The encapsulation of DNA molecules within biomimetic lipid nanocapsules.

Most of DNA synthetic complexes result from the self-assembly of DNA molecules with cationic lipids or polymers in an aqueous controlled medium. However, injection of such self-assembled complexes in medium like blood that differ from that of their formulation leads to strong instability. Therefore, DNA vectors that have physico-chemical properties and structural organisation that will not be sensitive to a completely different medium in terms of ionic and protein composition are actively sought. To this end, the goal here was to discover and optimize a nanostructured system where DNA molecules would be encapsulated in nanocapsules consisting in an oily core and a shell covered by PEG stretches obtained through a nanoemulsion process in the absence of organic solvent. This encapsulation form of DNA molecules would prevent interactions with external hostile biological fluid. The results show the entrapment of lipoplexes into lipid nanocapsules, leading to the formation of neutral 110 nm-DNA nanocapsules. They were weakly removed by the immune system, displaying an increased blood half-life, and improved carcinoma cell transfection, in comparison to the parent lipoplexes. Our results demonstrate that the fabrication of nanocapsules encapsulating hydrophilic DNA in an oily core that meet criteria for blood injection is possible.

Biological pacemaker engineered by nonviral gene transfer in a mouse model of complete atrioventricular block.

We hypothesized that a nonviral gene delivery of the hyperpolarization-activated HCN2 channel combined with the beta(2)-adrenergic receptor (ADRB2) would generate a functional pacemaker in a mouse model of complete atrioventricular block (CAVB) induced by radiofrequency ablation of the His bundle. Plasmids encoding HCN2 and ADRB2 mixed with tetronic 304, a poloxamine block copolymer, were injected in the left ventricular free wall (HCN2-ADRB2 mice). Sham mice received a noncoding plasmid. CAVB was induced 5 days later. Ventricular escape rhythms in HCN2-ADRB2 mice were significantly faster than in sham mice at day 15 after ablation and later. In HCN2-ADRB2 mice, QRS complexes were larger than in sham mice and characterized by abnormal axes. Immunostaining of GFP-HCN2 fusion protein showed an expression of HCN2 channel in left ventricular myocardium for at least 45 days after injection. In the mouse, CAVB induces progressive hypertrophy and heart failure leading to 50% mortality after 110 days. HCN2-ADRB2 mice survived 3 weeks longer than sham mice. Finally, beta-adrenergic input increased ventricular escape rhythms significantly more in HCN2-ADRB2 mice than in sham mice. In conclusion, nonviral gene transfer can produce a functional cardiac biological pacemaker regulated by sympathetic input, which improves life expectancy in a mouse model of CAVB.

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.

Self-assembly and characterization of layered double hydroxide/DNA hybrids.

The purpose of this study was to control the fabrication of new labile supramolecular assemblies by formulating associations of DNA molecules with inorganic layered double hydroxides (LDHs). The results show that LDH/DNA hybrids synthesized by a coprecipitation route involving the in situ formation of LDHs around DNA molecules acting as templates were characterized by a lamellar organization, with DNA molecules sandwiched between hydroxide layers, exhibiting a regular spacing of 1.96 nm. Our results indicate that labile complexes resulting from the association of nucleic acids and inorganic materials can be obtained not only by anion exchange but also by a direct self-assembly route.

Amphiphilic block copolymers promote gene delivery in vivo to pathological skeletal muscles.

We reported that amphiphilic block copolymers hold promise as nonviral vectors for the delivery of plasmid DNA, ranging from 4.7 to 6.2 kb, to healthy muscle for the production of local or secreted proteins. To evaluate the efficiency of these vectors to deliver large plasmid DNA molecules to pathological muscles, plasmid DNAs of various lengths were complexed with Lutrol or poloxamine 304 and injected intramuscularly into dystrophic muscles. Lutrol-DNA and poloxamine 304-DNA complexes promoted gene transfer into muscles of the naturally occurring mouse model for DMD (mdx) in a dose- and plasmid DNA size-dependent manner. For small plasmid DNAs encoding reporter genes, this improvement over naked DNA was smaller in mdx than in the wild-type control strain. By contrast, Lutrol enabled us to deliver the large plasmid (16.1 kb) encoding the rod-deleted dystrophin in mdx mouse muscle, whereas the same amount of naked DNA did not lead to dystrophin expression, under the same experimental conditions. Lutrol-treated mdx mice showed the production of dystrophin in large numbers of muscle fibers. More importantly, we also found that expressing dystrophin with Lutrol led to restoration of the dystrophin-associated protein complex. Thus, we conclude that block copolymers constitute a novel class of vectors for the delivery of large plasmid DNA not only to healthy muscles but also to pathological muscle tissues.

Nonionic amphiphilic block copolymers promote gene transfer to the lung.

