Development of a non-viral gene delivery vector based on the dynein light chain Rp3 and the TAT peptide

Autor(es)

Favaro, T. P. ^[1] ; de Toledo, M. A. S. ^[1] ; Alves, R. F. ^[2] ; Santos, C. A. ^[1] ; Beloti, L. L. ^[1] ; Janissen, R. ^[3] ; de la Torre, G. ^[4] ; Souza, A. P. ^[1] ; Azzoni, A. R. ^[2]

Afiliação do(s) autor(es)

^[1] Univ Estadual Campinas, Ctr Biol Mol & Engn Genet, Lab Anal Genet & Mol, Campinas, SP - Brazil

^[2] Univ Sao Paulo, Escola Politecn, Dept Engn Quim, BR-05508900 Sao Paulo - Brazil

^[3] Univ Estadual Campinas, Inst Fis Aplicada Gleb Wataghin, Campinas, SP - Brazil

^[4] Univ Estadual Campinas, Fac Engn Quim, Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Biotechnology; v. 173, p. 10-18, MAR 10 2014.
• Citações Web of Science: 10

Resumo

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. (AU)

• Processo FAPESP: 07/58323-9 - Estudo e desenvolvimento de proteínas de fusão para o transporte intracelular de vetores não virais através de proteínas motoras
• Beneficiário: Adriano Rodrigues Azzoni
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores