Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

Generation of cell hybrids via a fusogenic cell line.

BACKGROUND: Hybrids obtained by fusion between tumour cells (TC) and dendritic cells (DC) have been proposed as anti-tumour vaccines because of their potential to combine the expression of tumour-associated antigens with efficient antigen presentation. The classical methods used for fusion, polyethylene glycol (PEG) and electrofusion, are cytotoxic and generate cell debris that can be taken up by DC rendering the identification of true hybrids difficult. METHODS: We have established a stable cell line expressing a viral fusogenic membrane glycoprotein (FMG) that is not itself susceptible to fusion. This cell line has been used to generate hybrids and to evaluate the relevance of tools used for hybrid detection. RESULTS: This FMG-expressing cell line promotes fusion between autologous or allogeneic TC and DC in any combination, generating 'tri-parental hybrids'. At least 20% of TC are found to be integrated into hybrids. CONCLUSIONS: It is speculated that this tri-parental hybrid approach offers new possibilities to further modulate the anti-tumour effect of the DC/TC hybrids since it allows the expression of relevant immunostimulatory molecules by appropriate engineering of the fusogenic cell line.

Hypoxic-response elements in the oncolytic parvovirus Minute virus of mice do not allow for increased vector production at low oxygen concentration.

Vectors derived from the autonomous parvovirus Minute virus of mice, MVM(p), are promising tools for the gene therapy of cancer. The validation of their in vivo anti-tumour effect is, however, hampered by the difficulty to produce high-titre stocks. In an attempt to increase vector titres, host cells were subjected to low oxygen tension (hypoxia). It has been shown that a number of viruses are produced at higher titres under these conditions. This is the case, among others, for another member of the family Parvoviridae, the erythrovirus B19 virus. Hypoxia stabilizes a hypoxia-inducible transcription factor (HIF-1alpha) that interacts with a 'hypoxia-responsive element' (HRE), the consensus sequence of which ((A)/(G)CGTG) is present in the B19 and MVM promoters. Whilst the native P4 promoter was induced weakly in hypoxia, vector production was reduced dramatically, and adding HRE elements to the P4 promoter of the vector did not alleviate this reduction. Hypoxia has many effects on cell metabolism. Therefore, even if the P4 promoter is activated, the cellular factors that are required for the completion of the parvoviral life cycle may not be expressed.

Modulation of the metabolism and adverse effects of benzo[a]pyrene by a specific antibody: a novel host factor in environmental carcinogenesis?

The influence of specific antibodies on molecular and cellular mechanisms of activation, detoxification and biological activity of the ubiquitous carcinogen benzo[a]pyrene (B[a]P) was investigated using a monoclonal antibody. The antibody was shown to decrease cellular uptake and metabolic activation of B[a]P as demonstrated by higher recovery of unmetabolized B[a]P and decreased formation of end-point phenol metabolites in two types of target cells. Furthermore, strong antibody reactivity with 7,8-diol-B[a]P provided a second chance for interrupting metabolic activation by sequestration of this intermediate metabolite in the extracellular space. The biological relevance of B[a]P and 7,8-diol-B[a]P redistribution by antibody was demonstrated by reversion of B[a]P-induced inhibition of proliferation of human peripheral blood lymphocytes and by inhibition of CYP 1A1 induction in HepG2 cells. Remarkably, the antibody was still protective against B[a]P-induced immunotoxicity even after delayed addition, suggesting a more important role of metabolites in immunotoxicity than has been appreciated so far. Although B[a]P is activated to 7,8-diol-B[a]P in the same cells that are inhibited by this metabolite, the antibody completely restored lymphocyte proliferation indicating that extracellular trapping of the 7,8-diol-B[a]P is biologically highly effective. Thus, repartitioning of both B[a]P and its metabolites by the antibody may reduce their effective concentration in susceptible target organs and therefore relieve overloaded DNA repair mechanisms and inhibit carcinogen-induced P450 induction. These in vitro data also suggest that a natural or prophylactic antibody response against carcinogens may be associated with a reduced risk of cancer.

In vivo anti-tumour activity of recombinant MVM parvoviral vectors carrying the human interleukin-2 cDNA.

BACKGROUND: The natural oncotropism and oncotoxicity of vectors derived from the autonomous parvovirus, minute virus of mice (prototype strain) [MVM(p)], combined with the immunotherapeutic properties of cytokine transgenes, make them interesting candidates for cancer gene therapy. METHODS: The in vivo anti-tumour activity of a recombinant parvoviral vector, MVM-IL2, was evaluated in a syngeneic mouse melanoma model that is relatively resistant in vitro to the intrinsic cytotoxicity of wild-type MVM(p). RESULTS: In vitro infection of the K1735 melanoma cells prior to their injection resulted in loss of tumorigenicity in 70% of mice (7/10). Tumour-free mice were protected against a challenge with non-infected parental cells. In addition, MVM-IL2-infected tumour cells induced an anti-tumour activity on parental cells injected at a distant location. These non-infected tumour cells were injected either at the same time or 7 days before the injection of MVM-IL2-infected cells. In the latter setting, which mimics a therapeutic model for small tumours, 4/10 mice were still tumour-free after 4 months. CONCLUSIONS: Our results show that (i) the MVM-IL2 parvoviral vector efficiently transduces tumour cells; and (ii) the low multiplicity of infection (MOI = 1) used in our experiments was sufficient to elicit an anti-tumour effect on distant cells, which supports further studies on this vector as a new tool for cancer gene therapy.

