Different susceptibility of osteosarcoma cell lines and primary cells to treatment with oncolytic adenovirus and doxorubicin or cisplatin.

Despite improvements in treatment regimens for osteosarcoma (OS) patients, survival rate has not increased over the last two decades. New treatment modalities are therefore warranted. Preclinical results with conditionally replicative adenoviruses (CRAds) to treat OS are promising. One type of CRAd that was effective against OS cells is Ad5-Delta24RGD. In other types of cancer, CRAds have been shown to interact synergistically with chemotherapeutic agents. Chemotherapy for OS often includes doxorubicin and cisplatin. Therefore, we explored combination treatment of OS cell lines and primary OS cell cultures with Ad5-Delta24RGD and doxorubicin or cisplatin. On OS cell lines, combination treatment was additive to synergistic. Surprisingly, however, on seven of eight primary OS samples no such combination effects were observed. In contrast, in many cases chemotherapy even inhibited CRAd-mediated cell killing. The inhibitory effect of doxorubicin on Ad5-Delta24RGD in primary OS cells appeared to correlate with slow cell growth rate; reduced viral replication and absence of chemotherapy-induced G2 cell cycle arrest. Our results point to the possibility that, at least for OS, virotherapy and chemotherapy should best not be performed simultaneously. In general, our work underscores the importance of testing new genetic anticancer agents and treatment regimens on primary cancer specimens.

Towards integrating vectors for gene therapy: expression of functional bacteriophage MuA and MuB proteins in mammalian cells.

Bacteriophage Mu has one of the best studied, most efficient and largest transposition machineries of the prokaryotic world. To harness this attractive integration machinery for use in mammalian cells, we cloned the coding sequences of the phage factors MuA and MuB in a eukaryotic expression cassette and fused them to a FLAG epitope and a SV40-derived nuclear localization signal. We demonstrate that these N-terminal extensions were sufficient to target the Mu proteins to the nucleus, while their function in Escherichia coli was not impeded. In vivo transposition in mammalian cells was analysed by co-transfection of the MuA and MuB expression vectors with a donor construct, which contained a miniMu transposon carrying a Hygromycin-resistance marker (Hyg(R)). In all co-transfections, a significant but moderate (up to 2.7-fold) increase in Hyg(R) colonies was obtained if compared with control experiments in which the MuA vector was omitted. To study whether the increased efficiency was the result of bona fide Mu transposition, integrated vector copies were cloned from 43 monoclonal and one polyclonal cell lines. However, in none of these clones, the junction between the vector and the chromosomal DNA was localized precisely at the border of the Att sites. From our data we conclude that expression of MuA and MuB increases the integration of miniMu vectors in mammalian cells, but that this increase is not the result of bona fide Mu-induced transposition.

Ammonium sulphate precipitation of recombinant adenovirus from culture medium: an easy method to increase the total virus yield.

In the majority of the methods for purifying and concentrating recombinant adenoviruses (rAds) the virus that is associated with the helper cells is harvested, while the virus that is present in the cell-culture medium is discarded. During routine propagation of adenovirus type-5 vectors at optimised conditions we noted that, on average, 47% of the total amount of virus is present in the culture medium. To recover and concentrate these rAds from the medium, we devised a method, which is based on ammonium sulphate ((NH4)2SO4) precipitation. At 40% (NH4)2SO4 saturation, 95 +/- 6% of the available virus precipitates from the medium, while the majority of the protein (85%) remains in solution. In contrast to adenovirus precipitation with polyethylene glycol, the (NH4)2SO4 precipitation technique allows collection of precipitated rAds by filtration. We demonstrate here that (NH4)2SO4 precipitation of rAds from cell-culture medium is a simple and fast technique that can be used in combination with standard virus isolation methods to increase the yields of rAds.

Adenoviruses activate human dendritic cells without polarization toward a T-helper type 1-inducing subset.

