Virginie Escriou

NLS bioconjugates for targeting therapeutic genes to the nucleus

One of the major steps limiting non-viral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of transfected cells. Trafficking of nuclear proteins from the cytoplasm into the nucleus through nuclear pore complexes is mediated by the presence of nuclear localization sequences (NLS) on proteins. Viral DNA and RNA also require interaction with cellular machinery for efficient nuclear import. In this article, we review the various strategies used to provide plasmid DNA with nuclear localization sequences, and discuss the possibility of developing efficient gene delivery systems based on these strategies.

Formulated siRNAs targeting Rankl prevent osteolysis and enhance chemotherapeutic response in osteosarcoma models

The development of osteosarcoma, the most common malignant primary bone tumor is characterized by a vicious cycle established between tumor proliferation and paratumor osteolysis. This osteolysis is mainly regulated by the receptor activator of nuclear factor κB ligand (RANKL). Preclinical studies have demonstrated that Rankl blockade by soluble receptors is an effective strategy to prevent osteolytic lesions leading to osteosarcoma inhibition. A new therapeutic option could be to directly inhibit Rankl expression by small interfering RNAs (Rkl‐siRNAs) and combine these molecules with chemotherapy to counteract the osteosarcoma development more efficiently. An efficient siRNA sequence directed against both mouse and rat mRNAs coding Rankl was first validated in vitro and tested in two models of osteosarcoma: a syngenic osteolytic POS‐1 model induced in immunocompetent mice and a xenograft osteocondensant model of rat OSRGA in athymic mice. Intratumor injections of Rankl‐directed siRNAs in combination with the cationic liposome RPR209120/DOPE reduced the local and systemic Rankl production and protected bone from paratumor osteolysis. Although Rkl‐siRNAs alone had no effect on tumor development in both osteosarcoma models, it significantly blocked tumor progression when combined with ifosfamide compared with chemotherapy alone. Our results indicate that siRNAs could be delivered using cationic liposomes and thereby could inhibit Rankl production in a specific manner in osteosarcoma models. Moreover, the Rankl inhibition mediated by RNA interference strategy improves the therapeutic response of primary osteosarcoma to chemotherapy.

Advantages of bioluminescence imaging to follow siRNA or chemotherapeutic treatments in osteosarcoma preclinical models

Osteosarcoma is the most common malignant primary bone tumor for which pertinent preclinical models are still needed to develop new therapeutic strategies. As osteosarcoma growth is strongly supported by bone resorption, previous studies have inhibited the cytokine receptor activator of nuclear factor-κB ligand using antibodies or recombinant proteins. However, its expression has not yet been inhibited using genetic approaches using small interfering RNA. To optimize the delivery of small interfering RNA to its cellular target and demonstrate their efficiency in vivo, two new osteosarcoma models expressing the firefly luciferase enzyme were developed. These luciferase-expressing osteosarcomas showed conserved osteolytic and osteogenic activities in mice and were detectable by in vivo bioluminescence imaging. In comparison with measurement of tumor volume, bioluminescence analysis enabled earlier tumor detection and revealed extensive cell death in response to ifosfamide treatment. Finally, by targeting the luciferase expression into osteosarcoma, we established a protocol for in vivo administration of small interfering RNA combined with cationic liposome.

New synthetic glycolipids for targeted gene transfer: synthesis, formulation in lipoplexes and specific interaction with lectin.

Nonviral gene delivery systems are a promising approach for gene therapy applications, despite their low in vivo gene transfer efficiency. One approach to enhance this efficiency is to incorporate targeting elements into cationic lipid/DNA complexes (lipoplexes). Ligand-containing lipoplexes have to retain their efficiency while exposing accessible ligand on their surface. Physicochemical properties (particle size, surface charge, and efficacy of DNA complexation) of the lipoplexes largely determine their gene transfer efficiency. We synthesized glycolipids with various galactosylated head ligand and incorporated them into lipoplexes. We showed that incorporation of up to 33% mol of glycolipid did not change the physicochemical properties of lipoplexes. Some of our glycolipids yielded lipoplexes whose galactosyl heads were well exposed on the surface as demonstrated by a strong interaction with Ricinus communis agglutinin. Glycolipid-containing lipoplexes gave an efficient gene transfer on hepatocytes, although no ligand-targeted transfection could be observed.

SYNTHESIS OF NEW GALACTOSYL AND LACTOSYL CARBAMATE-CONTAINING GLYCOLIPIDS

An efficient synthesis of mono-, di- and tetrasaccharides linked to a lipid has been developed. Galactose or lactose was covalently coupled to a glycyldioctadecylamide, via well-defined chemical steps, in both an α and β anomeric configuration. The multiantennary galactosyl ligands were obtained using 1,3-diamino-2-propanol as a scaffold.

Lipid reagents for DNA transfer into mammalian cells

Publisher Summary Synthetic DNA delivery agents are of great interest as alternatives to viral vectors because they display potentially fewer risks in terms of immunogenicity and propagation. Essentially two chemical alternatives have been developed: cationic polymers such as poly (ethylene imine) and lipid reagents. Lipid reagents are among the most frequently used chemical vectors for DNA transfer into mammalian cells: their association with DNA leads to supra-molecular entities, that promote DNA penetration into the cells. As early as 1978, it was discovered that DNA could be encapsulated in lipidic particles such as liposomes. Few years later, it was shown that cationic lipids could strongly interact with DNA and condense it in small ionic particles called “lipoplexes”. Since this discovery at the end of the 80s, a large variety of cationic lipids were developed to optimize in vitro and in vivo DNA transfer. These reagents are yet defined as the most promising systems for DNA transfer, especially because no limitation of the size of transferred DNA occurs with these systems. Many cationic lipids are commercially available and used in vitro because of their high efficiency. Lipoplexes present low efficiency in vivo as compared to other systems such as viral vectors. Most of the lipid systems used for DNA transfer contain at least 2 or 3 lipid reagents, which are complexed with DNA. The main lipid reagent is cationic and therefore permits DNA condensation; a neutral lipid is often used to promote particle formation and to increase its efficiency for gene transfer. In many cases, a polyethylene glycol PEGylated lipid is also added in order to stabilize and optimize bio-distribution of the particle. The resulting supra-molecular assemblies may allow a selective and efficient in vivo DNA transfer to specific cells or tissues, provided that targeting properties are added. A lot of work was devoted to optimize DNA encapsulation, and condensation of DNA is yet easily obtained from commercially available lipid reagents. Nevertheless, the targeting of specific tissues with lipid reagents has to be enhanced as well as the optimization of intracellular traffic to the nucleus: both extra- and intra-cellular DNA traffic must be improved to enhance DNA transfer into mammalian cells.