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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Safety of genetically engineered retroviruses used for gene transfer

Background and objectives

Genetically engineered DNA molecules and viruses are being increasingly used for biotechnological applications. Among the most widely utilised are retroviral vectors - viruses which have been modified to provide a highly efficient means of transporting genes into various kinds of cells. Such genetically engineered retroviruses are being increasingly used in the medical field for vaccine production and gene therapy, in research to study gene function and regulation, and in industrial applications for the production of transgenic farm animals and bio-reactors.

The objective of the project was to accumulate basic information regarding cellular and viral mechanisms involved in modulating the behaviour of retroviral vector systems. This knowledge could then be used firstly as a basis for an assessment of the risk of using retroviral vectors for the production of transgenic animals and for human gene therapy, and secondly, to construct safe, efficient and targeted retroviral vector systems.


Approach and methodology

Retroviral vector systems consist of two components, the packaging cell line, providing the retroviral proteins, and the retroviral vector construct supplying the recombinant RNA that will be packaged into the retroviral particle. One part of this study addressed questions concerning the stability of the packaging cell line, allowing the identification of cellular factors causing either appearance of wild-type virus, or shut-down of protein expression. The stability of the second retroviral vector system component, the vector construct itself, was also investigated. These studies attempted to identify cellular factors affecting the expression of the retroviral vector, and to assess recombination of retroviral vectors with endogenous sequences. An important aspect of retroviral vector design is the ability to target expression of introduced genes to specific tissues or organs. The infectivity and expression of the vectors among various cell types was investigated both for avian and murine vectors.
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Main findings and outcome

A major safety issue raised in relation to retroviral vector systems is the risk of transferring to the host, uncharacterised genetic material originating in the donor cell. One such risk is the generation of recombinant viruses or vectors that will undergo multiple rounds of replication. These new structures may be derived by recombination of genetic material from the vector and the packaging construct, and may possibly also include retroviral genetic material endogenous to the packaging cell. Another risk is the transfer of endogenous virus-related sequences to the target cell. This study analysed these processes. One part of the work was aimed at the generation of safer packaging systems for avian leukosis virus-based vectors addressing all of the above issues. Parallel work on murine leukaemia virus-based vectors led to a new safety feature controlling a key step in the reverse transcription process needed for copying of the viral genome. Since the end of the contract, this safety feature has been further developed (published in 1997 with support from a subsequent European Community contract: BIO4-CT95-0100) and adopted for use in gene transfer systems based upon other retroviruses.

Work identifying determinants affecting the level and stability of expression of the retroviral vector revealed two types of influence relating to the site of integration. Such effects impose a level of uncertainty on precise control of retroviral vector expression in a target cell. However, the exact molecular mechanisms behind these effects are still unknown.

This study has also researched other aspects of improved design of retroviral vector systems. This includes some of the early work on tissue-specific targeting by retroviral vectors at the level of infection and expression. Modifications in the binding domain of an avian retrovirus envelope were found to redirect the host infection range, albeit at low efficiency. Cell-type specific gene delivery is considered crucial for development of safe and efficient protocols for in vivo delivery of retroviral vectors. Being of major international concern, these issues have been further pursued since the end of the contract. Part of our work on exploiting the features of another retrovirus group, the mouse mammary tumour viruses, for in vivo gene delivery, led to the surprising finding of a novel transcriptional promoter in these viruses. Work exploiting mouse mammary tumour viruses for controlled gene delivery is ongoing.

Finally, genes encoding drug-activating enzymes were tested as a safety and/or treatment modality in retroviral vectors, a development that has also been intensively pursued in subsequent years.
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Conclusions

The issues addressed in this study are of major importance. Safety and control of retroviral vectors are central issues in the development of these vectors for clinical use. Key points raised by the contract work concerning safety, efficiency and specificity of gene delivery have been pursued in subsequent developments and the tools have been refined.

 

Major publications

Duch M., Paludan K., Jørgensen P. and Pedersen F.S., “Lack of correlation between basal expression levels and susceptibility to transcriptional shut-down among single-gene murine leukemia virus vector proviruses”.
J. Virol.,
68, 1994, pp. 5596-5601.

Girod A., Drynda A., Cosset F.L., Verdier G., Ronfort C., “Homologous and non-homologous retroviral recombinations are both involved in the transfer by infectious particles of defective avian leukosis virus-derived transcomplementing genomes”.
J. Virol.,
70, 1996, pp. 5651-5657.

Günzburg W.H., Heinemann F., Wintersperger S., Miethke T., Wagner H., Erfle V., Salmons B., “Endogenous superantigen expression controlled by a novel promoter located in the MMTV long terminal repeat”.
Nature,
364, pp. 154-158.

Grignet-Debrus C., Calberg-Bacq C.M., “Potential of Varicella zoster virus thymidine kinase as a suicide gene in breast cancer cells”.
Gene Ther.,
4, 1997, pp. 560-569.

Valsesia-Wittmann S., Drynda A., Deleage G., Aumailley M., Heard J.M., Danos O., Verdier G., Cosset F.L., “Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors”.
J. Virol., 68, 1994, pp. 4609-4619.
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imageResearch project
 

Contract number
BIOT-CT91-0286

Period
October 1991 - September 1993

Coordinator
F. Skou Pedersen
University of Aarhus (DK)

 
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Partners


W.H. Günzburg
University of Veterinary Sciences
Vienna (AT)

J. Guardiola
Istituto Internazionale di Genetica e Biofisica CNR
Napoli (IT)

G. Verdier
Université Claude Bernard Lyon 1
Villeurbanne (FR)

C.-M. Calberg-Bacq
Université de Liège (BE)

 
 
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