Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.4.1 (cytosine deaminase)
747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are emerging as an effective and powerful therapeutic approach for cancer. Replication-competent HSV-1 vectors with mutations in genes that affect viral replication, neuropathogenicity, and immune evasiveness have been developed and tested for their safety and efficacy in a variety of mouse models. Evidence to-date following administration into the brain attests to their safety, an important observation in light of the neuropathogenicity of the virus. Phase I clinical traits of three vectors, G207, 1716, and NV1020, are either ongoing or completed, with no adverse events attributed to the virus. These and other HSV-1 vectors are effective against a myriad of solid tumors in mice, including glioma, melanoma, breast, prostate, colon, ovarian, and pancreatic cancer. Enhancement of activity was observed when HSV-1 vectors were used in combination with traditional therapies such as radiotherapy and chemotherapy, providing an attractive strategy to pursue in the clinic. Oncolytic HSV-1 vectors expressing "suicide" genes (thymidine kinase, cytosine deaminase, rat cytochrome P450) or immunostimulatory genes (IL-12, GM-CSF, etc.) have been constructed to maximize tumor destruction through multimodal therapeutic mechanisms. Further advances in virus delivery and tumor specificity should improve the likelihood for successful translation to the clinic.
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PMID:Oncolytic herpes simplex virus vectors for cancer virotherapy. 1252 36

The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in preclinical studies and is currently being assessed in phase I clinical trials. It is well established that there is an immune component to the bystander effect observed with other systems such as thymidine kinase and cytosine deaminase; however, such an effect has not previously been described using NR. We have preliminary data suggesting an immune bystander effect with NR to further examine these effects and their potential enhancement by cytokines, an adenoviral vector containing CMV-NR, an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF) was constructed. The NR-GM-CSF virus was validated in 2 experimental models and demonstrated increased therapeutic efficacy in the MC26 murine colorectal tumour model. These data illustrate that the combination of suicide gene therapy using NR and CB1954 with immune stimulation via GM-CSF gives an improved response compared to either modality alone and suggests that the immune component of this response may be beneficial in combating unresectable, metastatic disease and preventing tumour recurrence.
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PMID:Immune enhancement of nitroreductase-induced cytotoxicity: studies using a bicistronic adenovirus vector. 1253 26

The first large randomized phase III trial in gene therapy demonstrated no improvement in the survival of patients injected with packaging cells that produced conventional replication-defective retroviral vectors carrying the herpes simplex virus thymidine kinase gene, a disappointing result that was attributed to extremely poor levels of transduction efficiency. To circumvent this problem, we have developed a modified replication-competent retrovirus (RCR) that is capable of transducing human glioma cell lines A-172, U-87, T-98G, U-373, and U-138 and rat glioma cell lines C6 and 9L, over multiple infection cycles in vitro, resulting in a tremendous enhancement in transduction efficiency over conventional replication-defective retroviral vectors at the same dose. Whereas the transduction efficiency of conventional retroviral vectors injected into preestablished subcutaneous U-87 tumors at a dose of 1.0 x 10(5) transducing units (TU) was only 0.2% at 6 weeks postinjection, the same dose of RCR vector resulted in up to 97.2% transduction. When RCR vectors at a dose of 1.0 x 10(4) TU were injected into preestablished intracranial U-87 tumors, transduction efficiency at 2 and 3 weeks was 74 and 98.1%, respectively. Notably, however, intracranial injection of RCR vectors did not result in detectable infection of normal brain cells. Furthermore, using a sensitive polymerase chain reaction assay, no detectable RCR signal could be observed in any extracerebral tissues, including lung, liver, kidney, upper gastrointestinal tract (esophagus and stomach), lower gastrointestinal tract (colon and small intestine), skin, spleen, and bone marrow. Treatment of U-87 intracranial gliomas with RCR vectors carrying the yeast cytosine deaminase suicide gene followed by 5-fluorocytosine prodrug administration resulted in 100% survival over a 60-day follow-up period, compared with 0% survival of control groups receiving vector alone or prodrug alone. Our results demonstrate that RCR vectors can achieve therapeutically significant levels of transduction in malignant human gliomas, and that RCR vector spread after intratumoral injection is restricted to the tumor itself.
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PMID:Highly efficient and tumor-restricted gene transfer to malignant gliomas by replication-competent retroviral vectors. 1261 63

