Gynecological cancers present a tremendous disease burden worldwide. Endometrial cancer, the most common gynecological malignancy, is predominantly a disease of deranged glandular function. The mechanisms by which known environmental risk factors influence the mutational profile of endometrial cancer are poorly understood. Non-HPV vulvar cancer, on the other hand, is a very rare gynecological malignancy of vulvar squamous cells with little known about its pathogenesis. Surgical resection of vulvar cancer is associated with high post-surgical morbidity. Pivotal to improving treatment and outcomes for patients with gynecological cancers is an understanding of the molecular drivers unique to each tumor type. To inform our understanding of endometrial gland regulation, I began my investigations with an assessment of normal endometrial adenogenesis in vivo and present the first evidence implicating the necessity of Sox17 in endometrial gland development. My data suggest Sox17 mediates adenogenesis via a non-cell autonomous mechanism from within the stromal compartment of the endometrium. I then interrogated the contribution of SOX17 to dysregulated glandular function in Type I endometrial adenocarcinoma in vitro. My findings reveal an oncogenic role of SOX17 in the Ishikawa Type 1 endometrial cancer cell line, with homozygous loss of SOX17 impairing cellular proliferation, blunting the cancer phenotype of these cells. The majority of cancers, including gynecological cancers, develop from the accumulation of genetic mutations that occur sporadically in cells over time. The complexity and heterogeneity of solid tumors, however, renders the identification of mutations responsible for driving tumorigenesis difficult. The Sleeping Beauty (SB) insertional mutagenesis system can be used to streamline sporadic tumor formation and driver mutation identification. I present results from an initial attempt to develop an SB model of endometrial cancer and discuss ways in which the SB system can be harnessed to evaluate tumorigenesis in a variety of tissue types and microenvironmental contexts. Finally, I present an SB model of metastatic vulvar cancer. Primary tumors from this model resulted in the identification of 76 novel candidate drivers of vulvar cancer, with the ubiquitin-specific peptidase, Usp9x, the most commonly disrupted gene in our screen. I show data suggesting that differential expression of Usp9x isoforms may underlie Usp9x-mediated tumorigenesis and preliminary data demonstrating the relevance of USP9X to human vulvar cancer. Taken as a whole, these data contribute to our scientific understanding of gynecological tissue homeostasis and cancers, lay the foundation for the development of an SB model of endometrial cancer, and describe the first reported model system for studying HPV-naive vulvar cancer in vivo.

Die Gentherapie hämatopoetischer Stammzellen (HSCs) besitzt das Potenzial, verschiedene erbliche, nur symptomatisch behandelbare, Erkrankungen dauerhaft zu heilen. Die Mehrheit der aktuell angewandten Verfahren dazu, basiert auf der Isolation von hämatopoetischen Stammzellen, der ex vivo Modifikation dieser Zellen durch retrovirale Vektoren und der Reinfusion der modifizierten Zellen in den immunsupprimierten Patienten. Dieser Ansatz ist mit einer Reihe von Nachteilen verbunden, unter anderem einem teilweisen Verlust des Rekonstitutionsvermögens der Stammzellen nach ex vivo Kultur oder der Gefahr der Transformation durch Integration des retroviralen Vektorgenoms. Darüber hinaus sind aktuelle Gentherapieansätze mit hohen Kosten und großem logistischem Aufwand verbunden, was den Zugang zu diesen Behandlungen für potentielle Patienten stark einschränkt. Die vorliegende Arbeit verfolgt einen neuen Ansatz zur Gentherapie von HSCs, der auf der Mobilisierung von Stammzellen aus dem Knochenmark in den peripheren Blutstrom und der Transduktion dieser Stammzellen mit adenoviralen Vektoren basiert. Hierbei codieren die Vektoren sowohl ein Transgen als auch eine Integrationsmaschinerie. Der erste Teil der Arbeit belegt in einem humanen CD46-transgenen Mausmodell, dass adenovirale Vektoren der ersten Generation in der Lage sind, mobilisierte HSCs im Blut zu transduzieren und dass es den so transduzierten Stammzellen möglich ist, zurück ins Knochenmark zu migrieren und dort das Transgen zu exprimieren. Allerdings wurde im Verlauf von zwei Wochen ein Rückgang der Transgenexpression beobachtet. Um dies zu umgehen, wurde ein adenovirales Vektorsystem der dritten Generation genutzt, das eine hochaktive Sleeping Beauty Transposase, zum Zweck der Transgenintegration, codiert. Dieses System ermöglichte die stabile Genmodifikation mobilisierter hämatopoetischer Stammzellen nach intravenöser Injektion. Die Expression des Transgens konnte über längere Zeitspannen (bis 12 Wochen) beobachtet werden. Die modifizeirten Stammzellen waren darüber hinaus in der Lage, genmodifizierte Kolonien in vitro zu bilden und das hämatopoetische System letal bestrahlter Mäuse nach Knochenmarkstransplantation zu rekonstituieren. Es wurde somit gezeigt, dass HSCs nach in vivo Modifikation weiterhin funktional waren. / The gene therapy of hematopoietic stem cells holds the potential for curative treatment of several otherwise incurable inherited diseases. The majority of current gene therapy treatments relies on the collection of hematopoietic stem cells, their ex vivo modification with retroviral vectors and their transplantation into a myeloconditioned patient. This approach entails several disadvantages, including a reduction of stem cell engraftment potential after ex vivo culture and the potential danger of integrational mutagenesis. In addition, the high costs and complex logistics of this approach limit the access of patients to gene therapeutic regimens. This work explores an alternative approach to hematopoietic stem cell (HSC) gene therapy, termed stem cell in vivo transduction. This approach is based on the mobilization of HSCs from the bone marrow into the peripheral blood and the transduction of the stem cells with adenoviral vectors delivering a transgene as well as a transgene integration machinery. In the first part of this work, it was shown that first-generation adenoviral vectors could be used for the transduction of mobilized HSCs in the periphery of human CD46-transgenic mice. Further, the transduced HSCs were able to home back to the bone marrow and express the transgene. However, over the course of 14 days, a loss of transgene expression in HSCs was observed. To ameliorate these shortcomings, helper-dependent adenoviral vectors encoding a hyperactive Sleeping Beauty transposase for transgene integration were used for stable gene modification of hematopoietic stem cells following intravenous vector administration in mobilized human CD46-transgenic mice. Using this improved vector platform, gene marking of bone marrow HSCs could be observed for extended periods of time (up to 12 weeks). Further, the functionality of the modified HSCs was demonstrated both in colony-forming progenitor assays as well as through the transplantation of gene-modified HSCs into lethally irradiated recipients. Transplantation of modified HSCsled to long-term multi-lineage reconstitution showing that gene-modified stem cells were fully functional. Subsequently the safety of systemic vector administration in mobilized hosts as well as of the Sleeping Beauty-mediated transgene integration was assessed in human CD46- transgenic mice. Lastly, the stem cell in vivo transduction approach was employed in NOG mice transplanted with human CD34+ cells, as well as in Macaca nemestrina non-human primates.

Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition. Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play also a prominent role in plasmid DNA delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise β cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53 deficient HCT116p53-/-/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.