AMPA GluA1-flip targeted oligonucleotide therapy reduces neonatal seizures and hyperexcitability.

Glutamate-activated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) mediate the majority of excitatory neurotransmission in brain and thus are major drug targets for diseases associated with hyperexcitability or neurotoxicity. Due to the critical nature of AMPA-Rs in normal brain function, typical AMPA-R antagonists have deleterious effects on cognition and motor function, highlighting the need for more precise modulators. A dramatic increase in the flip isoform of alternatively spliced AMPA-R GluA1 subunits occurs post-seizure in humans and animal models.

Synaptotagmin-1 promotes the formation of axonal filopodia and branches along the developing axons of forebrain neurons.

Synaptotagmin-1 (syt1) is a Ca(2+)-binding protein that functions in regulation of synaptic vesicle exocytosis at the synapse. Syt1 is expressed in many types of neurons well before synaptogenesis begins both in vivo and in vitro. To determine if expression of syt1 has a functional role in neuronal development before synapse formation, we examined the effects of syt1 overexpression and knockdown on the growth and branching of the axons of cultured primary embryonic day 8 chicken forebrain neurons.

Oligonucleotide-mediated survival of motor neuron protein expression in CNS improves phenotype in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by homozygous mutation or deletion of the SMN1 gene encoding survival of motor neuron (SMN) protein, resulting in the selective loss of alpha-motor neurons. Humans typically have one or more copies of the SMN2 gene, the coding region of which is nearly identical to SMN1, except that a point mutation causes splicing out of exon 7 and production of a largely nonfunctional SMNDelta7 protein. The development of drugs that mitigate aberrant SMN2 splicing is an attractive therapeutic approach for SMA.

Formulation of polylactide-co-glycolic acid nanospheres for encapsulation and sustained release of poly(ethylene imine)-poly(ethylene glycol) copolymers complexed to oligonucleotides.

Antisense oligonucleotides (AOs) have been shown to induce dystrophin expression in muscles cells of patients with Duchenne Muscular Dystrophy (DMD) and in the mdx mouse, the murine model of DMD. However, ineffective delivery of AOs limits their therapeutic potential. Copolymers of cationic poly(ethylene imine) (PEI) and non-ionic poly(ethylene glycol) (PEG) form stable nanoparticles when complexed with AOs, but the positive surface charge on the resultant PEG-PEI-AO nanoparticles limits their biodistribution.

Polymersome delivery of siRNA and antisense oligonucleotides.

siRNA and antisense oligonucleotides, AON, have similar size and negative charge and are often packaged for in vitro delivery with cationic lipids or polymers-but exposed positive charge is problematic in vivo. Here we demonstrate loading and functional delivery of RNAi and AON with non-ionic, nano-transforming polymersomes. These degradable carriers are taken up passively by cultured cells after which the vesicles transform into micelles that allow endolysosomal escape and delivery of either siRNA into cytosol for mRNA knockdown or else AON into the nucleus for exon skipping within pre-mRNA.

PEG-PEI copolymers for oligonucleotide delivery to cells and tissues.

Inefficient delivery of antisense oligonucleotides (AO) to target cell nuclei remains as the foremost limitation to their usefulness. Copolymers of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) are extremely well-studied compounds that markedly improve the in vitro and in vivo delivery of AOs to cells and tissues. By varying the Mw of PEI, as well as the nature of PEG shielding, PEG-PEI-AO nanoparticulates can be prepared with a dynamic range of size, surface charge, and stability.

Functionalized PEG-PEI copolymers complexed to exon-skipping oligonucleotides improve dystrophin expression in mdx mice.

Exon-skipping oligonucleotides (ESOs) with 2'-O-methyl modifications are promising compounds for the treatment of Duchenne muscular dystrophy (DMD). However, the usefulness of these compounds is limited by their poor delivery profile to muscle tissue in vivo. We previously established that copolymers made of poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) enhanced ESO transfection in skeletal muscle of mdx mice, resulting in widespread distribution of dystrophin-positive fibers, but limited dystrophin expression by Western blot.

Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice.

Background: Exon skipping oligonucleotides (ESOs) of 2'O-Methyl (2'OMe) and morpholino chemistry have been shown to restore dystrophin expression in muscle fibers from the mdx mouse, and are currently being tested in phase I clinical trials for Duchenne Muscular Dystrophy (DMD). However, ESOs remain limited in their effectiveness because of an inadequate delivery profile. Synthetic cationic copolymers of poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) are regarded as effective agents for enhanced delivery of nucleic acids in various applications.

Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers.

Antisense oligonucleotides (AOs) with 2-O-methyl modifications can circumvent dystrophin mutations via exon skipping and, it is hoped, can become drugs for treatment of Duchenne muscular dystrophy (DMD). However, AO-based approaches are hindered by a lack of effective carriers to facilitate delivery of AOs to myonuclei. We examined whether copolymers composed of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) can enhance AO transfection in skeletal muscle of mdx mice.

Physiochemical properties of low and high molecular weight poly(ethylene glycol)-grafted poly(ethylene imine) copolymers and their complexes with oligonucleotides.

Inefficient delivery of antisense oligonucleotides (AOs) to target cell nuclei remains as the foremost limitation to their usefulness. Copolymers of cationic poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) have been well-studied for delivery of plasmids. However, the properties of PEG-PEI-AO polyplexes have not been comprehensively investigated.

Poly(ethylene imine)-poly(ethylene glycol) copolymers facilitate efficient delivery of antisense oligonucleotides to nuclei of mature muscle cells of mdx mice.

Antisense oligonucleotides (AO) can facilitate dystrophin expression via targeted exon skipping in cultured cells of Duchenne muscular dystrophy (DMD) patients and in the mouse model of DMD (mdx mice). However, the lack of effective means to deliver AO to myonuclei remains the foremost limitation to their usefulness in DMD gene therapy. In this study we show that copolymers of cationic poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) facilitated efficient cellular uptake and nuclear delivery of AO in mature skeletal muscle fibers isolated from mdx mice.

Sarcomere strain and heterogeneity correlate with injury to frog skeletal muscle fiber bundles.

Sarcomere length and first-order diffraction line width were measured by laser diffraction during elongation of activated frog tibialis anterior muscle fiber bundles (i.e., eccentric contraction) at nominal fiber strains of 10, 25, or 35% (n = 18) for 10 successive contractions.

Studies of myosin isoforms in muscle cells: single cell mechanics and gene transfer.

Myosin, the motor protein in skeletal muscle, is composed of two subunits, myosin heavy chain and myosin light chain. All vertebrates express a family of myosin heavy chain and myosin light chain isoforms that together are primary determinants of force, velocity, and power in muscle fibers. Therefore, appropriate expression of myosin isoforms in skeletal muscle is critical to proper motor function.

Desmin cytoskeletal modifications after a bout of eccentric exercise in the rat.

Desmin content and immunohistochemical appearance were measured in tibialis anterior muscles of rats subjected to a single bout of 30 eccentric contractions (ECs). Ankle torque was measured before EC and at various recovery times, after which immunohistochemical and immunoblot analyses were performed. Torque decreased by approximately 50% immediately after EC and fully recovered 168 h later (P < 0.

Influence of myosin isoforms on contractile properties of intact muscle fibers from Rana pipiens.

The myosin heavy chain (MHC) and myosin light chain (MLC) isoforms in skeletal muscle of Rana pipiens have been well characterized. We measured the force-velocity (F-V) properties of single intact fast-twitch fibers from R. pipiens that contained MHC types 1 or 2 (MHC1 or MHC2) or coexpressed MHC1 and MHC2 isoforms.