Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1997-04-15
2003-12-30
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S233100, C435S320100, C435S069100, C536S023720
Reexamination Certificate
active
06669942
ABSTRACT:
The present invention relates to new viral vectors, to their preparation and to their use in gene therapy. It also relates to pharmaceutical compositions containing the said viral vectors. More especially, the present invention relates to recombinant adenoviruses as vectors for gene therapy.
Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, 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.
Among these viruses, the adenoviruses display certain properties which are advantageous for use in gene therapy. In particular, they have a fairly broad host range, are capable of infecting resting cells and do not integrate in the genome of the infected cell. Adenoviruses are linear, double-stranded DNA viruses approximately 36 kb in size. Their genome comprises, in particular, an inverted repeat sequence (ITR) at their end, an encapsidation sequence, early genes and late genes (see FIG.
1
). The main early genes are the E1 (E1
a
and E1
b
), E2, E3 and E4 genes. The main late genes are the L1 to L5 genes.
In view of the properties of adenoviruses, mentioned above, the latter have already been used for in vivo gene transfer. To this end, different vectors derived from adenoviruses have been prepared, incorporating different genes (&bgr;-gal, OTC, &agr;-1AT, cytokines, and the like). In each of these constructions the adenovirus has been modified so as to render it incapable of replication in the infected cell. Thus, the constructions described in the prior art are adenoviruses from which the E1 (E1
a
and/or E1
b
) and, where appropriate, E3 regions have been deleted, in which regions a heterologous DNA sequence is inserted(Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161).
However, as in the case of all known viruses, the administration of a wild-type adenovirus induces a substantial immune response (Routes et al., J. Virol 65 (1991) 1450). This immunogenicity has also been observed following the administration of recombinant adenoviruses which are defective for replication (Yang et al., PNAS (1994) 4407). One of the major roles of the immune system consists, in effect, in destroying non-self or altered-self elements. The administration of a gene therapy factor of adenoviral origin introduces non-self units into the body. Similarly, cells infected with such a vector and expressing, as a result, an exogenous therapeutic gene become altered-self elements. Hence it is normal for the immune system to react against these vectors and infected cells as if they were foreign bodies. This immune response to infected cells constitutes a major obstacle to the development of these viral vectors, since (i) by inducing a destruction of the infected cells, it limits the period of expression of the therapeutic gene and hence the therapeutic effect, (ii) it induces a substantial concomitant inflammatory response, and (iii) it brings about rapid elimination of the infected cells after repeated injections. Thus, the expression of &bgr;-galactosidase encoded by a recombinant adenovirus administered in the muscle of immunocompetent mice is reduced to minimum levels 40 days after injection (Kass-Eisler et al., PNAS 90 (1993) 11498). Similarly, the expression of gene transferred by adenoviruses into the liver is significantly reduced after 4 months (Li et al., Hum. Gene Ther. 4 (1993) 403), and the expression of factor IX transferred by adenoviruses into hepatocytes of haemophilic dogs disappears 100 days after injection (Kay et al., PNAS 91 (1994) 2353).
Hence it would appear that the exploitation of vectors derived from adenoviruses in gene therapy entails the possibility of reducing the immune response to these vectors or the infected cells. This constitutes specifically the subject of the present invention. The present invention relates, in effect, to new vectors derived from adenoviruses displaying an immunogenicity which is greatly reduced or even eliminated. The vectors of the invention are hence especially suitable for gene therapy applications, in particular in man.
A first subject of the present invention relates to a defective adenovirus whose genome comprises a first recombinant DNA containing a therapeutic gene and a second recombinant DNA containing an immunoprotective gene.
The present invention is partly the outcome of the demonstration that it is possible to incorporate several genes of interest in adenoviruses, and to obtain a substantial expression of these different genes in the infected cells. The present invention is also the outcome of the construction of adenoviral vectors capable of incorporating several therapeutic genes under conditions permitting their optimal expression. It is also the outcome of the demonstration that coexpression in the infected cell of certain genes is capable of inducing an immunoprotective effect, and thus of leading the vectors of the invention and/or the infected cells to evade the immune system. The present invention thus provides viral vectors displaying immunological and therapeutic properties which are thoroughly advantageous for the purpose of their use in gene or cell therapy.
The recombinant DNAs according to the present invention are DNA fragments containing the gene in question (therapeutic or immunoprotective) and optionally signals permitting its expression, constructed in vitro and then inserted into the adenovirus genome. The recombinant DNAs used in the context of the present invention can be complimentary DNAs (cDNA), genomic DNAs (gDNA) or hybrid constructions consisting, for example, of a cDNA into which one or more introns might be inserted. They can also be synthetic or semi-synthetic sequences. These DNAs may be of human, animal, vegetable, bacterial, viral, and the like, origin. It is especially advantageous for cDNAs or gDNAs to be used.
The insertion of the genes in question in the form of recombinant DNAs according to the invention affords greater flexibility in the construction of the adenoviruses, and permits better control of the expression of the said genes.
Thus, the recombinant DNAs, (and hence the two genes of interest) incorporated in the adenoviral vectors according to the invention may be organized in different ways.
In the first place, they may be inserted at the same site of the adenovirus genome or at selected, different sites. In particular, the recombinant DNAs may be inserted at least partially in the E1, E3 and/or E4 regions of the adenovirus genome, replacing or in addition to viral sequences.
Next, they may each contain a transcription promoter, which may be identical or different. This configuration enables higher levels of expression to be obtained, and affords better control of the expression of the genes. In this case, the two genes may be inserted in the same orientation or in opposite orientations.
They may also constitute a single transcriptional entity. In this configuration the two recombinant DNAs are adjacent and positioned in such a way that both genes are under the control of a single promoter and give rise to a single premessenger
Lee Martin
Perricaudet Michel
Aventis Pharma S.A.
Salimi Ali R.
Wiley Rein & Fielding LLP
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