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. GluA1-flip produces higher gain AMPA channels than GluA1-flop, increasing network excitability and seizure susceptibility. Splice modulating oligonucleotides (SMOs) bind to pre-mRNA to influence alternative splicing, a strategy that can be exploited to develop more selective drugs across therapeutic areas. We developed a novel SMO, GR1, which potently and specifically decreased GluA1-flip expression throughout the brain of neonatal mice lasting at least 60 days after single intracerebroventricular injection. GR1 treatment reduced AMPA-R mediated excitatory postsynaptic currents at hippocampal CA1 synapses, without affecting long-term potentiation or long-term depression, cellular models of memory, or impairing GluA1-dependent cognition or motor function in mice. Importantly, GR1 demonstrated anti-seizure properties and reduced post-seizure hyperexcitability in neonatal mice, highlighting its drug candidate potential for treating epilepsies and other neurological diseases involving network hyperexcitability.

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. In vivo these neurons express syt1, and most have not yet extended axons. We present evidence that syt1 plays a role in regulating axon branching, while not regulating overall axon length. To study the effects of overexpression of syt1, we used adenovirus-mediated infection to introduce a syt1-YFP construct, or control GFP construct, into neurons. Syt1 levels were reduced using RNA interference. Overexpression of syt1 increased the formation of axonal filopodia and branches. Conversely, knockdown of syt1 decreased the number of axonal filopodia and branches. Time-lapse analysis of filopodial dynamics in syt1-overexpressing cells demonstrated that elevation of syt1 levels increased both the frequency of filopodial initiation and their lifespan. Taken together these data indicate that syt1 regulates the formation of axonal filopodia and branches before engaging in its conventional functions at the synapse.

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. A steric block antisense oligonucleotide (AO) has recently been developed that blocked an intronic splice suppressor element, and enhanced SMN2 exon 7 inclusion in SMA patient fibroblasts. Here, we show that periodic intracerebroventricular (ICV) delivery of this AO resulted in increased SMN expression in brain and spinal cord to as much as 50% of the level of healthy littermates. Real-time PCR of SMN2 transcripts confirmed the AO-mediated increase in full-length SMN. The AO-derived increase in SMN expression led to a concomitant improvement in bodyweight throughout the lifespan of the SMA animals. Treatment of SMA mice with AO also provided partial correction of motor deficits, manifest as improved righting response. Injections of a scrambled oligonucleotide had no effect on SMN expression or phenotype in the SMA mice. Our results validate that AOs that abrogate aberrant splicing of SMN2 are promising compounds for treating 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. We adapted a modified double emulsion procedure for encapsulating PEG-PEI-AO polyplexes into degradable polylactide-co-glycolic acid (PLGA) nanospheres. Formulation parameters were varied including PLGA molecular weight, ester end-capping, and sonication energy/volume. Our results showed successful encapsulation of PEG-PEI-AO within PLGA nanospheres with average diameters ranging from 215 to 240 nm. Encapsulation efficiency ranged from 60 to 100%, and zeta potential measurements confirmed shielding of the PEG-PEI-AO cationic charge. Kinetic measurements of 17 kDa PLGA showed a rapid burst release of about 20% of the PEG-PEI-AO, followed by sustained release of up to 65% over three weeks. To evaluate functionality, PEG-PEI-AO polyplexes were loaded into PLGA nanospheres using an AO that is known to induce dystrophin expression in dystrophic mdx mice. Intramuscular injections of this compound into mdx mice resulted in over 300 dystrophin-positive muscle fibers distributed throughout the muscle cross-sections, approximately 3.4 times greater than for injections of AO alone. We conclude that PLGA nanospheres are effective compounds for the sustained release of PEG-PEI-AO polyplexes in skeletal muscle and concomitant expression of dystrophin, and may have translational potential in treating DMD.

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. Polymersome-mediated knockdown appears as efficient as common cationic-lipid transfection and about half as effective as Lenti-virus after sustained selection. For AON, initial results also show that intramuscular injection into a mouse model of muscular dystrophy leads to the expected protein expression, which occurs along the entire length of muscle. The lack of cationic groups in antisense polymersomes together with initial tests of efficacy suggests broader utility of these non-viral carriers.

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. Each of these properties in-turn influences the transfection capacity of the PEG-PEI-AO polyplexes. In addition, PEG-PEI copolymers are readily functionalized for enhanced efficacy and specificity of cellular and tissue targeting. The synthesis and functionalization of PEG-PEI copolymers is remarkably simple and requires very little specialized equipment. Thus, PEG-PEI copolymers represent a tractable and adaptable oligonucleotide delivery system that can be customized and optimized to the investigators' specific application. This chapter describes the step-by-step synthesis of several PEG-PEI copolymers that are specifically formulated to provide effective delivery of AOs using both in vitro and in vivo applications. We describe the preparation of the PEG-PEI-AO polyplexes and provide examples showing transfection of cultured cells in vitro, as well as skeletal muscles in vivo using both local and systemic delivery.

