Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
1998-12-16
2002-11-26
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S320100
Reexamination Certificate
active
06485958
ABSTRACT:
The present invention relates to a new process for the production of recombinant adenoviruses. It also relates to the purified viral preparations produced according to this process.
Adenoviruses exhibit certain properties which are particularly advantageous for use as vector for the transfer of genes in gene therapy. In particular, they have a fairly broad host spectrum, are capable of infecting quiescent cells, do not integrate into the genome of the infected cell, and have not been associated, up until now, with major pathologies in man. Adenoviruses have thus been used to transfer genes of interest into the muscle (Ragot et al., Nature 361 (1993) 647), the liver (Jaffe et al., Nature genetics 1 (1992) 372), the nervous system (Akli et al., Nature genetics 3 (1993) 224), and the like.
Adenoviruses are viruses with a linear double-stranded DNA having a size of about 36 (kilobases) kb. Their genome comprises especially an inverted repeat sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes. The principal early genes are contained in the E1, E2, E3 and E4 regions. Among these, the genes contained in the El region in particular are necessary for viral propagation. The principal late genes are contained in the L1 to L5 regions. The genome of the Ad5 adenovirus has been completely sequenced and is accessible on a database (see especially Genebank M73260). Likewise, parts or even the whole of other adenoviral genomes (Ad2, Ad7, Ad12 and the like) have also been sequenced.
For their use in gene therapy, various vectors derived from adenoviruses have been prepared, incorporating various therapeutic genes. In each of these constructs, the adenovirus was modified so as to render it incapable of replicating in the infected cell. Thus, the constructs described in the prior art are adenoviruses from which the El region has been deleted, which region is essential for the viral replication and 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) (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. The E4 region is indeed involved in the regulation of the expression of the late genes, in the stability of the late nuclear RNAs, in the abolition of the expression of the host cell proteins and in the efficacy of replication of the viral DNA. Adenoviral vectors in which the E1 and E4 regions are deleted therefore possess a transcriptional background noise and an expression of viral genes which are highly reduced. Such vectors have been described for example in Applications WO 94/28152, WO 95/02697, PCT/FR96/00088). In addition, vectors carrying a modification at the level of the IVa2 gene have also been described (WO 96/10088).
The recombinant adenoviruses described in the literature are produced from different adenovirus serotypes. Indeed, various adenovirus serotypes exist whose structure and properties vary somewhat, but which exhibit a comparable genetic organization. More particularly, the recombinant adenoviruses may be of human or animal origin. As regards the adenoviruses of human origin, there may be mentioned preferably those classified in group C, in particular the adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Ad12). Among the various adenoviruses of animal origin, there may be mentioned preferably the adenoviruses of canine origin, and especially all the strains of the CAV2 adenoviruses [Manhattan strain or A26/61 (ATCC VR-800) for example]. Other adenoviruses of animal origin are cited especially in application WO 94/26914 incorporated into the present by reference.
In a preferred embodiment of the invention, the recombinant adenovirus is a group C human adenovirus. More preferably, it is an Ad2 or Ad5 adenovirus.
The recombinant adenoviruses are produced in an encapsidation line, that is to say a cell line capable of complementing in trans one or more functions deficient in the recombinant adenoviral genome. One of these lines is for example the line 293 into which a portion of the adenovirus genome has been integrated. More precisely, the line 293 is a human embryonic kidney cell line containing the left end (about 11-12%) of the serotype 5 adenovirus (Ad5) genome, comprising the left ITR, the encapsidation region, the E1, including E1a and E1b, region, the region encoding the pIX protein and a portion of the region encoding the pIVa2 protein. This line is capable of transcomplementing recombinant adenoviruses defective for the E1 region, that is to say lacking all or part of the E1 region, and of producing viral stocks having high titres. This line is also capable of producing, at a permissive temperature (32° C.), virus stocks comprising, in addition, the heat-sensitive E2 mutation. Other cell lines capable of complementing the E1 region have been described, based especially on human lung carcinoma cells A549 (WO 94/28152) or on human retinoblasts (Hum. Gen. Ther. (1996) 215). Moreover, the lines capable of transcomplementing several functions of the adenovirus have also been described. In particular, there may be mentioned lines complementing the E1 and E4 regions (Yeh et al., J. Virol. 70 (1996) 559; Cancer Gen. Ther. 2 (1995) 322; Krougliak et al., Hum. Gen. Ther. 6 (1995) 1575) and the lines complementing the E1 and E2 regions (WO 94/28152, WO 95/02697, WO 95/27071).
Recombinant adenoviruses are usually produced by introducing viral DNA into the encapsidation line, followed by lysis of the cells after about 2 or 3 days (kinetics of the adenoviral cycle being from 24 to 36 hours). After the lysis of the cells, the recombinant viral particles are isolated by caesium chloride gradient centrifugation.
For the carrying out of the process, the viral DNA introduced may be the complete recombinant viral genome, optionally constructed in a bacterium (WO 96/25506) or in a yeast (WO 95/03400), transfected into the cells. It may also be a recombinant virus used to infect the encapsidation line. The viral DNA may also be introduced in the form of fragments each carrying a portion of the recombinant viral genome and a zone of homology allowing, after introduction into the encapsidation cell, the viral genome to be reconstituted by homologous recombination between the various fragments. A conventional process for the production of adenoviruses thus comprises the following steps: the cells (for example the cells 293) are infected in a culture dish with a viral prestock in an amount of from 3 to 5 viral particles per cell (Multiplicity of Infection (MOI) =3 to 5), or transfected with the viral DNA. The incubation then lasts from 40 to 72 hours. The virus is then released from the nucleus by cell lysis, generally by several successive thawing cycles. The cellular lysate obtained is then centrifuged at low speed (2000 to 4000 rpm) and the supernatant (clarified cellular lysate) is then purified by centrifugation in the presence of caesium chloride in two steps:
A first rapid centrifugation of 1.5 hours on two caesium chloride layers of densities 1.25 and 1.40 flanking the virus density (1.34) so as to separate the virus from the proteins in the medium;
A second longer gradient centrifugation (from 10 to 40 hours depending on the rotor used), which constitutes the actual and sole virus purification step.
Generally, after the second centrifugation step, the virus band is predominant. Two fine, less dense bands are nevertheless observed whose examination by electron microscopy has shown that they are empty or broken viral particles in the case of the more dense band, and viral subunits (pentons, hexons) for the less dense band. After this step, the virus is harves
Blanche Francis
Guillaume Jean-Marc
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Mosher Mary E.
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