Human monocyte-derived dendritic cells (DC) infected with recombinant adenoviruses (rAd) are promising candidate vaccines for inducing protective immunity against pathogens and tumors. However, since some viruses are known to negatively affect DC function, it is important to investigate the interactions between rAd and DC. We now show that infection by rAd enhances the immunostimulatory capacity of immature human monocyte-derived DC through the upregulation of the costimulatory molecules CD80, CD86, and CD40 and the major histocompatibility complex class I and II molecules. Although rAd infection fails to induce the secretion of interleukin-12 (IL-12) and only marginally induces the expression of the DC maturation marker CD83, it acts in synergy with CD40 triggering in rendering DC fully mature. rAd-infected DC triggered through CD40 produce more IL-12 and are more efficient in eliciting T-helper type 1 responses than DC activated by CD40 triggering only. rAd lacking one or more of the early regions, E1, E2A, E3, and E4, which play an important role in virus-host cell interactions are equally capable of DC activation. Efficient DC infection requires a high multiplicity of infection (>1,000), a fact which can be attributed to the absence of the coxsackievirus and adenovirus receptor on this cell type. Despite the poor ability of DC to be infected by rAd, which may be improved by targeting rAd to alternative DC surface molecules, DC infected with all currently tested rAd constitute potent immunostimulators. Our study provides new insights into the interactions between two highly promising vaccine components, rAd and DC, and indicates that their combination into one vaccine may be very advantageous for the stimulation of T-cell immunity.

Photodynamic treatment of adenoviral vectors with visible light: an easy and convenient method for viral inactivation.

Recombinant adenovirus vectors are popular tools for gene transfer and gene therapy. However biosafety constraints require that all handling of the vectors and vector-containing samples is restricted to dedicated containment laboratories, unless they had undergone a validated virus-inactivation procedure, which decontaminates the samples from any active virus. In this study we evaluated the feasibility of photodynamic treatment (PDT) with visible light to inactivate recombinant adenovirus vectors in biological samples, with minimum associated effects on other biological activities. Several photosensitizers were tested for their capacity to inactivate a model human adenovirus vector, AdCMVLuc, upon illumination. Four photosensitizers (methylene blue (MB), rose bengal (RB), uroporphyrin (UP) and aluminum phthalocynine tetrasulphonate (AIPcS4)) could inactivate the adenovirus, as measured by expression of the luciferase reporter gene and by plaque assay. Of these, MB demonstrated to be the most effective sensitizer in phosphate-buffered saline (PBS), giving > 7 log10 inactivation of the adenovirus. DNA isolated from MB- and light-treated virions was inefficient as a template for transcription. Furthermore, Southern blot analysis revealed fragmentation of the viral DNA. Based on its preference for DNA, MB is suited for adenovirus inactivation in blood plasma. Spiking experiments in which AdCMVLuc was added to plasma samples demonstrated a reduction (> 4 log10-fold) of reporter gene expression to almost background levels. In contrast to MB, photodynamic treatment with RB, UP or AIPcS4 did not lead to DNA damage. Although alterations of the viral capsid could not be detected, the binding pattern of the particles to target cells was significantly changed. Taken together, our data demonstrate that PDT is an efficient, convenient and useful method for the inactivation of adenovirus vectors in biological samples.

[Gene therapy for hemophilia: feasible in principle but not yet clinically applicable]. / Gentherapie voor hemofilie: in principe uitvoerbaar, maar nog niet klinisch toepasbaar.

The current treatment of haemophiliacs consists of injection of concentrates of blood clotting factors VIII (haemophilia A) and IX (haemophilia B). The inconvenience of the multiple injections needed, and the risk of transmission of infectious agents (HIV, hepatitis) prompted the development of alternative therapies. Gene therapy aims at introducing functional factor VIII and IX genes into the body cells of patients in order to make these cells produce the desired clotting factors. There are two strategies for gene therapy: (a) in the laboratory cells of the patient may be provided with the desired gene, followed by reintroduction of the cells that now produce factor VIII, into the patient (ex vivo strategy); (b) vectors with the desired genes may be injected into the patient in order to induce the modification (in vivo strategy) For both routes, the formal proof-of-principle has been acquired recently in animal experiments: cells modified by factor VIII or IX genes will produce adequate concentrations of the clotting products in plasma and will correct the bleeding tendency. Before the clinical evaluation and widespread application of the technology can be considered many technical problems have to be solved.

Severe hepatic dysfunction after adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene and ganciclovir administration.

