The Diversity of Parvovirus Telomeres

Parvoviridae are small viruses composed of a 4–6 kb linear single-stranded DNA protected by an icosahedral capsid. The viral genes coding non-structural (NS), capsid, and accessory proteins are flanked by intriguing sequences, namely the telomeres. Telomeres are essential for parvovirus genome replication, encapsidation, and integration. Similar (homotelomeric) or different (heterotelomeric) at the two ends, they all contain imperfect palindromes that fold into hairpin structures. Up to 550 nucleotides in length, they harbor a wide variety of motifs and structures known to be recognized by host cell factors. Our study aims to comprehensively analyze parvovirus ends to better understand the role of these particular sequences in the virus life cycle. Forty Parvoviridae terminal repeats (TR) were publicly available in databases. The folding and specific DNA secondary structures, such as G4 and triplex, were systematically analyzed. A principal component analysis was carried out from the prediction data to determine variables signing parvovirus groups. A special focus will be put on adeno-associated virus (AAV) inverted terminal repeats (ITR), a member of the genus Dependoparvovirus used as vectors for gene therapy. This chapter highlights the diversity of the Parvoviridae telomeres regarding shape and secondary structures, providing information that could be relevant for virus-host interactions studies

Amphiphilic block copolymers enhance the cellular uptake of DNA molecules through a facilitated plasma membrane transport

Amphiphilic block copolymers have been developed recently for their efficient, in vivo transfection activities in various tissues. Surprisingly, we observed that amphiphilic block copolymers such as Lutrol® do not allow the transfection of cultured cells in vitro, suggesting that the cell environment is strongly involved in their mechanism of action. In an in vitro model mimicking the in vivo situation we showed that pre-treatment of cells with Lutrol®, prior to their incubation with DNA molecules in the presence of cationic lipid, resulted in higher levels of reporter gene expression. We also showed that this improvement in transfection efficiency associated with the presence of Lutrol® was observed irrespective of the plasmid promoter. Considering the various steps that could be improved by Lutrol®, we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol®. Microscopic examination of transfected cells pre-treated with Lutrol® confirmed that more plasmid DNA copies were internalized. Absence of cationic lipid did not impair Lutrol®-mediated DNA internalization, but critically impaired endosomal escape. Our results strongly suggest that in vivo, Lutrol® improves transfection by a physicochemical mechanism, leading to cellular uptake enhancement through a direct delivery into the cytoplasm, and not via endosomal pathways

RAAV WITH CHEMICALLY MODIFIED CAPSID

The invention is directed to the field of gene therapy, i.e. gene delivery into target cells, tissue, organ and organism, and more particularly to gene delivery via viral vectors. The inventors showed that it is possible by chemical coupling to modulate the coupling of a ligand in the surface of the capsid of AAV, for example AAV2 and AAV3b. In particular, the present invention relates to a recombinant Adeno-Associated Virus (rAAV) vector particle having at least one primary amino group contained in the capsid proteins, chemically coupled with at least one ligand L, wherein coupling of said ligand L is implemented through a bond comprising a -CSNH- bond and an optionally substituted aromatic moiety. Particularly, the inventors tested the chemical coupling of mannose ligand on AAV2 for subretinally injection to rats. The present invention further relates to a method for chemically coupling an Adeno-Associated Virus (AAV) vector particle with at least one ligand L and to a Recombinant Adeno-Associated Virus (rAAV) vector particle obtained by said method as well as a pharmaceutical composition comprising it and their corresponding medical use

Tyrosine conjugation methods for protein labelling

International audienceOver the last two decades, the development of chemical biology and the need for more defined protein conjugates have fostered active research on new bioconjugation techniques. In particular, a wide range of biorthogonal labelling strategies have been reported to functionalize the phenol side chain of tyrosines (Tyr). Tyr occur at medium frequency and are partially buried at the protein surface, offering interesting opportunities for site-selective labelling of the most reactive residues. Tyr-targeting has proved effective for designing a wide range of important biomolecules including antibody-drug conjugates, fluorescent or radioactive protein probes, glycovaccines, protein aggregates and PEG-conjugates. Innovative methods have also been reported for site-specific labelling with ligand-directed anchors and for specific affinity capture of proteins. This review will present and discuss these promising alternatives to the conventional labelling of the nucleophilic lysine and cysteine residues

