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

<div><p>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.</p></div

A single ICV injection of GR1 provided a long-lasting reduction in GluA1-flip expression without adverse effects on cognition.

<p>Mice were a given ICV injection of GR1 (4 μg) at P10, <b>(a)</b> Real-time PCR showed GR1 reduced GluA1-flip in the cortex and hippocampus at all time-points from 2–60 d after administration (n = 4–5; p < 0.001). <b>(b)</b> GluA1-flop expression levels, measured in same samples as in panel <b>a</b>, were only marginally different between the GR1-treated and saline-treated controls. <b>(c)</b> GR1-treated mice did not show impairment in Y maze performance when tested at P30, compared to saline-treated controls (p > 0.05; n = 8–9). <b>(d)</b> In the object recognition paradigm, GR1-injected mice showed similar preferences for exploring novel objects as saline-injected controls (p > 0.05 between groups; same cohort of mice as in panel <b>c</b>). GluA1-flip expression was analyzed by one-sample Student’s t-test with bonferoni correction, novel object preference by unpaired two-sample two-tailed t-tests, and Y-maze performance by Fischer’s exact test.</p

CA1 synaptic function measured extracellularly in P10 hippocampal slices from mice injected at P1, P3, and P5 with saline or GR1 (2 μg).

<p><b>(a).</b> Stimulus-response curves for fEPSPs at CA1 synapses showed a reduction in fEPSP amplitude in slices from GR1-treated mice (p < 0.01). <b>(b)</b> Paired-pulse facilitation was normal after GR1 treatment (p > 0.05). <b>(c)</b> LTP was normal in GR1-treated mice. Data shows % baseline slope before and after two high frequency trains. No difference was observed between slices from saline and GR1-treated mice (p > 0.05). <b>(d)</b> LTD was normal in GR1-treated mice. Data shows % baseline slope before and after a 15 min 1 Hz train. No difference was observed between slices from saline and GR1-treated mice (p > 0.05). In all panels <b>a-c</b>, n = 7 for saline and GR1-treated groups. In panel <b>d</b>, saline n = 8, GR1-treated n = 7. Asterisks indicate significant difference between GR1 and saline-treated groups. Two-factor ANOVA was performed on extracellular recording data.</p

Anti-seizure effects of GR1.

<p>GR1 protected neonatal mice from KA-induced seizures and prevented SE-induced increase in aEPSC amplitude. <b>(a)</b> Three 2 μg ICV doses of GR1 given at P1, P3, and P5 significantly reduced the percentage of mice progressing to severe seizure stages after a single dose of KA (3 mg/kg- IP) at P10, compared to saline-treated controls (n = 11 per group; p < 0.001 by Fisher’s exact test). <b>(b-c)</b> Total KA dose required to elicit SE and latency to reach individual seizures stages at P10 was significantly greater in mice treated with 2 μg (x3 doses) of GR1, compared to saline-treated controls (n = 6 per group; p < 0.05 and p < 0.01 respectively) as analyzed by two-factor ANOVA. <b>(d)</b> A single 4 μg dose of GR1, given 2 hours post-P10 SE, prevented the increased susceptibility to “double hit” KA seizure observed for saline-treated SE-experienced mice at P12, compared to naïve controls (*p < 0.05, n = 7 per group). <b>(e)</b> Whole-cell patch-clamp recordings of aEPSCs from CA1 pyramidal neurons in P12 mice treated as in panel <b>d</b>. Recordings from double hit SE-experience mice demonstrated a large increase in aEPSC amplitude compared to naïve (no SE) mice (p < 0.001) This SE-induced potentiation of aEPSCs was completely prevented by injection of 4 μg GR1 at 2 hr post-SE onset at P10 (n = 5–7 mice per group). Representative traces of maximal aEPSCs are shown for saline and GR1 treated mice post-SE (scale bars: 50 ms, 200 pA). Panels d and e were assessed by unpaired two-tailed t-test.</p

Development of GR1 SMO.

<p><b>(a)</b> Target binding location of top three SMO tested for splicing activity against GluA1 flip isoforms; 1. GR1-1 (yellow), 2. GR1-2 (blue), and 3. GR1-3 (green). <b>(1)</b> GR1-1 SMO was targeted to the 3’ splice site. <b>(2)</b> GR1-2 SMO was targeted to the 5’ splice site. Finally, <b>(3)</b> GR1-3 SMO was optimized from the GluR1-1 SMO at the 3’ splice site. Initial in vivo testing was performed in neonatal mice. Mice were injected with SMO (2 μg/ventricle) at postnatal (P) days P1, P3, and P5, tissues harvested at P6, and mRNA expression normalized to saline-injected mice (dotted line). <b>(b)</b> Testing of GluA1-flip targeting SMOs in P6 mice (n = 3–5). While all 3 candidate SMOs displayed some on-target splice modulation, only GR1-3 showed a nearly complete knockdown of GluA1-flip with no significant effect on GluA1-flop. Thus, GR1-3 was chosen as the therapeutic SMO to proceed forward in our studies, designated as GR1. (p < 0.05) <b>(c)</b> Schematic showing the mechanism of action of GR1 on GluA1 pre-mRNA splicing. The flip and flop exons are tandem 115 nucleotide exons which are spliced from GluA pre-mRNA in a mutually exclusive pattern. Through Watson-Crick base pairing, GR1 binds to the complementary site at the 3’ splice site of the GluA1-flip exon, sterically inhibiting the spliceosome from recognizing the intron/exon boundary and predicted exonic splice enhancer (ESE) motifs, causing exclusion of the GluA1-flip exon.</p