Efficacy of gene therapy-delivered cytosine deaminase is determined by enzymatic activity but not expression.

The potential utility of tumour-selective 5-fluorouracil treatment using attenuated Salmonella serovar typhimurium recombinant for cytosine deaminase (TAPET-CD) has been documented in experimental settings. The present data demonstrate that in vivo (19)F-magnetic resonance spectroscopy measurements allow the outcome prediction of this prokaryotic-based therapy, demonstrating the necessity of non-invasive real-time imaging techniques for treatment monitoring.

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo.

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration.

Non-invasive 19F MR spectroscopy of 5-fluorocytosine to 5-fluorouracil conversion by recombinant Salmonella in tumours.

The aim of this study was to evaluate the applicability of fluorine-19 magnetic resonance spectroscopy ((19)F MRS) for monitoring in vivo the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) after using an attenuated Salmonella Typhimurium strain recombinant to provide cytosine deaminase (TAPET-CD). The (19)F MRS measurements were done on mice bearing the human colon tumour xenograft (HCT116). The intratumoural conversion is greater when TAPET-CD/5-FC is delivered intratumourally (i.tu.) than when TAPET-CD is delivered intravenously (i.v.) and 5-FC intraperitoneally (i.p.). Repeat measurements of the same tumour also yielded important information on the tumour colonization by TAPET-CD through the correlated 5-FC to 5-FU conversion efficacy. The in vivo MRS spectra were confirmed by in vitro (19)F MRS of perchloric acid extracts of the tumour tissue. No 5-FU metabolites were detectable in vivo in the tumours. However, the in vitro measurements revealed, besides 5-FC and 5-FU, the presence of small amounts of catabolites. Finally, spectra obtained in vitro from liver extracts of tumour-bearing mice treated i.tu. with TAPET-CD/5-FC showed no 5-FU and only little amounts of catabolites. Our data illustrate most importantly the potential of (19)F MRS to monitor biologically-based treatments involving cytosine deaminase.

Tumor-specific gene delivery using genetically engineered bacteria.

The loco-regional control of cancer remains a major contributor to the treatment outcome for many cancer patients prescribed conventional radiotherapy or chemotherapy. Failure of treatment coupled with the realisation that cancer is essentially a genetic disease has led to development of many clinical protocols based on gene therapy. In this review, we will describe an alternative gene delivery system based on the use of non-pathogenic bacteria. Tumor regressions have been reported long ago in patients with bacterially infected tumors, suggesting that bacteria could target tumors and have local anti-tumor effects. The basis of this phenomenon is attributable to the unique properties of the tumor micro-environment. The presence of hypoxic and/or necrotic areas provides a haven for a number of anaerobic bacteria and over the past 60 years, several strains of anaerobic bacteria have been shown to localise within and cause cell lysis of experimental animal tumors. One of the most important strains in that context is Clostridium. Other bacteria have also been implicated in experimental anti-cancer settings. Of these, attenuated Salmonella strains capable of both selective amplification within tumors and expression of effector genes encoding therapeutic proteins are probably the most promising. We will discuss the potential advantages and the pitfalls of this alternative delivery approach. We will emphasize the importance of hypoxia in solid tumors and discuss the potential of radiation-inducible promoters and combined treatment modalities, involving vascular targeting and radiotherapy. We believe that this approach will act in a complementary way to current radiotherapy and chemotherapy treatments of solid tumors.

Clostridium spores for tumor-specific drug delivery.

