Membrane-active peptides from structural viral proteins

: identifying novel delivery vectors for gene therapy

Abstract

Membrane-active peptides provide wide therapeutic potential in several biomedical areas. Among these, cell-penetrating peptides (CPPs) are highly promising molecules in drug delivery, particularly when applied to gene therapy applications. This work aimed to identify novel CPP sequences in structural viral proteins using bioinformatics, followed by experimental validation. 270 structural viral proteins were screened for the existence of potential CPP sequences, which resulted in the identification of 2400 putative sequences. A subset of 14 viral CPPs was selected for in vitro testing as gene cargo vectors using a 15-mer ssDNA oligonucleotide as a model. Delivery efficiency was monitored by fluorescence spectroscopy, flow cytometry and confocal microscopy. Furthermore, biophysical assays were conducted to understand the physical-chemical properties required for effective CPP cellular delivery. As such, membrane dipole potential sensing, using di-8-ANEPPS, was employed to study the affinity of CPPs towards lipid membranes. Circular dichroism was used to infer about lipid membrane-induced CPP secondary structure. Moreover, conjugation between each viral CPP and oligonucleotides was evaluated by dynamic light scattering to infer about the proper formation of vector:cargo complexes. Six peptides demonstrated clear efficiency in delivering ssDNA into cells. Biophysical data showed that the molecular determinants required for an efficient CPP are dependent on CPP-lipid interactions and proper conjugation with the cargo to deliver. Thus, two CPPs were particularly efficient and should be considered for future development and characterization. Structural viral proteins are a viable source for new CPPs, which may also be explored for other membrane-active peptide biotechnologies, namely antimicrobial peptides.

Date of Award	27 Mar 2014
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Miguel Augusto Rico Botas Castanho (Supervisor)