Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/61556
Type: Artigo
Title: Development of a non-viral gene delivery vector based on the dynein light chain Rp3 and the TAT peptide
Author: Favaro, T. P.
Toledo, M. A. S. de
Alves, R. F.
Santos, C. A.
Beloti, L. L.
Janissen, R.
De la Torre, G.
Souza, A. P.
Azzoni, A. R.
Abstract: Gene therapy and DNA vaccination trials are limited by the lack of gene delivery vectors that combine efficiency and safety. Hence, the development of modular recombinant proteins able to mimic mechanisms used by viruses for intracellular trafficking and nuclear delivery is an important strategy. We designed a modular protein (named T-Rp3) composed of the recombinant human dynein light chain Rp3 fused to an N-terminal DNA-binding domain and a C-terminal membrane active peptide, TAT. The T-Rp3 protein was successfully expressed in Escherichia coli and interacted with the dynein intermediate chain in vitro. It was also proven to efficiently interact and condense plasmid DNA, forming a stable, small (similar to 100 nm) and positively charged (+28.6 mV) complex. Transfection of HeLa cells using T-Rp3 revealed that the vector is highly dependent on microtubule polarization, being 400 times more efficient than protamine, and only 13 times less efficient than Lipofectamine 2000 (TM), but with a lower cytotoxicity. Confocal laser scanning microcopy studies revealed perinuclear accumulation of the vector, most likely as a result of transport via microtubules. This study contributes to the development of more efficient and less cytotoxic proteins for non-viral gene delivery. (C) 2014 Elsevier B.V. All rights reserved.
Gene therapy and DNA vaccination trials are limited by the lack of gene delivery vectors that combine efficiency and safety. Hence, the development of modular recombinant proteins able to mimic mechanisms used by viruses for intracellular trafficking and nuclear delivery is an important strategy. We designed a modular protein (named T-Rp3) composed of the recombinant human dynein light chain Rp3 fused to an N-terminal DNA-binding domain and a C-terminal membrane active peptide, TAT. The T-Rp3 protein was successfully expressed in Escherichia coli and interacted with the dynein intermediate chain in vitro. It was also proven to efficiently interact and condense plasmid DNA, forming a stable, small (similar to 100 nm) and positively charged (+28.6 mV) complex. Transfection of HeLa cells using T-Rp3 revealed that the vector is highly dependent on microtubule polarization, being 400 times more efficient than protamine, and only 13 times less efficient than Lipofectamine 2000 (TM), but with a lower cytotoxicity. Confocal laser scanning microcopy studies revealed perinuclear accumulation of the vector, most likely as a result of transport via microtubules. This study contributes to the development of more efficient and less cytotoxic proteins for non-viral gene delivery.
Subject: Cadeia leve de dineína Rp3
Proteínas recombinantes
Vetores genéticos
Microtubos
Country: Holanda
Editor: Elsevier
Citation: Journal Of Biotechnology. Elsevier Science Bv, v. 173, n. 10, n. 18, 2014.
Rights: fechado
Identifier DOI: 10.1016/j.jbiotec.2014.01.001
Address: https://www.sciencedirect.com/science/article/pii/S0168165614000029
Date Issue: 2014
Appears in Collections:IFGW - Artigos e Outros Documentos
IB - Artigos e Outros Documentos
FEQ - Artigos e Outros Documentos

Files in This Item:
File SizeFormat 
000331207300003.pdf2.12 MBAdobe PDFView/Open


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.