Caracterización funcional de una nueva familia de péptidos bioactivos derivados de filamentos intermedios y estudio de sus posibles aplicaciones en salud humana

Abstract

This project started with a serendipitous discovery by the group of Dr. Joan Verdaguer (Universitat de Lleida) of a peptide called DIF-P derived from periferin, which belongs to a large family of intermediate filament (IF) derived peptides. These peptides have pro-inflammatory and cytotoxic activities (at concentrations higher than 50 [Mu]M) and present a core sequence of 9 amino acids that is highly conserved in all IFs. In this work, a comprehensive characterization of different activities (hemolytic and antimicrobial) was performed, in addition to the two mentioned above. In order to obtain a better cell penetration and internalization through biological membranes, the peptides studied underwent modifications at their carboxyl-terminal end, such as the addition of 8-arginine sequences (8R), which increases their activities. In addition, several difficulties were overcome, such as the low solubility of some of these peptides in saline media, which was finally solved by conjugation with the polymer Pluronic F-127 and its carboxylated derivative, a molecule that polymerizes forming micelles (m) and incorporates the cationic peptides, solubilizing them without any loss of activity. In turn, it was discovered that one of the identified peptides, called K18 because it is derived from FI keratin 18, after the addition of the 8R tails, shows greater hemolytic, cytotoxic and antimicrobial activity than the one initially discovered (DIF-P), so that the K18-8R peptide is the one we have mainly used in our experiments. Likewise, the pro-inflammatory activity of K18-8R(m) was further investigated and found to be optimal at concentrations of 5 [Mu]M. It has also been shown to activate the NLRP3 inflammasome in innate immune cells and to induce the production of IL-1beta, a process that is inhibited by specific inhibitors of this inflammasome. On the other hand, mechanistic studies have been carried out using electron microscopy assays on bacteria and it has been observed that K18-8R(m) is able to induce ruptures in the membrane of these bacteria. To corroborate this effect, confocal microscopy or fluorimetry assays using specific fluorochromes such as Calbryte have been performed, confirming that this peptide causes the outflow of intracellular Ca2+, supporting the idea that it causes some type of disruption in cell membranes, probably due to the formation of pores or other structures. Finally, a new activity of the peptide was discovered as a transduction-potentiating agent (PT), a line applicable to ex vivo gene therapy strategies. To study this activity, a large number of gene transduction experiments were performed using lentiviral vectors pseudotyped with the vesicular stomatitis virus G protein (VSV-G) and encoding a fluorescent protein (eGFP+) in both murine and human cell lines as well as primary cells: CD34+ hematopoietic progenitors, T cells and human NK cells. The results obtained with the K18-8R(m) peptide at concentrations between 1 and 3 [Mu]M are very promising, with up to an 8-fold increase in transduction, depending on the target cell type and experimental conditions used, and with results superior to those obtained with the best performing competitor product (LentiBoost) currently approved for clinical use. Finally, a new PT called K18-8R(m) has been discovered that could improve the efficiency and specificity of the genetic modification, reducing the doses required and contributing to the durability and success for the advancement of ex vivo gene therapy and its eventual vivo gene therapy and its eventual application in a wide variety of diseases.

Date of Award	5 Feb 2024
Original language	Spanish
Supervisor	Lluis Tort Bardolet (Director) & Jorge Barquinero Mañez (Director)