Insufficient blood supply of rapidly growing tumors leads to the presence of hypoxia, a well-known feature in solid tumors. Hypoxia is known to decrease the efficiency of currently used anti-cancer modalities like surgery, chemotherapy and radiotherapy. Therefore, hypoxia seems to be a major limitation in current anti-cancer therapy. The use of non-pathogenic clostridia to deliver toxic agents to the tumor cells takes advantage of this unique physiology. These strictly anaerobic, Gram-positive, spore-forming bacteria give, after systemic administration, a selective colonization of hypoxic/necrotic areas within the tumor. Moreover, they can be genetically modified to secrete therapeutic proteins like cytosine deaminase or tumor necrosis factor-alpha. The specificity of this protein delivery system can be further increased when expression is controlled by the use of a radio-inducible promoter, leading to increased spatial and temporal regulation of protein expression. This approach of bacterial vector systems to target protein expression to the tumor can be considered very safe since bacteria can be eliminated at any moment by the addition of proper antibiotics. The Clostridium-based delivery system thus presents an alternative therapeutic modality to deliver anti-tumor agents specifically to the tumor site. This high selectivity offers a major advantage in comparison with the classical gene therapy systems.

Hypoxia as a target for combined modality treatments.

There is overwhelming evidence that solid human tumours grow within a unique micro-environment. This environment is characterised by an abnormal vasculature, which leads to an insufficient supply of oxygen and nutrients to the tumour cells. These characteristics of the environment limit the effectiveness of both radiotherapy and chemotherapy. Measurement of the oxygenation status of human tumours has unequivocally demonstrated the importance of this parameter on patient prognosis. Tumour hypoxia has been shown to be an independent prognostic indicator of poor outcome in prostate, head and neck and cervical cancers. Recent laboratory and clinical data have shown that hypoxia is also associated with a more malignant phenotype, affecting genomic stability, apoptosis, angiogenesis and metastasis. Several years ago, scientists realised that the unique properties within the tumour micro-environment could provide the basis for tumour-specific therapies. Efforts that are underway to develop therapies that exploit the tumour micro-environment can be categorised into three groups. The first includes agents that exploit the environmental changes that occur within the micro-environment such as hypoxia and reduced pH. This includes bioreductive drugs that are specifically toxic to hypoxic cells, as well as hypoxia-specific gene delivery systems. The second category includes therapies designed to exploit the unique properties of the tumour vasculature and include both angiogenesis inhibitors and vascular targeting agents. The final category includes agents that exploit the molecular and cellular responses to hypoxia. For example, many genes are induced by hypoxia and promoter elements from these genes can be used for the selective expression of therapeutic proteins in hypoxic tumour cells. An overview of the various properties ascribed to tumour hypoxia and the current efforts underway to exploit hypoxia for improving cancer treatment will be discussed.

Insertion or deletion of the Cheo box modifies radiation inducibility of Clostridium promoters.

Radiation-inducible promoters are being used in many viral vector systems to obtain spatial and temporal control of gene expression. It was previously proven that radiation-induced gene expression can also be obtained in a bacterial vector system using anaerobic apathogenic clostridia. The effect of radiation inducibility was detected using mouse tumor necrosis factor alpha (mTNF-alpha) as a model protein under regulation of the radiation-inducible recA promoter. In this report, experiments are described in which this recA promoter was modified in order to increase radiation responsiveness. Incorporation of an extra Cheo box in the recA promoter region resulted in an increase in mTNF-alpha secretion from 44% for the wild-type promoter to 412% for the promoter with an extra Cheo box after a single irradiation dose of 2 Gy. Deletion of the Cheo box in the promoter region eliminated radiation inducibility. These results prove that the Cheo box in the recA promoter is indeed the radiation-responsive element. We also tested whether we could induce the constitutive endo-beta-1,4-glucanase promoter (eglA) via ionizing irradiation by introducing a Cheo box in the promoter region. While the use of the constitutive promoter did not lead to an increase in mTNF-alpha secretion after irradiation, the introduction of a Cheo box resulted in a 242% increase in mTNF-alpha secretion. Reverse transcriptase PCR of RNA samples isolated from irradiated and nonirradiated bacterial cultures demonstrated that the increase in secretion was the result of enhanced transcription of the mTNF-alpha gene.

Radio-responsive recA promoter significantly increases TNFalpha production in recombinant clostridia after 2 Gy irradiation.

