Entry targeted lentiviral vectors for the specific modification of distinct subsets of immune cells

Although pathogens are ubiquitously found in the environment, catching disease is quite scarce. This can be attributed to the action of immune cells, protecting the body from invading microorganisms. A central role in the induction and orchestration of adaptive immune responses is ascribed to antigen presenting cells (APCs), like dendritic cells (DCs), and CD4+ T lymphocytes. Due to their important functions, these cell types gained center stage in immunotherapy. Thereby, their specific modification is highly desirable for several applications, including prophylactic or therapeutic vaccination approaches and the fight against human immunodeficiency virus type 1 (HIV-1) infection. In recent years, especially viral vectors proved to be powerful tools for the modification of target cells. Among them, lentiviral vectors (LVs) are of particular interest as they are not only able to transduce dividing but also non-dividing cells. They can be retargeted to basically any receptor of choice by pseudotyping with (engineered) measles virus (MV) glycoproteins (GPs) hemagglutinin (H) and fusion protein (F). Beside transfer of genetic information, retroviral and lentiviral vectors are able to deliver heterologous proteins into their target cells when the cargo proteins are genetically fused to structural proteins of the vector particle (“protein transfer vectors”, PTVs). Taking advantage of this system, the present thesis aimed to employ lentiviral vectors for the specific modification of signaling lymphocyte activation molecule (SLAM)+ APCs and CD4+ T lymphocytes. Initially, production of PTVs was optimized to allow maximum gene and protein transfer into transduced cells. In order to achieve targeting of SLAM-positive APCs, PTVs were pseudotyped with truncated MVwt-GPs with an established tropism for SLAM. Beside SLAM, a hitherto unknown epithelial MV receptor was postulated, to whose identification as adherens junction protein Nectin-4 the present thesis contributed to characterize SLAM- target cell populations of MVwt-GPs. Indeed, upon pseudotyping of PTVs with MVwt-GPs, marker gene and GFP, Cre or ovalbumin (Ova) cargo protein delivery was completely restricted to SLAM- and Nectin-4-positive CHO cells and naturally SLAM-expressing B cell lines, whereas broad transduction was observed for VSV-G pseudotyped control vectors. Specific excision of the loxP-flanked cerulean open reading frame in receptor-positive indicator cell lines following PTV-mediated transfer of Cre recombinase exemplarily demonstrated cytoplasmic protein transfer and unimpaired functionality of the delivered cargo protein. Finally, transfer of the model antigen Ova by MVwt-GP and VSV-G pseudotyped PTVs into receptor-positive, murine DCs in vitro or after administration into human SLAM-transgenic and control mice in vivo was shown to stimulate antigen-specific T cells, and here especially CD8+ T lymphocytes. In contrast to SLAM-targeted PTVs, which relied on MVwt-GPs, specificity of CD4-targeted LV (CD4-LV) was achieved by pseudotyping with engineered MV-GPs, displaying a CD4-specific designed ankyrin repeat domain (DARPin) as binding domain on receptor-blinded H. H-αCD4 was well expressed on the surface of HEK-293T packaging cells allowing its efficient incorporation into CD4-LV particles and giving rise to high titer vector stocks with average yields > 10e8 transducing units/ml after concentration. In vitro, CD4-LV demonstrated absolute receptor specificity on cell lines and the naturally mixed population of primary peripheral blood mononuclear cells (PBMC), with transgene expression exclusively detectable in CD4+ but not CD4- cells. Of particular note, CD4-LV was capable to stably transduce not only activated but also resting primary CD4+ lymphocytes. Beside transduction of CD4+ T lymphocytes, also gene transfer into CD4+ human macrophages could be demonstrated in vitro. In vivo, following systemic injection of CD4-LV transferring a bicistronic gfp/luciferase reporter gene, luciferase activity in NOD-scid IL2Rγ-/- mice reconstituted with human PBMC or hematopoietic stem cells was mainly detected in lymphoid organs. Flow cytometric analyses of GFP-expression in lymphoid organs and blood confirmed the strict receptor-dependency of transduction events found in vitro also to apply in vivo. Interestingly, a clear preference for transduction of CD4+ T memory cells over naïve CD4+ T lymphocytes was observed. Concluding, this thesis clearly demonstrated suitability of (engineered) MV-GPs pseudotyped LV for specific protein and gene delivery into distinct subsets of immune cells. By the successful generation of SLAM- and CD4-targeted LVs, which allowed specific modification of SLAM+ APCs and CD4+ T lymphocytes, including even resting T lymphocytes, respectively, the broad applicability of MV-GP based cell entry targeting approaches for diverse cell surface receptors was emphasized. Both PTVs and CD4-LV are promising tools for basic research and gene and immunotherapy, opening up new avenues for a multitude of diverse applications