Design principle for construction of expression constructs...

Details Design principle for construction of expression constructs... Design principle for construction of expression constructs...

1999-05-12

2002-09-17

Priebe, Scott D. (Department: 1636)

Chemistry: molecular biology and microbiology

Vector, per se

C435S069100, C435S325000, C435S455000, C536S023100, C536S023500, C514S04400A

Reexamination Certificate

active

06451593

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention concerns a design principle for a minimalistic expression construct which contains no genetic information other than the information to be expressed, apart from promotor and terminator sequences which are necessary for the control of expression. Such minimal expression constructs are to be used for molecular-medical applications, specifically genetic vaccination, tumor therapy, and -prophylaxis.
The design principle is to be used for the construction of expression constructs for the expression of MHC-I or MHC-II presentable peptides, cytokines, or components of the regulation of the cell cycle, or for the synthesis of regulative RNA molecules and antisense RNA, ribozyme or mRNA-editing-RNA. Furthermore, an important aspect of the invention is that the construction principle allows for the covalent linking of the expression construct, e.g. with peptides, proteins, carbohydrates or glycopeptide ligands, as well as particles which allow for the transfer of the constructs into cells by ballistic transfer especially into dermis, muscle tissue, pancreas, and the liver.
2. Background Information
The invention is to be used especially in two related fields: somatic gene therapy and genetic vaccination. These two meet in the field of immuno gene therapy of oncological conditions. Whereas classical gene therapy intends to substitute missing or defective genes, immuno gene therapy intends to activate the immune system of the patient against tumor specific antigens. In malignant melanoma and some other tumors, a number of tumorspecific antigens have been identified which can be recognized by cytotoxic T-lymphocytes (Van den Eynde B. and Brichard V. G., Current Opinion in Immunology (1995) 7: 674-681). In most cases these are fragments of mutated proteins, which are either relevant for tumor development and promotion, or are fragments of proteins from a changed metabolism of the tumor cell (Stüber et al., Eur. J. Immunol. (1994) 24: 765-768). In the case of melanoma, the presented peptides often derive from proteins from the melanocyte-specific differentiation. Approaches which make use of the activation of the immune system against such tumor specific antigens are in need of methods which enable the antigenic epitopes to be overexpressed in non-tumor cells, such as antigen-presenting cells (macrophages, dendritic cells). Alternatively, genes which control the expression of peptide-presenting proteins, such as CIITA or ICSBP are of great importance.
Laboratory experiments and clinical studies, in which such peptides have been used for the induction or amplification of a tumor specific cytotoxic response, concentrate on conventional vaccination protocols, in which the corresponding peptides are being used (Strominger J., Nature Medicine, (1995) 1:1.179-1.183). Alternatively, antigen-presenting cells such as dendritic cells, were incubated with high concentrations of such peptides. Thereby, the peptides originally present on the MHC-complex were exchanged for tumor specific peptides (Grabbe et al., Immunology Today (1995) 16:117-121).
The term genetic vaccination (immunization) describes the utilization of an experimental finding which first was debated as a scientific artefact, but has recently been corroborated in a number of biomedical problems (Piatak et al., Science 259 (1993): 1745-1749). If an expression plasmid for mammalian cells is injected into skin or muscle, there is, albeit in very low efficiency, an expression of the corresponding gene close to the injection site. If the expression product is a protein alien to the organism (xenogenic or allogenic protein), uptake and presentation of fragments of the expressed protein (oligopeptide) by antigen-presenting cells (APC) takes place, probably by way of local inflammation, Depending on local cytokine patterns and the type of cells in which the plasmid is expressed (presentation by MHC-I or MHC-II), there is an induction of an immune reaction along the T
H1
or T
H2
pathway (Wang et al., Human Gene Therapy 6 (1995): 407-418), which eventually leads to the proliferation of cytotoxic T-cells or to the production of soluble antibodies. The transfection of dendritic cells with expression constructs for antigenic peptides ex-vivo is included in the term genetic vaccination in this context (Schadendorf et al. Molecular Medicine Today, 2 (1996); 144-145).
Such genetic vaccination avoids the numerous risks of conventional immunization approaches. Many approaches are known in gene therapy that are designed to effect therapeutic or prophylactic effects by the transfer of genetic information into cells. These approaches have not only been demonstrated in animal experiments, but also in numerous clinical studies in patients, an example being the so-called ballisto magnetic vector system (EP0686697 A2) for the transfection of conventional, plasmid-based expression constructs. The ballisto magnetic vector system was employed by the inventors of this application in three clinical phase I/Il studies for the production of interleukin-7 (IL-7), interleukin-12 (IL-12) or granulocyte-macrophage-colony stimulating factor (GM-CSF) expressing tumor cells. In the case of expression of IL-12, separate expression constructs for the genes of the two IL-12 subunits were transferred ballisto-magnetically at the same time.
With the maturing of this new discipline, however, the methodological repertoire for gene therapy demands critical inspection. A fundamental aspect of this question is the sequence information contained in conventionally employed DNA constructs . If such expression constructs are to be employed in a great number of patients, and possibly more than once, safety aspects, especially those related to immunological concerns, will come to bear heavily. The conventionally used expression constructs are derivatives of eucaryotic expression plasmids. These have two fundamental disadvantages: their size, which inhibits fast transport into the cell's nucleus, and the presence of sequences which are not needed for the intended use. Expression constructs used so far contain constitutively expressed genes, i.e. for resistance against cytostatica which serve as selection markers, and in some cases sequences for the episomal replication in the target cell. The expression of these genes leads to an unwanted background of transfected genetic information. Furthermore, apart from the promotor-gene-terminator structure which is to be expressed, these constructs carry at least the sequences needed for bacterial replication, since the plasmids are propagated in bacteria. These sequences are not needed for the intended use, either.
It is obvious that conventional expression constructs lead not only to the expression of the desired gene, but also to the biosynthesis of xenogenic proteins, even if their prokaryotic promotors show very low activity in mammalian cells. With longer or repeated application it can be assumed that the desired immune response is masked by such contaminating gene products, and significant immunological complications can occur.
Another problem in the application of gene therapeutic methods concerns the method by which the genetic material to be transferred is brought into the cell. For reasons of efficiency, immunological safety, and broad applicability across a wide spectrum of cell types, the method of ballistic transfer is preferred. A fundamental advantage of ballistic transfer, compared to alternative transfection methods, is that the method is applicable across a broad spectrum of different cells or tissues. Another disadvantage of methods currently used for the transfection of eukaryotic cells, such as electroporation or lipofaction, is that the treatment brings the substance to be transported only across the plasma membrane, the first barrier, which shields the cell from its environment. However, for most substances interacting with the regulative function of the cell, it is important to get from the cytoplasma across the nuclear membrane into the nucleus. This me

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