Generation of a caged lentiviral vector through an unnatural amino acid for photo-switchable transduction.

Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging-uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.

An oncolytic adenovirus regulated by a radiation-inducible promoter selectively mediates hSulf-1 gene expression and mutually reinforces antitumor activity of I131-metuximab in hepatocellular carcinoma.

Gene therapy and antibody approaches are crucial auxiliary strategies for hepatocellular carcinoma (HCC) treatment. Previously, we established a survivin promoter-regulated oncolytic adenovirus that has inhibitory effect on HCC growth. The human sulfatase-1 (hSulf-1) gene can suppress the growth factor signaling pathways, then inhibit the proliferation of cancer cells and enhance cellular sensitivity to radiotherapy and chemotherapy. I(131)-metuximab (I(131)-mab) is a monoclonal anti-HCC antibody that conjugated to I(131) and specifically recognizes the HAb18G/CD147 antigen on HCC cells. To integrate the oncolytic adenovirus-based gene therapy and the I(131)-mab-based radioimmunotherapy, this study combined the CArG element of early growth response-l (Egr-l) gene with the survivin promoter to construct a radiation-inducible enhanced promoter, which was used to recombine a radiation-inducible oncolytic adenovirus as hSulf-1 gene vector. When I(131)-mab was incorporated into the treatment regimen, not only could the antibody produce radioimmunotherapeutic effect, but the I(131) radiation was able to further boost adenoviral proliferation. We demonstrated that the CArG-enhanced survivin promoter markedly improved the proliferative activity of the oncolytic adenovirus in HCC cells, thereby augmenting hSulf-1 expression and inducing cancer cell apoptosis. This novel strategy that involved multiple, synergistic mechanisms, including oncolytic therapy, gene therapy and radioimmunotherapy, was demonstrated to exert an excellent anti-cancer outcome, which will be a promising approach in HCC treatment.

Regulation of gene expression in retrovirus vectors by X-ray and proton beam radiation with artificially constructed promoters.

BACKGROUND: We previously obtained an X-ray responsive promoter from 11 promoters that we constructed. In the present study, we aimed to determine the efficiency of our promoter construction method. In addition, the reactivity of the promoter to X-rays in vivo is also investigated. METHODS: Promoters constructed by linking the TATA box to randomly combined binding sequences of transcription factors activated by radiation were cloned to prepare a promoter library. Combinations of promoters and various genes were stably-transfected into HeLa cells to establish recombinant cell lines, which were then exposed to X-rays or a proton beam to observe gene expression enhancement with or without anti-oxidants. Tumors of luciferase-expressing recombinant cells on mice were exposed to X-rays and promoter activation was evaluated by detecting bioluminescence. As a model for in vitro suicide gene therapy, fcy::fur-expressing recombinant cells were exposed to X-rays before incubation with 5-fluorocytosin. Cell viability was determined with WST-8. RESULTS: Twenty-five of the 62 promoters in the library enhanced luciferase activity over five-fold, 6 h after receiving 10 Gy of X-ray irradiation, suggesting the effectiveness of our method. Luciferase activity in recombinant cells was enhanced by X-rays and, to a lesser extent, by a proton beam. Anti-oxidants attenuated the enhancement, suggesting the involvement of oxidative stress. Promoters were less reactive to X-rays in tumors on mice. In our suicide gene therapy model, survival of post-irradiated cells decreased dose-dependently with 5-fluorocytosin. CONCLUSIONS: Our method was efficient in generating radiation responsive promoters. Furthermore, we have successfully shown a potential therapeutic use for one of these promoters.

Radiation-induced upregulation of gene expression from adenoviral vectors mediated by DNA damage repair and regulation.

PURPOSE: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. METHODS AND MATERIALS: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. RESULTS: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. CONCLUSIONS: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

Enzyme treatment-free and ligation-independent cloning using caged primers in polymerase chain reactions.

