On Friday 7 October 2022, Alejandro Sanz Pérez will defend his PhD thesis "Utilizing Retroviral Particles as a Gene Delivery Platform for in vivo Imaging of Immune Cells upon Adoptive Cell Transfer and as a Model System for Developing New Means of Testing Viral Infections".
Time: 13:00
Place: Building 421, auditorium 71 & zoom: https://dtudk.zoom.us/meeting/register/u5wucemrrD8tHdeyBQgldWu5g9xg2pQStncy
Please be aware that the PhD defense may be recorded - This will also be informed at the beginning of the PhD defense.
Supervisor: Professor Thomas Lars Andresen
Co-Supervisor: Senior Researcher Anders Elias Andersen
Professor Anders Kristensen
Assessment committee:
Associate Professor Vasileios Bekiaris, DTU Health Technology
Associate Professor Maja Louise Arendt, University of Copenhagen – Section for Medicine, Oncology, and veterinary clinical pathology
Associate Professor Laura Mezzanotte, Erasmus University Medical Center (Erasmus MC)
Chairperson:
Senior Researcher, Jannik Larsen, DTU Health Technology
Abstract:
The scope of this PhD project was to utilize retroviral particles to obtain two different outcomes: the stable expression of reporter transgenes in primary murine T cells and the development of a novel viral testing method. Thus, the PhD was divided in Part I and Part II:
Part I focused on the optimization of the design, production, and transduction of retroviral particles for the obtainment of genetically engineered murine T cells for subsequent non-invasive in vivo imaging following adoptive transfer in mice. To do so, different reporter transgenes were designed in retroviral cassettes to enable efficient T cell imaging and tracking through various modalities: fluorescence, bioluminescence, and PET scanning. Subsequently, different approaches such as lentiviral and gamma-retroviral transduction, and CRISPR-Cas9 were optimized either directly on primary murine T cells or in murine bone marrow-derived stem cells. The latter was employed to achieve the repopulation of the hematopoietic lineage with cells expressing the given transgene. Overall, the completion of Part I rendered an optimized technique to induce the stable expression of different reporter transgenes in primary murine T cells. This set the context for a future thorough analysis of T cell kinetics upon adoptive transfer that could be used for the validation of different drug delivery platforms for cancer immune therapies, such as immune-gels or nanoparticles.
Part II harnessed retroviral particles as a model system to develop a particle detection and quantification method from virus-containing saliva based on z-stack fluorescence imaging and a Python-based software. This project was motivated during the beginning of the COVID-19 pandemic, when the public health restrictions were at their peak in Denmark, resulting in a collaboration with Odense University Hospital-Southern Denmark University (OUH-SDU) and DTU. A protocol for sample filtration and preparation was developed and optimized at DTU using HIV-1-derived lentivirus and heat-inactivated SARS-CoV-2 as a model system to mimic enveloped RNA-viruses. Thereafter, the protocol was transferred to OUH-SDU to be tested, first on intact SARS-CoV-2 particles, and later on infected people, both admitted patients and citizens. The successful completion of this project rendered a tool with potential of detecting respiratory viral infections. This could help in the decision-making of the pertinent public health measures from future pandemics before the specific tests are developed, or as a complementary viral testing method.