A major holdback to the wide application of gene therapy and DNA vaccination arises from the lack of an ideal vector, that is both safe and efficient. Although safer, nonviral vectors face a series of physical, enzymatic and diffusional barriers that limits the transgene's arrival to the nucleus of target cells. As a continuation of the work previously developed during the student's master's degree, the main objective of this project is to develop multifunctional nonviral vectors based on recombinant proteins, which can be evaluated in vitro as well as in vivo. We use the recombinant protein Rp3, a dynein light chain, aiming to explore the natural ability of molecular motors like dynein to carry cargoes from the periphery to inside the cell, through the microtubules network. Rp3 will be used fusioned to synthetic peptides: DNAbinding sequences, a cell-penetratin peptide (TAT) and a His Tag, and will be named T-Rp3. We expect to understand the mechanisms involved in the traffic of these vectors. By combining different techniques such as dynamic light scattering, zeta potential, interaction and transfection assays, as well as confocal microscopy, we will be able to correlate the physical-chemical properties of the pDNA:protein complex to the intracellular traffic and gene delivery efficiency. By transfecting mammalian cells we will be able to asses our vectors' efficiency to perform in vitro gene delivery, and finally, we will evaluate our vectors' potential through an in vivo model of cystic fibrosis (mice), performing immune histochemistry and quantification of repórter gene expression.
News published in Agência FAPESP Newsletter about the scholarship: