Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Implant or insert
Reexamination Certificate
1996-07-11
2002-10-15
Nguyen, Dave T. (Department: 1636)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Implant or insert
C435S325000, C435S382000, C435S395000, C435S397000, C435S400000, C435S455000, C435S456000, C435S320100, C536S023100, C536S023200, C536S023500
Reexamination Certificate
active
06464998
ABSTRACT:
The present invention relates to the field of gene and cell therapy. More especially, it relates to cell compositions intended to be implanted in vivo for delivering therapeutic substances in a sustained and controlled manner.
Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, and the like) by introducing genetic information into the body of an affected individual. This genetic information may be introduced either in vitro into a cell extracted from the body, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. Different techniques have been described for introducing this genetic information, 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 [Feigner et al., PNAS 84 (1987) 7413], the use of liposomes [Fraley et al., J. Biol. Chem. 255 (1980) 1043], 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.
Techniques involving direct administration of the gene in vivo have certain drawbacks, such as the non-selective dispersal of the gene throughout the body, its short half-life, the risks of immunological reaction or alternatively the lack of control of the gene after injection. For this reason, cell therapy offers an advantageous alternative, consisting in removing cells, modifying them ex vivo and then readministering them. As a result, only an identified cell population is modified by the therapeutic gene. However, after administration, the fate of the modified cells is not controlled. Similarly, it is no longer possible to stop the treatment. Lastly, this therapy implies that the cells to be treated can be removed from the body, manipulated ex vivo and readily reimplanted. As a result, it is limited in practice to blood cells.
The present invention provides an advantageous solution to these problems. The present invention relates, in effect, to compositions intended to be implanted in a body, comprising cells modified by a recombinant adenovirus comprising a heterologous DNA sequence coding for a therapeutic product, a gelling agent and a support to which the said cells are anchored.
The implantation of the compositions according to the invention affords many advantages compared to the prior art, and especially control of the number of cells implanted, control of the number of cells infected, measurement of the level of expression of the therapeutic gene before implantation, absence of immunological reaction associated with the direct injection of a virus, possibility of removing the implant at any time, and the like.
The implantation of genetically modified cells has already been envisaged in the prior art. Thus, Palmer et al. [PNAS 88 (1991) 1330] and Moullier et al. [Nature genetics 4 (1993) 154] have described the implantation of fibroblasts genetically modified by retroviruses. However, the use of retroviruses creates some problems limiting the applications of this technology. In particular, retroviruses are difficult to produce at high titres and, as a result, do not permit use at high multiplicities of infection. Retroviruses also have the drawback of not being able to incorporate large-sized fragments of heterologous DNA, thereby limiting the therapeutic applications. Lastly, retroviruses integrate into the genome of fibroblasts, which can contribute to the appearance of tumour cells after implantation. Moreover, the fibroblasts described by Palmer et al. are coated only in collagen, and the implants obtained do not have sufficient cohesion. As a result they gradually disintegrate in vivo, leading to an uncontrolled diffusion of the cells out of the implantation site.
The compositions according to the invention enable these drawbacks to be overcome. The present invention is partly the outcome of the demonstration that adenoviruses are capable of producing in vitro a very high-powered infection of cells in culture. Thus, it is possible to infect 100% of fibroblasts in culture. Moreover, it is also possible, by varying the multiplicity of infection, to obtain a large number of copies of adenovirus per cell (up to 100 copies), thereby enabling the therapeutic effect of the implants of the invention to be substantially increased. Furthermore, the adenoviruses of the invention may be produced at high titres, enabling not only primary cell cultures but also secondary cultures, previously cloned and stored, to be infected. The adenoviruses of the invention also have the advantage of not integrating into the genome of the cells they infect. As a result, the implants of the invention are less likely to induce the appearance of tumour cells. Furthermore, if the implanted cells divide, the adenovirus of the infection will be diluted over generations, and the character it confers on the infected cells will not be transmitted to the daughter cells. Lastly, the adenoviral vectors used in the present invention may be modified so as to incorporate very large-sized fragments of heterologous DNA. Thus, contrary to other viral vectors, it is, for example, possible to incorporate a large-sized heterologous gene such as that for factor VIII or for dystrophin. Furthermore, it is possible to incorporate, in addition to the therapeutic gene, a safety gene whose expression would permit, for example, the destruction of the infected cell.
The compositions according to the invention hence have many advantages compared to the systems described in the prior art, endowing them with much greater therapeutic potentials.
The compositions according to the invention may be made from different cell types, and in particular from fibroblasts, endothelial, epithelial or glial cells, hepatocytes, keratinocytes or alternatively myoblasts. Preferably, fibroblasts are used in the context of the invention.
In a preferred embodiment of the invention, autologous cells, that is to say ones removed from the patient in whom they will then be implanted, are used. However, in some cases, it can be advantageous to use allogeneic or xenogeneic cells, leading to a gradual rejection of the implant and thus giving it an effect which is limited in time. In particular, cells of murine origin may be implanted in man without any effect other than gradual rejection.
The cells used in the context of the invention can be primary cultures. In this case, they can be removed by any technique known to a person skilled in the art, and then cultured under conditions permitting their proliferation. As regards fibroblasts more especially, the latter may be readily obtained from biopsies, for example according to the technique described by Ham [Methods Cell. Biol. 21a (1980)255]. These cells may be used directly for the preparation of the compositions of the invention, or stored, for example by freezing, for the establishment of autologous banks with a view to subsequent use. Preferably, the compositions according to the invention comprise 10
5
to 10
10
cells. More preferably, they comprise 10
6
to 10
8
cells.
The cells in culture are then infected with recombinant adenoviruses to endow them with the desired therapeutic properties. Infection is carried out in vitro according to techniques known to a person skilled in the art. In particular, depending on the cell type used and the number of copies of virus desired per cell, a person skilled in the art can adapt the multiplicity of infection and, where appropriate, the number of infection cycles carried out. It is obvious that these steps must be performed under suitable sterility conditions for an in vivo adm
Beuzard Yves
Danos Olivier
Descamps Vincent
Heard Jean-Michel
Moullier Philippe
Aventis Pharma S.A.
Finnegan Henderson Farabow Garrett & Dunner LLP
Nguyen Dave T.
Nguyen Quang
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