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. In an attempt to improve ESO delivery and dystrophin expression, a new formulation of PEG-PEI copolymer was used, along with functionalized derivatives containing either the cell-penetrating peptide TAT (trans-activator of transcription), adsorbed colloidal gold (CG), or both TAT and CG. Tibialis anterior muscles were given three intramuscular injections of various PEG-PEI-ESO polyplexes (3 days apart; 5 microg of ESO per injection) and muscles were harvested 3 weeks after the first injection. Surface modifications of PEG-PEI copolymers with TAT showed the highest level of dystrophin recovery, with a 6-fold increase in dystrophin-positive fibers compared with ESO alone and up to 30% of normal dystrophin expression by Western blot. The adsorption of CG to either PEG-PEI or TAT-PEG-PEI copolymers showed no further improvement in dystrophin expression. Our data indicate that TAT-modified PEG-PEI copolymers are effective carriers for delivery of ESOs to skeletal muscle and are promising compounds for the therapeutic treatment of DMD.

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. RESULTS: We examined whether PEG-PEI copolymers can facilitate ESO-mediated dystrophin expression after intramuscular injections into tibialis anterior (TA) muscles of mdx mice. We utilized a set of PEG-PEI copolymers containing 2 kDa PEI and either 550 Da or 5 kDa PEG, both of which bind 2'OMe ESOs with high affinity and form stable nanoparticulates with a relatively low surface charge. Three weekly intramuscular injections of 5 microg of ESO complexed with PEI2K-PEG550 copolymers resulted in about 500 dystrophin-positive fibers and about 12% of normal levels of dystrophin expression at 3 weeks after the initial injection, which is significantly greater than for injections of ESO alone, which are known to be almost completely ineffective. In an effort to enhance biocompatibility and cellular uptake, the PEI2K-PEG550 and PEI2K-PEG5K copolymers were functionalized by covalent conjugation with nanogold (NG) or adsorbtion of colloidal gold (CG), respectively. Surprisingly, using the same injection and dosing regimen, we found no significant difference in dystrophin expression by Western blot between the NG-PEI2K-PEG550, CG-PEI2K-PEG5K, and non-functionalized PEI2K-PEG550 copolymers. Dose-response experiments using the CG-PEI2K-PEG5K copolymer with total ESO ranging from 3-60 microg yielded a maximum of about 15% dystrophin expression. Further improvements in dystrophin expression up to 20% of normal levels were found at 6 weeks after 10 twice-weekly injections of the NG-PEI2K-PEG550 copolymer complexed with 5 microg of ESO per injection. This injection and dosing regimen showed over 1000 dystrophin-positive fibers. H&E staining of all treated muscle groups revealed no overt signs of cytotoxicity. CONCLUSION: We conclude that PEGylated PEI2K copolymers are efficient carriers for local delivery of 2'OMe ESOs and warrant further development as potential therapeutics for treatment of DMD.

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. Single intramuscular injections of AO complexed with low Mw PEI2000(PEG550) copolymers into TA muscles of mdx mice resulted in widespread distribution of dystrophin-positive fibers at 3 weeks after injection, with no apparent cytotoxicity. Overall, injections of these low Mw polyplexes, which formed 250-nm aggregate particles, resulted in about sixfold more dystrophin-positive fibers than AO alone. Western analysis confirmed the dystrophin expression in these muscles. Surprisingly, injections of AO complexed with high Mw PEI25000(PEG5000) copolymers, which formed smaller nonaggregated particles, produced about threefold fewer dystrophin-positive fibers than injections of the low Mw polyplexes. We conclude that low Mw PEI2000(PEG550) copolymers function as high-capacity, nontoxic AO carriers suitable for in vivo transfection of skeletal muscle and are promising compounds for potential use in molecular therapy of DMD.

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. Therefore, we synthesized a series of PEG-PEI copolymers and evaluated their physiochemical properties alone and when complexed with AO. The M(w) of PEG was found to be the main determinant of polyplex size, via its influence on particle aggregation. DLS measurements showed that when PEG5000 was grafted to PEI2K and PEI25K, polyplex diameters were extremely small (range 10-90 nm) with minimal aggregation. In contrast, when PEG550 was grafted to PEI2K and PEI25K, polyplexes appeared as much larger aggregates (approximately 250 nm). As expected, the surface charge (zeta potential) was higher for polyplexes containing PEI25K than those containing PEI2K, but decreased with increased levels of PEG grafting. Surprisingly, within the physiological range (pH 7.5-5), the buffering capacity of all copolymers was nearly equivalent to that of unsubstituted PEI2K or PEI25K, and was barely influenced by PEGylation. The stability of polyplexes was evaluated using a heparin polyanion competition assay. Unexpectedly, polyplexes containing PEI2K showed stability equal to or greater than that of PEI25K polyplexes. The level of PEG grafting also had a dramatic effect on polyplex stability. The relationships established between molecular formulations and polyplex size, aggregation, surface charge, and stability should provide a useful guide for future studies aimed at optimizing polymer-mediated AO delivery in cell and animal studies. A summary of the relationships between polyplex structures and recent studies of their transfection capacity is provided.