A new simple scheme for constructing recombinant vectors that does not require any restriction enzyme, ligase, or any other special enzyme treatment has been developed. By using caged primers in PCR, unnatural sticky-ends of any sequence, which are sufficiently long for ligation-independent cloning (LIC), are directly prepared on the product after a brief UVA irradiation. Target genes and vectors amplified by this light-assisted cohesive-ending (LACE) PCR join together in the desired arrangement in a simple mixture of them, tightly enough to be repaired and ligated in competent cells.

[Effects of UV-induced DNA damage on vector ligation and transformation into bacterial cells].

OBJECTIVE: To study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells. METHODS: The expression vector was digested with the restriction enzyme SfiI, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed. RESULTS: The transformation efficiency of the DNA with a 5-min exposure to 302 nm UV was 60 colonies per nanogram of the DNA, as compared with 20400 for the DNA exposed to 365 nm UV. The time course experiment showed that prolonged DNA exposure to 365 nm UV light was associated with lowered transformation efficiency. DNA exposure for 30 min caused a reduction of the transformation efficiency to lower than 50% compared to that of DNA without UV exposure. But with a 15 min exposure, the DNA maintained a transformation efficiency more than 70%, which was sufficient for most molecular biology experiments. CONCLUSION: In construction of the expression vector, it is advisable to prevent the target DNA from UV exposure. When UV exposure is essential, we suggest that 365 nm UV be used and the exposure time controlled within 15 min.

Requirements for effective antitumor responses of TCR transduced T cells.

Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.

Radiation enhances adenoviral gene therapy in pancreatic cancer via activation of cytomegalovirus promoter and increased adenovirus uptake.

PURPOSE: Adenovirus-mediated gene therapy combined with radiation is expected to be a new approach to treat pancreatic cancer. However, there are no reports of definitive effects of radiation on adenovirus-mediated gene therapies. In the present study, we investigated the effect of radiation on the transduction efficiency of an adenovirus-based gene therapy. EXPERIMENTAL DESIGN: We used adenovirus expressing NK4 (Ad-NK4), an antagonist for hepatocyte growth factor, as a representative gene therapy. Pancreatic cancer cells preinfected with Ad-NK4 were irradiated, and NK4 levels in culture media of these cells were measured. We investigated cytomegalovirus (CMV) promoter activity and uptake of adenovirus in these cells. To examine the effect of radiation in vivo, Ad-NK4 was given to irradiated subcutaneous tumors in nude mice, and NK4 levels in tumors were measured. RESULTS: NK4 levels in culture media of irradiated cells were 4.5-fold (P < 0.01) higher than those of nonirradiated cells. Radiation enhanced activation of the CMV promoter and adenovirus uptake (P < 0.01), leading to increased levels of NK4. We found that activation of p38 mitogen-activated protein kinase and up-regulation of dynamin 2 may be involved in the radiation-induced activation of the CMV promoter and adenovirus uptake, respectively. NK4 levels in irradiated tumors were 5.8-fold (P = 0.017) higher than those in nonirradiated tumors. CONCLUSIONS: The present findings suggest that radiation significantly improves the efficiency of adenovirus-mediated gene transfer in pancreatic cancer and probably contributes to decreasing the dose of adenovirus required for gene transfer and controlling side effects of adenovirus infection in nonirradiated normal tissue.

Low-dose radiation response of the p21WAF1/CIP1 gene promoter transduced by adeno-associated virus vector.

In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (< 1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. This is mainly because the transiently transfected promoter of p53-target genes is not much sensitive to radiation. We examined the response of the p21 gene promoter to low-dose radiation when transduced into a human breast cancer cell line MCF-7 by use of recombinant adeno-associated virus (rAAV) vectors. It was shown that the p21 gene promoter transduced by rAAV vectors was more highly radiation-responsive than that transiently transfected by electroporation. A significant induction of the p21 gene promoter by radiation of low doses down to 0.2 Gy was observed. When cells were transduced with the p21 gene promoter-driven HSVtk gene by rAAV vector, they were significantly sensitized to repetitive treatment with low dose radiation (1 Gy) in the presence of the prodrug ganciclovir. It was therefore considered that the p21 gene promoter in combination with a rAAV vector is potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy.

