Biomedical subjects

Tim Clackson

Publications and source records attributed to Tim Clackson.

10 recordsLinked to original sources

PubMed2003

Regulation of gene expression by synthetic dimerizers with novel specificity.

New synthetic chemical inducers of dimerization, comprising homodimeric FKBP ligands with engineered specificity for the designed point mutant F36V, have been evaluated for inducing targeted gene expression in mammalian cells. Structure-activity studies indicated that high-affinity dimerizers such as AP1903 are ineffective, perhaps due to kinetic trapping of non-productive dimers, whereas lower-affinity molecules, exemplified by AP1889 and AP1966, potently activate transcription.

Cross-Linking Reagents↗

PubMed2003

Pharmacologically regulated production of targeted retrovirus from T cells for systemic antitumor gene therapy.

We aimed to use cell-based carriers to direct vector production to target sites for systemic therapy. We used T cells engineered to express a chimeric T cell receptor that can specifically recognize target cells expressing the tumor-associated carcinoembryonic antigen (CEA). These T cells were modified to produce a retrovirus under tight pharmacological control using the rapamycin-inducible transcriptional regulatory system. The retroviral vectors produced were transcriptionally targeted to CEA-expressing target cells. We found that vector production and transgene expression from these T cells in vitro was dependent on pharmacological induction and expression of CEA in target cells, respectively. Mice bearing metastatic tumors that received cell carriers delivering the HSVtk gene demonstrated a significant increase in survival, but only in response to pharmacological induction of vector production. Interestingly, the therapeutic effect required the presence of the tumor-specific chimeric receptor on T cells. Further studies demonstrated that systemic delivery of tumor-specific T cells to mice bearing metastatic tumors caused recruitment of nonspecific T cells to the tumor site. We hypothesize that this enhanced targeting to tumor sites is responsible for the efficiency of T cell-mediated retroviral gene transfer and that this principle can be used to enhance systemic therapies using immune-cell carriers.

Adenocarcinoma↗

PubMed2003

Regulated expression of erythropoietin from an AAV vector safely improves the anemia of beta-thalassemia in a mouse model.

In vivo gene transfer is being considered in the systemic delivery of therapeutic proteins. This report evaluates the use of AAV vectors administered into muscle to deliver erythropoietin (Epo) for the treatment of anemia in a mouse model of beta-thalassemia. Injection of vector expressing Epo from a constitutive promoter resulted in Epo overproduction and improved erythropoiesis. However, severe and lethal polycythemia developed. In order to titrate the expression of Epo to therapeutic and non-toxic levels, vectors were constructed to allow pharmacologic control of Epo transcription. Specifically, expression of Epo was dependent on the presence of a chimeric transcription factor that is activated by the orally available small molecule drug rapamycin. beta-thalassemic mice injected with vectors containing the regulated Epo gene failed to show any effect until they were administered a regimen of rapamycin, which led to the production of Epo and an increase in hematocrit values. Epo expression and its hematologic consequences were directly dependent on the dose of rapamycin and were completely reversed when rapamycin was withdrawn. The increase in hematocrit was associated with partial improvements in the abnormalities of red blood cell morphology. This study confirms the value of pharmacologic regulation of transgene expression in the development of safe and effective gene therapies in which biologically active secreted proteins are produced.

Animals↗

PubMed2002

Pharmacologically regulated in vivo selection in a large animal.

The inefficiency of gene transfer has greatly hindered gene therapy. In vivo selection may increase the frequency of genetically modified cells, thereby circumventing this critical limitation. Here we demonstrate regulated in vivo selection in a large animal. CD34(+) cells from 2 dogs were engineered to express a conditional derivative of the thrombopoietin receptor (F36Vmpl). Activation of the receptor through administration of a dimerizing drug, AP20187, produced reversible, drug-dependent rises in genetically modified red cells, white cells, and platelets in both animals, with minimal side effects. Cell growth switches could greatly enhance the efficacy and applicability of gene and cell therapy.

Animals↗

PubMed2002

CD28 costimulation and immunoaffinity-based selection efficiently generate primary gene-modified T cells for adoptive immunotherapy.

The introduction of an inducible suicide gene has been proposed as a strategy to exploit the antitumor reactivity of donor T cells after allogeneic hematopoietic stem cell transplantation but permit control of graft-versus-host disease. However, there are several obstacles to this approach that may impair the ability of T cells to function and survive in vivo. These include the requirement for in vitro activation or long-term culture to introduce the transgene and obtain therapeutic cell numbers, the toxicity of drug selection to enrich transduced cells, and the immunogenicity of the transgene-encoded products. Here we have developed a transduction and selection strategy for generating large numbers of polyclonal T cells transduced with a retroviral vector encoding the human low-affinity nerve growth factor receptor (LNGFR) for selection and a Fas-based suicide construct (LV'VFas). Ligation of CD28 in conjunction with a T-cell receptor signal permitted efficient transduction, substantially promoted T-cell growth, and contributed to the generation of gene-modified T cells that retained clonal diversity, functional properties, and a homing receptor profile similar to untransduced peripheral blood lymphocytes. Microbeads conjugated directly to antibody specific to LNGFR significantly improved the immunomagnetic selection of LV'VFas-modified T cells and assisted in scaling of the selection procedure to therapeutic cell numbers. Thus, these studies identified a strategy that requires only a brief ex vivo culture and does not use drug selection to obtain large numbers of functional gene-modified polyclonal T cells that can be used for adoptive immunotherapy.

