Nonviral gene transfer in human hematopoietic cells: Implications for immuno-gene therapy of cancer.

摘要

Ever since the development of gene transfer technology, the hematopoietic system has been an obvious and desirable target for gene therapy of both inherited and acquired diseases, accompanied with the initiation of multiple clinical protocols. All gene therapy strategies have two essential technical requirements, being efficient introduction of the relevant genetic material into the target cell and the expression of the transgene at therapeutic levels. To date, the most widely used and best understood vectors for gene transfer in hematopoietic cells are recombinant viral vectors. However, the weakness of current gene therapy protocols is attributable to the limitations of viral gene delivery, including safety, limited DNA insert size, production and packaging problems, replication-competent recombination and vector immunogenicity. For these reasons, nonviral gene transfer systems have become increasingly desirable in both fundamental and clinical areas of gene therapy research, especially in the field of gene-based. immunotherapy since genetic immunopotentiation strategies capitalize on the ability of the immune system to amplify the outcome of the gene transfer. In this thesis, efficiency of nonviral gene transfer was determined in various types of transformed and primary human hematopoietic. cells and potential implications for immunotherapy were investigated.;In a first part, we established high-level transgene expression in various human leukemic cell lines, activated primary T lymphocytes, and cultured CD34+ cells by electroporation-mediated gene delivery. Also, fresh unstimulated CD34+ cells were consistently transfected, even without preliminary CD34 cell purification. In addition, we show that an enhanced green fluorescent protein (EGFP) reporter gene can be used to select and clone stable transfectants of electroporated hematopoietic cells by consecutive FACSorting without concomitant drug selection.;In a next part we focus on the genetic modification of human dendritic cells (DC) and their potential use for immunotherapy of cancer. Gene transfer into DC would enable endogenous synthesis of the relevant antigen for subsequent major histocompatibility complex (MHC) class I processing and presentation to CD8+ cytotoxic T lymphocytes (CTL). As DC are the most potent antigen-presenting cells crucial for priming of naive T-cells, tumor antigen-loaded DC could be used for induction of tumor-specific (CTL). First, we described the in vitro generation of human dendritic cells (DC) derived from bone marrow progenitors in a two-stage system that enabled to investigate the effects of additional cytokines. Addition of interleukin (IL)-4 rather than interferon (IFN)-gamma improved phenotypical and functional characteristics of DC. Tumor necrosis factor (TNF)-alpha signalling was shown to be mediated by the TNF receptor 1. Next, we investigated the feasibility of gene transfer in different types of DC. Results show that CD34+ progenitor-derived DC and Langerhans cells (LC) were efficiently transfected by cDNA electroporation but not by cDNA lipofection. In contrast, monocyte-derived DC (Mo-DC) appeared relatively refractory to cDNA electroporation. Efficiency of electroporation clearly decreased when electroporation was performed at later stages during DC culture.;In a last step, we demonstrated that mRNA electroporation is much more effective and less toxic than plasmid cDNA electroporation in both leukemic cells and Mo-DC. Importantly, antigen-loading of the Mo-DC by mRNA electroporation was shown to be extremely effective for subsequent activation of antigen-specific CTL, in contrast to passive pulsing of antigen-encoding mRNA. Furthermore, mRNA transfection efficiency was comparable to retroviral transduction efficiency and was clearly correlated with the capacity to activate antigen-specific CTL. Therefore, mRNA transfection of DC provides an attractive approach to load DC with defined tumor antigens for future DC-based tumor vaccines.