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. 2020 Jan 8;28(1):235-253.
doi: 10.1016/j.ymthe.2019.08.018. Epub 2019 Sep 3.

dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice

Gaia Colasante  1 , Gabriele Lignani  2 , Simone Brusco  3 , Claudia Di Berardino  3 , Jenna Carpenter  2 , Serena Giannelli  3 , Nicholas Valassina  3 , Simone Bido  3 , Raffaele Ricci  3 , Valerio Castoldi  4 , Silvia Marenna  4 , Timothy Church  2 , Luca Massimino  3 , Giuseppe Morabito  3 , Fabio Benfenati  5 , Stephanie Schorge  2 , Letizia Leocani  4 , Dimitri M Kullmann  2 , Vania Broccoli  6
Affiliations

dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice

Gaia Colasante et al. Mol Ther. .

Abstract

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.

Keywords: Dravet syndrome; activatory CRISPR; epileptic encephalopathy; gene therapy.

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Figures

Figure 1
Figure 1
sgRNA Design and Screening for Stimulating Scn1a Gene Expression with the dCas9 Activation System in P19 Cells (A–C) Schematic representation of the Scn1a gene (A) with distal (B) and proximal (C) promoter regions; the positions of the sgRNAs selected for this screening are highlighted. (D) Experimental setting for the sgRNA screening in P19 cells and schematic representation of the constructs employed for cell lipofection. One day after plating, P19 cells were lipofected, and the subsequent day, GFP expression was ascertained. At 3 DIV, the cells were processed for RNA extraction. (E and F) qRT-PCRs for Scn1a mRNA levels performed on RNA extracted from P19 cells lipofected with dCas9VP160-T2A-GFP together with sgRNAs targeting the distal (E) or proximal (F) promoter. Data are normalized on the 18S rRNA and relative to sgCtrl-lipofected cells. sg1p induces significant upregulation of Scn1a compared with sgCtrl (n = 6, p < 0.0001, one-way ANOVA followed by Bonferroni multiple comparisons test). Data are shown as mean ± SEM, with dots representing individual samples.
Figure 2
Figure 2
dCas9-VP160/sg1P Potentiates Scn1a Gene Transcription in Primary Hippocampal Neurons (A) Schematic drawing depicting the experimental setting to deliver the Ctrl- and Scn1a-dCas9A system in primary neurons. Hippocampal neurons were derived from E17.5 embryos and, the day after plating, were co-transduced with two distinct lentiviruses (LVs) carrying Ef1a-dCas9-VP160- T2A-GFP and pU6-sg1P or pU6-sgCtrl guides, respectively. (B) Representative image of anti-GFP immunofluorescence at 10 DIV and quantification of GFP+ transduced neurons over the total cell number. Scale bar, 50 μm. (C) Representative FACS images of GFP+ neurons transduced with either the sgCtrl- or sg1P-dCas9 activation system. (D) qRT-PCR reveals the increased Scn1a transcriptional levels in hippocampal neurons infected with sg1P with respect to sgCtrl conditions (n = 6, p = 0.0002, Student’s t test). Data are shown as mean ± SEM, with dots representing individual samples. (E) Schematic setting of E17.5 neurons transduced with a LV carrying both the pU6-sgRNA cassette and dCas9-VP160-T2A-GFP sequence under control of the EF1alpha core promoter. (F) Anti-GFP immunofluorescence at 10 DIV and relative quantification of transduced GFP+ cells over total, showing that a single LV reaches a transduction efficiency of 75%. Scale bar, 50 μm. (G) qRT-PCR for Scn1a with primers amplifying the first (exon A) and second 5′ UTR exon (exon B). Data are normalized on 18S rRNA and expressed as relative to sgCtrl. Exon A, sg1P versus sgCtrl: p = 0.7353; exon B, sg1P versus sgCtrl: p < 0.0001; Student’s t test). (H) Left: western blot for Nav1.1 and Calnexin on protein lysates from Ctrl-dCas9A- and Scn1a-dCas9A-treated neurons at 10 DIV. Right: quantification obtained through densitometry and normalized on Calnexin levels and expressed as Scn1a-dCas9A relative to Ctrl-dCas9A (data are shown as mean ± SEM, with dots representing individual samples); n = 4; sg1P versus sgCtrl: p = 0.03, Student’s t test.