The use of so-called 'suicide' genes to activate prodrugs has been effective in animal models for several solid tumor types and is now in phase I and II clinical trials. We have exploited adenovirus vectors (Ad) for transfer and expression of the herpes simplex virus thymidine kinase (HSVtk) gene to render rat colorectal liver metastases sensitive to the anti-herpetic agent ganciclovir (GCV). The efficacy and toxicity of this enzyme-prodrug combination were tested after in situ transduction of rat colorectal tumor cells and after intraportal administration of the vector Ad.CMV.TK. Our results demonstrate the validity of the approach but reveal that hepatic expression of HSVtk, both in tumor bearing and in tumor-free rats, provokes severe liver dysfunction and mortality upon GCV administration. These data show, that in contrast to the common assumption, normally non-mitotic tissues too, can be affected by adenovirus-mediated HSVtk transfer and subsequent GCV treatment. Given the hepatotropic nature of systemically administered adenovirus type 2- and 5-derived vectors, it will be essential to monitor liver functions of patients included in all gene therapy trials involving adenoviral vectors with the HSVtk gene.

Factors impeding efficient expression of factor VIII complementary DNA minigenes.

Haemophilia A is a bleeding disorder that affects approximately 1 in 10,000 males. It is caused by a deficiency of functional blood-clotting factor VIII. Protein-replacement therapy has been effective as treatment, resulting in a vast improvement in the quality of life and dramatically increasing the life expectancy of patients. However, therapy with plasma-derived factor VIII has allowed the transmission of several human viruses, such as hepatitis viruses, human immunodeficiency virus and parvovirus B19. To date, the safety of the therapeutic agent is one of the key issues in haemophilia A treatment. The use of recombinant factor VIII in haemophilia therapy can avoid the dependence on blood-derived products and prevent the occurrence of transfusion-associated infections with blood-borne pathogens. Gene therapy could go further, and offers the prospect of one-time treatment which may, optimally, achieve a total cure of the disease. Therefore, haemophilia is an appealing and challenging target for somatic-cell gene therapy. On the basis of the phenotypic correction that is achieved upon infusion of factor VIII protein, it is expected that an increase in the factor VIII plasma level to 10% of the level found in healthy individuals would suffice to prevent the manifestation of the bleeding tendency. In this paper, we review the progress and the problems of gene therapy for haemophilia, with special focus on the problems specifically associated with the transfer and expression of human factor VIII complementary DNA.

Involvement of histamine in suckling-induced release of oxytocin, prolactin and adrenocorticotropin in lactating rats.

We have previously shown that histaminergic neurons participate in mediation of the prolactin (PRL), adrenocorticotropin (ACTH) and oxytocin responses to physiological stimuli such as stress and dehydration. Since suckling is a potent stimulus for the secretion of these three hormones, we investigated the mediating role of neuronal histamine in suckling-induced release of oxytocin, PRL and ACTH in conscious lactating rats. The animals were pretreated with the histamine synthesis inhibitor alpha-fluoromethylhistidine, the H1 receptor antagonist mepyramine, the H2 receptor antagonist cimetidine or the H3 receptor agonist R (alpha) methylhistamine, which by binding to H3 autoreceptors inhibits histamine release and synthesis. After the lactating rats were separated from their pups for 240 min, the pups were returned for a suckling period of 20 min. Thereafter the mothers were sacrificed by decapitation and trunk blood was collected for determination of hormones. Lactating rats not exposed to suckling served as controls. Suckling increased oxytocin 2-fold, PRL 50-fold and ACTH 5-fold. Blockade of histamine synthesis by alpha-fluoromethylhistidine or histamine release by R(alpha)methylhistamine reduced the suckling-induced secretion of the three hormones significantly. Blockade of postsynaptic H1 receptors by mepyramine inhibited the hormone responses to suckling, while the blockade of postsynaptic H2 receptors by cimetidine decreased the suckling-induced oxytocin and PRL release but did not affect the ACTH release. None of the compounds affected oxytocin, PRL or ACTH secretion in lactating mothers not exposed to suckling. In addition, suckling significantly increased the mRNA of the histamine synthesizing enzyme histidine decarboxylase in the ventrolateral tuberomammillary nucleus by 1.5-fold, indicating that the stimulus of suckling enhances the neuronal histamine synthesis. We conclude that suckling increases neuronal histamine synthesis and that histaminergic neurons by activation of postsynaptic H1 and H2 receptors are involved in the hypothalamic mediation of oxytocin, PRL and ACTH responses to suckling. These findings further substantiate a role of neuronal histamine in the neuroendocrine regulation of pituitary hormones in response to physiological stimuli.