Chemistry: molecular biology and microbiology – Vector – per se
Patent
1996-08-01
2000-08-29
Bui, Phuong T.
Chemistry: molecular biology and microbiology
Vector, per se
4352521, 4352523, 43525233, 4352528, 4352351, 4351723, 4242331, 514 44, 530350, A61K 39235, C07H 2104, C07K 14075, C12N 121
Patent
active
061107352
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for preparing a viral vector in vitro in a prokaryotic cell and to its application for producing an infectious viral particle intended for therapeutic use, and especially for use in gene therapy.
The possibility of treating human diseases by gene therapy has changed in a few years from the stage of theoretical considerations to that of clinical applications. The first protocol applied to man was initiated in the US in September 1990 on a patient who was genetically immunodeficient as a result of a mutation affecting the gene coding for adenine deaminase (ADA). The relative success of this first experiment encouraged the development of new gene therapy protocols for various genetic or acquired diseases (infectious diseases, and viral diseases in particular, such as AIDS, or cancers). The large majority of the protocols described hitherto employ viral vectors to transfer the therapeutic gene to the cells to be treated and to express it therein.
To date, retroviral vectors are among the ones most widely used on account of the simplicity of their genome. However, apart from their restricted capacity for cloning, they present two major drawbacks which limit their systematic use: on the one hand they chiefly infect dividing cells, and on the other hand, as a result of their integration at random in the genome of the host cell, the risk of insertional mutagenesis is not insignificant. For this reason, many scientific teams have endeavored to develop other types of vector, among which those originating from adenoviruses, adeno-associated viruses (AAV), poxviruses and herpesviruses may be mentioned. Generally speaking, their organization and their infection cycle are amply described in the literature available to a person skilled in the art.
In this connection, the use of adenoviral vectors has been seen to be a promising alternative. Adenoviruses have been demonstrated in many animal species, have a broad host range, have little pathogenicity and do not present the drawbacks associated with retroviruses since they replicate in resting cells and are nonintegrative. As a guide, their genome consists of a linear, double-stranded DNA molecule of approximately 36 kb carrying more than about thirty genes, both early genes necessary for viral replication (E1 to E4) and late structural genes (L1 to L5) (see FIG. 1).
Generally speaking, the adenoviral vectors are obtained by deletion of at least one portion of the viral genes (in particular of the E1 region essential for viral replication), which are replaced by the therapeutic genes. Consequently, they are propagated in a cell line, termed complementation line, which supplies in trans the deleted viral functions to generate a viral vector particle which is defective for replication but capable of infecting a host cell. The line 293, established from human embryonic kidney cells, which complements adenoviruses that are defective for the E1 function (Graham et al., 1977, J. Gen. Virol., 36, 59-72), is commonly used.
The techniques of preparation of adenoviral vectors are amply described in the literature (see, in particular, Graham and Prevec, Methods in Molecular Biology, Vol. 7; Gene Transfer and Expression Protocols; Ed: E. J. Murray, 1991, The Human Press Inc., Clinton, N.J.). One of the methods most often employed consists in generating the recombinant adenoviral vector in complementation cells transfected with a bacterial plasmid carrying the gene of interest subcloned within its adenoviral insertion region and the adenoviral genome. Generally, the latter is cleaved with a suitable restriction enzyme so as to reduce the infectivity of the parent viral DNA and to increase the efficiency of isolation of the recombinant adenoviruses. However, a substantial background of infectious viral particles of parenteral origin is nevertheless observed, which necessitates an especially arduous task of analysis of the plaques obtained (arduous from a time and cost standpoint, since each plaque has to be amplified and analyzed individually).
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Chartier Cecile
Degryse Eric
Bui Phuong T.
Transgene S.A.
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