Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-03-21
2001-03-13
Campell, Bruce R. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S455000, C435S456000, C435S325000, C514S04400A, C424S093210
Reexamination Certificate
active
06200798
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to new viral vectors, their preparation and their use in gene therapy. It also relates to the pharmaceutical compositions containing the said viral vectors. More particularly, 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 the introduction of a genetic information into the cell or affected organ. This genetic information can be introduced either in vitro or in 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, various techniques exist, among which various transfection techniques involving complexes of DNA and &Dgr;EAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), 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 the transfer of genes has appeared as a promising alternative to these physical transfection techniques. In this respect, various viruses have been tested for their capacity to infect certain cellular populations. In particular, the retroviruses (RSV, HMS, MMS and the like), the HSV virus, the adeno-associated viruses and the adenoviruses.
Among these viruses, adenoviruses present some advantageous properties for a use in gene therapy. Especially, 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 to date with major pathologies in man. Adenoviruses are viruses with linear double-stranded DNA of a size of about 36 kb. Their genome comprises especially an inverted repeat sequence (ITR) at their end, an encapsidation sequence, early genes and late genes (cf FIG.
1
). The principal early genes are the E1 (E1a and E1b), E2, E3 and E4 genes. The principal late genes are the L1 to L5 genes.
Given the properties of the abovementioned adenoviruses, the latter have already been used for the transfer of genes in vivo. To this end, various vectors derived from adenoviruses have been prepared, incorporating various genes (&bgr;-gal, OTC, a-1AT, cytokines and the like). In each of these constructs, the adenovirus was modified so as to render it incapable of replication in the infected cell. Thus, the constructs described in the prior art are adenoviruses from which there have been deleted the E1 (E1a and/or E1b) and optionally E3 regions 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). Nevertheless, the vectors described in the prior art have numerous disadvantages which limit their exploitation in gene therapy. In particular, all these vectors contain numerous viral genes whose expression in vivo is not desirable within the framework of a gene therapy. Furthermore, these vectors do not permit the incorporation of very large DNA fragments which may be necessary for certain applications.
SUMMARY OF THE INVENTION
The present invention makes it possible to overcome these disadvantages. The present invention indeed describes new recombinant adenoviruses which can be used in gene therapy, especially for the transfer and expression of genes in vivo. In particular, the adenoviruses of the invention allow an efficient transfer and a lasting expression in vivo of a gene of interest, while limiting the risks of production of viral proteins, of transmission of the virus, of pathogenicity and the like.
The present invention consists more particularly in the construction of recombinant adinoviruses in which at least one specific viral gene, designated IVa2, has been made nonfunctional. The IVa2 gene is a small gene situated in the left-hand part of the adenovirus genome. It overlaps in part with the end of the E2B gene in particular, which makes its manipulation difficult. The IVa2 gene appears to play the role of activator of transcription of the late genes in the replicative cycle of the adenovirus. Its presence therefore allows or promotes the expression of the virus proteins which are necessary for the formation of the viral particles.
The applicant has now shown that it is possible to inactivate this gene in the adenovirus genome. The applicant has moreover shown that this inactivation does not affect the properties of the adenovirus as gene therapy vector, namely its high power to infect cells, especially human cells, and its capacity to efficiently transfer a gene of interest into the said cells. Furthermore, compared with the prior art vectors, the vectors described in the present invention have an improved capacity to incorporate genes of interest resulting from the inactivation, by deletion, of the IVa2 gene, and are in particular substantially less immunogenic since the expression of the late genes which may be present in the recombinant adenoviruses according to the invention is considerably reduced or even abolished.
The vectors of the invention are therefore particularly advantageous since they allow the incorporation of genes of interest of large size (greater than 8 kb and capable of growing up to more than 11 kb) without particularly affecting the titers obtained, and since they possess very few expressed viral regions, which substantially reduces or even suppresses the risks inherent in the use of viruses as vectors in gene therapy such as immunogenicity, pathogenicity, transmission, replication, recombination and the like. The present invention thus provides viral vectors which are particularly adapted to the transfer and expression in vivo of desired DNA sequences.
A first subject of the present invention therefore relates to a defective recombinant adenovirus in which the IVa2 gene at least is inactivated.
The adenoviruses are said to be defective when they are incapable of replicating autonomously in the infected cells. Generally, the genome of the adenoviruses according to the present invention is therefore devoid of at least the sequences necessary for the replication of the said virus in the infected cell. These regions can be either removed (completely or partly), or rendered nonfunctional, or substituted by other sequences and especially by a heterologous nucleic acid sequence.
As indicated above, the applicant has now shown that it is possible to inactivate the IVa2 gene in the genome of the adenovirus without affecting the desired properties of this virus. More particularly, for the preparation of the vectors of the invention, the IVa2 gene can be inactivated by various techniques known to persons skilled in the art, and especially by suppression, substitution, deletion and/or addition of one or more bases. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or alternatively by treating with mutagenic agents. The said genetic modification(s) may be localized in the coding part of the gene, or outside the coding region, and for example in the regions responsible for the expression and/or transcriptional regulation of the said genes. The inactivation of the gene may therefore manifest itself by the production of an inactive protein because of structural or conformational modifications, by the absence of production, by the production of a protein having an altered activity, or alternatively by the production of the natural protein at an attenuated level or according to a desired mode of regulation.
Among the mutagenic agents which can be used for inactivation, there may be mentioned for example physical agents such as energetic radiations (X-, &ggr;- and ultraviolet rays and the like), or chemical agents capable of reacting with various functional groups of the bases of the DNA, and for example alkylating agents [ethyl methanesulphonate (EMS), N-methyl
Orsini Cecile
Perricaudet Michel
Vigne Emmanuelle
Yeh Patrice
Campell Bruce R.
Martin Jill D.
Rhone-Poulenc Rorer SA
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