One of the major problems with gene therapy today is the lack of tumour specificity. The use of anaerobic apathogenic clostridia as a gene transfer system can target anoxic areas within the tumour. These bacteria can be genetically modified to express therapeutic proteins such as TNFalpha locally in the tumour. As shown in our results, ionising irradiation can be used in clostridia to activate genes encoding cytotoxic agents under control of a radiation-inducible promoter. A 44% significant increase (P < 0.05) in TNFalpha secretion was seen 3.5 h after a single dose of 2 Gy. A second dose of 2 Gy was also capable of repeating gene activation and gave a significant increase of TNFalpha production of 42% (P < 0.05). These results provide evidence that spatial and temporal control of gene expression can be achieved using a radio-inducible promoter. Repetitive gene activation was feasible with a second dose of 2 Gy, indicating that fractionated radiotherapy could lead to repeated gene induction resulting in prolonged and enhanced protein expression. Gene targeting by ionising radiation could thus provide a new means of increasing the therapeutic ratio in cancer treatment.

Increasing specificity of anti-tumor therapy: cytotoxic protein delivery by non-pathogenic clostridia under regulation of radio-induced promoters.

BACKGROUND: Pathogenic clostridia, genetically engineered to express therapeutic genes, will specifically target hypoxic regions in tumors. This specificity can be further improved if expression of these genes is controlled by a radio-induced promoter, leading to spatial and temporal control of gene expression. MATERIALS AND METHODS: Following administration of Clostridium spores to tumor bearing rats, normal tissue and tumoral specimens were compared for colonization. Clostridium was genetically modified to express tumor necrosis factor a or cytosine deaminase. Expression of these proteins was assayed. Northern blot hybridizations were used to detect genes which are radio-induced. RESULTS: Clostridium gave a selective colonization of tumors. The recombinant clostridia expressed in vitro and in vivo TNF alpha and cytosine deaminase. Clostridial SOS-repair genes were induced at a dose of 2 Gy. CONCLUSIONS: Pathogenic Clostridium can be used for tumor specific delivery of therapeutic genes. The specificity can be improved via radio-induced promoters. Overall, this new gene delivery system can lead to an increase of the therapeutic ratio in cancer treatment.

Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum.

The presence of severe hypoxia and necrosis in solid tumors offers the potential to apply an anaerobic bacterial enzyme/prodrug approach in cancer treatment. In this context the apathogenic C. acetobutylicum was genetically engineered to express and secrete E. coli cytosine deaminase (CDase). Considerable levels of functional cytosine deaminase were detected in lysates and supernatants of recombinant C acetobutylicum cultures. After administration of the recombinant Clostridium to rhabdomyosarcoma bearing rats used as a model, cytosine deaminase could be detected at the tumor site. Moreover, following administration of the vascular targeting agent combretastatin A-4 phosphate significantly increased levels of cytosine deaminase were detected at the tumor site as a consequence of enlarged tumor necrosis and subsequently improved growth of C. acetobutylicum. The results provide evidence for the potential application of Clostrisdium-based therapeutic protein transfer to tumors in anticancer therapy.

Effect of TNP-470 (AGM-1470) on the growth of rat rhabdomyosarcoma tumors of different sizes.

Potential anticancer therapy with the fumagillin analog TNP-470 was investigated in the present project using subcutaneously growing rhabdomyosarcomas in rats. Specifically, influences of different tumor sizes at the start of treatment as well as dose/schedules were evaluated with this angiogenesis inhibitor. The results show a significant (p = < or = 0.01) reduction of the growth rate, even for relatively large-sized (> 7 cm3) tumors, when 50 mg/kg TNP-470 was used every other day for up to 3 or 5 injections. With 30 mg/kg TNP-470 injections, effects were seen only with tumors measuring < 7 cm3. The histologic examinations demonstrate an increase in necrosis, both in the center and in the peripheral part of TNP-470-treated tumors. Overall, both tumor volume and drug dose determine treatment outcome with the rat rhabdomyosarcoma. The results suggest that angiogenesis inhibitors could represent a valid component in the treatment of progressive tumor growth, also of large tumors as often encountered in clinics. The antivasculature therapy might also improve hypoxia/necrosis-related therapeutic approaches.

