Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-12-11
2002-06-25
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S320100, C435S455000, C536S023100, C536S023700
Reexamination Certificate
active
06410273
ABSTRACT:
The present invention relates to the preparation of DNA, in particular plasmid DNA. It relates more especially to the production of bacterial plasmid DNA which can be used in gene therapy, in plasmid, supercoiled or relaxed minicircle or linear form, and whose immunogenic properties are reduced or even eliminated. The invention also relates to microorganisms which can be used for the production of DNA, as well as to pharmaceutical compositions.
Gene therapy consists in correcting a deficiency or an abnormality by introducing genetic information into the affected cell or organ. This information may be introduced either in vitro into a cell extracted from the organ and then reinjected into the body, or in vivo, directly into the target tissue. Being a negatively charged, high molecular weight molecule, DNA has difficulties in passing spontaneously through the phospholipid cell membranes. Various vectors are hence used in order to permit gene transfer: viral vectors on the one hand, natural or synthetic chemical and/or biochemical vectors on the other hand. Viral vectors (retroviruses, adenoviruses, adeno-associated viruses, etc.) are very effective, in particular for passing through the membranes, but present a number of risks, such as pathogenicity, recombination, replication, immunogenicity, etc. Chemical and/or biochemical vectors enable these risks to be avoided (for reviews, see Behr, 1993, Cotten and Wagner, 1993). They are, for example, cations (calcium phosphate, DEAE-dextran, etc.) which act by forming precipitates with DNA, which precipitates can then be “phagocytosed” by the cells. They can also be liposomes in which the DNA is incorporated and which fuse with the plasma membrane. Synthetic gene transfer vectors are generally cationic lipids or polymers which complex DNA and form therewith a particle carrying positive surface charges. These particles are capable of interacting with the negative charges of the cell membrane and then of crossing the latter. As examples of such vectors, dioctadecylamidoglycylspermine (DOGS, Transfectam™) or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA, Lipofectin™) may be mentioned. Chimeric proteins have also been developed: they consist of a polycationic portion which condenses the DNA, linked to a ligand which binds to a membrane receptor and gives rise to the complex in the cells by endocytosis. It is thus theoretically possible to “target” a tissue or certain cell populations in order to improve the in vivo bioavailability of the transferred gene.
The plasmids currently used in gene therapy generally carry (i) an origin of replication, (ii) a marker gene such as a gene for resistance to an antibiotic (kanamycin, ampicillin, etc.) and (iii) one or more transgenes with sequences necessary for their expression (enhancer(s), promoter(s), polyadenylation sequences, etc.). This type of plasmid is, for example, currently used in gene therapy in the context of clinical trials such as the treatment of melanoma, Nabel et al., 1992, or in the context of experimental studies.
The use of plasmid DNA in gene therapy creates, however, a number of problems.
In particular, it entails the possibility of producing large amounts of DNA of pharmacological purity. In effect, in these gene therapy techniques, the medicament consists of the DNA itself, and it is essential to be able to manufacture, in appropriate amounts, DNAs having suitable properties for therapeutic use in man. In this connection, various methods of production and/or purification have been described in the prior art, enabling the quality of the plasmid DNA to be improved (PCT/FR95/01468; FR96 03519).
Moreover, the use of DNA carrying genes for resistance to antibiotics or functional origins of replication can also have some drawbacks, linked, in particular, to their dissemination in the body. Various approaches have also been developed to limit these drawbacks (PCT/FR96/00274, FR95 10825).
Another drawback of the plasmid DNAs used hitherto lies in their origin. They are, in effect, molecules produced essentially in prokaryotic organisms (bacteria) or lower eukaryotic organisms (yeasts), which potentially possess motifs which are immunogenic in man. The immunological properties of DNA are still relatively unknown. Bacterial DNA in mice leads i) to the synthesis of antibodies that recognize double-stranded and single-stranded bacterial DNA, which has made immunization possible, but do not react with mammalian double-stranded DNA, and ii) to the stimulation of macrophage and cytokine production (D. Pisetsky “The Immunologic Properties of DNA”, J. Immunol. 156 (1996) 1). The DNA macromolecule is thus said to be immunogenic. Moreover, a macromolecule can also lead to a stimulation of the immune system without being immunogenic (for example, foreign body leading to a cell-mediated immune response). The first evidence suggesting that bacterial DNA leads to an immune response was described by Pisetsky et al. (1991 J. Immunol. 147 p.1759). They showed that the DNA of three bacterial species can stimulate the proliferation of mouse lymphocytes, whereas the DNA extracted from three animal species does not lead to this stimulation. Then, Yamamoto et al. (1992 Microbiol. Immunol. 36 p.983) observed that the bacterial DNA of six species leads in the spleen cells of BALB/c mice to an increase in “natural killer” NK activity and to the induction of interferon production. However, the DNA extracted from ten vertebrate species does not lead to any of these responses. In addition, Krieg et al. reported in 1995 (Nature vol.374 p.546) that a genomic DNA fragment of
E. coli
induces in vitro the proliferation of murine B cells and the secretion of IgM immunoglobulins, whereas this same bacterial DNA treated in vitro with a CpG methylase does not induce such a response. Krieg et al. also showed that, in the presence of unmethylated DNA, interferon-&ggr; is produced, and acts as a factor which costimulates the differentiation of B cells by modulating the production of IL-6 by the B cells (Krieg et al. 1996 J. Immunol. 156 p.558). Furthermore, an oligonucleotide possessing an unmethylated CpG motif and flanked at the 5′ end by 2 purines and at the 3′ end by 2 pyrimidines leads in vivo to a coordinated secretion of interleukins IL-6 and IL-12 and of interferons-&ggr; by NK cells (IFN-&ggr;), B cells (IL-6 and IL-12) and CD4
+
T lymphocytes (IL-6 and IFN-&ggr;) (Krieg et al. 1996 Proc. Natl. Acad. Sci. USA 93 p.2879).
The plasmid DNA used to date in gene therapy is essentially produced in prokaryotic cells, and hence displays a methylation profile comparable to that of bacterial genomic DNA. It has, in addition, been demonstrated that plasmid DNA which has been injected into muscle or into the liver, and then extracted, retains the prokaryotic methylation profile (Wolf et al. 1992 Hum. Mol. Genet. 1 p.363; Malone et al. 1995 J. Biol. Chem. 269 p.29903). As a result, the bacterial plasmid DNA used has considerable potential for stimulation of the immune system.
Hence it would be especially advantageous to be able to have at one's disposal plasmid DNA having immunological properties which are reduced or even eliminated. It would also be especially advantageous to be able to have at one's disposal a method that enables plasmid DNAs of this type to be produced on a scale which is compatible with an industrial utilization.
The present invention provides a solution to these problems. The Applicant directed its attention, in effect, to the immunogenic properties of bacterial DNA. The Applicant has now developed a method which enables pharmaceutical grade plasmid DNAs, potentially lacking undesirable immunogenic effects, to be produced. The Applicant also showed that the methylation of some residues of of DNA enabled the immunogenic potential of plasmid DNAs to be reduced without affecting their capacity to transfect cells and to express a nucleic acid of interest therein.
One aspect of the invention is to prepare DNAs, in particular plasmid DNAs, of therape
Cameron Beatrice
Crouzet Joël
Aventis Pharma S.A.
Chen Shin-Lin
Clark Deborah J. R.
Wiley Rein & Fielding LLP
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