Hypoxia- and radiation-activated Cre/loxP 'molecular switch' vectors for gene therapy of cancer.

Although a significant negative prognostic factor, tumor hypoxia can be exploited for gene therapy. To maximize targeting within the tumor mass, we have developed synthetic gene promoters containing hypoxia-responsive elements (HREs) from the erythropoietin (Epo) gene as well as radiation-responsive CArG elements from the early growth response (Egr) 1 gene. Furthermore, to achieve high and sustained expression of the suicide gene herpes simplex virus thymidine kinase (HSVtk), our gene therapy vectors contain an expression amplification system, or 'molecular switch', based on Cre/loxP recombination. In human glioma and breast adenocarcinoma cells exposed to hypoxia and/or radiation, the HRE/CArG promoter rapidly activated Cre recombinase expression leading to selective and sustained HSVtk synthesis. Killing of transfected tumor cells was measured after incubation with the prodrug ganciclovir (GCV; converted by HSVtk into a cytotoxin). In vitro, higher and more selective GCV-mediated toxicity was achieved with the switch vectors, when compared with the same inducible promoters driving HSVtk expression directly. In tumor xenografts implanted in nude mice, the HRE/CArG-switch induced significant growth delay and tumor eradication. In conclusion, hypoxia- and radiation-activated 'molecular switch' vectors represent a promising strategy for both targeted and effective gene therapy of solid tumors.

Ionizing radiation: a genetic switch for cancer therapy.

Gene therapy of cancer represents a promising but challenging area of therapeutic research. The discovery of radiation-inducible genes led to the concept and development of radiation-targeted gene therapy. In this approach, promoters of radiation-inducible genes are used to drive transcription of transgenes in the response to radiation. Constructs in which the radiation-inducible promoter elements activate a transgene encoding a cytotoxic protein are delivered to tumors by adenoviral vectors. The tumoricidal effects are then localized temporally and spatially by X-rays. We review the conceptual development of TNFerade, an adenoviral vector containing radiation-inducible elements of the early growth response-1 promoter upstream of a cDNA encoding human tumor necrosis factor-alpha. We also summarize the preclinical work and clinical trials utilizing this vector as a treatment for diverse solid tumors.

Interaction of measles virus vectors with Auger electron emitting radioisotopes.

A recombinant measles virus (MV) expressing the sodium iodide symporter (NIS) is being considered for therapy of advanced multiple myeloma. Auger electrons selectively damage cells in which the isotope decays. We hypothesized that the Auger electron emitting isotope 125I can be used to control viral proliferation. MV was engineered to express both carcinoembryonic antigen and NIS (MV-NICE). Cells were infected with MV-NICE and exposed to 125I with appropriate controls. MV-NICE replication in vitro is inhibited by the selective uptake of 125I by cells expressing NIS. Auger electron damage is partly mediated by free radicals and abrogated by glutathione. In myeloma xenografts, control of MV-NICE with 125I was not possible under the conditions of the experiment. MV-NICE does not replicate faster in the presence of radiation. Auger electron emitting isotopes effectively stop propagation of MV vectors expressing NIS in vitro. Additional work is necessary to translate these observations in vivo.

Radiation therapy: activation for gene transcription and the development of genetic radiotherapy-therapeutic strategies in oncology.