The use of radiation-induced bacterial promoters in anaerobic conditions: a means to control gene expression in clostridium-mediated therapy for cancer.

Nuyts, S., Van Mellaert, L., Theys, J., Landuyt, W., Lambin, P. and Anné, J. The Use of Radiation-Induced Bacterial Promoters in Anaerobic Conditions: A Means to Control Gene Expression in Clostridium-Mediated Therapy for Cancer. Radiat. Res. 155, 716-723 (2001). Apathogenic clostridia, which have been genetically engineered to express therapeutic genes, will specifically target hypoxic and necrotic regions in tumors. This specificity can be improved further if the expression of these genes is controlled by a radiation-induced promoter, leading to spatial and temporal control of gene expression. We isolated two radiation-inducible genes of the SOS repair system of Clostridium. Northern blot experiments confirmed radiation activation of the recA and recN genes at a dose of 2 Gy. The promoter region of these genes was isolated and used to regulate expression of the lacZ gene under anaerobic conditions. For the recA promoter, a significant increase of beta-galactosidase activity of 20-30% was seen after 2 Gy irradiation. The recN promoter did not show a significant induction and had a 50-100 times lower basal expression. Treatment of the recombinant clostridial cultures with the cytostatic agent mitomycin C also resulted in a significant increase of beta-galactosidase activity that was under the control of recA or recN promoter. Oxygen does not appear to be necessary in the activation of the SOS repair system by irradiation as tested with Escherichia coli since recA-deficient and recA-containing strains showed similar survival after treatment with UV and ionizing radiation in the presence or absence of oxygen.

Improvement of Clostridium tumour targeting vectors evaluated in rat rhabdomyosarcomas.

Previous studies have demonstrated the feasibility of using apathogenic clostridia as a promising strategy for hypoxia-specific tumour targeting. The present study shows that the use of the vascular targeting compound combretastatin A-4 phosphate could significantly (P<0.001) increase the number of Clostridium vegetative cells in rat rhabdomyosarcomas with sizes between 0.2 cm(2) and 3 cm(2). Furthermore, this study showed that administration of metronidazole for a 9-day period was sufficient to eliminate systemically administered Clostridium from the tumour. Moreover, previous Clostridium spore administration did not effect tumour colonisation, regardless of the immune response status of the host.

In vivo antitumor effect of vascular targeting combined with either ionizing radiation or anti-angiogenesis treatment.

PURPOSE: Interference with the tumor blood vessels through anti-angiogenesis or vascular targeting can indirectly suppress tumor growth. Vascular targeting of solid tumors, using tubulin-compromising agents, seems a promising and selective novel treatment. We aimed to evaluate the potential (hypothesis-based) benefit from combinations of vascular targeting using combretastatin A-4 phosphate (combreAp) with either ionizing radiation or anti-angiogenesis. METHODS AND MATERIALS: Rhabdomyosarcoma tumor pieces were inplanted subcutaneously (s.c.) in the lower flank region of syngeneic adult WAG/Rij rats. Tumors were grown until different sizes and stratified for the various treatment groups: small (1-3 cm3), medium (3.1-7 cm3), and large (7.1-14 cm3). CombreAp was injected i.p.; injections of TNP-470 were s.c. in the neck area. Localized single-dose (8 Gy) irradiations of tumors were done under Nembutal anesthesia, always 1 day before a single combreAp (25 mg/kg) injection. The TNP-470 treatment (3 times 30 mg/kg in 1 week) started 1 day after a double (8 days interval between both) combreAp administration. Tumor responses were evaluated by the growth delay assay, and statistical significance of tumor growth change was computed. RESULTS: Large tumors responded better to combreAp treatment given alone than did the smaller ones, confirming our previous data with this tumor model. Combining irradiation with combreAp also resulted in a tumor size-dependent growth delay. With small and medium tumor volumes, a similar response was measured after the combination treatment when compared with irradiation only. Large tumors, however, showed a strong (at least additive) increase of the growth delay with the combined therapy; the difference in tumor growth between the two treatment groups was very significant (p < 0.0001). m When TNP-470 was combined with combreAp, no significant lengthening of the growth delay, irrespective of the tumor size, was present with the applied schedule. CONCLUSION: The current data show a significant advantage in the combination of combreAp with irradiation in rhabdomyosarcomas having a large size (7-14 cm3) at treatment. Such a benefit in tumor response was not observed with the smaller tumors, seemingly because irradiation as such was very effective. No significant gain in growth delay for any tumor size was observed when TNP-470, showing efficacy on its own specifically with tumors measuring <7 cm3, was added to the combreAp treatment. This presumably reflects only little angiogenesis during the first week of rhabdomyosarcoma regrowth after the combreAp treatment.

