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Author Dieste Izquierdo, Arnau |
Abstract The advances in gene therapy have allowed the birth of new treatments against monogenic disorders, such as Duchenne muscular dystrophy. However, in order to make these treat-ments successful, the delivery vector must be safe, effective and stealth. Adeno-associated viruses, the main type of viral vector, fulfill the first two requisites but not the last one, as they are easily detected and neutralized by pre-existing serum antibodies. To avoid this recogni-tion, it has been previously proposed their encapsulation inside extracellular vesicles, result-ing in the so-called vexosomes, which could hide the virus from the immune system. So far, this has only been achieved by producing viruses and vesicles in the same donor cell, but nowadays the production of viruses is specific to a limited number of cell lines, and thus, it is not possible the encapsulation inside vesicles coming from the target cells of a disease. In fact, it is interesting to use a donor cell line that simulates the target cells, since the released extracellular vesicles are described in the bibliography to be naturally vectorized to the same lineage they come from, thus avoiding the need of adding usually expensive targeting moie-ties. In order to overcome this limitation, in this project it has been designed a method that allows the exogenous encapsulation of adeno-associated viruses inside previously isolated vesicles from a myoblast cell line. For this purpose, four techniques traditionally used to load small molecules have been selected and they have been optimized to be applicable to viruses with their confirmation by a proof-of-concept study using GFP protein as the element to be loaded. Finally, the production of vexosomes has been carried out, studying both the encap-sulation and transduction efficiencies. It has been found that extrusion and freeze-thaw are two promising techniques that allow a successful encapsulation of the viruses inside vesicles, while electroporation and permeabili-zation through surfactants seem to fail in this purpose. Moreover, the in vitro testing has showed optimistic results since the vexosomes produced through freeze-thaw, and especially extrusion, have exhibited significant transduction efficiencies when applying them to the my-oblast cell line. |
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Director Fornaguera i Puigvert, Cristina |
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Degree IQS SE - Undergraduate Program in Biotechnology |
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Date 2022-06-07
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