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Receptor-Targeted Viral Vectors for Basic and Medical Research Receptor-targeted viral vectors for basic and medical research Vom Fachbereich Biologie der Technischen Universität Darmstadt zur Erlangung des akademischen Grades eines Doctor rerum naturalium genehmigte Dissertation von Diplom Biochemikerin Anke Muth geb. Rasbach aus Friedberg (Hessen) 1. Referentin: Prof. Dr. Beatrix Süß 2. Referent: Prof. Dr. Harald Kolmar 3. Referent: Prof. Dr. Christian Buchholz Tag der Einreichung: 18.12.2014 Tag der mündlichen Prüfung: 06.03.2015 Darmstadt 2015 D 17 Die vorliegende Arbeit wurde unter der Leitung von Prof. Dr. Christian Buchholz in der Arbeitsgruppe „Molekulare Biotechnologie und Gentherapie“ am Paul-Ehrlich-Institut in Langen angefertigt. Die Betreuung seitens der Technischen Universität Darmstadt erfolgte durch Prof. Dr. Beatrix Süß vom Fachbereich Biologie. Parts of this thesis have been published Rasbach A, Abel T, Münch RC, Boller K, Schneider-Schaulies J & Buchholz CJ (2013). The Receptor Attachment Function of Measles Virus Hemagglutinin Can Be Replaced with an Autonomous Protein That Binds Her2/neu While Maintaining Its Fusion-Helper Function. Journal of Virology (IF1 4.648) 87: 6246-6256 Münch RC2, Muth A2, Muik AG, Friedel T, Schmatz J, Dreier B, Trkola A, Plückthun A, Büning H & Buchholz CJ (2015). Off-target-free gene delivery by affinity-purified receptor- targeted viral vectors. Nature Communications (IF 10.742), 6: 6246 Münch RC, Janicki H, Völker I, Rasbach A, Hallek M, Büning H & Buchholz CJ (2013). Displaying high-affinity ligands on adeno-associated viral vectors enables tumor cell-specific and safe gene transfer. Molecular Therapy (IF 6.425) 21: 109–118 Kneissl S, Abel T, Rasbach A, Brynza J, Schneider-Schaulies J & Buchholz CJ (2012). Measles virus glycoprotein-based lentiviral targeting vectors that avoid neutralizing antibodies. PLoS ONE (IF 3.534) 7: e46667 Friedel T, Hanisch LJ, Muth A, Honegger A, Abken H, Plückthun A, Buchholz CJ & Schneider IC (2015). Receptor-targeted lentiviral vectors are exceptionally sensitive towards the biophysical properties of the displayed single-chain Fv. Protein Engineering, Design and Selection (IF 2.319), Epub ahead of print Zhou Q2, Uhlig KM, Muth A, Kimpel J, Lévy C, Münch RC, Seifried J, Trkola A, Coulibaly C, von Laer D, Wels WS, Hartwig UF, Verhoeyen E & Buchholz CJ. Exclusive transduction of human CD4+ T cells upon systemic delivery of CD4-targeted lentiviral vectors. Manuscript in revision. 1 Impact Factor 2013 Journal Citation Report (Thomson Reuters, 2014) 2 shared first authors I Oral presentations at international conferences Rasbach A & Buchholz CJ. Cell surface receptor targeting expanded: from oncolytic measles viruses to lentiviral and AAV vectors. European Congress of Virology 2013, Lyon, France Rasbach A, Kirsch N, Abel T, Münch RC, Boller K, Schneider-Schaulies J, Reuter A & Buchholz CJ. Measles virus-pseudotyped lentiviral vectors as tool to understand the fusion complex. Measles Minisymposium 2013, Annecy, France Rasbach A, Abel T, Münch RC, Boller K, Schneider-Schaulies J & Buchholz CJ. Receptor attachment is not essential for the measles virus hemagglutinin membrane fusion-helper function. Annual Meeting of the Society of Virology 2013, Kiel, Germany Poster presentations at international conferences Muth A, Münch RC, Muik AG, Plückthun A, Büning H & Buchholz CJ. Adeno-associated viral vectors for targeting circulating tumor cells. UCT Science Day 2014, Frankfurt, Germany Rasbach A, Münch RC, Kalicki J, Büning H & Buchholz CJ. Second Generation Entry Targeted AAV Vectors: Reduced Off-Targeting, Flexibility in Receptor Choice and Enhanced Gene Delivery. American Society of Gene & Cell Therapy Meeting, 2014, Washington DC, USA Rasbach A, Münch RC, Muik AG, Plückthun A, Büning H & Buchholz CJ. Adeno- associated viral vectors for targeting disseminated and circulating tumor cells. German Cancer Consortium DKTK Retreat 2014, Heidelberg, Germany Rasbach A, Abel T, Münch RC, Boller K, Schneider-Schaulies J & Buchholz CJ. The receptor attachment function of measles virus hemagglutinin can be replaced with an autonomous protein that binds Her2 while maintaining its fusion-helper function. European Congress of Virology 2013, Lyon, France II Rasbach A & Buchholz CJ. Cell surface receptor targeting expanded: from oncolytic measles viruses to lentiviral vectors. Measles Minisymposium 2013, Annecy, France Rasbach A, Abel T, Münch RC, Schneider-Schaulies J & Buchholz CJ. A second generation lentiviral vector targeting system displays targeting ligands on a type I transmembrane protein. German Society of Gene Therapy Meeting 2012, Frankfurt, Germany III Summary/Zusammenfassung Summary Viral vectors are powerful tools for a steadily increasing number of applications in gene therapy