Enhancing natural killer cells proliferation and cytotoxicity using imidazole-based lipid nanoparticles encapsulating interleukin-2 mRNA.

mRNA applications have undergone unprecedented applications-from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production.

Impact of net charge, targeting ligand amount and mRNA modification on the uptake, intracellular routing and the transfection efficiency of mRNA lipopolyplexes in dendritic cells.

Targeting mRNA formulations to achieve cell specificity is one of the challenges that must be tackled to mettle their therapeutic potential. Here, lipopolyplexes (LPR) bearing tri-mannose-lipid (TM) are used to target mannose receptor on dendritic cells. We investigated the impact of the net charge and percentage of TM units on the binding, uptake, transfection efficiency (TE) and RNA sensors activation.

CFTR and dystrophin encoding plasmids carrying both luciferase reporter gene, nuclear import specific sequences and triple helix sites.

Duchenne Muscular Dystrophy and Cystic Fibrosis are two major monogenetic diseases which could be treated by non-viral gene therapy. For this purpose, plasmid DNA (pDNA) coding for the functional genes requires its equipment with signal molecules favouring its intracellular trafficking and delivery in the nucleus of the target cells. Here, two novel constructions of large pDNAs encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and full-length dystrophin (DYS) genes are reported.

Reversible stabilization of DNA/PEI complexes by reducible click-linkage between DNA and polymer. A new polyplex concept for lowering polymer quantity.

Nonviral transfection of mammalian cells can be performed with electrostatic complexes (polyplexes) between a plasmid DNA (pDNA) encoding a foreign gene and a cationic polymer. However, an excess of the cationic polymer is required for pDNA condensation and polyplexes formation, which generate in vivo toxicity. Here, we present a new concept of polyplexes preparation aiming to reduce the polymer quantity.

Synthesis of Double Hydrophilic Block Copolymers Poly(2-isopropyl-2-oxazoline-b-ethylenimine) and their DNA Transfection Efficiency.

Gene delivery is now a part of the therapeutic arsenal for vaccination and treatments of inherited or acquired diseases. Polymers represent an opportunity to develop new synthetic vectors for gene transfer, with a prerequisite of improved delivery and reduced toxicity compared to existing polymers. Here, the synthesis in a two-step's procedure of linear poly(ethylenimine-b-2-isopropyl-2-oxazoline) block copolymers with the linear polyethylenimine (lPEI) block of various molar masses is reported; the molar mass of the poly(2-isopropyl-2-oxazoline) (PiPrOx) block has been set to 7 kg mol .

DNA minicircles as novel STAT3 decoy oligodeoxynucleotides endowed with anticancer activity in triple-negative breast cancer.

Decoy technology is a versatile and specific DNA oligonucleotide-based targeting strategy of pathogenic transcription factors (TFs). Chemical modifications of linear decoy oligonucleotides have been made to decrease nuclease sensitivity because of the presence of free ends but at the cost of new limitations that affect their use as therapeutic drugs. Although a short DNA minicircle is a phosphodiester nucleic acid without free ends, its potential therapeutic activity as a TF decoy oligonucleotide has not yet been investigated.

Selective attachment of a microtubule interacting peptide to plasmid DNA via a triplex forming oligonucleotide for transfection improvement.

In nonviral gene therapy approaches, the linkage of signal molecules to plasmid DNA (pDNA) is of interest for guiding its delivery to the nucleus. Here, we report its linkage to a peptide (P) mediating migration on microtubules by using a triplex-forming oligonucleotide (TFO). pDNA of 5 kbp and 21 kbp containing 6 and 36 oligopurine • oligopyrimidine sites (TH), respectively, inserted outside the luciferase gene sequence were used.

mRNA Lipoplexes with Cationic and Ionizable α-Amino-lipophosphonates: Membrane Fusion, Transfection, mRNA Translation and Conformation.

