Gene therapy aims to revert diseased phenotypes by the use of both viral and nonviral gene delivery systems. Substantial progress has been made in making gene transfer vehicles more efficient, less toxic, and nonimmunogenic and in allowing long-term transgene expression. One of the key issues in successfully implementing gene therapies in the clinical setting is to be able to regulate gene expression very tightly and consistently as and when it is needed. The regulation ought to be achievable using a compound that should be nontoxic, be able to penetrate into the desired target tissue or organ, and have a half-life of a few hours (as opposed to minutes or days) so that when withdrawn or added (depending on the regulatable system used) gene expression can be turned "on" or "off" quickly and effectively. Also, the genetic switches employed should ideally be nonimmunogenic in the host. The ability to switch transgenes on and off would be of paramount importance not only when the therapy is no longer needed, but also in the case of the development of adverse side effects to the therapy. Many regulatable systems are currently under development and some, i.e., the tetracycline-dependent transcriptional switch, have been used successfully for in vivo preclinical applications. Despite this, there are no examples of switches that have been employed in a human clinical trial. In this review, we aim to highlight the main regulatable systems currently under development, the gene transfer systems employed for their expression, and also the preclinical models in which they have been used successfully. We also discuss the substantial challenges that still remain before these regulatable switches can be employed in the clinical setting.
In spite of preclinical efficacy and recent randomized, controlled studies with adenoviral vectors expressing herpes simplex virus-1 thymidine kinase (HSV1-TK) showing statistically significant increases in survival, most clinical trials using single therapies have failed to provide major therapeutic breakthroughs. Because glioma is a disease with dismal prognosis and rapid progression, it is an attractive target for gene therapy. Preclinical models using microscopic brain tumor models (e.g., V 0.3 mm 3 ) may not reflect the pathophysiology and progression of large human tumors. To overcome some of these limitations, we developed a syngeneic large brain tumor model. In this model, administration of single therapeutic modalities, either conditional cytotoxicity or immunostimulation, fail. However, when various immunostimulatory therapies were delivered in combination with conditional cytotoxicity (HSV1-TK), only the combined delivery of fms-like tyrosine kinase ligand (Flt3L) and HSV1-TK significantly prolonged the survival of large tumor-bearing animals (z80%; P V 0.005). When either macrophages or CD4 + cells were depleted before administration of viral therapy, TK + Flt3L therapy failed to prolong survival. Meanwhile, depletion of CD8 + cells or natural killer cells did not affect TK + Flt3L efficacy. Spinal cord of animals surviving 6 months after TK + Flt3L were evaluated for the presence of autoimmune lesions. Whereas macrophages were present within the corticospinal tract and low levels of T-cell infiltration were detected, these effects are not indicative of an overt autoimmune disorder. We propose that combined Flt3L and HSV1-TK adenoviralmediated gene therapy may provide an effective antiglioma treatment with increased efficacy in clinical trials of glioma. (Cancer Res 2005; 65(16): 7194-204)
Purpose: In preparation for a phase I clinical trial using a combined cytotoxic/immunotherapeutic strategy with adenoviruses (Ad) expressing Flt3L (Ad-Flt3L) and thymidine kinase (Ad-TK) to treat glioblastoma (GBM), we tested the hypothesis that Ad-TK+GCV would be the optimal tumor-killing agent in relation to efficacy and safety when compared with other proapoptotic approaches. Experimental Design: The efficacy and neurotoxicity of Ad-TK+GCV was compared with Ads encoding the proapoptotic cytokines [tumor necrosis factor-a, tumor necrosis factor^related apoptosis-inducing factor (TRAIL), and Fas ligand (FasL)], alone or in combination with Ad-Flt3L. In rats bearing small GBMs (day 4), only Ad-TK+GCV or Ad-FasL improved survival. Results: In rats bearing large GBMs (day 9), the combination of Ad-Flt3L with Ad-FasL did not improve survival over FasL alone, whereas Ad-Flt3L combined with Ad-TK+GCV led to 70% long-term survival. Expression of FasL and TRAIL caused severe neuropathology, which was not encountered when we used Ad-TK+/-Ad-Flt3L. In vitro, all treatments elicited release of high mobility group box 1protein (HMGB1) from dying tumor cells. In vivo, the highest levels of circulating HMGB1 were observed after treatment with Ad-TK+GCV+Ad-Flt3L; HMGB1 was necessary for the therapeutic efficacy of AdTK+GCV+Ad-Flt3L because its blockade with glycyrrhizin completely blocked tumor regression. We also showed the killing efficacy of Ad-TK+GCV in human GBM cell lines and GBM primary cultures, which also elicited release of HMGB1. Conclusions: Our results indicate that Ad-TK+GCV+Ad-Flt3L exhibit the highest efficacy and safety profile among the several proapoptotic approaches tested. The results reported further support the implementation of this combined approach in a phase I clinical trial for GBM.
