Episomally replicating vector, its preparation and use

Details Episomally replicating vector, its preparation and use Episomally replicating vector, its preparation and use

1999-10-05

2002-06-25

Crouch, Deborah (Department: 1632)

Chemistry: molecular biology and microbiology

Vector, per se

C435S091400

Reexamination Certificate

active

06410314

ABSTRACT:

The present invention relates to stably episomally replicating vectors, comprising at least one scaffold/matrix attached region (S/MAR) and at least one viral or eukaryotic origin of replication (ORI), cells comprising these, processes for their preparation, and their use, in particular as a medicament or diagnostic.
At present, vectors are widely used in research and therapy. In this context, vectors are used in particular for transfecting or for transforming eu- and prokaryotic cells or cell systems and, in these, bringing effectors into action which code, for example, for pharmaceutically/medicinally relevant proteins or peptides, but also for proteins necessary for replicating the vectors themselves. Effectors are understood in general as meaning substances which produce a particular effect of metabolic or therapeutic nature in the host cell. Customary effectors are nucleic acids coding for proteins or peptides, ribozymes or antisense RNAs and antisense DNAs.
Vectors are of particular importance in gene therapy. The fundamental object of gene therapy is the introduction of nucleic acids into cells in order to express an effector gene. Three fundamental problems exist here in gene therapy, a) the introduction of the gene (gene delivery), b) the maintenance of the gene (gene maintenance) and c) the expression of the gene (gene expression). In this context, just the maintenance of the gene and thus the stable and persistent expression of genes is a basic condition for successful gene therapy, which until now has not been solved very satisfactorily. The prerequisite for this is therefore the use of suitable vectors. In this context, in gene therapy in-vitro and in-vivo processes are differentiated in principle. In in-vitro processes, cells are removed from the body and transfected ex vivo with vectors in order then to be introduced into the same or into another body again. In in-vivo gene therapy, vectors are administered systemically—e.g. via the blood stream. However, local application, in which a gene-therapy vector is applied locally in the tissue, for example in an affected section of vessel, is also possible (see, for example, WO 95/27070).
Thus, for the local application of a therapeutic gene in a selected case, for example, various strategies were developed based on modified balloon catheters, which are intended to permit direct administration of a substance or of a gene into the vascular wall. After a local administration using a double balloon catheter, Nabel, E. G. et al. (1990) Science, 249, 1285, for example, were able to detect a transient expression of the &bgr;-galactosidase gene in transfected cells of the femoral artery of the pig by means of liposomal and retroviral transfection.
Vectors are used in particular for the optimization of tissue-specific expression, which is used for the therapy of chronic diseases and hereditary diseases such as diabetes, hemophilia, ADA, muscular dystrophy, familial hypercholesterolemia or rheumatism, but can also be employed in acute diseases, such as vascular disorders—arteriosclerosis or its sequelae (stenosis, restenosis, cardiac infarcts)—and in tumors. Finally, the expression of genes and thus in particular the intracellular formation of therapeutic proteins and peptides, which on account of pathological or genetic modification are not or are no longer present to an adequate extent in the target organism, e.g. insulin or, in vascular cells, factor VII, etc., can also take place by means of a tissue-directed gene transfer.
An essential aim of somatic gene therapy is therefore to incorporate a therapeutic gene specifically into the target cells of the body after systemic or local administration and to express the therapeutic gene in these cells, without at the same time, however, inducing a transformation of the target cell or an immune response.
Up to now, there are two classes of vectors available for this: the viral vectors, where a differentiation has to be made here between a) episomally replicating vectors and b) vectors integrating into the DNA, and the nonviral vectors, in which c) a stable transfection is achieved by random insertion (integrating) or d) (transient) only a temporary transfection is present. The random integration into the host genome in approaches using integrating vectors can, depending on the integration point, lead both to insertion mutagenesis and to so called “silencing”, in which no reading or expression of the inserted gene takes place. Transient expression vectors are limited in their life in terms of time, not stable and in some cases also subject to integration, but sometimes also transform the host cell. Their most important disadvantage, however, is that they often have to be repeatedly used on account of the limited expression associated with the short-lived nature. These vectors thus cause considerable problems just with respect to the effectiveness, reproducibility and safety necessary here.
The viral, episomally replicating vectors group does not have these disadvantages, as they are not integrated into the host genome and are retained in self-replicating form in the host cell. The term episomally replicating is understood here as meaning that the vector is not integrated into the genome of the host cell, but exists in parallel, is also replicated during the cell cycle and in the course of this the vector copies—depending on the number of the copies present before and after cell division—are distributed statistically in the resulting cells. Plasmid vectors, for example the pGFP-C1 vector (Clontech UK Ltd.), which have been optimized for research and other application purposes by alterations, are derived from the viral vectors. At present, only a few vectors are known which—starting from viral origins—replicate episomally in a few eukaryotic cells, e.g. SV40, BPV or EBV vectors. The replication origin of these vectors, however, requires interaction with one or more virally encoded trans-acting factors. These factors are also necessary for the stability of the vectors, but often lead to immortalization and transformation of the host cell or induce an immune response in the body (Ascenzioni et al. (1997) Cancer Letters 118, 135-142).
The eukaryotic virus SV40 (simian virus) thus replicates episomally in monkey cells and in some mammalian cells and cell lines. For this, the virus needs the so-called “large T antigen” for its existence in the host cell. The functions of the “large T antigen” are of crucial importance for the replication of the virus in the cell. The “large T antigen” binds, inter alia, to the viral DNA in the region of the origin of replication, and initiates its replication there (Mohr et al. (1987) EMBO J. 6, 153-160). Beside these activities which are important for the virus, the “large T antigen”, however, also affects cellular functions. It is bound, inter alia, to proteins which are involved in the regulation of the cell cycle (cyclin, tubulin, cdc2). Infections with SV40 or transfections with vectors which carry genes coding for SV40 “large T antigen” can therefore lead to the immortalization of primary cells and induce tumor formation in animals (Fried, M. (1965) Proc. Natl. Acad. Sci. USA, 53, 486-491; Eckhart, W. (1969) Virology, 38, 120-125; Di Mayorca et al. (1969) Virology, 830, 126-133).
WO 98/27200 discloses a construct containing a human or mammalian replication origin cloned in a circular vector, which—without being integrated into the host genome—replicates episomally in human cells. Cossons N. et al. (1997) J. Cell. Biochem. 67, 439-450 describe vectors that contain a matrix attachment region (MAR) and different mammalian replication origin cloned in a circular vector. However, the episomal replication can only be maintained by selection pressure with selective antibiotics (G418) and even then occurs only with limited effectiveness. In fact, the stability per generation was only 80% under selective pressure. Therefore, no stable maintenance of the episomally replicating vector was observed. While the use of selective antibiotics like G418 is feasible f

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