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.

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.