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Engineering of oncolytic adenoviruses for delivery by mesenchymal stem cells to pancreatic cancer

Hammer, Katharina

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Abstract

Alternative treatment strategies for pancreatic cancer are urgently needed as all available therapies fail to work efficiently. Oncolytic adenoviruses are promising agents as they can be engineered to specifically replicate in tumor cells and subsequently lyse them (oncolysis). Oncolytic adenoviruses have shown an excellent safety profile in phase 1 and 2 clinical trials. However, these studies revealed a need for improvement of the therapeutic efficiency of the viral agents. The critical issues were the limited efficiency of tumor cell lysis and the poor delivery of the virus to the tumor site after systemic administration due to clearance by neutralizing antibodies and liver sequestration. The delivery problem was approached by using mesenchymal stem cells (MSCs), isolated from human bone marrow, as carrier cells to shield the virus from nonspecific uptake and neutralization. MSCs are established carriers to mediate targeted delivery of oncolytic adenoviruses to tumor sites in animal models. In this study, genetic modifications of oncolytic adenovirus were explored for improvement of the viral agent in the context of MSC delivery. The overall aim of the study was to identify those modifications, which mediated optimized adenovirus transduction and replication in MSCs, allowed unimpeded migration of infected MSCs for the time required for tumor homing, and mediated improved killing of the pancreatic cancer cells. From a set of capsid variants, performed transduction experiments identified the fiber chimera 5/3, derived from adenovirus serotype 5 fiber with the cell binding domain of serotype 3, to consistently mediate most efficient transduction in MSCs as well as in established and low-passage pancreatic tumor cells. Ad5/3 served as capsid background in the subsequently investigated set of modified, replication-competent, oncolytic adenoviruses. The modifications included: i. the deletion of the viral gene E1B19K aiming at improved virus replication and release kinetics, ii. the transgene insertions of the shedded death ligand TRAIL to mediate bystander killing, and iii. the transgene insertion of the prodrug-converting enzyme FCU1, which mediates conversion of the systemically administered prodrug 5-Fluorocytosine to the chemotherapeutic agent 5-Fluorouracil, for additional tumor cell killing. Infection of MSCs with the E1B19K-deleted or TRAIL-modified viruses resulted in dramatically improved virus replication and release kinetics compared to a matching control virus. Further, it was shown that MSCs infected with the E1B19K-deleted or TRAIL-modified viruses maintained their migration properties over 2 days. This time span corresponds to the reported time MSCs need in vivo to home to a tumor after systemic administration. From these observations it was concluded that the investigated modifications have the potential to improve MSC-mediated virus delivery. Subsequently, the E1B19K-deleted, TRAIL-expressing, and FCU1-expressing viruses were tested in established pancreatic tumor cell lines for improved killing. All modified viruses showed enhanced tumor cell killing in a subset of cell lines. Further, virus spread and killing was investigated in low-passage pancreatic tumor cell cultures, as a clinically relevant model, and in MiaPaCa-2 spheroids, to mimic viral behavior in a 3D tumor structure. Also in these models, the results indicated that the analyzed virus modifications are suited to achieve more efficient tumor cell killing. In the course of this study, virus mutants were identified, which possessed improved replication and release kinetics in MSCs, allowed unimpeded MSC migration, and showed enhanced pancreatic tumor cell killing abilities. Therefore, strategies to improve oncolytic adenoviruses for MSC-mediated delivery to pancreatic tumor sites were derived, which can contribute to improve clinical efficiency of adenovirotherapy.

Item Type: Dissertation
Supervisor: Müller, Prof. Dr. Martin
Date of thesis defense: 4 November 2014
Date Deposited: 17 Nov 2014 11:02
Date: 2014
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
570 Life sciences
Uncontrolled Keywords: Oncolytic Adenovirus, Pancreatic Cancer, Carrier Cells, prodrug activation, TRAIL
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