Kurt Zatloukal

Transferrinfection: a highly efficient way to express gene constructs in eukaryotic cells.

The expression of antisense RNA is a powerful means of extinguishing unwanted gene activity in the eukaryotic cell (see this volume). This approach has worked particularly well in plantst4 and animals5 in which antisense-producing gene constructs can be inserted into the germ cell. In man, germ cell manipulation is not yet possible. The problem arises as to how somatic cells can be targeted with genes generating antisense RNAs. Apart from retroviral vectors, a multitude of techniques exist for introducing DNA into eukaryotic somatic cells (ref. 6 and references therein). During the last few years, we and others7J have been interested in adapting the cellular mechanisms of receptor-mediated endocytosis of macromolecules for the importation of DNA molecules into cell^.^-^* The principle of the method is to covalently connect a polycation, usually polylysine, to a ligand for a receptor on the cell surface. DNA, usually in the form of cloned genes, is then bound ionically to the polylysine. During importation of the DNA by receptor-mediated endocytosis the ligand binds to the receptor, and during internalization of both of these entities the DNA is thought to become colocalized, first in the coated pits and later in the endosome. The further fate of the DNA can be particularly well studied when reporter genes such as the firefly luciferase gene or the bacterial P-galactosidase gene, driven from a viral enhancer/promoter, are employed. It can be predicted that DNA in the endosome is directed to the lysosomal compartment, where it can be expected to be destroyed by resident nucleases. Despite this, apparently a small amount of DNA does escape destruction, so that reporter gene activity is observed in a few cells of a cell population (see below). Because the genes are expressed, it is probably safe to assume that some of the DNA has reached the cell nucleus. When transferrin is used as a ligand, the transfection procedure is called “transferrinfection.” Consistent with the postulated chain of events during transferrinfection, we find that the addition of the lysosomatropic agent chloroquine, which prevents acidification and concomitant activation of lysosomal degradative enzymes, greatly enhances expression of the chosen reporter gene.“ This enhancement can be spectacular, as for instance in the human erythroleukemic cell line K562 in which subsequent to the

Phase I study to the immunotherapy of metastatic malignant melanoma by a cancer vaccine consisting of autologous cancer cells transfected with the human IL-2 gene.

Vienna: 30 November 1994 Würzburg: 7 February 1995 Freiburg: 7 February 1995 Registration of protocol with state authority AMB: 7 November 1994 Bfarm: 30 October 1994 Registration of product with federal authority (Paul-Ehrlich-Institut) 30 December 1994 Therapeutic gene Human interleukin-2 Regulatory element(s) CMV promoter Vector constructed by Institute of Molecular Pathology, Vienna, Austria Vector produced by Bender & Co., Vienna, Austria Transfer method Receptor-mediated, adenovirus enhanced gene transfer (transferinfection) in vitro Packaging cells N/A Target cells Autologous human malignant melanoma cells Indication Metastatic malignant melanoma (stage IV) First patient enrolled 15 December 1994 (Vienna) Patient number 12 evaluable patients Expected accrual rate 3 patients per month

Clusterin expression in cholestasis, hepatocellular carcinoma and liver fibrosis

Aims:  Clusterin shares functional properties with small heat shock proteins. In contrast to other heat shock proteins, it is present in the extracellular space. Its expression is altered in various diseases. The aim was to evaluate the presence and distribution of clusterin in liver diseases associated with cholestasis, in fibrosis and in hepatocellular carcinoma.

Tumor vaccines: effects and fate of IL-2 transfected murine melanoma cells in vivo.

We have previously demonstrated the general usefulness of the adenovirus-enhanced transferrinfection (AVET) in the generation of IL-2 producing tumor vaccines. By optimizing different parameters of the transfection protocol we were able to transform the poorly immunogenic M-3 mouse melanoma cell line into a potent immunogen. A long-lasting immunity was demonstrated after administration of the IL-2 releasing vaccine, since immunized animals successfully rejected native M-3 melanoma cells even after a period of more than 6 months. We also demonstrated that in vivo administration of such a vaccine is safe since transmission of the transfected IL-2 gene in host organs was not detected. IL-2 production ceased 2 days after injection because the engineered cells were destroyed. However, RT-PCR analysis of the site of vaccine injection suggests that IL-2 exerts its effects not only directly but also by inducing a set of other immunomodulator cytokines in situ that are probably indispensable in inducing a host response. We conclude that AVET of IL-2 into tumor cells is a safe and efficient method for the generation of tumor vaccines.

The Generation of Tumor Vaccines by Adenovirus-Enhanced Transferrinfection of Cytokine Genes into Tumor Cells

The major obstacle to immunotherapy of cancer is the poor immunogenicity of tumors, especially in man. This is somewhat surprising since the large number of genetic alternations found in advanced cancers should give rise to peptide neo-epitopes capable of being recognized in the context of MHC-I molecules by cytotoxic lymphocytes (Lurquin et al., 1989). Indeed, tumor-associated (Groen et al., 1987) and tumor-specific antigens (van der Bruggen and van den Eynde, 1992) which should be targets for immunological attack have recently been identified. This leads to the suspicion that foreign antigens are indeed present on most, or at least many, tumor cells and that tumor cells are not rejected as foreign because the response of the immune system to the presented foreign antigen is inadequate (Fearon et al., 1990).

Receptor-mediated gene delivery into mammalian cells

We have developed gene transfer systems which use the receptor- mediated endocytosis route to import DNA into mammalian cells. DNA gene constructs have been complexed with a polylysine-conjugated ligand (such as transferrin) for uptake via receptor-mediated endocytosis and polylysine-conjugated, endosome-disruption agents (such as replication-defective adenoviruses or peptides derived from the N-terminal sequence of influenza virus hemagglutinin) which allow cytoplasmic entry of the DNA. These complexes have been delivered to and expressed at very high level in a large proportion of target cells (up to 80% in primary fibroblasts, primary myoblasts or primary human melanoma cells).