Cationic liposomes are attractive carriers for mRNA delivery. Here, mRNA lipoplexes (LX) were prepared with the cationic lipids α-aminolipophosphonate () or imidazolium lipophosphoramidate () associated with various α-aminolipophosphonates co-lipids comprising protonable groups (imidazole or pyridine) and . Physicochemical parameters of liposomes and their membrane fusion activity were measured.

LentiRILES, a miRNA-ON sensor system for monitoring the functionality of miRNA in cancer biology and therapy.

A major unresolved challenge in miRNA biology is the capacity to monitor the spatiotemporal activity of miRNAs expressed in animal disease models. We recently reported that the miRNA-ON monitoring system called RILES (RNAi-inducible expression Luciferase system) implanted in lentivirus expression system (LentiRILES) offers unique opportunity to decipher the kinetics of miRNA activity , in relation with their intracellular trafficking in glioblastoma cells. In this study, we describe in detail the method for the production of LentiRILES stable cell lines and employed it in several applications in the field of miRNA biology and therapy.

Intracellular Routing and Recognition of Lipid-Based mRNA Nanoparticles.

Messenger RNA (mRNA) is being extensively used in gene therapy and vaccination due to its safety over DNA, in the following ways: its lack of integration risk, cytoplasmic expression, and transient expression compatible with fine regulations. However, clinical applications of mRNA are limited by its fast degradation by nucleases, and the activation of detrimental immune responses. Advances in mRNA applications, with the recent approval of COVID-19 vaccines, were fueled by optimization of the mRNA sequence and the development of mRNA delivery systems.

DNA nuclear targeting sequences for enhanced non-viral gene transfer: An and study.

An important bottleneck for non-viral gene transfer commonly relates to translocation of nucleic acids into the nuclear compartment of target cells. So-called 3NFs are optimized short nucleotide sequences able to interact with the transcription factor nuclear factor κB (NF-κB), which can enhance the nuclear import of plasmid DNA (pDNA) carrying such motifs. In this work, we first designed a consistent set of six pDNAs featuring a common backbone and only varying in their 3NF sequences.

In Vivo bone tissue induction by freeze-dried collagen-nanohydroxyapatite matrix loaded with BMP2/NS1 mRNAs lipopolyplexes.

Messenger RNA (mRNA) activated matrices (RAMs) are interesting to orchestrate tissue and organ regeneration due to the in-situ and sustained production of functional proteins. However, the immunogenicity of in vitro transcribed mRNA and the paucity of proper in vivo mRNA delivery vector need to be overcome to exert the therapeutic potential of RAM. We developed a dual mRNAs system for in vitro osteogenesis by co-delivering NS1 mRNA with BMP2 mRNA to inhibit RNA sensors and enhance BMP-2 expression.

CEBA: A new heterobifunctional reagent for plasmid DNA functionalization by click chemistry.

Here, we report the synthesis of 3,6,9-trioxaundecan-1-{4-[(2-Chloroethyl)Ethylamino)]-Benzylamino},11-Azide (CEBA). CEBA alkylates the N7 of guanine of DNA thanks its chloroethyl group and can be coupled by a strain-promoted azide-alkyne cycloaddition to an alkynylated molecule. The optimization of the alkylation level of pDNA reveals that the expression of the encoded gene is preserved when it is randomly modified with at most 1 CEBA molecule per 150 bp.

Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation.

Application of messenger RNA (mRNA) for bone regeneration is a promising alternative to DNA, recombinant proteins and peptides. However, exogenous in vitro transcribed mRNA (IVT mRNA) triggers innate immune response resulting in mRNA degradation and translation inhibition. Inspired by the ability of viral immune evasion proteins to inhibit host cell responses against viral RNA, we applied non-structural protein-1 (NS1) from Influenza A virus (A/Texas/36/1991) as an IVT mRNA enhancer.

Periodic Mesoporous Organosilica Nanoparticles with BOC Group, towards HIFU Responsive Agents.

Periodic Mesoporous Organosilica Nanoparticles (PMONPs) are nanoparticles of high interest for nanomedicine applications. These nanoparticles are not composed of silica (SiO). They belong to hybrid organic-inorganic systems.

