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Understanding the potential of pdmaema-based vectors for gene therapy of the retina / Diogo Miguel Barros Bitoque ; orient. Gabriela Araújo Silva

Main Author Bitoque, Diogo Miguel Barros Secondary Author Silva, Gabriela Araújo Language Inglês. Country Portugal. Publication Lisboa : NOVA Medical School, Faculdade de Ciências Médicas, 2018 Description 130 p. : il. ; 30 cm Dissertation Note or Thesis: Tese de Doutoramento, Mecanismos de Doença, Medicina Regenerativa, Universidade Nova de Lisboa
Topical name RPE
Gene therapy
Gene transfer
Vector trafficking
Academic Dissertation
CDU 616 Classification Medicina
Mecanismos de Doença
Medicina Regenerativ
Online Resources Click here to access the eletronic resource List(s) this item appears in: Teses NL
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Monografia Biblioteca NMS|FCM
BIT1 TeseD-2018 Presencial/Restrito 20180157NL

Tese de Doutoramento, Mecanismos de Doença, Medicina Regenerativa, Universidade Nova de Lisboa

It is known that an efficient gene therapy vector must overcome several steps to be able to express the gene of interest: (I) enter the cell by crossing the cell membrane; (II) escape the endo-lysosomal degradation pathway; (III) release the genetic material; (IV) traffic through the cytoplasm and enter the nucleus; and last (V), enable gene expression to synthetize the protein of interest. In recent years, we and others have demonstrated the potential of poly(2-(N,N’- dimethylamino)ethylmethacrylate) (PDMAEMA) as a gene therapy vehicle. Further optimization of gene transfer efficiency requires the understanding of the intracellular pathway of PDMAEMA. Therefore the goal of this study was to determine the cellular entry and intracellular trafficking mechanisms of our PDMAEMA vectors and determine the gene transfer bottleneck. For this, we have produced rhodamine-labeled PDMAEMA polyplexes that were used to transfect retinal cells and the cellular localization determined by co-localization with cellular markers. Our vectors quickly and efficiently cross the cell membrane, and escape the endo-lysosomal system by 24h. We have observed the PDMAEMA vectors to concentrate around the nucleus, and the DNA load to be released in the first 24h after transfection. These results allow us to conclude that although the endolysosomal system is an important obstacle, PDMAEMA gene vectors can overcome it. The nuclear membrane, however, constitutes the bottleneck to PDMAEMA gene transfer ability.

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