For the development of efficient and safe gene therapy protocols for clinical application it is desirable to determine the tissue dose of vector-mediated therapeutic gene expression noninvasively in vivo. The herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk) has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK) expression by positron emission tomography (PET). Using bicistronic or multicistronic gene-expressing cassettes with tk as the PET marker gene, the quantitative analysis of tk gene expression may indirectly indicate the distribution and the level of expression of linked and proportionally coexpressed genes. Here, we describe the construction and functional evaluation of HSV-1 amplicon vectors mediating proportional coexpression of HSV-1-tk as PET marker gene and the enhanced green fluorescent protein gene (gfp) as proof of principle and cell culture marker gene and the Escherichia coli cytosine deaminase (cd) as therapeutic gene. Several double-/triple-gene constructs expressing HSV-1-tk, gfp, and E. coli cd were engineered based on gene fusion or the use of an internal ribosome entry site (IRES). Functional analysis in cell culture (green fluorescent protein [GFP] fluorescence and sensitivity to the prodrugs ganciclovir [GCV] and 5-fluorocytosine [5-FC]) and Western blots were carried out after infection of proliferating rat 9L gliosarcoma and human Gli36 glioma cells with helper virus-free packaged HSV-1 amplicon vectors. To study the ability of PET to differentiate various levels of tk expression noninvasively in vivo, retrovirally transduced and selected populations of rat F98 and human Gli36dEGFR glioma cells with defined levels of proportionally coexpressed tk and gfp genes were grown as subcutaneous tumors in nude rats and nude mice, and tk imaging by PET was performed. To study HSV-1 amplicon vector-mediated gene coexpression in vivo, HSV-1 amplicon vectors bearing coexpression constructs were injected (4 x 10(7) to 1 x 10(8) transducing units) into subcutaneously growing Gli36dEGFR gliomas in nude animals, and tk imaging was performed 24 hr later. All vector constructs mediated GFP expression and sensitized 9L and Gli36 cells toward GCV- and 5-FC-mediated cell killing in a drug dose-dependent manner, respectively. The levels of gene expression varied depending on the location of the genes within the constructs indicating the influence of the IRES on the level of expression of the second gene. Moreover, functional proportional coexpression of the PET marker gene HSV-1-tk and the linked therapeutic E. coli cd gene was observed. In selected tumor cell populations, subtle IRES-dependent differences of tk gene expression could be noninvasively distinguished by PET with good correlation between quantitative assays for IRES-dependent attenuated GFP and TK expression in culture and in vivo. After infection of subcutaneously growing gliomas with HSV-1 amplicon vectors, various levels of TK expression were found ranging from 0.011-0.062 percentage injected dose per gram (%ID/g). These values were 4.0- to 5.7-fold lower than positive control tumor cells. TK expression could be imaged by PET in vivo even with the tk gene located at the weak position downstream from the IRES. In conclusion, these HSV-1 amplicon vectors carrying HSV-1-tk as PET marker gene and any linked therapeutic gene will serve an indirect noninvasive assessment of the distribution of therapeutic gene expression by PET. Monitoring the correlation between primary transduction and therapeutic efficiency of a given vector is highly desirable for the development of safe and efficient gene therapy and vector application protocols in clinical applications.
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PMID:Improved herpes simplex virus type 1 amplicon vectors for proportional coexpression of positron emission tomography marker and therapeutic genes. 1263 7

To test the concept that a replication-competent retrovirus carrying a suicide gene could have potential utility in the control of the natural virus infection in mammalian species, we constructed derivatives of a feline leukemia virus (FeLV) that is commonly associated with leukemia-lymphomas in this species. The FeLV, Rickard strain, subgroup A (FRA) genome contained at the 3' end of the envgene, an insert of an internal ribosomal entry site (IRES) linked to cDNA sequence of either herpes simplex virus thymidine kinase (HSV-TK) or a truncated HSV-TK (HSV-ATK) or yeast cytosine deaminase (CD). These constructs were transfected into feline fibroblast cells (H927). The viruses produced were determined to be replication-competent. The stable propagation of the full-length transgene was, however, dependent on the size of the insert, IRES-CD being the smallest in size (1031 bp) exhibiting maximal stability for at least up to six months. The protein products of the transgenes could be detected, despite the appearance of deleted proviruses at late passages. The transduced cells were susceptible to cytotoxic killing when the appropriate prodrug, ganciclovir (GCV), acyclovir (ACV) or 5-fluorocytosine (5-FC) was added to the culture medium. H927 cells, infected with another subgroup of FeLV, namely, FeLV-B or FeLV-C, could be superinfected by the FRA-suicide gene viruses and thus, subjected to killing. Interestingly, at an early stage of infection by the parental FRA, H927 cells could also be reinfected by the same subgroup FRA constructs to induce the suicide effect. Among the three constructs, the vector with the CD gene was determined to be superior to others in terms of stability, therapeutic index and bystander effect in the cell culture test system. While the in vivo correlates of the therapeutic effect in the feline model remain to be determined, our results do encourage investigation of the same concept in the control of HTLV and, perhaps even, HIV infection in humans.
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PMID:A potential therapeutic strategy to combat leukemia virus infection. 1267 28

