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Evaluation of modular proteins for gene and drug delivery

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Author(s):
Marianna Teixeira de Pinho Favaro
Total Authors: 1
Document type: Doctoral Thesis
Institution: Universidade Estadual de Campinas, Instituto de Biologia
Defense date:
Abstract

Although delivering exogenous genes to a cell may seem a prosing approach, this strategy is still limited by the lack of ideal vectors, which are both efficient and safe. The several physical, enzymatical and diffusional barriers offered by the cells highly limit the delivery of the transgene to its target inside the cell, therefore the development of carriers could allow the improvement of gene therapy and DNA vaccination protocols. In this sense, the present work proposes the employment of modular multifunctional proteins to act as gene delivery vectors. Since the viruses recurrently use the microtubules network to reach the nucleus, it would be possible to mimic such strategy by using molecular motors as carriers. For that, dynein¿s light chain Rp3 was modified by the addition of a DNA binding domain and a TAT peptide, resulting in a protein that self assembles in nanoparticles that Interact with pDNA, forming stable and positively charged complexes. Such complexes efficiently transfect HeLa cells through mechanisms highly dependent on the microtubules network, indicating that this protein is capable of mimicking viral strategies to internalize exogenous genes with low toxicity and low pro-inflammatory profile. This cationic protein is capable of promoting the delivery of both pDNA and dsRNA, quickly leading to a perinuclear accumulation. In vivo assays in a DNA vaccine model indicate, however, that this vector still faces some obstacles that could not be anticipated from the in vitro assays. Although seemingly different, targeted delivery of drugs faces many obstacles similar to the ones from gene delivery, from the arrival to the target cell up to adequate internalization. A group of proteins composed by GFP and arginine-rich sequences developed for drug delivery protocols was characterized here. Results indicate that minor modifications to the sequence may affect the formation of nanoparticles. Oligomerization, as well as the amount of arginines in the sequence, may influence the internalization pathway, which can vary from unspecific mechanisms to receptor-dependent internalization through CXCR4. The application of modular proteins was shown to be a promising strategy that allows the fine-tuning of protein¿s features, as well as its internalization efficiency (AU)

FAPESP's process: 12/18850-8 - Development and in vitro and in vivo evaluation of gene delivery vectors based on dynein light chain Rp3 and synthetic peptides
Grantee:Marianna Teixeira de Pinho Favaro
Support type: Scholarships in Brazil - Doctorate