Clostridium as a tumor-specific delivery system of therapeutic proteins.

The feasibility of gene therapy strategies in cancer treatment still has important pitfalls. Transfer of therapeutic proteins to the hypoxic/necrotic 'extracellular' microenvironment of solid tumors, based on the engineering of nonpathogenic clostridia is proposed as an alternative methodology. Using the rat rhabdomyosarcoma R1 in vivo tumor model, we demonstrated that Clostridium species colonized the tumors, whereas proliferation of these bacteria was absent in normal tissues. C. acetobutylicum was genetically engineered to express and secrete either mTNF-alpha or the E. coli cytosine deaminase. Quantitative in vitro data showed stability of the vectors, and significant levels of biologically active therapeutic proteins in lysates and supernatants of recombinant clostridia. Administration of either of these recombinant Clostridium strains to tumor-bearing rats resulted in the presence of active proteins in the tumor tissue. Based on these data and supported by its selective colonization pattern and safety, the Clostridium gene transfer system offers a potential application in anti-cancer therapies.

Vascular targeting of solid tumours: a major 'inverse' volume-response relationship following combretastatin A-4 phosphate treatment of rat rhabdomyosarcomas.

Tumour-specific vascularisation may be therapeutically approached in two different ways: by antiangiogenic treatments specifically directed to dividing and migrating endothelial cells, or by agents that target principally the inadequate and ill-structured tumour vasculature. Combretastatin A-4 phosphate (combreAp), a recently synthesised prodrug (OXiGENE, Lund, Sweden), is a vascular targeting agent of the latter kind. We evaluated the effect of a single intraperitoneal (i.p.) combreAp injection on the growth of rhabdomyosarcomas syngeneic in WAG/Rij rats. Different tumour volume groups, ranging between 0.1 and 27 cm(3), were selected to assess the relationship between the size at treatment time and the response to combreAp. A double combreAp treatment (2x25 mg/kg) was investigated within the same overall aim: the relationship between growth delay and tumour size. Our results show that the systemic administration of combreAp induces a clear-cut differential growth delay in the solid rat rhabdomyosarcomas: with very large tumours (>/= 14 cm(3)), a 17.6-fold stronger effect was measured than with very small tumours (<1 cm(3)). This is the 'inverse' of the volume-response seen with the conventional therapeutic approaches (radiotherapy, chemotherapy or surgery). These combreAp antitumour responses were observed without treatment limiting systemic toxicity in the rats. With clinical digital subtraction angiography, using microsurgical cannulation of a major tumour draining vessel, and with histopathology, we demonstrate that growth delay is related to an early (within 3-6 h) and extensive breakdown of tumour blood vessels. The experiments involving a second injection also indicate a volume-dependent effect of combreAp in reducing the regrowth rate of small or large rhabdomyosarcomas. This significant differential volume-response obtained with 'selective' vascular targeting, stronger in larger tumours than smaller ones, suggests the potential of broadening the therapeutic window.

