Nabila Laroui

Seasonal influenza causes up to 650,000 deaths annually and remains a persistent pandemic threat due to zoonotic strains crossing the species barrier. Current antivirals, which target neuraminidase or the viral polymerase, have limited efficacy and rapidly select for resistant variants, highlighting the urgent need for new therapeutic strategies. The heterotrimeric influenza polymerase (FluPol), comprising PA, PB1, and PB2 subunits, is essential for viral replication and harbors virus-specific protein-protein interfaces that are potential drug targets. We focused on the highly conserved PA-PB1 interface, where the N-terminal peptide of PB1 binds the PA C-terminal domain with high affinity. Disrupting this interaction abrogates polymerase function, halting viral replication. Using phage display, we identified PB1-derived peptides with enhanced affinity for PA and characterized their binding via biophysical methods and X-ray crystallography. Lead peptides efficiently disrupted the PB1-PA interaction and inhibited polymerase activity in cell-based assays. To address peptide delivery challenges, we expressed these inhibitors intracellularly from synthetic mRNA formulated in lipid nanoparticles, achieving robust inhibition of viral replication in cultured cells. This work establishes intracellularly expressed peptide inhibitors as a viable antiviral strategy and provides a generalizable framework for targeting essential protein-protein interactions of influenza and other RNA viruses.

Le développement de thérapies géniques ciblées représente un défi majeur en cancérologie qui se heurte souvent à des problèmes de délivrance. Les systèmes de délivrance actuels génèrent de nombreux effets indésirables, dont beaucoup sont dus à des interactions non spécifiques. Ainsi, l’utilisation de nano-vecteurs pour la délivrance ciblée de siRNA devrait permettre de répondre à ces besoins cliniques. L’objectif général de cette thèse porte sur la conception et l’étude de nano-vecteurs de siRNA pour cibler et traiter des pathologies cancéreuses. Le premier axe de recherche est consacré à l’étude de peptides modifiés pour la délivrance du siRNA. Ce travail de thèse visait à améliorer la vectorisation et l’internalisation du siRNA dans les cellules cibles. Pour cela, deux types de peptides ont été développés : i) des CPP (Cell Penetrating Peptides) fonctionnalisés par une séquence de ciblage dérivée de la laminine ̶ ciblant les cellules du cancer du sein̶ et ii) des peptides agrafés qui contraignent une structure secondaire en hélice α permettant d’augmenter la résistance à la protéolyse et favoriser l'internalisation cellulaire. Le deuxième axe s’est orienté vers le développement d’une nouvelle génération de vecteurs obtenus par auto-assemblage. Deux stratégies ont été explorées: i) une approche d’assemblage dynamique covalent pour développer des polymères dynamiques covalents (PDC) obtenus par un processus d’adaptation à la cible siRNA et ii) une approche d’assemblage supramoléculaire de porphyrines cationiques induits par le siRNA. La première stratégie a permis, via la fonctionnalisation des PDC par du D-mannose, de démontrer une vectorisation ciblée de cellules du cancer colorectal et la seconde stratégie a conduit au premier exemple de thérapie duale combinant siRNA et thérapie photo-dynamique basé sur des petites molécules.

The perspective of using messenger RNA (mRNA) as a therapeutic molecule first faced some uncertainties due to concerns about its instability and the feasibility of large-scale production. Today, given technological advances and deeper biomolecular knowledge, these issues have started to be addressed and some strategies are being exploited to overcome the limitations. Thus, the potential of mRNA has become increasingly recognized for the development of new innovative therapeutics, envisioning its application in immunotherapy, regenerative medicine, vaccination, and gene editing. Nonetheless, to fully potentiate mRNA therapeutic application, its efficient production, stabilization and delivery into the target cells are required. In recent years, intensive research has been carried out in this field in order to bring new and effective solutions towards the stabilization and delivery of mRNA. Presently, the therapeutic potential of mRNA is undoubtedly recognized, which was greatly reinforced by the results achieved in the battle against the COVID-19 pandemic, but there are still some issues that need to be improved, which are critically discussed in this review.

Dynamic covalent libraries enable exploring complex chemical systems from which bioactive assemblies can adaptively emerge through template effects. In this work, we studied dynamic covalent libraries made of complementary bifunctional cationic peptides, yielding a diversity of species from macrocycles to polymers. Although polymers are typically expressed only at high concentration, we found that siRNA acts as a template in the formation of dynamic covalent polymers at low concentration in a process guided by electrostatic binding. Using a glycosylated building block, we were able to show that this templated polymerization further translates into the multivalent presentation of carbohydrate ligands, which subsequently promotes cell uptake and even cell‐selective siRNA delivery.

Dynamic covalent libraries enable exploring complex chemical systems from which bioactive assemblies can adaptively emerge through template effects. In this work, we studied dynamic covalent libraries made of complementary bifunctional cationic peptides, yielding a diversity of species from macrocycles to polymers. Although polymers are typically expressed only at high concentration, we found that siRNA acts as a template in the formation of dynamic covalent polymers at low concentration in a process guided by electrostatic binding. Using a glycosylated building block, we were able to show that this templated polymerization further translates into the multivalent presentation of carbohydrate ligands, which subsequently promotes cell uptake and even cell‐selective siRNA delivery.

Small interfering RNAs (siRNAs) are promising molecules for developing new therapies based on gene silencing; however, their delivery into cells remains an issue. In this study, we took advantage of stapled peptide technology that has emerged as a valuable strategy to render natural peptides more structured, resistant to protease degradation and more bioavailable, to develop short carriers for siRNA delivery. From the pool of stapled peptides that we have designed and synthesized, we identified non-toxic vectors that were able to efficiently encapsulate siRNA, transport them into the cell and induce gene silencing. Remarkably, the most efficient stapled peptide (JMV6582), is composed of only eight amino-acids and contains only two cationic charges.

Cell‐penetrating peptides (CPPs) are one of the most efficient vectors for achieving cellular uptake of different cargos. However, their application in cancer therapy is greatly limited due to the lack of tissue selectivity, as they are widely distributed in most tissues following in vivo administration. To introduce specificity and improve tumor targeting, a combination of a cell‐penetrating peptide (CADY) and a targeting ligand, the laminin receptor binding peptide (YIGSR) to target the 37/67 kDa laminin receptor, known to be upregulated in most cancer cells and to be correlated with the invasiveness and metastatic potential, is formulated. The targeting CPP named “Y‐CADY” does not induce any change in the physico‐chemical properties (secondary structure, small interference RNA (siRNA) complexation, size, and charge) of CADY. Interestingly, Y‐CADY is more efficient than CADY for gene silencing in cell lines, and more potent for targeting and for inducing apoptosis of tumor xenografts in zebrafish embryos. The present study demonstrates that the novel targeting CPP “Y‐CADY” is efficient for gene silencing and can serve as a vector to target cancer cells in vivo.