of genetically engineered retroviruses used for gene transfer
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.
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
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.
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.
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October 1991 - September 1993
F. Skou Pedersen
University of Aarhus (DK)
University of Veterinary Sciences
Istituto Internazionale di Genetica e Biofisica CNR
Université Claude Bernard Lyon 1
Université de Liège (BE)