Encapsulated cells producing cytochrome P450

Details Encapsulated cells producing cytochrome P450 Encapsulated cells producing cytochrome P450

1999-11-18

2003-04-01

Priebe, Scott D. (Department: 1636)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C424S093200, C424S093100, C435S320100, C435S325000, C435S455000, C514S04400A

Reexamination Certificate

active

06540995

ABSTRACT:

BACKGROUND OF THE INVENTION
The anti-cancer drugs used to treat tumours are in most cases applied systemically and spread through the whole body of the patient. The high systemic dose of such drugs required for cancer treatment is combined with unpleasant side-effects for the patient.
In an attempt to circumvent this problem, cancer-prodrugs that have to be metabolized or activated in the body before they become cytotoxic have been used. Unfortunately, human tumours that contain appropriate high levels of the activating enzymes are rare. The main site for activation of prodrugs is the liver and to ensure that a tumour, at a distant site, receives a sufficient dose of the activated drug, the amount of activated prodrug produced in the liver has to be quite high and again this leads to toxic side effects for the patient.
One strategy by which these problems of high systemic concentration of activated drugs could be circumvented would be to provide means for activation of the prodrug directly in or near the site of the tumour. This strategy would require that tumour cells, or cells at the site of a tumour are genetically transformed to produce high amounts of the enzymes required for metabolizing the cancer prodrugs. Retroviral vectors are ideally suited for the stable delivery of genes to cells since the retrovirus is able to integrate the DNA form of its genome into the genome of the host cell and thus all daughter cells of an infected cell will carry the retroviral vector carrying the therapeutic gene. A further advantage is that most retroviruses only infect dividing cells and they are therefore ideal gene delivery vehicles for tumour cells.
Retroviral vectors are the most commonly used gene transfer vehicles for the clinical trials that have been undertaken to date. Most of these trials have, however, taken an ex vivo approach where the patient's cells have been isolated, modified in culture and then reintroduced into the patient.
The delivery of genes in vivo introduces a variety of new problems. First of all, and above all, safety considerations have to be addressed.
A major concern for eventual in vivo gene therapy, both from a safety stand point and from a purely practical stand point, is the targeting of the expression. It is clear that therapeutic genes carried by vectors should not be indiscriminately expressed in all tissues and cells, but rather only in the requisite target cell. This is especially important when the genes to be transferred are such prodrug activating genes designed to ablate specific tumour cells. Ablation of other, non-target cells would obviously be very undesirable.
The essentially random integration of the proviral form of the retroviral genome into the genome of infected cells has posed a serious ethical problem because such random integration may lead to activation of proto-oncogenes and thus lead to the development of a new cancer. Most researchers would agree that the probability of a replication defective retrovirus, such as all those currently used, integrating into or near a cellular gene involving in controlling cell proliferation is vanishingly small. However, it is generally also assumed that the explosive expansion of a population of replication competent retroviruses from a single infection event, will eventually provide enough integration events to make such a phenotypic integration a very real possibility.
Retroviral vector systems are optimized to minimize the chance of replication competent virus being present. It has however, been well documented that recombination events between components of the retroviral vector system can lead to the generation of potentially pathogenic replication competent virus and a number of generations of vector systems have been constructed to minimize this risk of recombination (Salmons, B. and Günzburg, W. H.,
Human Gene Therapy,
4(2):129-41 (1993).
Retroviral vector systems consist of two components:
1) The retroviral vector itself is a modified retrovirus (vector plasmid) in which the genes encoding for the viral proteins have been replaced by therapeutic genes. Since the replacement of the genes encoding for the viral proteins effectively cripples the virus it must be rescued by the second component in the system which provides the missing viral proteins to the modified retrovirus.
The second component is:
2) A cell line that produces large quantities of the viral proteins, however lacks the ability to produce replication competent virus. This cell line is known as the packaging cell line and consists of a cell line transfected with one or more plasmids carrying the genes enabling the modified retroviral vector to be packaged.
To generate a recombinant retroviral particle, the retroviral vector is transfected into the packaging cell line. Under these conditions the modified retroviral genome including the inserted therapeutic gene is transcribed from the retroviral vector and packaged into the modified retroviral particles. These recombinant retroviral particles are then used to infect tumour cells during which the vector genome and any cytotoxic gene becomes integrated into the target cell's DNA. A cell infected with such a recombinant viral particle cannot produce new vector virus since no viral proteins are present in these cells but the DNA of the vector carrying the therapeutic is integrated in the cell's DNA and can now be expressed in the infected cell.
A number of retroviral vector systems have been previously described that should allow targeting of the carried cytotoxic genes (Salmons, B. and Günzburg, W. H.
Human Gene Therapy,
4(2):129-41 (1993)). Most of these approaches involve either limiting the infection event to predefined cell types or using heterologous promoters to direct expression of linked heterologous therapeutic genes to specific tumour cell types. Heterologous promoters are used which should drive expression of linked genes only in the cell type in which this promoter is normally active or/and additionally controllable. These promoters have previously been inserted, in combination with the therapeutic gene, in the body of the retroviral vectors, in place of the gag, pol or env genes.
The retroviral Long Terminal Repeat (LTR) flanking these genes carries the retroviral promoter, which is generally non-specific in that it can drive expression in many different cell types (Majors, J. (1990). in “Retroviruses—Strategies of replication (Swanstrom, R. and Vogt, P. K., Eds.): Springer-Verlag, Berlin: 49-92). Promoter interference between the LTR promoter, and heterologous internal promoters, such as the tissue specific promoters, described above, has been reported. Additionally, it is known that retroviral LTR's harbour strong enhancers that can, either independently, or in conjunction with the retroviral promoter, influence expression of cellular genes near the site of integration of the retrovirus. This mechanism has been shown to contribute to tumourigenicity in animals (van Lohuizen, M. and Berns, A. (1990),
Biochim. Biophys. Acta,
1032:213-235). These two observations have encouraged the development of Self-Inactivating-Vectors (SIN) in which retroviral promoters are functionally inactivated in the target cell (WO 94/29437). Further modifications of these vectors include the insertion of promoter gene cassettes within the LTR region to create double copy vectors (WO 89/11539). However, in both these vectors the heterologous promoters inserted either in the body of the vector, or in the LTR region are directly linked to the therapeutic gene.
The previously described SIN vector mentioned above carrying a deleted 3′LTR (WO 94/29437) utilizes in addition a heterologous promoter such as that of Cytomegalovirus (CMV), instead of the retroviral 5′LTR promoter (U3-free 5′LTR) to drive expression of the vector construct in the packaging cell line. A heterologous polyadenylation signal is also included in the 3′LTR (WO 94/29437).
A variety of cytotoxic genes carried by retroviral vectors have already been tested. These genes encode

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