DNA/amphiphilic block copolymer nanospheres promote low-dose DNA vaccination.

Intramuscular (i.m.) DNA vaccination induces strong cellular immune responses in the mouse, but only at DNA doses that cannot be achieved in humans. Because antigen expression is weak after naked DNA injection, we screened five nonionic block copolymers of poly(ethyleneoxide)-poly(propyleneoxide) (PEO-PPO) for their ability to enhance DNA vaccination using a beta-galactosidase (betaGal) encoding plasmid, pCMV-betaGal, as immunogen. At a high DNA dose, formulation with the tetrafunctional block copolymers 304 (molecular weight [MW] 1,650) and 704 (MW 5,500) and the triblock copolymer Lutrol (MW 8,600) increased betaGal-specific interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) responses 2-2.5-fold. More importantly, 704 allowed significant reductions in the dose of antigen-encoding plasmid. A single injection of 2 microg pCMV-betaGal with 704 gave humoral and ELISPOT responses equivalent to those obtained with 100 microg naked DNA and conferred protection in tumor vaccination models. However, 704 had no adjuvant properties for betaGal protein, and immune responses were only elicited by low doses of pCMV-betaGal formulated with 704 if noncoding carrier DNA was added to maintain total DNA dose at 20 microg. Overall, these results show that formulation with 704 and carrier DNA can reduce the dose of antigen-encoding plasmid by at least 50-fold.

Therapeutic relevance of osteoprotegerin gene therapy in osteosarcoma: blockade of the vicious cycle between tumor cell proliferation and bone resorption.

Osteosarcoma is the most frequent primary bone tumor that develops mainly in the young, the median age of diagnosis being 18 years. Despite improvement in osteosarcoma treatment, survival rate is only 30% at 5 years for patients with pulmonary metastases at diagnosis. This warrants exploration of new therapeutic options, and among them, osteoprotegerin (OPG), a naturally occurring protein that inhibits bone resorption, is very promising in blocking the vicious cycle between bone resorption and tumor proliferation that takes place during tumor development in bone site. As OPG binds and inhibits the activity of tumor necrosis factor-related apoptosis-inducing ligand, the truncated form of murine OPG 1-194 was used. The cDNA encoding OPG was administered by gene transfer using replication-defective adenoviral vector or was associated with an amphiphilic polymer in two models of rodent osteosarcoma. In both models, OPG gene transfer was effective in preventing the formation of osteolytic lesions associated with osteosarcoma development, in reducing the tumor incidence and the local tumor growth, leading to a 4-fold augmentation of mice survival 28 days postimplantation. On the contrary, OPG did not prevent the development of pulmonary metastasis alone, suggesting that bone environment is necessary for OPG therapeutic efficacy. Because OPG has no direct activity on osteosarcoma cells in vitro (cell binding, cell proliferation, apoptosis, or cell cycle distribution), we show that OPG exerts indirect inhibitory effect on tumor progression through the inhibition of RANKL whose production is enhanced in bone tumor environment, leading to osteolysis inhibition as reflected by osteoclast number decrease.

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.

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.

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.

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.