Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-12-07
2003-10-07
Nguyen, Dave T. (Department: 1633)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S091400, C435S320100, C435S325000, C435S455000
Reexamination Certificate
active
06630322
ABSTRACT:
The present invention relates to circular and replicative DNA molecules which can be used in gene therapy. The invention also describes a particularly efficient method for their generation in situ from a corresponding viral vector.
Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression and the like) by the introduction of genetic information into the affected organ or cell.
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, various techniques exist, among which are different transfection techniques involving vectors of different types. They may be naturally occurring or synthetic chemical and/or biochemical vectors, on the one hand, or viral vectors on the other. As examples of viral vectors, there may be mentioned especially the 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), of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), liposomes (Fraley et al., J.Biol.Chem. 255 (1980) 10431) and the like. However, their use involves especially the possibility of producing large quantities of DNA of pharmacological purity.
Viral vectors (retroviruses, adenoviruses, adeno-associated viruses and the like) are very efficient compared to the chemical and/or biochemical vectors previously described, especially for crossing the membranes. Among these viruses, the adenoviruses exhibit most particularly advantageous properties for use in gene therapy. In particular, they have a fairly broad host spectrum, are capable of transducing dividing cells or quiescent cells and the adenovirus genome persists in extrachromosomal form; furthermore, they have so far not been associated with major pathologies in man. On the other hand, the use of retroviruses whose genome randomly integrates into the genome of the infected cell is limited to dividing cells. The adenoviruses have thus been used to transfer genes of interest into quiescent muscle (myotubes; Ragot et al., Nature 361 (1993) 647)), hepatic (Jaffe et al., Nature genetics 1 (1992) 372), nerve (Akli et al., Nature genetics 3 (1993) 224) and epithelial bronchial (Rosenfeld et al., 1992) cells and the like. However, in rapidly renewable cells, a gradual loss of expression of the transgene by a dilution effect during cell divisions is observed.
The improvement of adenoviral vectors and the development of new generations of vectors have been aimed at reducing the residual potential risks of pathogenicity as well as the potential risks of immunogenicity linked to the replication of the vector, the recombination of its genome and the expression of viral proteins.
To avoid such risks as much as possible, the viral vector constructs currently proposed are modified so as to make the said vectors incapable of autonomously replicating in the target cell. They are said to be defective. Generally, the genome of the defective viruses therefore lacks at least sequences necessary for the replication of the said virus in the infected cell. Thus, in the specific case of adenoviruses, the constructs described in the prior art are adenoviruses deleted of E1 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). Other constructs comprise a deletion at the level of the E1 region and of a non-essential part of the E4 region (WO94/12649), or a modified genomic organization (FR 94 13355).
However, the risk of recombinations generating replicative viral particles or transcomplementations in vivo by E1-type cellular functions remains during the production of these defective viral vectors. It is clear that the use in gene therapy of vectors thus contaminated may have very damaging consequences such as, for example, inducing a viral propagation and causing an uncontrolled dissemination with risks of an inflammatory reaction and of an immune response directed against the viral proteins, and the like.
Moreover, the enhancement of the stability of the expression of the transgene in the transduced cells by the adenoviral vector, and more particularly in dividing cells, remains a major problem to be solved. Consequently, a real need for transfer vectors which would essentially manifest the advantages of each of the vectors described above, namely good transfection properties based, for example, on those of the viral vectors and in particular those of the adenoviruses and a perfect safety resulting in particular in an absence of generation of replicative viral particles in vivo, a risk which is inexistant with plasmids or non-viral vectors, remains up until now in gene therapy.
The object of the present invention is precisely to propose a new concept of gene transfer which meets the abovementioned requirements.
The present invention consists especially in the in situ generation, via a viral vector, of therapeutic replicative circular DNA molecules which are advantageous from the point of view of the stability of expression of the transgene and of safety. Indeed, they are free of any sequence of the viral genome capable of inducing an inflammatory-type immune response as well as a specific response directed against the viral proteins which may have a deleterious effect on the body and limit the duration of expression of the transgene.
The subject of the present invention is thus replicative circular DNA molecules comprising at least:
(i) one or more genes of interest with the sequences necessary for their expression,
(ii) a replication origin functional in mammalian cells, and
(iii) a sequence resulting from the site-specific recombination between two sequences recognized by a recombinase.
The present invention stems in particular from the development of a process and of specific constructs which are particularly efficient for the production of these therapeutic DNA molecules. More particularly, the process according to the invention consists in the production of the therapeutic DNA molecules defined above from a viral vector.
Surprisingly, the applicant has thus demonstrated that it is possible to generate in situ, from a viral vector and by site-specific recombination, a circular DNA molecule with a therapeutic and replicative character. Furthermore, in an advantageous embodiment, the transgene and the replication origin are inactive in the viral vector, their activity being dependent on the site-specific recombination event.
Still more advantageously, the recombination event is induced conditionally by the expression of recombinase, thereby offering a high level of control over the expression of the gene of interest and over the replication of the episomal molecules produced.
Such a procedure is particularly advantageous from the therapeutic point of view:
it takes advantage of the good transfection capacities generally manifested by the viral vectors compared to the non-viral vectors,
it considerably reduces the risks of viral contamination, of local inflammatory reaction as well as of antiviral immune response, given the small quantity of viral vectors used. The latter is introduced only in a proportion necessary for the generation of the therapeutic DNA molecule,
it makes it possible to broaden the field of application of some viral vectors: thus, the adenoviral vectors have limited applications in proliferative cells such as haematopoietic stem cells. The present invention makes it possible to exploit their infectivity in order to generate, in proliferative cells, stable replicative circular molecules.
The DNA molecule as claimed therefore has the ability to efficiently ensure the transfer, into the intended cells, of the therapeutic gene(s) which it contains.
To do this, it contains a replication origin characterized especially by the fact that it is functional in mammalian and human cells. Advantageously, the replication origin used is a conditio
Leblois-Prehaud Hélène
Perricaudet Michel
Yeh Patrice
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Nguyen Dave T.
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