Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-08-08
2001-07-17
Salimi, Ali (Department: 1643)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S320100, C435S069100, C435S235100, C435S252300
Reexamination Certificate
active
06261807
ABSTRACT:
The present invention relates to a novel process for preparing recombinant adenoviruses and to the use of these adenoviruses in gene therapy. It also relates to prokaryotic plasmids which are suitable for preparing these adenoviruses.
Gene therapy consists in correcting a deficiency or an anomaly (mutation, aberrant expression, etc.) by introducing genetic information into the affected cell or organ. This genetic information can be introduced either in vitro, into a cell which has been removed from the organ, with the modified cell then being reintroduced into the organism, or directly in vivo into the appropriate tissue. A variety of techniques exist with regard to this second case, including various transfection techniques involving complexes of DNA and DEAE dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), and of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), etc. More recently, the use of viruses as vectors for transferring genes has come to be seen as a promising alternative to these physical transfection techniques. In this regard, various viruses have been tested for their ability to infect specific cell populations. These viruses include, in particular, retroviruses (RSV, EMS, MMS, etc.), the HSV virus, adeno-associated viruses and adenoviruses.
Of these viruses, the adenoviruses exhibit certain properties which are attractive for use in gene therapy. In particular, they have a relatively wide host range, are able to infect quiescent cells, do not integrate into the genome of the infected cell and have not to date been associated with serious pathologies in man. As a result, adenoviruses have been used for transferring relevant genes into muscle (Ragot et al., Nature 361 (1993) 647), liver (Jaffe et al., Nature genetics 1 (1992) 372), the nervous system (Akli et al., Nature genetics 3 (1993) 224), etc.
The adenoviruses are viruses which have a linear double-stranded DNA of a size of approximately 36 kb. Their genome encompasses, in particular, an inverse repeat (ITR) sequence at each end, an encapsidation sequence (Psi), early genes and late genes (cf. FIG.
1
). The principal early genes are contained in the regions E1, E2, E3 and E4. Of these, the genes contained in the E1 region are required for viral propagation. The principal late genes are contained in the regions L1 to L5. The genome of the adenovirus Ad5 has been sequenced in its entirety and is available on database (see, in particular, Genebank M73260). Similarly, parts, if not the whole, of other adenoviral genomes (Ad2, Ad7, Ad12, etc.) have also been sequenced.
In view of the properties of the adenoviruses as mentioned above, and in view of the fact that it is possible to obtain high viral titres, these viruses have already been used for transferring genes in vivo. To this end, a variety of vectors have been prepared which derive from adenoviruses and which incorporate various genes (&bgr;-gal, OTC, a-1AT, cytokines, etc.). In each of these constructs, the adenovirus has been modified in such a way as to render it unable to replicate following gene transfer. Thus, the constructs described in the prior art are adenoviruses in which the E1 and, possibly, E3 regions are deleted and into the sites of which the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Other constructs contain a deletion within the E1 region and of a non-essential part of the E4 region (WO94/12649), or a modified genomic organization (FR 94 13355).
Nevertheless, industrial and therapeutic exploitation of the adenoviruses is still limited by the current methods for preparing these recombinant viruses.
Thus, the adenoviruses are produced by transfecting the DNA of the recombinant virus into a competent encapsidating cell line. This transfection can be a straightforward transfection, when a construct is available which carries the whole of the genome of the recombinant virus, or, as is more often the case, a co-transfection of several DNA fragments which supply the different parts of the recombinant viral genome. In this case, the procedure involves one or more steps of homologous recombination between the different constructs in the encapsidating cell line in order to generate the DNA of the recombinant virus. In order to carry out one or other of these methods, it is necessary, therefore, to have available appropriate constructs which carry all or parts of the genome of the recombinant adenovirus which it is desired to produce.
Various methods, which are described in the prior art, exist for preparing these constructs in vitro. The technique which is most generally employed consists in isolating the viral DNA and then modifying it in vitro using standard molecular biological methods (digestion, ligation, etc.). The constructs which are obtained are then purified and used for transfecting the encapsidating lines. However, this technique involves producing stocks of virus and purifying viral DNA for each construct or for any manipulation of the DNA of the recombinant virus.
Another technique is based on using a plasmid which carries a part of the genome of the recombinant virus and which is co-transfected with a virus which supplies the part of the genome which is lacking. Nevertheless, as indicated above, this method involves recombination in the encapsidating line and the availability of an appropriate supplementary virus. In order to remedy these drawbacks, it has been proposed that prokaryotic plasmids be employed for preparing the viral DNAs which are to be used for the transfection. In particular, Bett et al. (PNAS 91 (1994) 8802) describes the construction of a plasmid which replicates in
E. coli
and which contains a modified adenoviral genome (plasmid pBHG10). More precisely, this plasmid carries an adenoviral genome in which the E1, E3 and Psi regions are deleted, which is circularized by joining the ITR sequences, and which encompasses a part of plasmid pBR322, which part is inserted within the 188-1339 region of the genome of the adenovirus. While this plasmid can be replicated in E. coli and manipulated for inserting genes of interest, it still suffers from drawbacks. In particular, its use for producing virus involves employing a second plasmid which at least supplies the left-hand region of the viral genome. Other plasmids of this type, suffering from similar drawbacks, have been described, for example by Graham (EMBOJ. 3(12) (1984) 2917). These plasmids also require a recombination step to be carried out in the encapsidating cells
In particular, these technologies require different plasmids to be used in order to enable manipulation to be carried out in different regions of the adenoviral genome. Furthermore, the recombinant virus is only obtained following homologous recombination of the genomic fragments which have been co-transfected into the encapsidating cells. This accordingly limits the frequency with which the recombinant viruses are obtained, and the overall process is slow.
There exists, therefore, within the prior art, a clear need to have available suitable plasmids, which can easily be manipulated and amplified in vitro, for preparing recombinant adenoviral genomes. It is also important that the genomes which are produced in this way should be practically devoid of regions which derive from the plasmid and which are able (i) to induce an immune response, (ii) to encode resistance proteins and (iii) to reduce the capacity of the virus as a vector.
The present invention enables these drawbacks to be remedied. Thus, the present invention describes plasmids which meet all these requirements and consequently permit rapid and efficient clonal production of recombinant adenoviruses which can be used therapeutically.
More specifically, the invention relates, in the first instance, to a prokaryotic plasmid which encompasses a recombinant adenovirus genome which is flanked by one or more restriction sites
Crouzet Joel
Naudin Laurent
Orsini Cecile
Vigne Emmanuelle
Yeh Patrice
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Rhone-Poulenc Rorer S.A.
Salimi Ali
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