target gene, following exposure to the selected ligand;

wherein the target gene encodes thrombospondin, angiostatin, a soluble receptor for VEGF, a dominant negative form of VEGF, a tumor-specific antigen or a cytokine;

which method comprises administering to said mammalian host an effective amount of a selected ligand capable of binding to the chimeric responder protein to effect observable expression of the target gene.

6. A method for treating or preventing MS episodes in a mammalian host organism containing cells which:

(a) contain, and are capable of expressing, a first DNA construct or pair of first DNA constructs encoding chimeric responder protein molecules comprising (i) at least one receptor domain capable of binding to a selected ligand and (ii) another protein domain, heterologous with respect to the receptor domain, but capable, upon multimerization of the responder protein molecules, of triggering the activation of transcription of a target gene tinder the transcriptional control of a transcriptional contiol element responsive to said multimerization; and

(b) contain a target gene under the expresssion control of a transcriptional control element responsive to said multimerization;

(c) express the target gene, following exposure to the selected ligand;

wherein the target gene encodes beta interferon;

which method comprises administering to said mammalian host an effective amount of a selected ligand capable of binding to the chimeric responder protein to effect observable expression of the target beta-interferon gene.

7. A method for treating or preventing HIV infection in a mammalian host organism containing cells which:

(a) contain, and are capable of expressing, a first DNA construct or pair of first DNA constructs encoding chimeric responder protein molecules comprising (i) at least one receptor domain capable of binding to a selected ligand and (ii) another protein domain, heterologous with respect to the receptor domain, but capable, upon multimerization of the responder protein molecules, of triggering the activation of transcription of a target gene tinder the transcriptional control of a transcriptional control element responsive to said multimerization; and

(b) contain a target gene tmder the expresssion contiol of a transcriptional control element responsive to said multimerization;

(c) express the target gene, following exposure to the selected ligand;

wherein the target gene encodes a ribozyme or antisense message directed against an HIV nucleotide sequence; which method comprises administering to said mammalian host an effective amount of a selected ligand capable of binding to the chimeric responder protein to effect observable expression of the target gene.