Production of an effective vaccine and long-term treatment against human immunodeficiency virus (HIV) is elusive. In this thesis two different techniques were used in an attempt to insert HIV-neutralising monoclonal antibody (IgG1b12) sequences into a simian retroviral gene therapy agent pseudo-typed with vesicular stomatitis virus glycoprotein. Genes were encoded in either a poxvirus split-vector system or a baculovirus expression system. Both systems aim to produce replication incompetent pseudotyped virus like particles with simian origin. It is believed that the resulting non-infectious artificial lentivirus particles enter neighbouring cells, penetrate the nucleus and insert genetic material (the antibody gene) into the mammalian genome. The poxvirus split-vector system used in this project was a Vaccinia Retroviral Hybrid Vector, where recombinant modified vaccinia Ankara (MVA) is used to deliver the simian immunodeficiency virus (SIV) like particles into mammalian cells. However, the MVA system failed to express proteins of interest due to the instability of genetic insertion into the recombinant MVA genome. As an alternative strategy, two different BacMam systems were used to allow the production of VLPs, where mammalian cells are co-transduced with different recombinant baculoviruses (rBVs). VLPs were expressed either under the control of T7 RNA polymerase system or under the cytomegalovirus immediate early gene promoter. The results from the first BacMam system indicated that the T7 RNA polymerase system was not suitable to express detectable levels of proteins. The results indicated that translation of the produced mRNA by T7 promoter is inefficient, most likely because of the absence of RNA 5’ cap structure. To overcome this hybrid BV–T7 system limitation, a different system was developed. Proteins of interest from the second BacMam system were successfully expressed and detected using western blot analysis. VLPs were generated and visualised under electronic microscope. IgG1b12 was secreted in the supernatant of the transduced mammalian cells. Mammalian cells were successfully transduced with multiple different recombinant BVs simultaneously. The study establishes the feasibility of antibody gene transfer, and demonstrates the use of SIV like particles production to transduce mammalian cells using BacMam technology. The technique may have application for use as an immunotherapy of HIV infection as well providing long-acting prevention of HIV infection for those not yet infected with HIV.

Application of corona ions produced in air to B16F10 murine melanoma cells in vitro and in animal models resulted in the transport of molecular therapeutics across the cell membrane. This work presents the development of new methods for drug and gene delivery based upon similar principles as the traditional electrode driven membrane destabilization processes known as electroporation. This was achieved with non-contact corona ion deposition that temporarily increased the permeability of cell membranes. Interaction of corona charge with biological cells was studied and their potential for molecular delivery was established. Molecular delivery was first demonstrated in vitro using tracer molecules followed by in vitro delivery of the cytotoxic drug bleomycin. Building upon these results, the delivery of bleomycin coincident with ion deposition was xxi shown to significantly slow the growth of very aggressive solid tumors in animal models, compared to drug alone or no treatment. Delivery of plasmid DNA to cells in the skin of animal models indicated that application of corona ions (both positive and negative) to live tissue produced a four to six fold increase in gene expression. As this is the first significant study of the interaction and impact of corona ions on the delivery of drug and plasmid DNA to biological cells, numerous fundamental investigations were performed and discussed. A charge dose dependence was observed and physical mechanistic models were proposed. A model of cell resealing time constant following corona ion exposure was developed and demonstrated a reasonable prediction of experimental findings. The foundation laid by this work may enable continued exploration and use of corona ion deposition in the future as a new and promising physical method for drug and gene delivery.