and basic research. In recent years, many proof-of-concept studies and clinical trials resulted in important progress and benefit for patients. Strategies to customize viral vectors will be key to their future success. This thesis deals with viral vectors that were engineered to use a cell surface receptor of choice for cell entry. Such vectors can mediate a restricted gene delivery to a cell type defined by the targeted receptor. The first part of this thesis investigates lentiviral vectors (LVs) carrying engineered glycoproteins of measles virus (MV) to better understand the molecular events during particle cell entry that proceeds via membrane fusion. The second part focuses on adeno-associated viral vectors (AAV) which are non-enveloped particles. AAVs were engineered to assess their potential in detecting rare epithelial cell adhesion molecule (EpCAM)-positive tumor cells. The first step of MV infection is attachment mediated by the hemagglutinin (H) to one of its receptors which is thought to produce conformational changes in the membrane fusion protein (F) that trigger insertion of its fusion peptide into the target cell membrane. This fusion triggering model was challenged in this thesis by uncoupling the receptor attachment and the fusion-helper function of H by introducing mutations that made it blind to its normal receptors. An artificial receptor attachment protein specific for Her2/neu was incorporated into the membranes of pseudotyped lentiviral particles as a separate transmembrane protein along with H and F. Surprisingly, these particles entered efficiently into Her2/neu-positive SK-OV-3 as well as CHO-Her2 cells. Cell entry was pH-independent, required high-affinity receptor binding and was strictly dependent on the presence of H and F. H-specific monoclonal antibodies as well as mutations in H interfering with H/F interactions blocked cell entry. The vector particles mediated stable and specific transfer of reporter genes into Her2/neu-positive human tumor cells also in vivo, while exhibiting improved infectivity and higher titers as compared to conventional Her2/neu-targeted vectors displaying the targeting ligand on H. Additionally, several other targeting ligands were tested in this setting. Extending the current model of MV cell entry, the data suggest that receptor binding of H is not required for its fusion-helper function but particle-cell contact in general may be sufficient to induce the conformational changes in the H/F complex and to activate membrane fusion. Towards applying AAV targeting technology for the detection, modification or elimination of rare cells expressing EpCAM, which is a marker of circulating tumor cells (CTC), AAVs displaying designed ankyrin repeat proteins (DARPins) specific for EpCAM were generated. IV Summary/Zusammenfassung The DARPin Ec1 was displayed on the particle surface and mediated EpCAM-specific gene transfer into receptor-positive cell lines which was further confirmed by competition experiments with the extracellular domain of EpCAM. Transduction was clearly receptor density-dependent and non-malignant EpCAM-positive primary cells were only poorly transducable ex vivo and in vivo. Performance of vector particles was further enhanced by ion metal affinity chromatography purification. To assess the potency of EpCAM-AAV in detecting rare EpCAM-positive tumor cells, low amounts of MDA-MB-453 tumor cells were mixed with isolated human PBMC. EpCAM-AAV tracked basically all of the tumor cells, even when these made up only 0.3% of the cell mixture. In a next step, the conditions of circulating tumor cells in whole blood were mimicked. As few as 150 tumor cells present in 7.5 milliliters of human blood were readily detected by EpCAM-AAV suggesting that targeted AAVs form a new tool for the detection of rare tumor cells. The work of this thesis clearly demonstrates that receptor targeting of enveloped and non- enveloped viral vectors is a flexible and versatile technique. Highlighting that both LVs and AAVs can be used not only as gene therapy vectors but also in virological research and diagnostics, these targeting systems open up new possibilities for a broad spectrum of applications. V Summary/Zusammenfassung Zusammenfassung Virale Vektoren haben sich als nützliche Hilfsmittel für eine immer größer werdende Zahl von Anwendungen in der Gentherapie aber auch in der Grundlagenforschung etabliert. In den letzten Jahren wurde die Weiterentwicklung dieser Vektoren von vielen Machbarkeits- und klinischen Studien vorangetrieben. Strategien um virale Vektoren so anzupassen, dass sie spezifische und individuelle Kriterien erfüllen, werden der Schlüssel zu ihrem weiteren Erfolg sein. Dies könnte zum Beispiel durch Ansteuern eines definierten Rezeptors und somit einen auf bestimmte Zelltypen begrenzten
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