Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-11-04
2003-02-18
Wilson, Michael C. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S325000, C536S024100, C530S350000, C424S450000, C514S04400A
Reexamination Certificate
active
06521449
ABSTRACT:
The present invention relates to a gene construct, a pharmaceutical preparation and their use.
The beginning of the era of gene therapy in medicine has been marked by the successful gene transfer of the adenosine desaminase gene to a child with severe immunodeficiency. To date findings coming mainly from animal experiments indicate that this form of therapy is not only useful in the correction of genetically caused diseases but also in the therapy of malignant neoplasias (Culver and Blaese, 1994).
The methods up to now being in the test phase are aimed at either a direct destruction or at least “normalization” of the tumor cell, or at the activation of an immune reaction directed against the tumor. The destruction by the transfer of so-called suicide genes or the normalization by the transfer of tumor suppressor genes requires the gene transfer being performed with high efficiency. The direct intratumour transfer of murine cell lines producing retroviruses containing the suicide gene thymidine kinase of herpes virus has been already performed in the case of multiform glioblastoma (Culver and Van Gilder, 1994).
As long as there is no efficient system available to achieve targeted gene transfer to all tumor cells in vivo, approaches involving the immune system in tracking down and destroying all tumor cells seem the most promising. However, a prerequisite of these approaches is the fundamental capability of the immune system to recognize the tumor cells by means of tumor-specific antigens, which appear on tumor cells and not on normal cells (Boon et al., 1994). For example, these tumor-specific antigens include viral gene products (e. g. gene products of the human papilloma virus in genital tumors) or mutationally altered oncogene products (e. g. the ras gene product or the tumor-specific bcr-abl fusion protein). Further suitable candidates for tumor specific antigens are the so-called idiotypes, i. e. immunoglobulins or T cell receptors on the cellular surfaces of B or T cell derived tumors. Recently, the identification of a plurality of tumor-associated antigens has been carried out, for example in malignant melanoma. These genes, however, are not exclusively expressed by the tumor, but to a small extent also by other somatic cells, such as melanocytes. The knowledge of tumor-specific or tumor-associated antigens, respectivly, is probably about to increase sharply because of the recent successful recovery and analysis by biochemical methods of the peptides presented by a tumor MHC complex (Mandelboim et al.; Cox et al., 1994).
Already in the mid-eighties, using an approach employing experiments on laboratory animals tumor cells were observed to loose their tumorigenicity in the syngeneic animal if the tumor cells were transfected with a cytokine expression vector (e. g. IL-2) by. gene transfer (Pardoll, 1993). This effect has also been observed in the case of a mixture of modified (i.e., cells that contained the expression vector) and non-modified cells. The local production of an immunostimulatory cytokine in a subset of the tumor cells is obviously capable of causing an immune reaction directed against the wild-type tumor. One of the most extensive studies of this kind using the murine malignant melanoma model B-16 showed retroviruses transducing GM-CSF, IL-4, and IL-6 to be most effective (Dranoff et al., 1993). The outcome of these observations was that a number of clinical studies on the subject of intratumour transfer of cytokines have been entered or are presently being entered worldwide (Foa et al., 1994). Generally, the protocols entail an ex vivo gene transfer into tumor cells which have been established in vitro for a short time period. In most of the protocols amphotrophic retroviruses are employed as vectors. After viral gene transfer, the cells are reimplanted into the patients. They can be irradiated prior to reimplantation. These approaches are, however, strongly limited by the technical difficulties of culturing the tumor cells in vitro even for a short time period. Therefore, a modification of this approach entails transducing or transfecting, respectively, either tumor infiltrating lymphocytes (TIL) (Treisman et al., 1995) or autologous fibroblast cells instead of the tumor cells themselves.
A further approach also referring to observations obtained from animal experiments in the eighties has been developed by Gary Nabel and already converted into a clinical protocol (Nabel et al., 1993; Plautz et al., 1993). This approach assumes that artificial allogenization of a subset of the tumor cells by the transfer of transplantation antigens may be sufficient to induce an immune reaction of the organism against the unmodified tumor cell. This protocol entails the direct transfer of an HLA B7 expression construct into the tumor using liposomes. Repeated injection of the HLA B7 gene construct into skin metastases of a moribund patient brought about regression of another untreated metastasis and of a pulmonary mestastasis, respectively (Nabel et al., 1993).
Many in vivo tumor cells lack the B7 surface antigen mediating co-stimulatory signals for T cell recognition. Therefore, attempts are made to stimulate the production of this signalling molecule in tumor cells by gene transfer (June et al., 1994; Li et al., 1994).
The aforementioned approaches to solve the problems bear the following disadvantages:
Retroviral Vectors
An advantage of amphotropic retroviruses is that integration of the proviral DNA into the target cell and the viral promoter/-enhancer combination generally permit a stable expression level during several cell divisions. The essential step of integration, however, bears the danger of insertional mutagenesis. Moreover, so-called “packaging” cell lines produce relatively small amounts of recombinant virus which to date fail to be enriched because of their lability. Therefore, direct intratumour gene transfer is only possible using virus producing cells. Release of infectious virus in the target organism, however, may lead to infection of other dividing cells, such as intestinal epithelium or hematopoietic stem cells, after hematogenous transmission.
Direct Intratumour DNA Transfer
The liposome-mediated direct incorporation of DNA has been demonstrated successfully using the endothelium of large blood vessels (Ohno et al., 1994). An advantage of this approach is that it lacks the risk of insertional mutagenesis as well as of the undesirable remote effect; but this approach achieves only transient expression of the incorporated gene construct in dividing tissue because the DNA generally fails to be integrated or replicated.
Cell Culture of Tumor Cells and Ex VIVO Transfection
One of the main technical obstacles is the in vitro culturing of tumor cells of every single patient. The performance of gene transfer into tumor cells cultured for a relatively short time period requires extraordinary experimental skills and is successful only in a portion of the cases. To date, infection with recombinant retroviruses represents the technique of choice for a gene transfer into this kind of cells.
EBV, EBV-derived Vectors, and EBV-immortalized Cells
EBV is present in lymphoblastoid cell lines (LCLs) in a state of latency. That means only a very small percentage of the infected cells produces infectious virus. In the state of latency, only six nuclear localized proteins (EBNA1, 2, 3A, B, C, LP) and two membrane-bound proteins (LMP and TP) of the virus are expressed. Generally, the EBV genome is present in the infected cell in episomal form in 10 to 100 copies. In the state of latency, the replication of the viral genome starts at an origin of replication (orip) (Yates et al., 1984). Maintenance of the episomal replication further requires binding of the EBNAl protein to the oriP (Yates et al., 1984). The EBV-derived vectors consist of pBR sequences, oriP, an EBNA-1 expression cassette, and a selection marker specific for eukaryotic cells (e. g. the hygromycin resistance gene). Furthermore, these vectors have the capacity for additional 20 to 30 kb of foreign sequences
Hörtnagel Konstanze
Mücke Susanne
Polack Axel
Wolf Jürgen
Bingham & McCutchen LLP
GSF-Forschungszentrum für Umwelt und Gesundheit GmbH
Maher David W.
Wilson Michael C.
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