Cell-Selective Adeno-Associated Virus-Mediated SCN1A Gene Regulation Therapy Rescues Mortality and Seizure Phenotypes in a Dravet Syndrome Mouse Model and Is Well Tolerated in Nonhuman Primates

Dravet syndrome (DS) is a developmental and epileptic encephalopathy caused by monoallelic loss-of-function variants in the SCN1A gene. SCN1A encodes for the alpha subunit of the voltage-gated type I sodium channel (NaV1.1), the primary voltage-gated sodium channel responsible for generation of action potentials in GABAergic inhibitory interneurons. In these studies, we tested the efficacy of an adeno-associated virus serotype 9 (AAV9) SCN1A gene regulation therapy, AAV9-REGABA-eTFSCN1A, designed to target transgene expression to GABAergic inhibitory neurons and reduce off-target expression within excitatory cells, in the Scn1a+/− mouse model of DS. Biodistribution and preliminary safety were evaluated in nonhuman primates (NHPs). AAV9-REGABA-eTFSCN1A was engineered to upregulate SCN1A expression levels within GABAergic inhibitory interneurons to correct the underlying haploinsufficiency and circuit dysfunction. A single bilateral intracerebroventricular (ICV) injection of AAV9-REGABA-eTFSCN1A in Scn1a+/− postnatal day 1 mice led to increased SCN1A mRNA transcripts, specifically within GABAergic inhibitory interneurons, and NaV1.1 protein levels in the brain. This was associated with a significant decrease in the occurrence of spontaneous and hyperthermia-induced seizures, and prolonged survival for over a year. In NHPs, delivery of AAV9-REGABA-eTFSCN1A by unilateral ICV injection led to widespread vector biodistribution and transgene expression throughout the brain, including key structures involved in epilepsy and cognitive behaviors, such as hippocampus and cortex. AAV9-REGABA-eTFSCN1A was well tolerated, with no adverse events during administration, no detectable changes in clinical observations, no adverse findings in histopathology, and no dorsal root ganglion-related toxicity. Our results support the clinical development of AAV9-REGABA-eTFSCN1A (ETX101) as an effective and targeted disease-modifying approach to SCN1A+ DS.


Immunohistochemistry image analysis
A 1-mm square region of the somatosensory cortex was selected from each animal. This raw data image was spectrally unmixed with Akoya InForm software using a spectral library constructed from single stained slides stained in parallel in the same assay. A separate spectral library was generated and used for the first and second multiplex data sets. A binary image of the nuclei in the DAPI channel was created manually from all images from the first data set (4 test and 4 control). These 'ground-truth' binary masks were used to train a StarDist neural network segmentation model which was used with the Fiji plugin to perform nuclei segmentation for all the DAPI-stained images. 1 Validation of this model using a 'ground-truth' image not part of the original training set yielded a precision and recall of 98% each. In Fiji, once nuclei regions were identified they were overlayed on each marker image and regions were visually identified as positive or negative for a respective marker. During region identification each marker image was inspected separately and not overlaid with any other marker. Inspection was performed on a 27" 4K UHD monitor with the image zoom increased to 200%. Images were grayscale and not contrast adjusted. Counts per image for nuclear region GFP or marker status were found and percentages were calculated using the total nuclei count. The ratio of percent double-positive GFP with a respective marker divided by percent total GFP positive was found for each marker in addition to the ratio of double-positive divided by percent total marker positive regions.

Serum and CSF anti-AAV9 and anti-eTF antibody assays
Anti-eTF SCN1A binding IgG antibodies were determined by ELISA. Briefly, 96-well High Binding ELISA microplates were coated with 100 μL/well of recombinant eTF SCN1A (a transgene encoded protein, diluted in 50 mM carbonate-bicarbonate buffer, pH 9.6) and incubated at 4°C overnight. Plates were washed with 1X PBS-T) buffer and blocked with 3% bovine serum albumin in PBS for 1 hour. One hundred microliters of diluted serum or CSF sample was added to each well and incubated for 1 hour. After wash, detection antibody (HRP-Goat anti-Monkey IgG) was added, incubated for 1 hour, and washed. The color was developed by adding 100 μL of TMB Substrate Solution to each well for 3 minutes and followed by 100 μL of stop solution. The optical density (OD) was measured at 450/640 nm.
For the anti-AAV9 neutralizing antibody assay, heat-inactivated sera or CSF samples were continuously diluted 2-fold with Dulbecco's Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum and preincubated with AAV9-CMV-NLuc vector at 37°C for 60 minutes. Twenty-five microliters of the mixture were added to 96-well plates containing 1.5E4 of 293AAV cells to reach a final multiplicity of infection of 10,000 vg/cell (titer determined by qPCR), followed by an incubation at 37°C in a 5% CO 2 incubator for 48 hours. Luciferase expression was measured with a luciferase assay kit (Promega, Madison, WI, USA). The neutralizing antibody titer was determined as the serum dilution at which 50% of AAV reporter gene expression is permitted, compared to no serum controls.

Supplementary Table S1. Primers for RT-qPCR in vitro
Target gene Forward primer Reverse primer S5

Supplementary Table S4. Pre-and post-dose anti-AAV9 neutralizing antibody (NAb) titer in cynomolgus monkey serum and CSF.
Six cynomolgus monkeys with no detectable serum anti-AAV9 NAb were dosed with either AAV9-RE GABA -eTF SCN1A or vehicle control. Serum samples were taken on Day -1 (prior to dosing) and weekly until the end of study (Day 27-29). CSF samples were taken on Day 1 (pre-dose) and Day 24-29, before the end of study. At Day 15, serum levels of anti-AAV9 NAb were not increased in vehicle-treated animals but were increased in AAV9-RE GABA -eTF SCN1A -treated animals, and remained positive at the end of the study with one animal showing further elevated serum titer. CSF samples from pre-dose and Day 24 (vehicle control group) or Day 29 (ETX101-treated group) all tested negative (titer ≤1:5) for anti-AAV9 NAb.