Therapy of anemia in kidney failure, using plasmid encoding erythropoietin.

Numerous studies using erythropoietin (EPO) gene delivery vectors, either viral or nonviral, have shown uncontrolled EPO expression leading to transient or sustained erythrocytosis and, more recently, severe autoimmune anemia. Therefore, there is a need to develop other EPO gene delivery systems that allow sustained and adjustable expression of EPO. We have examined a new approach of delivering plasmid encoding mouse EPO cDNA into mouse skeletal muscle, using an amphiphilic block copolymer. Repeated injections of low doses of block copolymer-EPOcDNA formulations increased hematocrit in a dose-dependent manner for more than 9 months, without any initial overshoot. Low doses of block copolymer-EPOcDNA formulations prevented autoimmune anemia in immunocompetent Swiss mice and prevented or reversed chronic anemia in an acquired mouse model of renal failure. We conclude that repeated injections of low doses of block copolymer-DNA formulations that do not induce (1) inflammation at the injection site, (2) overexpression of EPO, or (3) the production of anti-EPO neutralizing auto-antibodies hold promise for in vivo expression of therapeutic proteins, in particular for systemic delivery.

Physicochemical parameters of non-viral vectors that govern transfection efficiency.

Gene therapy is based on the vectorization of nucleic acids to target cells and their subsequent expression. Cationic lipids and polymers are the most widely used vectors for the delivery of DNA into cultured cells. Nowadays, numerous reagents made of these cationic molecules are commercially available and used by researchers from the academic and industrial field. By contrast their evaluations in preclinical programs have revealed that their use for in vivo applications will be more problematic than their massive use in vitro. This is mostly due to the physicochemical properties of cationic vectors/DNA complexes, which are the result of their mode of interaction. Indeed, these cationic vectors interact through electrostatic forces with negatively charged DNA. This results in the formation of highly organized positively charged supramolecular structures where DNA molecules are condensed. Association of DNA with cationic lipids under a micellar or liposomal form leads to lamellar organization with DNA molecules sandwiched between lipid bilayers. Although the lamellar phase is the common described structure, as evidenced by small-angle X-ray scattering and electron microscopy, some cationic lipid combined with a hexagonal forming lipid could also result with DNA in an inverted hexagonal structure. Despite a lot of effort, the precise mechanism of gene transfer with cationic vector is still ill-defined. Here, our objective was to overview the main relationships between the physico chemical properties of cationic lipid/DNA complexes and their transfection efficiency. An overview of a new class of vectors consisting of amphiphilic block copolymers designed for in vivo delivery is also presented and discussed.

Galactosylated multimodular lipoplexes for specific gene transfer into primary hepatocytes.

BACKGROUND: Numerous synthetic cationic vectors have been synthesized and are successfully used for in vitro gene transfer but an excess of positive charges can lead to cytotoxicity and does not enable specific transfection. METHODS: We decided to develop alternative molecular systems consisting of neutral, colloidally stable bioassemblies equipped with ligands for specific cell targeting. Consequently, we directed our efforts toward the development of a multimodular non-viral gene delivery system consisting of a condensed core of DNA with cationic liposomes of bis(guanidinium)-tren-cholesterol and an external corona of poly(ethylene oxide) stretches harbored by the steric stabilizers used to stabilize lipoplexes colloidally. A ligand capable of cell targeting by receptor-mediated endocytosis was covalently linked at the poly(ethylene oxide) extremity of steric stabilizers. Steric stabilizers were functionalized by a one-step enzymatic galactosylation to develop new supramolecular assemblies of lipoplexes able to target asialoglycoprotein receptors located on primary hepatocytes. RESULTS: Cryo-TEM and fluorescence experiments showed that DNA was condensed within lamellar complexes whose size ranged between 100 to 300 nm in diameter. Bis(guanidinium)-tren-cholesterol-DNA lipoplexes, colloidally stabilized by galactosylated steric stabilizers at a galactosylated steric stabilizer/DNA ratio of 300, led to specific transfection of primary hepatocytes whereas ungalactosylated steric stabilizer did not transfect. CONCLUSIONS: Our findings confirm the receptor-mediated endocytosis pathway of galactosylated multimodular lipoplexes. Thus, we conclude that the fabrication of a multimodular assembly harboring a ligand without non-specific interaction with cell membranes is possible and a highly promising system to transfect other primary or cultured cells specifically through a receptor-dependent mechanism.

Self-assembled lamellar complexes of siRNA with lipidic aminoglycoside derivatives promote efficient siRNA delivery and interference.

RNA interference requires efficient delivery of small double-stranded RNA molecules into the target cells and their subsequent incorporation into RNA-induced silencing complexes. Although current cationic lipids commonly used for DNA transfection have also been used for siRNA transfection, a clear need still exists for better siRNA delivery to improve the gene silencing efficiency. We synthesized a series of cationic lipids characterized by head groups bearing various aminoglycosides for specific interaction with RNA. siRNA complexation with such lipidic aminoglycoside derivatives exhibited three lipid/siRNA ratio-dependent domains of colloidal stability. Fluorescence and dynamic light-scattering experiments showed that cationic lipid/siRNA complexes were formed at lower charge ratios, exhibited a reduced zone of colloidal instability, and had smaller mean diameters compared with our previously described guanidinium-based cationic lipids. Cryo-transmission electron microscopy and x-ray-scattering experiments showed that, although the final in toto morphology of the lipid/siRNA complexes depended on the aminoglycoside type, there was a general supramolecular arrangement consisting of ordered lamellar domains with an even spacing of 67 A. The most active cationic lipid/siRNA complexes for gene silencing were obtained with 4,5-disubstituted 2-deoxystreptamine aminoglycoside derivatives and were characterized by the siRNA being entrapped in small particles exhibiting lamellar microdomains corresponding to siRNA molecules sandwiched between the lipid bilayers. These results clearly show that lipidic aminoglycoside derivatives constitute a versatile class of siRNA nanocarriers allowing efficient gene silencing.