Various pulmonary disorders, including cystic fibrosis, are potentially amenable to a treatment modality in which a therapeutic gene is directly delivered to the lung. Current gene delivery systems, either viral or nonviral, need further improvement in terms of efficiency and safety. We reported that nonionic amphiphilic block copolymers hold promise as nonviral gene delivery systems for transfection of muscular tissues. To evaluate the efficiency of these vectors in the lung, intratracheal instillation or aerosolization of reporter genes complexed with Lutrol or PE6400 was performed. Lutrol-DNA and, to a lesser extent, PE6400-DNA complexes promoted efficient gene transfection into mouse airways in a dose-dependent manner. This improvement over naked DNA was observed irrespective of the reporter gene. Lutrol enabled us to deliver significantly higher DNA amounts than current nonviral vectors, with even greater increases in gene expression and without the formation of colloidally unstable complexes. Time course studies showed that Lutrol-DNA complexes permitted prolonged gene expression for up to 5 days whereas with poly(ethylenimine) (PEI)-DNA polyplexes, expression peaked on days 1-2 postinstillation, was strongly reduced by day 5, and reached background levels on day 7. Aerosolized delivery of Lutrol-DNA complexes, a less invasive approach to deliver genes to the lung, gave 5- to 15-fold higher reporter gene expression compared with PEI-DNA polyplexes administered via the same delivery route. After intratracheal instillation of Lutrol-DNA complexes, histochemical staining for beta-galactosidase expression showed the presence of large blue areas. Histopathological analysis showed that Lutrol alone did not elicit inflammation, and that the inflammatory response after intratracheal instillation of Lutrol-DNA complexes was reversible and was observed only with the highest amounts of DNA. We also found that Lutrol can efficiently deliver genes to the airways of cystic fibrosis mice. Thus, we conclude that Lutrol is a highly promising vector for gene delivery to the lung.

Inducible production of erythropoietin using intramuscular injection of block copolymer/DNA formulation.

BACKGROUND: We have previously shown that intramuscular injection of plasmid DNA formulated with a non-ionic amphiphile synthetic vector [poly(ethylene oxide)(13)-poly(propylene oxide)(30)-poly(ethylene oxide)(13) block copolymer; PE6400] increases reporter gene expression compared with naked DNA. We have now investigated this simple non-viral formulation for production of secreted proteins from the mouse skeletal muscle. METHODS: Plasmids encoding either constitutive human secreted alkaline phosphatase or murine erythropoietin inducible via a Tet-on system were formulated with PE6400 and intramuscularly injected into the mouse tibial anterior muscle. RESULTS: PE6400/DNA formulation led to an increased amount of recombinant alkaline phosphatase secreted from skeletal muscle as compared with naked DNA. In the presence of doxycycline, a single injection of 10 microg plasmid encoding inducible murine erythropoietin formulated with PE6400 significantly increased the hematocrit, whereas the same amount of DNA in the absence of PE6400 had no effect. The increase in the hematocrit was stable for 42 days. The tetracycline-inducible promoter permitted pharmacological control of hematocrit level after DNA intramuscular injection. However, 4 months post-injection the hematocrit returned to its pre-injection value, even in the presence of doxycycline. This phenomenon was likely caused by an immune response against the tetracycline-activated transcription factor. CONCLUSIONS: Intramuscular injection of plasmid DNA formulated with PE6400 provides an efficient and simple method for secretion and production of non-muscle proteins.

Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer.

Over the past decade, numerous nonviral cationic vectors have been synthesized. They share a high density of positive charges and efficiency for gene transfer in vitro. However, their positively charged surface causes instability in body fluids and cytotoxicity, thereby limiting their efficacy in vivo. Therefore, there is a need for developing alternative molecular structures. We have examined tetrabranched amphiphilic block copolymers consisting of four polyethyleneoxide/polypropyleneoxide blocks centered on an ethylenediamine moiety. Cryo-electron microscopy, ethidium bromide fluorescence and light and X-ray scattering experiments performed on vector-DNA complexes showed that the dense core of the nanosphere consisted of condensed DNA interacting with poloxamine molecules through electrostatic, hydrogen bonding and hydrophobic interactions, with DNA molecules also being exposed at the surface. The supramolecular organization of block copolymer/DNA nanospheres induced the formation of negatively charged particles. These particles were stable in a solution that had a physiological ionic composition and were resistant to decomplexation by heparin. The new nanostructured material, the structure of which clearly contrasted with that of lipoplexes and polyplexes, efficiently transferred reporter and therapeutic genes in skeletal and heart muscle in vivo. Negatively charged supramolecular assemblies hold promise as therapeutic gene carriers for skeletal and heart muscle-related diseases and expression of therapeutic proteins for local or systemic uses.