Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Avian cell – per se
Reexamination Certificate
1999-02-12
2001-07-03
Wortman, Donna C. (Department: 1643)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Avian cell, per se
C435S455000, C435S467000
Reexamination Certificate
active
06255108
ABSTRACT:
The present invention relates to avian cell lines and their derivatives.
It is not possible to establish cell lines spontaneously from organs taken from avian species, as can be done in the case of some organs derived from mammalian species.
The only available cell lines to date were obtained using the transforming properties of certain avian viruses which possess oncogenic properties, such as the retroviruses of the avian leukosis group or Marek's disease virus, or certain chemical molecules such as methylcholanthrene and diethylnitrosamine.
For the most part, these cell lines are considerably transformed, which renders them unsuitable for multiplying vaccine viruses.
Authors are adopting a novel approach which consists in introducing into cells a vector which does not exhibit any oncogenic character but which is able to integrate, into these cells, a gene which is selected for its capacity to induce immortalization.
The first tests were carried out using vectors which integrate avian retrovirus genes such as erbA and erbB.
French Patent Application FR-A-2 596 770 proposes an immortalization method in which a culture of avian or mammalian cells is infected with a vector or a system which, while not being oncogenic for the said cells, is able to integrate a gene selected from v-myb, v-ets and v-erbA into these cells. The AMV, E26 and XJ12 viruses, with this latter being a virus derivative of the AEV virus in which the oncogenic v-erB gene has been deleted, can be appropriate vectors.
In practice, while these tests made it possible to obtain established cell lines from cells of the haematopoietic cell line, they did not give the expected results in the case of chick embryo cells in adherent culture, such as fibroblasts or epithelial cells.
It was possible to obtain untransformed avian cell lines of the myeloblastoid type (blood cells) using the oncogene myb (International Patent Application WO91/18971).
In parallel, authors have proposed using the early t and T genes of the simian virus SV40 for immortalizing cells derived from different mammalian tissues (D. S. Neufeld et al., Molecular and Cellular Biology, Aug. 1987, 2794-2802, O. Kellermann and F. Kelly, Differentiation 1986, 32: 74-81 and French Patent Application FR-A-2 649 721).
For its part, French Patent Application FR-A-2 649 721 proposes a method of conditional immortalization which, it is claimed, can be used for any cell type and in any species, with the aim in this case being that of remedying the drawback of the high degree of specificity of the conventional approaches (limitation to particular species and/or to particular cell types): transformation of cells with a transforming virus (adenovirus, Epstein-Barr virus, certain papovaviruses such as the SV40 virus or polyoma virus; for example, the SV40 virus is indicated as only transforming rodent cells and human cells); transfection with constructs which contain a transforming gene which is linked to a viral promoter; transfection with a transforming gene which is linked to a cellular promoter. The choice of this patent application falls on a construct which combines a DNA fragment from the regulatory sequence of vimentin and a DNA fragment which encodes an immortalizing gene, which construct can be the T antigen of the SV40 virus under the control of the inducible promoter of vimentin. This document never mentions the avian species.
The actual use of such viral oncogenes has never been described in the avian species, apart from the use of the 12S form of the E1A protein of human adenovirus 5, which made it possible to immortalize quail epithelial cells (Guilhot et al. (1993), Oncogene 8: 619-624).
Contrary to all expectations, the inventors succeeded in producing immortal, untransformed avian cell lines.
More generally, the inventors have found that it was possible to prepare immortal, untransformed avian cell lines which are resistant to apoptosis even from cells of avian tissues, that is to say from cells other than circulating blood cells or haematopoietic cells.
The present invention therefore relates to immortal, untransformed avian cells which are resistant to apoptosis and which derive, in particular, from avian tissues, that is to say from cells other than blood cells or haematopoietic cells, in particular fibroblasts and epithelial cells, for example from embryos.
The present invention more especially relates to an untransformed, immortal avian cell line which is selected from the group consisting of:
cell line TDF-2A bcl-2, which is deposited in the CNCM (Collection Nationale de Cultures de Microorganismes de l'Institut Pasteur [Pasteur Institute National Collection of Microorganism Cultures]) under reference number I-1709
cell line TCF-4.10, which is deposited in the CNCM under reference number I-1710
cell line TCF-4.10 bcl-2, which is deposited in the CNCM under reference number I-1711
bcl-2 denotes that the cells of the cell line functionally integrate the bcl-2 gene, which confers on them resistance to apoptosis (WO-A-93/20200, which is hereby incorporated by reference).
The invention naturally covers the cells which are derived from these cell lines. By this, it is to be understood that it is not only the cells as deposited in the CNCM under the indicated references which are covered but also the cells which constitute the progeny of these deposited cells, i.e., on the one hand, those which are obtained by simple multiplication and which may undergo mutations during these multiplications and, on the other hand, those which are obtained after deliberate modification, which are then termed the derived cells, and, of course, also those which have undergone the two types of modification.
The invention therefore also covers the derived cells which are obtained by modifications of the above cells. These modifications may consist in:
Inserting one or more expression cassettes, each of which comprises one or more nucleotide sequences encoding a molecule of industrial relevance, with these expression cassettes being able to produce this molecule following insertion into the cells of the invention. The skilled person is fully conversant with the technique. Molecules of industrial relevance which may be mentioned, in particular, are viral subunits of the peptide, protein or glycoprotein type, in particular for use in a vaccine or a diagnostic reagent, protein molecules such as hormones, etc.
Chronically infecting with a virus which is able to multiply in these cells, for virus or vaccine production purposes, with or without prior modification of the sensitivity towards this virus. The infection may also not be chronic but carried out on a batch of cells which is selected for the viral multiplication.
(The modifications described below are to be understood as preferably and advantageously being combined with the preceding two types of modification).
Introducing survival or anti-apoptotic genes other than bcl-2, such as the genes which encode the human adenovirus p19E1B (Rao et al. (1992), Proc. Natl. Acad. Sci. USA 89: 7742-7746) the Epstein Barr virus LMP-1 (Gregory et al. (1991), Nature 349: 612-614) and BHRF1 (Pearson et al. (1987), Virology 160: 151-161), the herpes simplex virus ICP34.5 (Chou and Roizman (1992), Proc. Natl. Acad. Sci. USA 89: 3266-3270) and the baculovirus p35 (Clem et al. (1991), Science 254: 1388-1390) proteins in order to render these cell lines more resistant to the culture conditions, in particular for maintaining confluence.
Overexpressing genes which are involved in controlling the cell cycle using vectors which are suitable for increasing the rate of proliferation. Thus, it has been demonstrated that, in certain cases, overexpressing cyclin-encoding genes leads to the cell cycle being shortened and therefore to the rate of proliferation being increased (Rosenberg et al. (1995), Oncogene 10: 1501-1509; Quelle et al. (1993), Genes and Dev. 7: 1559-1571).
Modifying the viral sensitivity spectrum of the cell lines by integrating genes which encode receptors for the viruses of interest, with a view to multiplying
Bouquet Jean-Francois
Cleuziat Catherine
Desmettre Philippe
Samarut Jacques
Larson & Taylor PLC
Merial
Wortman Donna C.
Zeman Robert A.
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