Unit:
Specialty Molecular Biomedicine Mechanisms of Disease, Molecular and Cellular Therapies, and BioinnovationLibrary of the School of Health Sciences
Supervisors info:
Μαρία Τσουμακίδου, Ερευνήτρια Β', ΙΒΚ, ΕΚΕΒΕ "Αλέξανδρος Φλέμιγκ"
Γεώργιος Κόλλιας, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Χριστόφορος Νικολάου, Ερευνητής Β', ΙΒΚ, ΕΚΕΒΕ "Αλέξανδρος Φλέμιγκ"
Original Title:
Optimization of in vivo gene editing in lung tumors using viral vectors
Translated title:
Optimization of in vivo gene editing in lung tumors using viral vectors
Summary:
Over the last 10 years, the development of genome engineering tools has evolved, opening opportunities for in vivo applications. The emergence of the CRISPR technology combined with the action of Cas9 and a guide RNA has shown great therapeutic potential by repurposing elements that can correct disease-causing mutations and eventually advance to the clinical setting. These components need to be transferred successfully within the cells to exert their role. As a result, researchers have also focused on investigating different modes and vehicles of delivery. In the context of cancer, CRISPR-based technologies have ameliorated our understanding when it comes to diagnosis and treatment. With the generation of knockout (KO) models and manipulation of endogenous genetic elements, CRISPR has offered insights regarding driver mutations and responses to treatment.
In the present study, we aim to perform the first in-house in vivo characterization of gene editing potential in the lung of healthy and tumor-bearing mice. Initially, we employ a 3rd generation lentiviral system that allows us to see the transduction efficiency via a fluorescent marker. We are able to infer the successful targeting of fibroblasts. At our next step, we perform an in vitro screening of 3 sgRNAs targeting Tgfbr1. The selection of the gene is based on the fact that Tgfb is highly expressed on cancer-associated fibroblasts and has been shown to convert them into a highly protumorigenic state called myofibroblasts. We work on fibroblasts previously isolated from tumor-bearing murine lungs that have been transduced to express constitutively Cas9. To produce the sgRNA virus, we use a 2nd generation lentiviral system. After the in vitro transduction with the sgRNAs, we select the one that shows the highest deletion efficiency of the protein in the cell line and perform the final in vivo trial. The animal model of use employs the TetOn system for the induction of the Cas9, meaning it requires the administration of doxycycline. Using this model, we can successfully transduce fibroblasts in the tumor setting and observe a deletion effect in the protein level of the receptor. Our data also show a reduction in the mRNA of the receptor, supporting that this in vivo gene editing trial has been successful.
As this is the first in vivo gene editing study in the lab and to our knowledge in the fibroblasts in the lung cancer setting, we believe that these are promising results. They lay the foundation for exploration and leave room for optimization of the application. In essence, they demonstrate the capability of in-house genome engineering tools and suggest that therapeutic applications using animal models have strong potential.
Main subject category:
Health Sciences
Keywords:
In vivo, Lung cancer, Genome engineering, Lentivirus, CRISPR/Cas9, Tgfbr1, Fibroblasts
File:
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THESIS_MANETA.pdf
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File access is restricted until 2025-11-25.