The potential therapeutic gain of radiation-associated gene therapy with the suicide gene cytosine deaminase.

PURPOSE: To estimate the concentration of 5-fluorocytosine (5-FC), necessary for conversion to 5-fluorouracil (5-FU) in tumours transduced with the gene cytosine deaminase (CD), to achieve clinically significant radiosensitization to radiotherapy. MATERIALS AND METHODS: Starting with a tumour control probability (TCP) of 37% from radiotherapy of 66 Gy in 2 Gy fractions, estimates were made of increase in TCP expected from sensitizer enhancement ratios (SER) of 1.1, 1.2, etc. SER values for 5-FU were obtained from a literature review. Clinical toxicity of 5-FC is also reviewed. RESULTS: 5-FU has been reported to be an effective radiosensitizer if maintained for several days after each irradiation at concentrations of 0.6-0.9 microg/ml in surrounding medium. 5-FC is well tolerated by patients at concentrations of 25-100 microg/ml (average 60 microg/ml) for 6 weeks in standard antifungal treatment. Sufficient 5-FU should be available if conversion efficiency from 5-FC is 1-3%. SER values of 1.1 to 1.2 should be achievable with daily 2 Gy fractions. In vitro and xenograft experiments are reviewed and they do not contradict the conclusions. CONCLUSIONS: Increases in tumour control of 20 to 40% can be expected, which should be detectable in a 2-arm randomized trial of 260 (for 20%) or 60 (for 40%) patients.

Stable Escherichia coli-Clostridium acetobutylicum shuttle vector for secretion of murine tumor necrosis factor alpha.

Recombinant plasmids were constructed to secrete mouse tumor necrosis factor alpha (mTNF-alpha) from Clostridium acetobutylicum. The shuttle plasmids contained the clostridial endo-beta1, 4-glucanase (eglA) promoter and signal sequence that was fused in frame to the mTNF-alpha cDNA. The construction was first tested in Escherichia coli and then introduced in C. acetobutylicum DSM792 by electroporation. Controls confirmed the presence and stability of the recombinant plasmids in this organism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an in vitro cytotoxic assay were used to monitor expression and secretion of mTNF-alpha during growth. Significant levels of biologically active mTNF-alpha were measured in both lysates and supernatants. The present report deals with investigations on the elaboration of a gene transfer system for cancer treatment using anaerobic bacteria.

Colonisation of Clostridium in the body is restricted to hypoxic and necrotic areas of tumours.

The use of gene therapy is one of the most recent molecular strategies for the treatment of cancer. It is essential, however, to have an efficient transfer system by which the desired gene can be delivered to the correct environment. The experiments described in this report investigate apathogenic Clostridium as a possible vector to transfer a specific gene product into the extracellular microenvironment of the tumour which is hypoxic/necrotic in parts, using WAG/Rij rats with transplantable rhabdomyosarcomas as a model. Our data show that Clostridium, after systemic administration of at least 10(7) spores, specifically colonises the hypoxic/necrotic areas of our tumour model, the most efficient species being C. acetobutylicum (NI-4082) and C. oncolyticum. Although spores were also detected in normal tissues for up to 4 weeks, they did not germinate in these tissues. We conclude that it seems likely that these bacteria can be used as a selective transfer system into the extracellular environment of tumours which have hypoxic regions. This strategy would be more tumour-specific than various other strategies that are currently being investigated in anti-cancer gene therapy.

[Psychopathy and drug addiction]. / Psychopathie et toxicomanie.

The author presents a study comparing psychopathic personality and drug addiction with regard to diagnosis, aethiopathogenia and treatment. Two third of drug addicts have associated personality disorders related to psychopathic disorders. Treatment on Therapeutic Communities provides a good alternative to both treatments in out patients or in open care units. The results obtained support the value of such a system in discussions about the taking over of patients in Social Defense Institutes and in jails.