Adjuvant hyperthermia can improve treatment outcome for locally recurrent breast cancer (LRBC). Previously, we demonstrated that infection of human breast cancer cells with a recombinant adenovirus expressing beta-galactosidase from the human hsp70b gene promoter (Ad.70b.betagal) results in 50- to 800-fold increases in reporter gene expression following heat treatment (30 minutes at 43 degrees C). Here, we describe a heat-directed suicide gene therapy strategy based on an adenoviral vector (Ad.70b.CDTK) in which expression of the dual prodrug-activating E. coli cytosine deaminase/herpes simplex virus thymidine kinase (CDTK) fusion gene is under the control of the hsp70b promoter. Treatment of T47D and MCF-7 breast cancer cells with mild hyperthermia (43 degrees C/30 minutes) and prodrugs (100 microg/ml 5-fluorocytosine and 10 microg/ml ganciclovir) following infection with Ad.70b.CDTK (10-100 PFU/cell) resulted in 30- to 60-fold decreases in clonogenic survival relative to control cultures treated with heat or prodrugs alone. Clonogenic survival declined even further (up to 240-fold) following heat treatment at 41.5 degrees C for 120 minutes. A decreased clonogenic survival was accompanied by tumor cell apoptosis. These results demonstrate that this combined treatment strategy can be highly effective against heat- and radiation-resistant breast tumor cells and supports the continued development of heat-directed CDTK suicide gene therapy strategies for LRBC.
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PMID:Heat-directed suicide gene therapy for breast cancer. 1267 2

Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)-herpes simplex virus type 1 thymidine kinase (HSV1-tk) fusion gene (CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells). The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.
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PMID:Imaging expression of cytosine deaminase-herpes virus thymidine kinase fusion gene (CD/TK) expression with [124I]FIAU and PET. 1292 Aug 59

With the goal of optimizing adenovirus-mediated suicide gene therapy for prostate cancer, we have developed a method based on the human sodium iodide symporter (hNIS) that allows for noninvasive monitoring of adenoviral vectors and quantification of gene expression magnitude and volume within the prostate. A replication-competent adenovirus (Ad5-yCD/mutTK(SR39)rep-hNIS) coexpressing a therapeutic yeast cytosine deaminase (yCD)/mutant herpes simplex virus thymidine kinase (mutTK(SR39)) fusion gene and the hNIS gene was developed. Ad5-yCD/mutTK(SR39)rep-hNIS and a replication-defective hNIS adenovirus (rAd-CMV-FLhNIS) were injected into contralateral lobes of the dog prostate and hNIS activity was monitored in live animals following administration of Na(99m)TcO(4) using gamma camera scintigraphy. Despite the close proximity of the urinary bladder, (99m)TcO(4)(-) uptake was readily detected in the prostate using viral dose levels (10(10) to 10(12) viral particles) that have been safely administered to humans. Due to its rapid clearance and short physical half-life (6 h), it was possible to obtain daily measurements of (99m)TcO(4)(-) uptake in vivo, allowing for dynamic monitoring of reporter gene expression within the prostate as well as biodistribution throughout the body. High-resolution autoradiography of prostate sections coupled with 3D reconstruction of gene expression demonstrated that the magnitude and volume of gene expression could be quantified with submillimeter resolution. Implementation of the GENIS (gene expression of Na/I symporter) technology in the clinic will facilitate optimization of future human gene therapy trials.
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PMID:GENIS: gene expression of sodium iodide symporter for noninvasive imaging of gene therapy vectors and quantification of gene expression in vivo. 1294 25

