Development and characterization of non-viral vectors based in proteins and liposomes to gene delivery.

Author(s)

Rafael Ferraz Alves

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Escola Politécnica (EP/BC)
• Defense date: 2013-10-24

Examining board members

Adriano Rodrigues Azzoni; Renato Mancini Astray; Viviane Maimoni Gonçalves

• Advisor: Adriano Rodrigues Azzoni

Abstract

One of the major challenges on the development of efficient protocols for gene therapy and DNA vaccination is the low efficiency of gene transfer by non viral vectors. This is mainly attributed to the fact that, during the traffic to target cells nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The objective of this work was the development and characterization of new multifunctional non-viral vectors, based on lipids and proteins, able to delivery efficiently the foreign pDNA (plasmid DNA) to the nucleus of mammalian cells. A model pDNA containing the reporter gene GFP was complexed to protamine or the recombinant protein (TRP3), forming binary complexes (BC). In addition, we studied the ability of the cationic liposomes (EPC:DOPE:DOTAP) to encapsulate this binary complexes to form pseudo-ternary complexes (PTCs). The studies of size (DLS) and zeta potential revealed that both proteins were able to condense pDNA to form small complexes (BCS and PTCs) (~100 nm) and positively charged (+11,9 mV a +36,8 mV), both interesting characteristics for transfections. However, the CPTs formed by TRP3 was that showed the highest transfections level (25,3%). The cytotoxicity assays indicated the BCs had a low effect on the cell viability. On the other hand, the biggest effect on cell death was found when PTCs were used. The results indicated the TRP3 associated to liposomes (PTCs) increased the delivery efficiency due to differences in the intracellular trafficking, suggesting a synergic effect between these different molecules in the vector in order to overcome the barriers found inside the cell. (AU)

• FAPESP's process: 11/15189-6 - Development and Characterization of "Artificial Viruses" for Gene Delivery in Gene Therapy and DNA Vaccination Studies
• Grantee: Rafael Ferraz Alves
• Support Opportunities: Scholarships in Brazil - Master