L’électrochimie-click pour la fonctionnalisation de surfaces virales, bactériennes ou de cellules eucaryotes

International audienceNo abstract availabl

Enzyme-triggered PEGylated siRNA-nanoparticles for controlled release of siRNA

A key goal of our recent research efforts has been to develop novel 'triggerable nanoparticle' systems with real potential utility in vivo. These are designed to be stable from the point of administration until a target site of interest is reached, then triggered for the controlled release of therapeutic agent payload(s) at the target site by changes in local endogenous conditions or through the application of some exogenous stimulus. Here we describe investigations into the use of enzymes to trigger RNAi-mediated therapy through a process of enzyme-assisted nanoparticle triggerability. Our approach is to use PEG2000-peptidyl lipids with peptidyl moieties sensitive to tumour-localized elastase or matrix metalloproteinase-2 digestion, and from these prepare putative enzyme-triggered PEGylated siRNA-nanoparticles. Our results provide initial proof of concept in vitro. From these data, we propose that this concept should be applicable for functional delivery of therapeutic nucleic acids to tumour cells in vivo, although the mechanism for enzyme-assisted nanoparticle triggerability remains to be fully characterized.</p

The Diversity of Parvovirus Telomeres

Parvoviridae are small viruses composed of a 4–6 kb linear single-stranded DNA protected by an icosahedral capsid. The viral genes coding non-structural (NS), capsid, and accessory proteins are flanked by intriguing sequences, namely the telomeres. Telomeres are essential for parvovirus genome replication, encapsidation, and integration. Similar (homotelomeric) or different (heterotelomeric) at the two ends, they all contain imperfect palindromes that fold into hairpin structures. Up to 550 nucleotides in length, they harbor a wide variety of motifs and structures known to be recognized by host cell factors. Our study aims to comprehensively analyze parvovirus ends to better understand the role of these particular sequences in the virus life cycle. Forty Parvoviridae terminal repeats (TR) were publicly available in databases. The folding and specific DNA secondary structures, such as G4 and triplex, were systematically analyzed. A principal component analysis was carried out from the prediction data to determine variables signing parvovirus groups. A special focus will be put on adeno-associated virus (AAV) inverted terminal repeats (ITR), a member of the genus Dependoparvovirus used as vectors for gene therapy. This chapter highlights the diversity of the Parvoviridae telomeres regarding shape and secondary structures, providing information that could be relevant for virus-host interactions studies.</jats:p

Self-assembling complexes between binary mixtures of lipids with different linkers and nucleic acids promote universal mRNA, DNA and siRNA delivery

Protein expression and RNA interference require efficient delivery of DNA or mRNA and small double stranded RNA into cells, respectively. Although cationic lipids are the most commonly used synthetic delivery vectors, a clear need still exists for a better delivery of various types of nucleic acids molecules to improve their biological activity. To optimize the transfection efficiency, a molecular approach consisting in modifying the chemical structure of a given cationic lipid is usually performed, but an alternative strategy could rely on modulating the supramolecular assembly of lipidic lamellar phases sandwiching the nucleic acids molecules. To validate this new concept, we synthesized on one hand two paromomycin-based cationic lipids, with either an amide or a phosphoramide linker, and on the other hand two imidazole-based neutral lipids, having as well either an amide or a phosphoramide function as linker. Combinations of cationic and helper lipids containing the same amide or phosphoramide linkers led to the formation of homogeneous lamellar phases, while hybrid lamellar phases were obtained when the linkers on the cationic and helper lipids were different. Cryo-transmission electron microscopy and fluorescence experiments showed that liposomes/nucleic acids complexes resulting from the association of nucleic acids with hybrid lamellar phases led to complexes that were more stable in the extracellular compartment compared to those obtained with homogeneous systems. In addition, we observed that the most active supramolecular assemblies for the delivery of DNA, mRNA and siRNA were obtained when the cationic and helper lipids possess linkers of different natures. The results clearly show that this supramolecular strategy modulating the property of the lipidic lamellar phase constitutes a new approach for increasing the delivery of various types of nucleic acid molecules

Liposome-based Formulation for Intracellular Delivery of Functional Proteins

International audienceThe intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal) enzyme and the anti-cytokeratin8 (K8) antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP)-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies