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.

Respiratory evoked potentials and occlusion elicited sympathetic skin response.

INTRODUCTION: Neurophysiological study of respiratory structures usually relies upon diaphragm electromyography and phrenic nerve conduction study, which do not assess the afferent sensory pathways. OBJECTIVE: To assess the feasibility of respiratory evoked potentials (REPs) and sympathetic skin responses (SSRs) elicited by inspiratory occlusion. METHODS: REPs and SSRs were studied in 12 healthy adults. REPs were elicited by inspiratory occlusions triggered by the physician within 1 s after the onset of a respiratory effort. They were recorded from C3, C4 and Cz needle electrodes (referenced to Fz). Each individual trial consisted of two superimposed 30-sweep averaged responses to inspiratory occlusions. SSRs were recorded from surface electrodes placed on the subject's hand and elicited by similar inspiratory occlusions. RESULTS: Reproducible REPs and SSRs were obtained in all subjects. Mean latencies of initial P1 and N1 cortical responses were 41 and 72 ms, respectively. SSRs were similar to those usually elicited by peripheral nerve electrical stimulation. CONCLUSION: Brief occlusion of inspiration induces cortical and sympathetic activation, both are easily recordable. Since REPs are considered to be the neurophysiological substrate of certain types of respiratory sensations and are altered in different chronic respiratory diseases, they, in addition to SSR, represent attractive new techniques that may provide better understanding of respiratory dysfunction.

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.