Radiotherapy is a widely used local and regional modality for the treatment of cancer. Despite improved methods of radiation delivery, local recurrence accounts for treatment failure in most patients. Radiosensitizers have been studied as one approach for improving the effectiveness of radiotherapy. Few agents, however, have been shown to selectively increase the anti-tumor effects of radiation. Advances in our understanding of how cells respond molecularly to ionizing radiation have provided opportunities for the development of new approaches that selectively enhance radiotherapy of tumors. Cells respond to ionizing radiation with the activation of specific early and later response genes. These findings led us to the concept that promoters from these genes could be used to drive therapeutic transgenes introduced into irradiated tumor cells. In this strategy, designated genetic radiotherapy, radiation is combined with gene therapy, another local/regional modality, to spatially and temporally control transgene expression in the irradiated field. Tumor necrosis factor-alpha (TNF-alpha) was selected as the transgenic protein for its potent anti-tumor activity and synergistic interactions with ionizing radiation. The radio-inducible elements from the early growth response-1 (EGR-1) gene promoter have been inserted upstream to a cDNA encoding TNF-alpha and integrated into a replication-defective adenovirus (Ad.EGR-TNF). Preclinical studies have shown that tumors infected with Ad.EGR-TNF respond to radiation with induction of TNF-alpha expression and substantial increases in anti-tumor activity. Importantly, local production of TNF-alpha is not associated with the limiting toxicities encountered when this cytokine was administered systemically. Based on these findings, radiation in combination with intratumoral administration of a second generation Ad.EGR-TNF (TNFerade) has completed Phase I evaluation and has entered Phase II clinical trials.

Photochemical control of the infectivity of adenoviral vectors using a novel photocleavable biotinylation reagent.

We have explored a novel strategy for controlling the infectivity of adenoviral vectors. This strategy involves a method whereby the infectivity of adenoviral vectors is neutralized by treatment of viral particles with a water-soluble, photocleavable biotinylation reagent. These modified viral vectors possess little to no infectivity for target cells. Exposure of these modified viral vectors to 365 nm light induces a reversal of the neutralizing, chemical modification, resulting in restoration of infectivity to the viral vectors. The light-directed transduction of target cells by photoactivatable adenoviral vectors was demonstrated successfully both in vitro and in vivo. This photochemical infectivity trigger possesses great potential, both as a research tool and as a novel tactic for the delivery of gene-transfer agents, since the infectivity of adenoviral vectors can be controlled externally in a versatile manner.

Ionizing radiation does not alter the antitumor activity of herpes simplex virus vector G207 in subcutaneous tumor models of human and murine prostate cancer.

Viral gene therapy against malignant tumors holds great promise for tumors that are susceptible to the oncolytic activity of viruses. One advantage of oncolytic viral therapy is that it can potentially be combined with other therapies, such as radiotherapy, to obtain an enhanced tumor response. In the case of prostate cancer, herpes simplex virus-mediated therapies have been shown to be highly effective in animal models; however, studies of the efficacy of combined viral and radiation therapy have not yet been reported. In this study, we have combined G207, a multimutated HSV type 1 vector, with external beam radiation therapy of prostate tumors grown subcutaneously in mice. We examined both the human LNCaP tumor in athymic mice and the mouse transgenic TRAMP tumor in either athymic mice or its syngeneic host, C57BL/6 mice. Virus was delivered either intravenously, in the case of LNCaP, or intratumorally, in the case of TRAMP. We found that individually, either G207 or radiation was effective in delaying tumor growth in these models. However, delivering the treatments simultaneously did not produce an enhanced effect.

Enhanced S phase delay and inhibition of replication of an undamaged shuttle vector in UVC-irradiated xeroderma pigmentosum variant.