In den letzten zwei Jahrzehnten wurden T-Lymphozyten verstärkt als Zielzellen für genetische Modifikationen eingesetzt, mit der Absicht vererbbare oder erworbene Krankheiten zu behandeln. Vor kurzem konnte unsere Arbeitsgruppe zeigen, dass alloantigen-spezifische, genmodifizierte T-Lymphozyten ein enormes Potential besitzen, als Transport- Systeme für therapeutische Gene in allogene Transplantate zu dienen. Diese T-Lymphozyten migrieren und akkumulieren spezifisch in das allogene Transplantat, in welchem es zu einer lokalen und starken T-Zell aktivierungsabhängigen Expression des Transgens kommt. In der vorliegenden Arbeit wurde das Potential von viralem IL-10 als therapeutisches Transgen untersucht. Dazu wurde zunächst in der gemischten Lymphozyten-Kultur (MLR) eine Lewis T-Zelllinie spezifisch für Ratten-DA Alloantigene generiert. Während der MLR wurden die Lymphozyten retroviral transduziert, so dass sie vIL-10 stabil exprimieren. Der Zytokin-Level der TvIL-10-Lymphozyten liegt 48h nach der T-Zellaktivierung zwischen 1-2ng/m. Die vIL-10 transgenen T-Lymphozyten zeigen einen CD4+CD25+ Phänotyp und sezernieren neben vIL-10 auch Ratten IL-10 und IFN-g aber kein IL-4, ähnlich wie T-regulatorische Zellen Typ 1 (Treg1). Zunächst wurden die vIL-10 transgenen T-Lymphozyten hinsichtlich ihres Potentials untersucht, die alloantigen-spezifische Immunantwort in vitro zu modulieren. Dazu wurden 5% vIL-10 transgener T-Lymphozyten zu einer MLR hinzugegeben. Zuvor wurden die naiven Lewis T-Lymphozyten mit einem Membranfarbstoff markiert, um die Proliferation und die Zytokin-Expression dieser Zellen zu untersuchen. Im Vergleich zu Kontroll-MLRs ohne transgene oder mit EGFP-transgenen Lymphozyten konnte eine signifikante Inhibierung der Proliferation als auch der INF-g Expression von naiven T-Lymphozyten detektiert werden. Trotz dieses großen Potentials in vitro, führte der adoptive Transfer der vIL-10 transgenen Zellen allein oder in Kombination mit Cyclosporin (0,5mg/kg/Tag) nicht zu einer Verlängerung der Transplantatüberlebenszeit im allogenen Herztransplantationsmodell. Diese Daten zeigen, dass die Überexpression von vIL-10 im Transplantat eines starken Abstoßungsmodells nicht zur Verlängerung der Transplantatüberlebenszeit führt. Außerdem kann das in vitro gezeigte regulatorische Potential dieser T-Zellen nicht zwangsläufig auf ihr in vivo Potential übertragen werden. / During last two decades T lymphocytes have become key targets for genetic modification in order to treat inherited or acquired human diseases. Recently, we demonstrated the capacity of allospecific gene-engineered T lymphocytes as a transport shuttle for therapeutic transgenes into allografts. These lymphocytes migrate and accumulate specifically in allografts where alloantigen-driven T cell activation strongly enhances local expression of the gene of interest. In this study, the influence of viral IL-10 as a therapeutic transgene was addressed. Lewis T cell lines specific for DA rat alloantigens were generated in a modified mixed lymphocyte reaction protocol (MLR). During MLR, lymphocytes were genetically modified to express vIL-10 using a retroviral gene expression system. The cytokine level in the supernatant of TvIL-10-lymphocytes varied between 1-2 ng/ml 48h after T-cell activation. Like T regulatory 1 (Treg1) cells, vIL-10 transgenic T lymphocytes express the phenotype CD4+25+ and secrete, in addition to vIL-10, rat IL-10 and IFN-g but no IL-4. First we evaluated the potential of vIL-10 transgenic T cell lines to modulate alloantigen-specific immune responses in vitro. Small numbers of TvIL-10-lymphocytes were added to MLR (less than 5%). Naive cells were stained with membrane dyes to trace proliferation and to analyze cytokine expression. In comparison to control MLR with no transgenic cells or equal numbers of TEGFP-lymphocytes, the proliferation as well as the production of IFN-g of naive responder cells were significantly diminished. Despite this regulatory capacity in vitro, the vIL-10 transgenic T lymphocytes were not able, either alone or in combination with suboptimal cyclosporine (0,5mg/kg/day), to prolong the survival of DA rat cardiac allografts in LEW recipients. These data demonstrate that intragraft IL-10 overexpression is not sufficient to prolong allograft survival in a high-responder strain combination and that the regulatory capacity of T cells in vitro does not predict their in vivo efficiency.