Glioblastoma multiforme is an intracranial tumor that has very poor prognosis. Patients usually succumb to their disease 6 to 12 months after they are diagnosed despite very aggressive treatment modalities. We tested the efficacy of a potent differentiation and proliferation factor for the professional antigen-presenting dendritic cells (DCs), i.e., Flt3L, for its potential role as a novel therapy for gliomas. We investigated the ability of recombinant adenoviral vectors encoding human soluble Flt3L (hsFlt3L) to improve the survival of Lewis rats bearing intracranial syngeneic CNS-1 gliomas. We show that RAdhsFlt3L can improve survival in a dose-dependent manner. Seventy percent of rats survive when treated with 8 x 10(7) pfu RAdhsFlt3L (P < 0.0005). In addition we demonstrate in both naive Lewis rats and C57BL/6 mice the presence of increased numbers of cells bearing DC markers (OX62 and MHCII, in rats, or CD11C, 33D1, MHCII, and F4/80, but not DEC205, in mice) in sites of brain delivery of RAdhsFlt3L. These results show that expression of hsFlt3L in the brain leads to the presence of cells displaying DC markers. We demonstrate that treatment with hsFlt3L leads to inhibition of tumor growth and significantly increased life span of animals implanted with syngeneic CNS-1 glioma cells. Animals that had survived for long periods, i.e., 6 months, had eliminated the implanted tumors after neuropathological analysis; on the other hand, some of the 3-month survivors still appeared to harbor brain tumors. Our results have profound implications for immune-mediated brain tumor therapy and also suggest the ability to recruit DC-like cells within the brain parenchyma in response to the local expression of Flt3L from adenoviral vectors.
Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM.
The main challenge of gene therapy is to provide long-term, efficient transgene expression. Long-term transgene expression from first generation adenoviral vectors (Advs) delivered to the central nervous system (CNS) is elicited in animals not previously exposed to adenovirus (Ad). However, upon systemic immunization against Ad, transgene expression from a first generation Adv is abolished. High-capacity Advs (HC-Advs) provide sustained very long-term transgene expression in the brain, even in animals pre-immunized against Ad. In this study, we tested the hypothesis that a HC-Adv in the brain would allow for long-term transgene expression, for up to 1 year, in the brain of mice immunized against Ad prior to delivery of the vector to the striatum. In naïve animals, the expression of beta-galactosidase from Adv or HC-Adv was sustained for 1 year. In animals immunized prior to vector delivery, expression from a first generation Adv was abolished. These results point to a very long-term HC-Adv-mediated transgene expression in the brain, even in animals that had been immunized systemically against Ad before the delivery of HC-Adv into the brain. This study therefore indicates the utility of HC-Adv as a powerful gene therapy vector for chronic neurological disorders, even in patients who had been pre-exposed to Ad prior to gene therapy.
Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor in humans. Systemic immunity against gene therapy vectors has been shown to hamper therapeutic efficacy; however, helper-dependent high-capacity adenovirus (HC-Ad) vectors elicit sustained transgene expression, even in the presence of systemic anti-adenoviral immunity. We engineered HC-Ads encoding the conditional cytotoxic herpes simplex type 1 thymidine kinase (TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Flt3L). Flt3L expression is under the control of the regulatable Tet-ON system. In anticipation of a phase I clinical trial for GBM, we assessed the therapeutic efficacy, biodistribution, and clinical and neurotoxicity with escalating doses of HC-Ad-TetOn-Flt3L + HC-Ad-TK in rats. Intratumoral administration of these therapeutic HC-Ads in rats bearing large intracranial GBMs led to long-term survival in ~70% of the animals and development of antiglioma immunological memory without signs of neuropathology or systemic toxicity. Systemic anti-adenoviral immunity did not affect therapeutic efficacy. These data support the idea that it would be useful to develop HC-Ad vectors further as a therapeutic gene-delivery platform to implement GBM phase I clinical trials.
The disseminated characteristics of human glioblastoma multiforme (GBM) make it a particularly difficult tumor to treat with long-term efficacy. Most preclinical models of GBM involve treatment of a single tumor mass. For therapeutic outcomes to translate from the preclinical to the clinical setting, induction of an antitumor response capable of eliminating multifocal disease is essential. We tested the hypothesis that expression of Flt3L (human soluble FMS-like tyrosine kinase 3 ligand) and TK (herpes simplex virus type 1-thymidine kinase) within brain gliomas would mediate regression of the primary, treated tumor mass and a secondary, untreated tumor growing at a distant site from the primary tumor and the site of therapeutic vector injection. In both the single-GBM and multifocal-GBM models used, all saline-treated control animals succumbed to tumors by day 22. Around 70% of the animals bearing a single GBM mass treated with an adenovirus expressing Flt3L (AdFlt3L) and an adenovirus expressing TK (AdTK + GCV) survived long term. Approximately 50% of animals bearing a large primary GBM that were implanted with a second GBM in the contralateral hemisphere at the same time the primary tumors were being treated with AdFlt3L and AdTK also survived long term. A second multifocal GBM model, in which bilateral GBMs were implanted simultaneously and only the right tumor mass was treated with AdFlt3L and AdTK, also demonstrated long-term survival. While no significant difference in survival was found between unifocal and multifocal GBM-bearing animals treated with AdFlt3L and AdTK, both treatments were statistically different from the saline-treated control group (p < 0.05). Our results demonstrate that combination therapy with AdFlt3L and AdTK can eradicate multifocal brain tumor disease in a syngeneic, intracranial GBM model.
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