Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Reexamination Certificate
1999-05-18
2002-05-14
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
C424S093200, C435S069100, C435S069800, C435S070100, C435S235100, C435S320100, C435S325000, C435S455000, C435S456000, C435S457000, C435S462000, C514S04400A, C536S023100, C536S023720
Reexamination Certificate
active
06387670
ABSTRACT:
The present invention relates to a method for the production of recombinant viruses. It also relates to constructs used for carrying out this method, the producing cells, and the viruses thus produced. These viruses can be used as vector for the cloning and/or expression of genes in vitro, ex vivo or in vivo.
Vectors of viral origin are widely used for the cloning, transfer and expression of genes in vitro (for the production of recombinant proteins, for carrying out screening tests, for studying the regulation of genes and the like), ex vivo or in vivo (for the creation of animal models, or in therapeutic approaches). Among these viruses, there may be mentioned in particular adenoviruses, adeno-associated viruses (AAV), retroviruses, herpesviruses or vaccinia viruses.
The Adenoviridae family is widely distributed in mammals and birds and comprises more than one hundred different serotypes of nonenveloped double-stranded DNA viruses possessing a capsid of icosahedral symmetry (Horwitz, In: Fields B N, Knipe D M, Howley P M, ed. Virology. Third edition ed. Philadelphia: Raven Publishers, 1996: 2149-2171). In addition to its safety, the adenovirus has a very broad cellular tropism. Unlike the retrovirus, whose cycle is dependent on cell division, it can infect actively dividing cells such as quiescent cells and its genome is maintained in episomal form. Furthermore, it can be produced at high titres (10
11
pfu/ml). These major assets of it one makes a most preferred vector for the cloning and expression of heterologous genes. The group C adenoviruses, particularly types 2 and 5, as well as the CAV-2-type canine adenoviruses, whose molecular biology is best known, are the source of the vectors currently used.
The adenovirus has a linear genome of 36 kb, terminating at each of these ends with inverted terminal repeat (ITR) sequences of 103 bp comprising a replication origin as well as an encapsidation signal situated near the left ITR (Shenk, Adenoviridae: The Viruses and Their Replication. In: Fields B N, Knipe D M, Howley P M, ed. Virology. Philadelphia: Raven publishers, 1996: 2111-2148). Three families of genes are expressed during the viral cycle:
The immediate-early genes (E1, E2, E3 and E4) which are involved in the regulation of cellular genes allowing in particular the entry of the cell into the S phase (E1A) and the inhibition of apoptosis (E1B). They are also involved in the regulation of early or late viral genes at the level of the transcription, splicing or transport of the messenger RNAs (E1A, E2A, E4). They also play a role in replication and in escaping the immune response.
The delayed-early genes (pIX and IVa2) are linked to the regulation of transcription of the late genes (IVa2) or to the assembling of the virion (pIX).
The late genes (L1 to L5) are transcribed from the strong promoter (MLP). A primary transcript of 28 kb makes it possible to generate the transcripts corresponding to the various structural proteins (core, penton, hexon) and nonstructural proteins participating in the assembling and in the maturation of the viral particles, by alternative splicing and the use of 5 polyadenylation sites.
Adenoviral vectors have been used for the cloning and expression of genes in vitro (Gluzman et al., Cold Spring Harbor, N.Y. 11724, p. 187), for the creation of transgenic animals (WO95/22616), for the transfer of genes into cells ex vivo (WO95/14785; WO95/06120) or for the transfer of genes into cells in vivo (see in particular WO93/19191, WO94/24297, WO94/08026).
As regards the adeno-associated viruses (AAV), they are relatively small DNA viruses which become integrated into the genome of the cells which they infect, in a stable and relatively site-specific manner. They are capable of infecting a broad spectrum of cells, without inducing any effect on cell growth, morphology or differentiation. Moreover, they do not seem to be involved in pathologies in man. The genome of the AAVs has been cloned, sequenced and characterized. It comprises about 4700 bases and contains, at each end, an inverted terminal repeat (ITR) region of about 145 bases which serves as replication origin for the virus. The remainder of the genome is divided into 2 essential regions carrying the encapsidation functions: the left part of the genome, which contains the rep gene involved in the viral replication and the expression of the viral genes; the right part of the genome, which contains the cap gene encoding the virus capsid proteins.
The use of vectors derived from AAVs for the transfer of genes in vitro and in vivo has been described in the literature (see in particular WO91/18088; WO93/09239; U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488 528).
As regards the retroviruses, they are integrative viruses which selectively infect dividing cells. They therefore constitute vectors of interest for cancer or restenosis applications for example. The genome of retroviruses essentially comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). The construction of recombinant vectors and their use in vitro or in vivo has been widely described in the literature: see in particular Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP 178220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, and the like.
For their use as recombinant vectors, various constructs derived from viruses have been prepared, incorporating various genes of interest. In each of these constructs, the viral genome was modified so as to make the virus incapable of autonomously replicating in the infected cell. Thus, the constructs described in the prior art are viruses which are defective for certain regions of their genome which are essential for replication. In particular, as regards adenoviruses, the first-generation constructs exhibit a deletion in/of the E1 region, which is essential for viral replication, at the level of which the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Moreover, to enhance the properties of the vector, it has been proposed to create other deletions or modifications in the adenovirus genome. Thus, a heat-sensitive point mutation was introduced into the ts125 mutant, making it possible to inactivate the 72 kDa DNA binding protein (DBP) encoded by the E2 region (Van der Vliet et al., 1975). Other vectors comprise a deletion of another region essential for the viral replication and/or propagation, the E4 region. Adenoviral vectors in which the E1 and E4 regions are deleted have highly reduced transcription background noise and viral gene expression. Such vectors have been described, for example, in applications WO94/28152, WO95/02697, WO96/22378. Moreover, vectors carrying a modification at the level of the IVa2 gene have also been described (WO96/10088). In addition, so-called “minimum adenovirus” or “pseudo-adenovirus” vectors (or alternatively Ad&Dgr;) containing only the regions necessary in cis for the production of the virus (ITR and encapsidation sequences) and lacking any coding viral sequence have also been described (WO94/12649, WO94/28152, WO95/02697), although their production remains very difficult, as explained below.
As regards the AAVs, the vectors described generally lack the entire coding regions Rep and Cap, which are replaced by nucleic acids of interest.
In the recombinant vectors derived from retroviruses, the gag, pol and env genes are generally deleted, completely or in part, and replaced by a heterologous nucleic acid sequence of interest. Moreover, the recombinant retroviruses may comprise modifications at the level of the LTRs in order to suppress the transcriptional activity, as well as large encapsidation sequences, comprising part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639).
Given their defective character in relation to the replication, the production of these various recombinant viruses involves the possibility of transcomplementing the functions deleted from the genome. The transcomplementation is precisely the
Leblois-Prehaud Hélène
Perricaudet Michel
Vigne Emmanuelle
Yeh Patrice
Aventis Pharma S.A.
Stucker Jeffrey
Wiley Rein & Fielding LLP
Winkler Ulrike
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