Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-06-30
2003-12-02
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S458000, C435S461000, C536S023100, C536S023200, C536S023500, C536S024100
Reexamination Certificate
active
06656734
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a composition which can be used for introducing a therapeutically active substance into a target cell, in particular a vertebrate cell, more specifically a mammalian cell. More specifically, the present invention relates to the use of this composition for preparing a vector for transferring a therapeutically active substance, in particular a polynucleotide, into a target cell.
BACKGROUND OF THE INVENTION
Genetic diseases are due, in particular, to dysfunction in the expression of specific genes or to the expression of non-functional mutated polypeptides. Cystic fibrosis, for example, is regarded as the most frequently occurring of the lethal genetic diseases (1/2000) with a mean life expectancy of from 25 to 30 years, and is characterized by substantial thickening of the secretions derived from the mucous membranes, by chronic pulmonary attacks and by insufficiency of the exocrine pancreas. Said disease is associated with disturbances in the transport of electrolytes, in particular chloride, across the epithelial membrane, which disturbances are the consequence of mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene (Rommens, Science 245 (1989), 1059-1066; Rosenfeld, Chest 109 (1996), 241-252). The therapeutic solution which appears to be most suitable for this type of disorder is that of transferring the gene encoding a functional CFTR polypeptide into the target cells in order to correct the observed cellular dysfunction. Within the context of this approach, also termed gene therapy, several authors indicate that the cells of the respiratory epithelium are a target of choice, especially as a result of its accessibility, in particular by means of intrapulmonary delivery, for example by instillation into the lungs. It has been shown that a level of expression of approximately 5 to 7% of that of the normal CFTR gene has to be obtained in the target cells for restoration of the electrolyte transport to be observed. Similarly, several publications describe the possibility of eliminating tumours or, failing that, of delaying their progress, by using the technique of transferring genes into the cancerous target cells. Several approaches have been considered, in particular transferring immunostimulatory genes (immunotherapy) which are able to induce or activate a cell-mediated immune response against the tumour; as examples of the therapeutic uses of such genes, mention may be made of the administration of genes which encode cytokines; transferring cytotoxic genes which confer toxicity on cells which express them, for example the tk gene of type 1 herpes simplex virus (HSV-1); or transferring anti-oncogenes, that is tumour suppressor genes, such as the retinoblastoma gene or the p53 gene, or polynucleotides which are able to inhibit the activity of an oncogene, such as antisense molecules or ribozymes which are able to degrade the specific messenger RNAs of the oncogenes.
Over the course of the last 30 years, a large number of tools have been developed for introducing various heterologous genes into cells, in particular mammalian cells. These different techniques can be divided into two categories. The first category relates to physical techniques such as microinjection, electroporation or particle bombardment which, although effective, are to a large extent limited to in vitro applications, the implementation of which is cumbersome and delicate. The second category involves techniques relating to molecular and cell biology, where the gene to be transferred is combined with a biological or synthetic vector which promotes the introduction of the said material.
The vectors which are currently most effective are viral vectors, in particular adenoviral or retroviral vectors. The techniques which have been developed are based on the natural properties which these viruses possess for traversing cell membranes, evading degradation of their genetic material and enabling their genome to penetrate into the nucleus. These viruses have already been the subject of a large number of studies, and some of them are already employed experimentally as gene vectors in man with a view, for example, to vaccination, immunotherapy or therapy which is aimed at compensating for a genetic deficiency. Nevertheless, this viral approach suffers from a large number of limitations, in particular on account of the restricted cloning capacity within the viral genome, of the risks of infectious viral particles which have been produced being disseminated in the host organism and in the environment, of the risk of artefactual mutagenesis resulting from insertion in the host cell, in the case of retroviral vectors, and of the fact that immune and inflammatory responses are powerfully induced in vivo during therapeutic treatment, thereby substantially limiting the number of administrations which can be envisaged (McCoy, Human Gene Therapy 6 (1995), 1553-1560; Yang, Immunity 1 (1996), 433-442). These many drawbacks, in particular within the context of using viral vectors in man, have led several groups to develop alternative systems for transferring polynucleotides.
Several non-viral methods are available at the present time. Mention may be made, for example, of coprecipitation with calcium phosphate, use of cationic lipids such as DOTMA (Feigner, PNAS 84 (1987), 7413-7417), DOGS or Transfectam (Behr, PNAS 86 (1989), 6982-6986), DMRIE and DORIE (Felgner, Methods 5 (1993), 67-75), DC-CHOL (Gao, BBRC 179 (1991), 280-285), DOTAP (McLachlan, Gene Therapy 2 (1995), 674-622) or Lipofectamine; the use of receptors which mimic viral systems (for a review, see Cotten, Current Opinion in Biotechnology 4 (1993), 705-710); and the use of polymers such as polyamidoamine (Haensler, Bioconjugate Chem. 4 (1993), 372-379). However, although promising, these techniques suffer from a number of limitations, in particular their low level of in vivo efficacy, which substantially limits their application within the, context of a gene therapy. Furthermore, some of these techniques are either limited to in vitro applications, in particular on account of the toxic character of the molecule employed (polybrene may be mentioned as an example), or on account of inflammatory reactions in response to the introduction of these compounds (in the case of cationic lipids, for example; Scheule, Human Gene Therapy 8 (1997), 689-707), or are difficult to control, as in the case of receptors where a large quantity of the nucleic material is trapped in vesicles during endocytosis and is not therefore any longer available for the therapy. Finally, these techniques are relatively sensitive to environmental factors and long and delicate development is required in order to adapt them to the target cells or to the chosen mode of administration and, more particularly, in order to transfer them from the in vitro model to an in vivo application.
Wolff (Science 2478 (1990), 1465-1468) have described an attractive and simple system for introducing a polynucleotide into muscle cells, which system consists simply in injecting the purified polynucleotide, which is not associated with any other compound facilitating its introduction into the target cells, by the intramuscular route. The results which have more recently been obtained by means of intratracheal injection (Meyer, Gene Therapy 2 (1995), 450-460) or by means of injection into the arteries (Riessen, Human Gene Therapy 4 (1993), 749-758) confirm the interest which such a system affords. Nevertheless, the levels at which the genes which have been introduced into the tissues are expressed are still too limited to enable this technique to be implemented within the context of an efficient gene therapy, in particular in association with pulmonary disorders. Some studies suggest alternative methods in order to improve the introduction of this type of polynucleotide into cells. For example, the patent application WO95/26718 relates to methods of introducing genetic material into cells comprising the steps of contacting said cells with a ge
Bischoff Rainer
Kolbe Hanno
Schughart Klaus
Burns Doane Swecker & Mathis L.L.P.
Nguyen Dave T.
Schnizer Richard
Transgene S.A.
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