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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - DYNAP (Dynamic Penetrating Peptide Adaptamers)

Teaser

Summary

In the DYNAP project we have applied chemistry to investigate and develop new models to understand the intracellular deliver of macromolecules with biological activity. The project aims to the preparation of peptides bearing dynamic covalent bonds to identify new candidates with improved delivery efficiency and reduced toxicity. Our training as synthetic chemist has allowed us to solve all the technical challenges that we have faced during the preparation of these peptides. We have developed a straightforward solid phase synthetic method using orthogonal protecting groups to incorporate different dynamic reactive groups such as alkoxyamine, hydrazide, thiols, etc. We have also used organic synthesis to prepare other multiple connectors and different ligands that we have then anchored to the peptide structure. Our synthetic efforts have allowed us to prepare complex peptide and organic structures with a minimal number of synthetic steps. In this convergent methodology, the peptide scaffold is modulated by the modification with the corresponding pendants by the dynamic bond formation (i.e. hydrazone bond formation). Therefore, we can prepare different amphiphilic molecules that we can combine with different cargos of biological interest for intracellular delivery (i.e. siRNA, plasmid DNA, Cas9). Along the last two-three years we have considerably progressed in the different objectives proposed in the DYNAP project. The DYNAP project studies the importance of the secondary structure and the folding of penetrating peptides for their penetrating and endosomal escaping capacities. The approach is based in the connection of peptide scaffolds with different pendant tails. This strategy allowed us to prepare libraries of amphiphilic complex peptides with a little synthetic effort and a full control over the structural integrity. Using this methodology we have achieved several breakthrough in the filed of nucleic acids and proteins. For instance, we have been able to demonstrate for the first time the use of penetrating peptides for the supramolecular delivery of the Cas9 nuclease for gene edition. The result generate during the first half of the project have shown a strong market transference potential and the patents and MTAs signed certify its applicability and potential social impact. We continue with our efforts towards the understanding of the molecular underpinnings behind the penetrating capacities. However, we are also now trying to reach a higher level of risk by approaching the delivery of new challenging cargos such as therapeutic antibodies and by the design of new protein nanoparticles for anticancer vaccines. We have also recently started a project to quantify the amount delivered and to simultaneously track it with spatiotemporal resolution.

Work performed

The DYNAP project studies the importance of the secondary structure and the folding of penetrating peptides for their penetrating and endosomal escaping capacities. The approach is based in the connection of peptide scaffolds with different pendant tails. This strategy allowed us to prepare libraries of amphiphilc complex peptides with a little synthetic effort and a full control over the structural integrity. Using this methodology we have achieved several breakthroughs in the filed of nucleic acids and protein delivery. For instance, we have been able to demonstrate for the first time the use of penetrating peptides for the supramolecular delivery of the Cas9 nuclease for gene edition. The result generate during the first half of the project have shown a strong market transference potential and the patents and MTAs signed certify its applicability and potential social impact. We continue with our efforts towards the understanding of the molecular underpinnings behind the penetrating capacities. In summary, the work performed during the first half of the project has been mainly focused in the synthetic efforts towards the development of a robust strategy for the preparation dynamic penetrating peptides. This work has delivered a robust methodology that we can now apply to prepare complex peptide and peptides hybrids to perform a wide range of different functional applications. We have also focused in learning and improving the level of biological characterization and experimental cell biology of the group and we have implemented many different techniques to the group biological experimental arsenal. We now know how to study in detail the different localization of macromolecular cargos inside the cells and we have also improved the comparison and quantification of the delivered payloads. Our work in new fluorescent probes has also been focused in the synthetic part of it and we have learned a lot in terms of preparation, purification and physicochemical characterization of fluorescent probes.