Autonomous parvovirus vectors: preventing the generation of wild-type or replication-competent virus.

The preferential expression of autonomous parvoviruses in tumour cells and their oncolytic activity has attracted attention to the potential use of these viruses as vectors for cancer gene therapy. Moreover, they are non-pathogenic in adult animals and they seem to be associated with low or no immunogenicity. Other interesting features are their episomal replication and high stability. Vectors derived from the autonomous parvoviruses MVM(p) or H1 express proteins that can directly or indirectly interfere with tumour development. They retain cis- and trans-acting sequences required for viral DNA amplification; the transgene replaces part of the capsid coding genes. Their development has been hampered by low titres and contamination with replication-competent virus (RCV) that is generated through homologous recombination with helper plasmids. Several approaches have been used to avoid recombination between vectors and helpers. In most instances, reducing the homology up- or downstream of the transgene in either the vector or the helper did not significantly affect RCV production. However, completely eliminating homology downstream of the transgene, splitting VP genes on different helpers or pseudotyping vectors resulted in the production of RCV-free stocks. Although VP-containing particles could sometimes be identified in these stocks by in situ hybridisation, they did not amplify and are therefore not true RCV. The integration of capsid-coding sequences into packaging cells also reduced contamination by RCV and allowed for the amplification of vectors through serial infections. Great progress has been made recently towards the generation of truly RCV-free stocks of vectors derived from autonomous parvoviruses H1 and MVMp. Combining these new vectors with a new packaging cell line should greatly facilitate their development.

A new genetic method to generate and isolate small, short-lived but highly potent dendritic cell-tumor cell hybrid vaccines.

Fusion of tumor cells with antigen-presenting cells (APCs) has been proposed for the preparation of cancer vaccines. However, generation of these hybrids, using physical or chemical methods such as electrofusion or polyethylene glycol (PEG), has been difficult to standardize. Characterization of cell fusion has also been problematic because of difficulties in differentiating fusion from cell aggregation, leakage of cellular dyes and dendritic-cell (DC) phagocytosis of tumor material. In this report, we describe a new method to generate hybrid cell vaccines, based on gene transfer of a viral fusogenic membrane glycoprotein (FMG) into tumor cells, and incorporate a genetic method by which true hybrid formation can be unambiguously detected. We describe a new class of tumor cell-DC hybrid that can be rapidly isolated after cell fusion. These hybrids are highly potent in in vitro antigen presentation assays, target lymph nodes in vivo and are powerful immunogens against established metastatic disease.

A novel method for the titration of recombinant virus stocks by ELISPOT assay.

The development of vectors for gene therapy requires the definition of quality control parameters such as titration, contamination, transduction efficiency and biological effects in defined model systems. For most viral vectors, the classical titration by plaque formation is not applicable, because vectors are defective for replication and packaging cell lines are not always available. In particular, for vectors derived from the autonomous parvovirus MVM(p), the titration method used currently is based on the amplification of the viral genome inside an infected cell, which can then be revealed with a specific radioactive probe (J. Virol. 63 (1989) 1023). In situ hybridization allows to titrate wild-type virus as well as vectors, using probes that are specific for the substituted viral genes or for the transgene, respectively. This method is, however, time consuming, making the simultaneous titration of large numbers of samples difficult. The use of a radioactive probe requires an adequate facility. An ELISPOT method that allows for rapid titration of up to 23 vector stocks in one 96 well dish was devised. This method is based on the actual expression of the transgene. Compared to in situ hybridization, titers obtained by the ELISPOT method were in general equivalent or higher. However, for some vector stocks the ELISPOT titers were repeatedly lower, indicating that in situ hybridization does not give an accurate measure of transducing units. Our model system is recombinant parvovirus MVM expressing human IL2, but the method should be adaptable to other vectors expressing transgenes that are secreted and for which antibodies are available.

Construction and production of oncotropic vectors, derived from MVM(p), that share reduced sequence homology with helper plasmids.

The production of currently available vectors derived from autonomous parvoviruses requires the expression of capsid proteins in trans, from helper sequences. Cotransfection of a helper plasmid always generates significant amounts of replication-competent virus (RCV) that can be reduced by the integration of helper sequences into a packaging cell line. Although stocks of minute virus of mice (MVM)-based vectors with no detectable RCV could be produced by transfection into packaging cells; the latter appear after one or two rounds of replication, precluding further amplification of the vector stock. Indeed, once RCVs become detectable, they are efficiently amplified and rapidly take over the culture. Theoretically RCV-free vector stocks could be produced if all homology between vector and helper DNA is eliminated, thus preventing homologous recombination. We constructed new vectors based on the structure of spontaneously occurring defective particles of MVM. Based on published observations related to the size of vectors and the sequence of the viral origin of replication, these vectors were modified by the insertion of foreign DNA sequences downstream of the transgene and by the introduction of a consensus NS-1 nick site near the origin of replication to optimize their production. In one of the vectors the inserted fragment of mouse genomic DNA had a synergistic effect with the modified origin of replication in increasing vector production.