Xeroderma pigmentosum variant (XP-V) cells are defective in bypass replication of UVC-induced thymine dimers in DNA because they lack a novel DNA polymerase (polymerase eta). In this study the effects of UVC on S phase cells were compared in fibroblasts derived from normal donors (IDH4) and XP-V patients (CTag) and immortalized by expression of the SV40 large T antigen. These transformed fibroblasts did not activate the G(1) checkpoint or inhibit replicon initiation when damaged by UVC or gamma-rays. The transformed XP-V cells (CTag) retained the increased sensitivity to UVC-induced inhibition of DNA strand growth previously observed with their diploid counterpart. Cell cycle progression analyses showed that CTag cells displayed a stronger S phase delay than transformed fibroblasts from normal individuals (IDH4) after treatment with only 2 J/m(2) UVC. Low doses of UVC also caused a lag in CTag cell proliferation. The extent of replication of an episomal DNA (pSV011), not previously exposed to radiation, was measured after the host cells were irradiated with 1-3 J/m(2) UVC. Replication of pSV011 was barely affected in irradiated IDH4 cells. Plasmid replication was inhibited by 50% in irradiated CTag cells and this inhibition could not be accounted for by increased killing of host cells by UVC. These results suggest that even in transformed cells UVC induces DNA damage responses that are reflected in transient cell cycle arrest, delay in proliferation and inhibition of episomal DNA replication. These responses are enhanced in CTag cells, presumably because of their bypass replication defect. The accumulation of replication complexes blocked at thymine dimers and extended single-stranded regions in chromosomal DNA might sequester replication factors that are needed for plasmid and chromosomal replication. Alternatively, aberrant replication structures might activate a signal transduction pathway that down-regulates DNA synthesis.

Diminished DNA repair and elevated mutagenesis in mammalian cells exposed to hypoxia and low pH.

The tumor microenvironment is characterized by regions of fluctuating and chronic hypoxia, low pH, and nutrient deprivation. It has been proposed that this unique tissue environment itself may constitute a major cause of the genetic instability seen in cancer. To investigate possible mechanisms by which the tumor microenvironment might contribute to genetic instability, we asked whether the conditions found in solid tumors could influence cellular repair of DNA damage. Using an assay for repair based on host cell reactivation of UV-damaged plasmid DNA, cells exposed to hypoxia and low pH were found to have a diminished capacity for DNA repair compared with control cells grown under standard culture conditions. In addition, cells cultured under hypoxia at pH 6.5 immediately after UV irradiation had elevated levels of induced mutagenesis compared with those maintained in standard growth conditions. Taken together, the results suggest that cellular repair functions may be impaired under the conditions of the tumor microenvironment, causing hypermutability to DNA damage. This alteration in repair capacity may constitute an important mechanism underlying the genetic instability of cancer cells in vivo.

Photoactivatable retroviral vectors: a strategy for targeted gene delivery.

We have explored a novel strategy for the targeting of retroviral vectors to particular sites or cell types. This strategy involves a method whereby the infectivity of a retroviral vector is neutralized by treatment of viral particles with a photocleavable, biotinylation reagent. These modified viral vectors possess little to no infectivity for target cells. Exposure of these modified viral vectors to long-wavelength UV light induces a reversal of the neutralizing, chemical modification resulting in restoration of infectivity to the viral vector. This infectivity 'trigger' possesses great potential, both as a research tool and as a novel tactic for the targeting of gene-transfer agents, since it would become possible to direct both the time and location of a viral infection in a versatile manner.

Multiple mutations in a shuttle vector modified by ultraviolet irradiation, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, and aflatoxin B(1) have different properties than single mutations and may be generated during translesion synthesis.

Shuttle vector-based systems are extensively employed to study the mutational properties of various mutagens in mammalian cells. Such vectors are designed for the detection of point mutations, that is small deletions and single base and tandem substitutions. However, mutant target genes carrying two or more point mutations, referred to as multiple mutations, can also be found in various proportions depending on the mutagen and the cells used. To evaluate the frequency and characteristics of multiple mutations, we used a system where the plasmid, pYZ289, was treated by ultraviolet irradiation, aflatoxin B(1) or (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene before transfection into mouse fibroblast cells. The kinds of mutations and the mutational spectra were different for single and multiple mutations. In addition, in at least 75% of the cases, mutations of multiples appeared to arise in the same strand. Furthermore, mutational spectra for multiple mutations were different for 5' and 3' members of multiple sets. These observations suggest that multiple mutations arise via a different mechanism than single mutations. Moreover, these findings suggest that multiples arise during translesion DNA synthesis and involve an error-prone polymerase able to introduce a base opposite misinstructive or noninstructional DNA lesions and subject to subsequent misincorporation errors.