Animals↗

PubMed2002

JAK2, complemented by a second signal from c-kit or flt-3, triggers extensive self-renewal of primary multipotential hemopoietic cells.

Defining signals that can support the self-renewal of multipotential hemopoietic progenitor cells (MHPCs) is pertinent to understanding leukemogenesis and may be relevant to developing stem cell-based therapies. Here we define a set of signals, JAK2 plus either c-kit or flt-3, which together can support extensive MHPC self-renewal. Phenotypically and functionally distinct populations of MHPCs were obtained, depending on which receptor tyrosine kinase, c-kit or flt-3, was activated. Self-renewal was abrogated in the absence of STAT5a/b, and in the presence of inhibitors targeting either the mitogen-activated protein kinase or phosphatidylinositol 3' kinase pathways. These findings suggest that a simple two-component signal can drive MHPC self-renewal.

Animals↗

PubMed2002

A system for small-molecule control of conditionally replication-competent adenoviral vectors.

Replication-competent adenoviral vectors are potentially far more efficient than replication-defective vectors. However, for reasons of safety, there is a need to restrict viral replication both spatially, by limiting replication to certain cell types, and temporally. To control replication temporally, we have developed a system, based on the small-molecule dimerizer rapamycin, for regulating the replication of adenoviral vectors. In this system, one adenoviral vector, AdC4, expresses transcription factors whose activity is regulated by the non-immunosuppressive rapamycin analog AP21967. A second vector, Ad(Z12-I-E1aE1b19k), contains E1 genes placed downstream of binding sites for the regulated transcription factor. Co-infection of several cell lines by the vector pair leads to dimerizer-dependent E1 expression and an increase in viral replication, as shown by Southern blots and replication assays. Furthermore, expression of a reporter gene from a replication-defective vector, Ad-GM-CSF, can be augmented by up to 18-fold by co-infection with the pair of conditionally replicating vectors in the presence of dimerizer. Similar results are obtained when the vectors are directly injected into subcutaneous HT1080 xenograft tumors in nude mice. We believe that vectors based on this principle will be a useful additional tool to enhance efficiency and safety of gene delivery for anti-cancer therapy.

Adenoviridae↗

PubMed2002

Pharmacological regulation of protein expression from adeno-associated viral vectors in the eye.

The control, over time and space, of the levels of therapeutic proteins is crucial for successful retinal gene therapy. We tested the ability of adeno-associated viral vectors (AAV) delivered intraocularly to release a secreted protein (erythropoietin (Epo) used as a marker) in the eye, either constitutively or in a pharmacologically regulated manner using the dimerizer-inducible transcriptional regulatory system. Following delivery of a constitutively expressing vector to the intravitreal or subretinal space of nude rats, Epo protein was detected in both the anterior chamber and vitreous fluids. A dual-vector system inducible by the dimerizer rapamycin and expressing Epo was administered into the subretinal space in an attempt to achieve pharmacologic control of trangene expression in the eye. Before induction with rapamycin, the intraocular Epo level was negligible. However, following a systemic administration of rapamycin, Epo was detected in the anterior chamber, peaking on day 3 and returning to baseline 2-3 weeks after withdrawal of the drug. Peak-induced Epo in the anterior chamber was proportional to the dose of rapamycin and was not detected in serum. Similar results were obtained following subretinal administration of the vectors in one nonhuman primate. The rapamycin inducible system promises to be useful for developing gene therapies for inherited retinal degeneration and ocular neovascularization.

Animals↗

PubMed2002

Dimerizer-regulated gene expression.

Control of gene expression using small molecules is a powerful research tool and has clinical utility in the context of regulated gene therapy. Use of chemical inducers of dimerization, or dimerizers, for this purpose has several advantages, including tight regulation, modularity to facilitate iterative improvements, and assembly from human proteins to minimize immune responses in clinical applications. Recent developments include the use of the rapamycin-based dimerizer system to regulate the expression of endogenous genes, the generation of new chemical dimerizers based on FK506, dexamethasone and methotrexate, and progress towards the clinical use of adeno-associated virus and adenovirus vectors regulated by rapamycin analogs.

Animals↗

PubMed2002

Regulation of endogenous gene expression with a small-molecule dimerizer.

Artificial transcription factors containing designer zinc-finger DNA-binding domains (DBDs) have been used to activate or repress expression of a growing number of endogenous genes. We have combined targeted zinc-finger DBD technology with a dimerizer-regulated gene expression system to permit the small-molecule control of endogenous gene transcription. We constructed a dimerizer-responsive transcription factor that incorporates an artificial zinc-finger DBD targeted to the promoter of the human VEGF gene. Introduction of this activator into human cells allowed expression of the chromosomal VEGF gene to be induced by a small-molecule dimerizer compound consisting of a nonimmunosuppressive rapamycin analog. We found that by directly regulating zinc-finger protein (ZFP) activity, we could circumvent difficulties encountered in the generation of cell lines stably expressing conventional unregulated activators. Dimerizer-dependent VEGF induction was rapid, tight, and dose dependent, and resulted in VEGF protein expression levels several-fold greater than those produced by the natural hypoxic response.

Cell Line↗