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. Confocal analysis of dual fluorescently tagged PEG-PEI-AO polyplexes, 24 hr after transfection, showed that the copolymer and AO were colocalized within punctate membrane- associated structures. Importantly, AO was efficiently translocated into myonuclei, whereas the copolymer was mostly excluded. The morphology of all transfected myofibers was perfectly maintained with no indication of damage or cytotoxicity. Quantitative fluorescence analysis showed that transfection with PEG-PEI-AO resulted in a 6-fold higher uptake of AO into myonuclei compared with transfections of AO alone. Interestingly, transfections with rhodamine-labeled PEG-PEI copolymers yielded an approximately 2- fold higher uptake of AO into myonuclei compared with transfections of unlabeled copolymers. Attempts to further increase AO delivery by addition of insulin-transferrin-selenium (ITS) to the medium showed no further improvement in AO delivery. Dose-response analysis indicated saturation of endocytotic uptake of the polyplex. Overall, we conclude that PEG-PEI copolymers represent high-capacity, nontoxic carriers for efficient delivery of AO to nuclei of mature myofibers.

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. Tetanic tension, measured just before each eccentric contraction, differed significantly among strain groups and changed dramatically during the 10-contraction treatment (P < 0.01). Average maximum tetanic tension for the three groups measured before any treatment was 203.7 +/- 6.8 kN/m2, but after the 10-eccentric contraction sequence decreased to 180.3 +/- 3.8, 125.1 +/- 7.8, and 78.3 +/- 5.1 kN/m2 for the 10, 25, and 35% strain groups, respectively (P < 0.0001). Addition of 10 mM caffeine to the bathing medium decreased the loss of tetanic tension in the 10% strain group but had only a minimal effect on either the 25 or 35% strain groups. Diffraction pattern line width, a measure of sarcomere length heterogeneity, increased significantly with muscle activation and then continued to increase with successive stretches of the activated muscle. Line width increase after each stretch was significantly correlated with the lower yield tension of the successive contractile record. These data demonstrate a direct association and, perhaps, a causal relationship between sarcomere strain and fiber bundle injury. They also demonstrate that muscle injury is accompanied by a progressive increase in sarcomere length heterogeneity, yielding lower yield tension as injury progresses.

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. Myosin isoform expression is highly plastic and undergoes significant changes in response to muscular injury, muscle disuse, and disease. Therefore, myosin isoform function and plasticity are highly relevant to clinical orthopaedic research, musculoskeletal surgery, and sports medicine. Muscle from frogs offers a special opportunity to study the structural basis of contractile protein function because single intact fibers can be isolated that maintain excellent mechanical stability, allowing for high-resolution studies of contractile performance in intact cells. The current authors summarize recent studies defining the myosin isoforms in muscle from frogs and the relationship between myosin isoforms and mechanical performance of intact single muscle cells. Preliminary studies also are described that show the potential for simple plasmid-based in vivo gene transfer approaches as a model system to elucidate the structural basis of muscle protein function in intact cells.

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 < 0.001). Loss of desmin staining was maximal 12 h after EC and recovered by 72 h. Immunoblots unexpectedly demonstrated a significant increase in the desmin-to-actin ratio by 72 h after EC (P < 0.01) and was still increasing after 168 h (P < 0.0001). These data demonstrate a relatively rapid qualitative loss of desmin immunostaining immediately after a single EC bout but a tremendous quantitative increase in desmin content 72-168 h later. This dynamic restructuring of the muscle's intermediate filament system may be involved in the mechanism of EC-induced muscle injury and may provide a structural explanation for the protective effects observed in muscle after a single EC bout.

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. Velocities were measured between two surface markers that spanned most of the fiber length. MHC and MLC isoform content was quantified after mechanics analysis by SDS-PAGE. Maximal shortening velocity (V(max)) and velocity at half-maximal tension (V(P 50)) increased with percentage of MHC1 (%MHC1). Maximal specific tension (P(o)/CSA, where P(o) is isometric tension and CSA is fiber cross-sectional area) and maximal mechanical power (W(max)) also increased with %MHC1. MHC concentration was not significantly correlated with %MHC1, indicating that the influence of %MHC1 on P(o)/CSA and W(max) was due to intrinsic differences between MHC isoforms and not to concentration. The MLC3-to-MLC1 ratio was not significantly correlated with V(max), V(P 50), P(o)/CSA, or W(max). These data demonstrate the powerful relationship between MHC isoforms and F-V properties of the two most common R. pipiens fiber types.