Final results

As a result of the first half of the project we have developed new dynamic amphiphilic peptides for the intracellular delivery of different nucleic acids and nucleases for genetic edition. These discoveries have gone beyond the state of the art as they have generated new kind of penetrating peptide vehicles with the capacity of delivering new cargos with potential therapeutic interest.1-5 For instance we have demonstrated, for the first time, the delivery of Cas9 using a penetrating peptide in a full supramolecular strategy using a penetrating peptide vehicle.3 We have extended this methodology in order to incorporate targeting ligands and we have discovered and protected new glycopeptide hybrids for enhanced cytosolic release and reduced toxicity.6 We have prepared the first polyhydrazides scaffolds to anchor different pendant tails to prepare in situ different amphiphlic polymers to screen for the delivery of nucleic acids (siRNA and plasmid DNA)4,5 across a consistent degree of polymerization. We have also advanced in the synthesis and characterization of novel fluorescent probes for the study of the internalization pathway. We are also now trying to reach a higher level of risk by approaching the delivery of new challenging cargos such as therapeutic antibodies and by the design of new protein nanoparticles for anticancer vaccines. We have also recently started a project to quantify the amount delivered and to simultaneously track it with spatiotemporal resolution.

(1) Aqueous Synthesis and in-Situ Rapid Screening of Amphiphilic Polymers; Priority Date: 17 December 2015; Priority Application Number: ES201531831; International Application Published as WO 2017/102894 A1; US Patent Application US 16/062,536; European Patent Application EP 16822404.6. Aqueous Synthesis and In-situ Rapid Screening of Amphiphilic Polymers; priority date: 17 December 2015; priority application number: ES201531831; International Application published as WO 2017/102894 A1; US Patent Application US 16/062,536; European Patent Application EP 16822404.6.
(2) Louzao, I.; GarcÃa-FandiÃ±o, R. G. X. A.-F. X.; Montenegro, J. Hydrazone-Modulated Peptides for Efficient Gene Transfection. J. Mat. Chem. B 2017, 5 (23), 4426â€“4434.
(3) LostalÃ©-Seijo, I.; Louzao, I.; Juanes, M.; Montenegro, J. Peptide/Cas9 Nanostructures for Ribonucleoprotein Cell Membrane Transport and Gene Edition. Chem. Sci. 2017, 8 (12), 7923â€“7931.
(4) Priegue, J. M.; Crisan, D. N.; MartÃnez-Costas, J.; Granja, J. R.; Fernandez-Trillo, F.; Montenegro, J. In Situ Functionalized Polymers for siRNA Delivery. Angew. Chem. Int. Ed. 2016, 55 (26), 7492â€“7495.
(5) Priegue, J. M.; LostalÃ©-Seijo, I.; Crisan, D.; Granja, J. R.; Fernandez-Trillo, F.; Montenegro, J. Different-Length Hydrazone Activated Polymers for Plasmid DNA Condensation and Cellular Transfection. Biomacromolecules 2018, 19 (7), 2638â€“2649.
(6) Cell Penetrating Peptides; Priority Date: April 2018; Priority Application Number EP18382216.8. Not Published.
(7) Pazo, M.; FernÃ¡ndez-Caro, H.; Priegue, J. M.; LostalÃ©-Seijo, I.; Montenegro, J. Tuning the Properties of Penetrating Peptides by Oxime Conjugation. Synlett 2017, 28, 924â€“928.
(8) Juanes, M.; LostalÃ©-Seijo, I.; Granja, J. R.; Montenegro, J. Supramolecular Recognition and Selective Protein Uptake by Peptide Hybrids. Chem. Eur. J. 2018, 24 (42), 10689â€“10698.
(9) Reina, J.; Rioboo, A.; Montenegro, J. Glycosyl Aldehydes: New Scaffolds for the Synthesis of Neoglycoconjugates via Bioorthogonal Oxime Bond Formation. Synthesis 2018, 50 (04), 831â€“845.
(10) Pazo, M.; Juanes, M.; LostalÃ©-Seijo, I.; Montenegro, J. Oligoalanine Helical Callipers for Cell Penetration. Chem. Commun. 2018, 54 (50), 6919â€“6922.