Gene therapy is a promising treatment option for cancer. Ideally, a therapeutic gene is delivered specifically into tumor cells sparing the neighboring normal cells. For this purpose gene delivery vectors are designed that can recognize structures, which are exclusively expressed on tumor cells (i.e. the tumor-associated antigens -TAA-). Retroviral vectors are commonly used for gene therapy by modifying the envelope protein responsible for the recognition of the target cell. The Vesicular Stomatitis Virus G protein (VSV-G) is a well-liked choice for pseudotyping the retroviral vectors since it confers on the viral particle stability to allow concentration to high titers necessary for the clinical applications. However, the main drawback of VSV-G, the ubiquitously expressed receptor and thus the broad target range, hinders the use of this protein for targeted gene therapy. In this thesis, we aimed to modify the VSV-G for targeted gene therapy against Prostate Stem Cell Antigen (PSCA) -expressing tumors. Therefore we followed two approaches. The first approach comprised of the fusion of a single-chain antibody fragment against PSCA to the N-terminus of VSV-G. In the second approach the VSV-G was modified by insertion of a small epitope. We could demonstrate that two positions in the N-terminal region of VSV-G protein permit insertion of a ten amino acid long epitope. These mutant VSV-G proteins were successfully assembled into retroviral particles. We demonstrated that the mutant retroviral particles can be used for targeting to PSCA-positive cells using nanobeads. The nanobeads were chemically coupled to antibodies against the epitope in the VSV-G protein and PSCA on the tumor cell. These bispecific nanobeads allowed the recruitment of mutant retroviral particles to the PSCApositive cells. Our results point out the potential of these mutant retroviral particles in targeted gene delivery. Further studies will be necessary to assess the efficiency of in vivo targeted gene therapy using these mutant retroviral particles.

Gene therapy is a promising treatment option for cancer. Ideally, a therapeutic gene is delivered specifically into tumor cells sparing the neighboring normal cells. For this purpose gene delivery vectors are designed that can recognize structures, which are exclusively expressed on tumor cells (i.e. the tumor-associated antigens -TAA-). Retroviral vectors are commonly used for gene therapy by modifying the envelope protein responsible for the recognition of the target cell. The Vesicular Stomatitis Virus G protein (VSV-G) is a well-liked choice for pseudotyping the retroviral vectors since it confers on the viral particle stability to allow concentration to high titers necessary for the clinical applications. However, the main drawback of VSV-G, the ubiquitously expressed receptor and thus the broad target range, hinders the use of this protein for targeted gene therapy. In this thesis, we aimed to modify the VSV-G for targeted gene therapy against Prostate Stem Cell Antigen (PSCA) -expressing tumors. Therefore we followed two approaches. The first approach comprised of the fusion of a single-chain antibody fragment against PSCA to the N-terminus of VSV-G. In the second approach the VSV-G was modified by insertion of a small epitope. We could demonstrate that two positions in the N-terminal region of VSV-G protein permit insertion of a ten amino acid long epitope. These mutant VSV-G proteins were successfully assembled into retroviral particles. We demonstrated that the mutant retroviral particles can be used for targeting to PSCA-positive cells using nanobeads. The nanobeads were chemically coupled to antibodies against the epitope in the VSV-G protein and PSCA on the tumor cell. These bispecific nanobeads allowed the recruitment of mutant retroviral particles to the PSCApositive cells. Our results point out the potential of these mutant retroviral particles in targeted gene delivery. Further studies will be necessary to assess the efficiency of in vivo targeted gene therapy using these mutant retroviral particles.

Ziel der Arbeit war, zu untersuchen, ob der gezielte Einsatz gentherapeutischer Methoden zu einer Verhinderung der Abstoßung allogener Transplantate bzw. zu einer Verhinderung der Induktion des Ischämie-/Reperfusionsschadens in verschiedenen Transplantationsmodellen der Ratte beitragen kann. Dabei wurden zwei Schwerpunkte gesetzt: Zum einen wurde auf den ex-vivo Gentransfer von therapeutischen Molekülen direkt in das Transplantat mit Hilfe von rekombinanten Adenoviren fokussiert. Zum anderen wurde das Potenzial von retroviral modifizierten, allospezifischen T-Zellen als Träger therapeutischer Gene zur Verhinderung der Transplantatrejektion untersucht. Diese Habilitationsschrift umfasst dreizehn Originalartikel in internationalen Zeitschriften, sechs Übersichtsartikel (Reviews) und vier Manuskripte, die bereits zur Veröffentlichung eingereicht sind. / The aim of the research was to investigate, whether the specific use of gene therapeutic methods can play a role in the prevention of allogeneic graft rejection or in the prevention of the induction of ischemia/reperfusion damage in various rat transplantation models. Doing this there were to main focusses: First we concentrated on the ex-vivo gene transfer of therapeutic molecules directly into the graft, which was done using recombinant adenoviruses. Then we investigated the potential of retrovirally modified, allospecific T-cells as carriers for therapeutic genes for the prevention of graft rejection. This publication consists of thirteen original papers in international journals, six reviews and four manuscripts that have been submitted for publication.