Abstract
Gene therapy is a medical intervention involving the delivery of genetic material. The aim is to induce the host cell to produce new proteins in situ, which will have a therapeutic effect. Gene therapy has a wide range of applications, including vaccines, gene editors and monoclonal antibodies. Adenoviral vectors (AdV) are the most commonly used delivery system for gene therapy due to a number of beneficial characteristics: (i) extensive research provides a clear understanding of their interaction with human host factors, (ii) the ability to efficiently transduce cells with the appropriate surface markers, (iii) adenoviral vectors do not integrate into the human genome, thereby reducing the risk of unintended mutagenesis, (iv) they are relatively stable to physical influences, allowing storage over long periods, (v) they have a large packaging capacity, suitable for the delivery of multiple transgenes. A disadvantage of the most commonly used adenoviral vector, human adenovirus serotype C5 (HAdV5), is its high liver tropism and certain interactions with blood components, which can lead to the neutralization of the vector. However, the immunogenic property can also be used as an advantage: Adenoviral vectors show great promise for vaccine vector development due to their ability to induce T- and B-cell responses while exhibiting relatively low pathogenicity in humans. The best clinical examples to date are the COVID-19 adenoviral vaccines based on human and non-human AdV serotypes. The potential of adenoviruses to induce maturation of dendritic cells (DCs) and thus act as vaccine boosters makes them popular for vaccination approaches. The large coding capacity of HAdV5 allows the delivery of multiple payloads beyond the antigen itself. However, the HAdV5 has limited transduction efficiency to dendritic cells (DCs) and therefore requires very high viral loads. Therefore, the objective of this doctoral thesis is to investigate the potential of transducing dendritic cells with adenoviral vectors (HAdV5) for potential applications in tumor vaccination. To exploit the full potential of HAdV5, we use a versatile platform of modular retargeting adapters to enhance transduction into specific cell types, including challenging host cells. We achieve this by using stable trimerized protein clamps that bind with high avidity to the HAdV5 fiber knob, covering the CAR epitope and effectively reducing natural transduction via CAR. Transduction can now be redirected to surface cell markers via fused retargeting moieties such as DARPins, scFvs, peptides or small molecules. This allows transduction of a wide range of cell types with very low vector loads (Chapter 3). In addition, we were also able to use high-capacity vectors (HC-HAdV5) carrying multiple gene cassettes with a maximum capacity of up to 37 kbp (Chapter 4). In the main part of this thesis, we combine those two achievements and significantly improve targeted tumor vaccination by HAdV5 and HC-HAdV5 (Chapter 5). Using rational design, we construct a dual adapter for DC-SIGN and CD11c and demonstrate successful targeting of AdV5 to human and murine DCs. Our in vivo characterization examining draining lymph nodes clearly demonstrates the enhanced transduction of DCs by retargeted adenoviral vectors, without off-targeting to more abundant cell types (e.g. B or T lymphocytes). Furthermore, a tumor vaccination study demonstrates the advantageous co-expression of T cell stimulatory cytokines (IL-2v or IL-21) locally in lymph nodes alongside a potent tumor antigen. The autocrine and paracrine immune stimulation of IL-2v or IL-21, co-expressed by the same vaccine vector, showcases a potent vaccination effect at an exceptionally low dose of administered HC-HAdV5 (30x lower viral load). By targeting DCs and direct in situ production of the antigen in this host cell (including IL2v and IL-21 secretion), the population of cytotoxic T cells derived against the encoded antigen specifically multiplied. In addition, the antibody response against the encoded antigen was improved without increasing IgG levels against the hexon protein. Lymph node-directed gene therapy with significantly reduced vector loads avoids the potential systemic toxicity of stimulating payloads. Our proposed low-dose DC-targeted vaccine offers an effective solution for patients and also minimizes adenovirus-related side effects, with the potential to significantly reduce the cost of cell-based therapies. The robust immunogenicity of HC-HAdV5 with its large coding capacity (37 kbp DNA) opens up exciting possibilities for future therapeutic combination strategies. A collaborative project extends the platform to the transduction of human T cells in vitro and in vivo (Chapter 6). Like DCs, T cells are difficult to transduce with untargeted HAdV5. Targeting T cells in vivo has great potential for the generation of CAR T cells. We furthermore demonstrate the potential for pulmonary delivery of HAdV5 (Chapter 8). Targeted gene transfer into airway tissue shows potential for improved therapies of respiratory diseases (e.g. SARS-CoV-2)
Weiss, Fabian