The primary study objective was to determine the safety of intraprostatic administration of a replication-competent, oncolytic adenovirus containing a cytosine deaminase (CD)/herpes simplex virus thymidine kinase (HSV-1 TK) fusion gene concomitant with increasing durations of 5-fluorocytosine and valganciclovir prodrug therapy and conventional-dose three-dimensional conformal radiation therapy (3D-CRT) in patients with newly diagnosed, intermediate- to high-risk prostate cancer. Secondary objectives were to determine the persistence of therapeutic transgene expression in the prostate and to examine early posttreatment response. Fifteen patients in five cohorts received a single intraprostatic injection of 10(12) viral particles of the replication-competent Ad5-CD/TKrep adenovirus on day 1. Two days later, patients were administered 5-fluorocytosine and valganciclovir prodrug therapy for 1 (cohorts 1-3), 2 (cohort 4), or 3 (cohort 5) weeks along with 70-74 Gy 3D-CRT. Sextant needle biopsy of the prostate was obtained at 2 (cohort 1), 3 (cohort 2), and 4 (cohort 3) weeks for determination of the persistence of transgene expression. There were no dose-limiting toxicities and no significant treatment-related adverse events. Ninety-four percent of the adverse events observed were mild to moderate and self-limiting. Acute urinary and gastrointestinal toxicities were similar to those expected for conventional-dose 3D-CRT. Therapeutic transgene expression was found to persist in the prostate for up to 3 weeks after the adenovirus injection. As expected for patients receiving definitive radiation therapy, all patients experienced significant declines in prostate-specific antigen (PSA). The mean PSA half-life in patients administered more than 1 week of prodrug therapy was significantly shorter than that of patients receiving prodrugs for only 1 week (0.6 versus 2.0 months; P < 0.02) and markedly shorter than that reported previously for patients treated with conventional-dose 3D-CRT alone (2.4 months). With a median follow-up of only 9 months, 5 of 10 (50%) patients not treated with androgen-deprivation therapy achieved a serum PSA < or = 0.5 ng/ml. The results demonstrate that replication-competent adenovirus-mediated double-suicide gene therapy can be combined safely with conventional-dose 3D-CRT in patients with intermediate- to high-risk prostate cancer. The shorter than expected PSA half-life in patients receiving more than 1 week of prodrug therapy may suggest a possible interaction between the oncolytic adenovirus and/or double-suicide gene therapies and radiation therapy.
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PMID:Phase I study of replication-competent adenovirus-mediated double-suicide gene therapy in combination with conventional-dose three-dimensional conformal radiation therapy for the treatment of newly diagnosed, intermediate- to high-risk prostate cancer. 1461 51

The efficacy of single and combination suicide gene therapy was evaluated using a Herpes simplex virus thymidine kinase/ganciclovir system and Escherichia coli cytosine deaminase/5-fluorocytosine system on the rat prostate tumor cell line R3327 AT-1. The wild-type R3327 AT-1 cell line was transfected with a bifunctional fusion gene CDglyTK, which had the advantage that the resulting R3327 AT-1/CDglyTK cell line has the same amount of cytosine deaminase and thymidine kinase molecules. The percentage of viable R3327 AT-1/CDglyTK cells after 96 h incubation with 0.1 micro g/ml ganciclovir or 10 micro g/ml 5-fluorocytosine were 85% and 52% of controls, respectively. The cell viability when both suicide genes systems were activated was 43%. For in vivo analysis, Copenhagen rats were injected subcutaneously with R3327 AT-1 or R3327 AT-1/CDglyTK cells and treated with 30 mg/kg ganciclovir, 500 mg/kg 5-fluorocytosine, or both prodrugs together. A survival of 83% with the thymidine kinase/ganciclovir and 57% with the CD/5-FC could be observed. Only co-administration of thymidine kinase- and cytosine deaminase-specific prodrugs resulted in a 100% recurrence-free survival of the Copenhagen rats with a Dunning R3327 AT-1/CDglyTK prostate tumor and showed an additive cytotoxic effect. Calculation of the degree of activation and the potential of activation can be used to predict the success of a suicide gene therapy. In our case, the cytosine deaminase/5-fluorocytosine system had a low degree of activation (value 40), which is also found in the low response to 5- fluorocytosine in vivo (57% tumor free).
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PMID:Cytosine deaminase versus thymidine kinase: a comparison of the antitumor activity. 1464 29


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