Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-01-12
2002-03-05
Crouch, Deborah (Department: 1633)
Chemistry: molecular biology and microbiology
Vector, per se
C435S455000, C435S456000
Reexamination Certificate
active
06352856
ABSTRACT:
The invention relates to a chimeric poxvirus comprising the vector components of a retroviral (defective) genome. Furthermore, the invention relates to a chimeric poxvirus comprising the vector components and packaging components for producing retroviral defective viruses, as well as the use of the chimeric poxvirus according to the invention for gene or tumor therapy.
A central object of gene therapy is the stable genetic modification of target cells, and hitherto primarily viral vectors based on replication-deficient adenoviruses and retroviruses have been constructed for application in gene therapy.
The in vivo gene transfer with adenoviral vectors is very efficient, since particularly with adenoviral vectors, a large number of different cells can be transduced. However, in adenovirus infections, the DNA is not integrated into the host genome, but is present in episomal form. Moreover, a cellular immune reaction can be triggered which restricts the foreign gene expression (Li et al., 1993, Hum. Gene Ther. 4: 403-409). An attenuation of this reaction is possible by means of vectors in which the adenovirus E3 region has not been deleted (Ilan et al., 1997. Proc. Natl. Acad. Sci. USA 94: 2587‥2592). These modifications allow for a transient expression of the therapeutic gene lasting for months. A permanent action, however, cannot be obtained with adenovirus vectors, since, as stated above, these vectors are not integrated into the genome of the host cell in a stable manner.
In contrast thereto, retroviral vectors are capable of integrating foreign genes into the genome of the host cell in a stable manner. Retroviruses belong to the few presently known viruses which have mechanisms for integrating foreign DNA into the genome of the host cell and thus are capable of permanently transforming the latter. On account of this property, they are frequently used in gene therapy approaches. For this purpose, in techniques used so far, foreign genes have been cloned into the proviral DNA of retroviruses, wherein, however, replication-deficient retroviruses have been used, in which at least one of the retroviral genes encoding for the packaging function (gag-pol or env) has been inactivated or deleted. These constructs contain the packaging signal psi and the flanking Long Terminal Repeat (LTR) sequences of the provirus. These transcription units are introduced into the cells with the plasmid vectors by conventional transfection techniques and commonly transcribed in RNA in the nucleus under the control of the LTR promoter. On account of the packaging signal psi, such an RNA is accepted as genomic viral RNA and packaged in retroviral particles.
To form infectious retroviral particles, in the simplest case, the gene products gag-pol and env must be present. These genes can be inserted into the cells also without the packaging signal via plasmid transfection, or they may be provided via a helper virus. For preparing retroviral particles, also cell lines in which the viral genes for the packaging components gag, pol and/or env are integrated in a stable manner and which express the same, can be transduced. Such cell lines are termed “packaging” lines and are described e.g. in WO 97/35996.
Plasmid constructs have been described, so-called “plasmoviruses”, which encode all the necessary components for the formation of non-replicating retroviral particles (Noguiez-Hellin et al. 1996, Proc. Natl. Acad. Sci. USA 93: 4175-4180).
The use of replication-deficient retroviruses does, however, not completely prevent the risk that the retroviral DNA recombines with the helper genes, whereby it may come to the production of replication-competent viruses which possibly may have a tumorigenic potential.
Although the transformation of cells with retroviral vectors is highly efficient in vitro, difficulties are encountered at present in gene-therapeutical applications. Thus, transfection techniques as are required in the production of retroviral vectors via plasmid constructs, can only be carried out in vitro. The direct administration of the plasmid constructs for production of the defective retroviral particles in the body is extremely inefficient. Likewise, retroviral particles in vitro can be produced only up to comparatively low titers. On account of their instability, concentration of the particles is possible also to a limited extent only. The low titers and the instability of retroviral particles thus frequently prevent the efficient transformation in vivo. Methods, such as the direct injection of the particles into the target organ, or after tissue removal, ex vivo-transformation and implantation of transformed cells are being attempted at present. Also, the targeted transduction of certain target organs, the so-called “targeting”, is very difficult in case of retroviral vectors. It has been attempted to change the tropism of the particles by heterologous enveloping proteins (pseudotyping).
The wide-spread retroviral vectors based on simple retroviruses, such as, e.g., Moloney Murine Leukemia Virus (MLV), are able to transduce only cells in the division phase. Cell growth in many fully grown organs is, however, very low. In tests for hepatic gene therapy, it has thus been attempted i.a. to obtain growing and thus transformable tissue by a partial removal of the liver. Likewise, retroviral vectors based on complex retroviral viruses, such as, e.g., HIV (lentiviral vectors) have been constructed, since the latter can also transform non-dividing cells (Reiser et al., 1996. Proc. Natl. Acad. Sci. USA 93: 15266-15271).
To correct the defective genes in case of gene therapy, an efficient insertion of the corresponding genes as well as long-term expression of the genes are particularly important. New approaches for preparing vectors for the stable in vivo-transduction thus take advantage of the principle of using viruses as the carriers for retroviral defective particles. In doing so, particularly the large insertion potential with DNA viruses, such as herpes virus and adenovirus, are utilized. The furthest-developed approach of chimeric virus systems is the preparation of defective retroviral particles by co-infection of two recombinant adenoviruses which encode the retroviral vector and the packaging function, respectively, the retroviral vector and the packaging proteins being introduced via the adenovector system, whereby these cells become transient retroviral production cells, and the retroviral vector particles formed therewith can infect the neighbouring cells (Feng et al., 1997. Nat. Biotechn. 15:866-870, Bilbao et al., 1997. FASEB J. 11:624-634).
This system has, however, several decisive disadvantages: The accepting capacity of foreign genes of adenovirus vectors is restricted to a few kilobases so that it is difficult to unite all the functions for producing a defective retrovirus (packaging components and retroviral vector components) in one adenovirus. Such a construct is, however, the prerequisite for an efficient in vivo gene therapy. Moreover, both adenovirus and retroviral genes and genomes are transcribed in the nucleus of the host cell and replicated, respectively. This topographic vicinity makes recombination to wild type retroviruses probable. In principle, this system also does not allow for a deletion of important retroviral transcription control regions, since the latter are necessary for the transcription in the nucleus. Thus, the adenovirus/retrovirus system cannot be attenuated to the desired degree, in terms of safety technique. The same holds for the herpes simplex amplicon system; so far, the latter has only been described for the expression of retroviral structural proteins, wherein cells containing a lacZ provirus have been infected with an HSV amplicon vector containing the packaging components, and thus retroviral lacZ particles have been obtained (Savard et al., 1997. J. Virol. 71:4111-4117).
A disadvantage both with adenoviral and with retroviral vectors is in particular that introns for improving the foreign gene expression or for stabilising vector RNAs in the t
Dorner Friedrich
Falkner Falko-Guenter
Holzer Georg
Baxter Vaccine AG
Crouch Deborah
Heller Ehrman White & McAuliffe
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