Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-12-19
2002-07-16
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S235100, C435S456000, C435S069100
Reexamination Certificate
active
06420170
ABSTRACT:
The present invention relates to new viral vectors, to their preparation and to their uses. It also relates to pharmaceutical compositions containing the said viral vectors.
Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expressions, and the like) by introducing genetic information into the cell or organ affected. This genetic information may be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. In this second case, different techniques exist, including various techniques of transfection 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), and the like.
More recently, the use of viruses as vectors for gene transfer has been seen to be a promising alternative to these physical transfection techniques. In this connection, different viruses have been tested for their capacity to infect certain cell populations. This applies especially to retroviruses (RSV, HMS, MMS, and the like), the HSV virus, adeno-associated viruses and adenoviruses.
As regards adenoviruses more especially, the latter are linear double-stranded DNA viruses approximately 36 kb in size. Their genome comprises, in particular, an inverted sequence (ITR) at each end, an encapsidation sequence, early genes and late genes (see FIG.
1
). The main early genes are contained in the E1, E2, E3 and E4 regions. Among them, the genes contained in the E1 region (E1a and E1b, in particular) are necessary for viral replication. The E4 and L5 regions, for example, are involved in viral propagation, and the main late genes are contained in the L1 to L5 regions. The Ad5 adenovirus genome has been sequenced completely and is available on a database (see, in particular, Genebank M73260). Similarly, portions, or in some cases the whole, of the genome of adenoviruses of different serotypes (Ad2, Ad7, Ad12, and the like) have also been sequenced. These viral vectors advantageously display a fairly broad host range, are capable of infecting quiescent cells, do not integrate in the genome of the infected cell and have not been hitherto associated with significant pathologies in man. In view of their properties, they have already been used for gene transfer in vivo. To this end, different vectors derived from adenoviruses have been prepared, incorporating different genes (&bgr;-gal, OTC, &agr;
1
-AT, cytokines, and the like).
Naturally, all of these viral vectors contain numerous viral genes whose expression is, on the other hand, not desirable in gene therapy. It is essential to control in vivo the non-expression of wild-type viral genes and/or of proteins which are derived therefrom and which are liable to induce an immune and/or inflammatory response which is undesirable or even thoroughly deleterious with respect to the body being treated.
For these purposes, the viral vector constructions currently proposed are modified so as to render the said vectors incapable of replicating autonomously in the target cell. They are said to be defective. Generally, the genome of defective viruses hence lacks at least the sequences necessary for replication of the said virus in the infected cell. These regions may be either removed (wholly or partially), or rendered non-functional, or replaced by other sequences, and in particular by a sequence coding for a molecule of therapeutic interest. Preferably, the defective virus nevertheless retains the sequences of its genome which are necessary for encapsidation of the viral particles.
In the particular case of recombinant adenoviruses, the constructions described in the prior art are generally adenoviruses from which the E1 (E1a and/or E1b) and possibly E3 regions have been deleted, in which regions the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Other constructions contain a deletion in the E1 region and of a non-essential portion of the E4 region (WO 94/12649). These defective recombinant adenoviruses may be prepared in different ways, employing or otherwise a competent cell line capable of complementing all the defective functions essential for replication of the recombinant adenovirus. At the present time, the vectors derived from adenoviruses are generally produced in a complementation line (line 293) in which a portion of the adenovirus genome has been integrated. More specifically, line 293 contains the left-hand end (approximately 11-12%) of the adenovirus serotype 5 (Ad5) genome, comprising the left-hand ITR, the encapsidation region and the E1 region, including E1a, E1b and a portion of the region coding for the pIX protein. This line is capable of trans-complementing recombinant adenoviruses which are defective for the E1 region, that is to say lacking all or part of the E1 region, necessary for replication.
However, during the production of these defective viral vectors, it is not possible to rule out completely the possibility of recombinations generating replicative viral particles, or in vivo trans-complementations by E1 type cellular functions. It is obvious that this type of event is completely incompatible with their subsequent use in gene therapy. The presence in vivo of replicative viral particles may have highly deleterious consequences, such as, for example, the induction of a viral propagation and production of an uncontrolled dissemination with risks of inflammatory reaction, recombination, and the like.
Concomitantly, it is essential to prevent in vivo the expression of corresponding viral proteins. Although the latter do not necessarily display a toxic character with respect to the cell, they are also highly undesirable since they are also liable to induce immune system responses of the inflammation type and/or fevers which are detrimental to the body being treated (D. Y. Schwarz, (1995), P.N.A.S. 92, 1401-1405; J. F. Engelhardt, (1994), Human Gene Therapy, 5, 1217-1229 and (1994) P.N.A.S. 91, 6196-6200; Y. Yang, (1994), Immunity, 1, 433-442, (1995) J; Virol., 69, 2004-2015 and Nature Genetics, (1994) 7, 362-369).
The objective of the present invention is specifically to provide an approach enabling these drawbacks to be remedied, and the invention proves most especially useful for preparing batches of adenovirus type viruses displaying enhanced safety since, in particular, they lack replicative viral particles.
Unexpectedly, the Applicant demonstrated that it was possible, using a novel promoter system, to control effectively the expression of viral gene, which expression is effective in vitro during viral production but, on the other hand, subsequently ineffective in vivo when the said recombinant viruses are used therapeutically.
More specifically, the present invention relates to a recombinant adenovirus in which the expression of at least one homologous or heterologous gene of viral origin is controlled by an inducible promoter.
For the purposes of the present invention, inducible promoter is understood to mean any promoter whose activity is initiated by the presence of an external chemical and/or biological agent, which agent, in the context of the present invention, displays, in addition, low or even zero toxicity. “External” is understood to mean that the chemical and/or biological agent does not naturally exist in the cells treated with the claimed adenovirus.
As inducible promoters capable of being employed according to the present invention, traditional promoters such as those responding to heavy metals (CRC Boca Raton, Fla. (1991), 167-220; Brinster et al. Nature (1982), 296, 39-42), to thermal shocks, to hormones (Lee et al. P.N.A.S. USA (1988), 85, 1204-1208; (1981), 294, 228-232; Klock et al. Nature (1987), 329, 734-736; Israël and Kaufman, Nucleic Acids Res. (1989), 17, 2589-2604) or to chemical ag
Latta Martine
Orsini Cecile
Perricaudet Michel
Prost Edouard
Vigne Emmanuelle
Aventis Pharma S.A.
Guzo David
Wiley Rein & Fielding LLP
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