Temperature-Sensitive Amphiphilic Non-Ionic Triblock Copolymers for Enhanced In Vivo Skeletal Muscle Transfection.

It is reported that low concentration of amphiphilic triblock copolymers of pMeOx-b-pTHF-b-pMeOx structure (TBCPs) improves gene expression in skeletal muscle upon intramuscular co-injection with plasmid DNA. Physicochemical studies carried out to understand the involved mechanism show that a phase transition of TBCPs under their unimer state is induced when the temperature is elevated from 25 to 37 °C, the body temperature. Several lines of evidences suggest that TBCP insertion in a lipid bilayer causes enough lipid bilayer destabilization and even pore formation, a phenomenon heightened during the phase transition of TBCPs.

Gene transfer to skeletal muscle using hydrodynamic limb vein injection: current applications, hurdles and possible optimizations.

Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached.

Comparative binding and uptake of liposomes decorated with mannose oligosaccharides by cells expressing the mannose receptor or DC-SIGN.

Mannose Receptor (MR) and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) are two mannose-specific targets for antigens carried by liposomes but DC-SIGN is more specific of DCs. Here, DC targeting is addressed by using DPPC/DOPE liposomes decorated with a series of diether lipids with a polar head of either a mannose (Man), tri-antenna of α-d-mannopyranoside (Tri-Man), [Manα1-3(Manα1-6)Man] (Man-tri), pseudo-Man (PMan) or pseudo-Man (PMan). Liposomes decorated with Man-Tri show the highest binding and internalization in cells expressing DC-SIGN and in human monocytes-derived DCs.

Amino-acid functionalized porous silicon nanoparticles for the delivery of pDNA.

Porous silicon nanoparticles as a novel platform in gene therapy, have shown to be an efficient vehicle for the delivery of nucleic acids in cells. For the first time, a family of porous silicon nanoparticles has been produced featuring an amino-acid functionalized cationic external surface aiming at pDNA complexation. The amino acid-based pDNA nanocarriers, displaying an average diameter of 295 nm, succeeded in transfection of HEK293 cells with an efficiency 300 times superior to "bare" porous silicon nanoparticles.

Improved histidinylated lPEI polyplexes for skeletal muscle cells transfection.

Linear Polyethylenimine (lPEI) is an efficient cationic polymer for transfecting cells, both in vitro and in vivo, but poses concerns regarding cytotoxicity. Histidinylated lPEI (His-lPEI) exhibits also high transfection efficiency but lower cytotoxicity than lPEI. For the first time, we tested polyfection efficiency of polyplexes comprising both lPEI and His-lPEI.

Dendritic Cell Targeting mRNA Lipopolyplexes Combine Strong Antitumor T-Cell Immunity with Improved Inflammatory Safety.

In vitro transcribed mRNA constitutes a versatile platform to encode antigens and to evoke CD8 T-cell responses. Systemic delivery of mRNA packaged into cationic liposomes (lipoplexes) has proven particularly powerful in achieving effective antitumor immunity in animal models. Yet, T-cell responses to mRNA lipoplexes critically depend on the induction of type I interferons (IFN), potent pro-inflammatory cytokines, which inflict dose-limiting toxicities.

Poly(N-methylvinylamine)-Based Copolymers for Improved Gene Transfection.

Poly(N-methylvinylamines) with secondary amines can form complexes with plasmid DNA (pDNA) and provide transfection efficiency in HeLa cells in the same order as linear polyethyleneimine but with higher cell viability. Chemical modifications of poly(N-methylvinylamine) backbones are performed to further improve transfection efficiency while maintaining low degree of cytotoxicity. In a first type of polymer, primary amino groups are incorporated via a copolymerization strategy.

Cationic gas-filled microbubbles for ultrasound-based nucleic acids delivery.

The use of ultrasound has gained great interest for nucleic acids delivery. Ultrasound can reach deep tissues in non-invasive manner. The process of sonoporation is based on the use of low-frequency ultrasound combined with gas-filled microbubbles (MBs) allowing an improved delivery of molecules including nucleic acids in the insonified tissue.