title: Development of a non-viral gene delivery vector based on the dynein light chain Rp3 and the TAT peptide.
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 (∼100nm) and positively charged (+28.6mV) 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™, 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.
authors.affiliation: Laboratório de Análise Genética e Molecular, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: M T P
authors.initials: MT
authors.lastname: Favaro
authors.affiliation: Laboratório de Análise Genética e Molecular, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: M A S
authors.initials: MA
authors.lastname: de Toledo
authors.affiliation: Departamento de Engenharia Química, Escola Politécnica, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: R F
authors.initials: RF
authors.lastname: Alves
authors.affiliation: Laboratório de Análise Genética e Molecular, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: C A
authors.initials: CA
authors.lastname: Santos
authors.affiliation: Laboratório de Análise Genética e Molecular, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: L L
authors.initials: LL
authors.lastname: Beloti
authors.affiliation: Instituto de Física Aplicada "Gleb Wataghin", Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: R
authors.initials: R
authors.lastname: Janissen
authors.affiliation: Faculdade de Engenharia Química, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: L G
authors.initials: LG
authors.lastname: de la Torre
authors.affiliation: Laboratório de Análise Genética e Molecular, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: A P
authors.initials: AP
authors.lastname: Souza
authors.affiliation: Departamento de Engenharia Química, Escola Politécnica, Universidade de São Paulo, São Paulo, SP, Brazil. Electronic address: adriano.azzoni@poli.usp.br.
authors.forename: A R
authors.initials: AR
authors.lastname: Azzoni
mesh_terms: D056445:Cytoplasmic Dyneins / Q000235:genetics* / Q000378:metabolism; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000494:pharmacology; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism; D018716:Molecular Mimicry; D010455:Peptides / Q000378:metabolism*; D011479:Protamines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000008:administration & dosage* / Q000378:metabolism; D014162:Transfection
chemical_list: D015696:Gene Products, tat; D008055:Lipids; C086724:Lipofectamine; D010455:Peptides; D011479:Protamines; D011994:Recombinant Proteins; D056445:Cytoplasmic Dyneins
keywords: Dynein light chain Rp3; Gene delivery; Non-viral protein vectors; TAT