Figure 3
Figure 3
Global Gene Expression analysis of Transduced Neurons Confirms the High Specificity Profile of the Scn1a-dCas9A System (A) Schematic view of the experimental setting to perform gene expression profiling of Ctrl-dCas9A- and Scn1a-dCas9A-treated primary neurons. E17.5 embryo-derived neurons were transduced with single LVs at DIV 1 expressing either the Ctrl-dCas9A or Scn1a-dCas9A elements and processed for RNA extraction 48 h later (DIV 3). (B) Volcano plots showing the log10 p value as a function of log2 fold changes in gene expression in Scn1a-dCas9A-treated neurons with respect to Ctrl-dCas9A. Scn1a is shown as a red dot. Yellow dots represent off-target genes in the left panel and other Scna genes in the right panel. All other genes are shown as gray dots. (C) qRT-PCRs for profiling the expression of predicted off-targets (genes Prp4, BC024978, Olfr919, and Plrg1; left panel) or other Scna genes (Scn2a, Scn3a, Scn4a, Scn5a, Scn7a, Scn8a, Scn9a, and Scn11a; right panel). Plotted values are normalized on 18S rRNA and expressed as relative to sgCtrl-treated samples (value = 1, data not shown). n = 6; sg1p versus sgCtrl p < 0.0004, Student’s t test. Data are shown as mean ± SEM, with dots representing individual samples.
Figure 4
Figure 4
Scn1a-dCas9A Increases Neuronal Excitability in Cortical Immature Wild-Type Interneurons (A) Schematic drawing showing the timeline of transduction with LVs expressing the dCas9A systems on primary wild-type GAD67-GFP neurons and their subsequent functional analysis. (B) Representative images of a patch-clamp-recorded interneuron expressing both GFP under the GAD67 promoter and tdTomato, reflecting the active Scn1a-dCas9A system. Scale bar, 25 μm. (C) Representative current-clamp traces of APs induced by a single current step in dCas9A (black trace, sgCtrl) or Scn1a-dCas9A interneurons (blue trace, sg1P). (D) Firing frequency versus injected current for Ctrl- and Scn1a-dCas9A-transduced interneurons (Ctrl-dCas9A, n = 11; Scn1a-dCas9A, n = 15). (E) Histogram of the maximum frequency reached by interneurons during the current step protocol (p = 0.03, Mann-Whitney U test). (F) Experimental design of activity clamp in primary neuronal cultures in the presence of 4AP (Materials and Methods). (G and H) Representative full traces (G) and magnified traces (H) for the activity clamp protocol in Ctrl-dCas9A (black trace, sgCtrl) and Scn1a-dCas9A (blue trace, sg1P) interneurons. (I) Activity clamp analysis for the number of events during the full traces (left) and cumulative plot for AP frequency (right) (Ctrl, n = 10; Scn1a-dCas9A, n = 12; p = 0.0009, unpaired Student`s t test).
Figure 5
Figure 5
Scn1a-dCas9A Stimulates Scn1a Basal Expression and Nav1.1 Protein Levels in Scn1a+/− Hippocampal Neurons (A) Schematic drawing of the dCas9A treatments in E17.5 Scn1a+/− primary neurons. Neurons were transduced with either the Ctrl-dCas9A or Scn1a-dCas9A system 1 day after plating and processed for RNA and protein extraction at 22–25 DIV. (B and C) qRT-PCRs for Scn1a transcriptional levels performed on RNA extracted from Ctrl-dCas9A- or Scn1a-dCasA-treated Scn1a+/+(B) and Scn1a+/− (C) primary neurons. Plotted data are expressed as relative to Ctrl-dCas9A. Scn1a+/+: n = 18, p = 0.068; Scn1a+/−: n = 4, p < 0.0001; Student’s t test. (D) Binary alignment map (BAM) within the mouse Scn1a transcript (NM_001313997.1). The red box indicates the region amplified and sequenced with high coverage. Ctrl-dCas9A and Scn1a-dCas9A variant allele frequency (VAF) tracks show the observed VAF. Ctrl-dCas9A and Scn1a-dCas9A read tracks display a sample of about 30 different sequencing reads per sample; nucleotides diverging from the reference genome are highlighted. (E) Western blot for Nav1.1 and Calnexin on protein lysates from adult (P45) Scn1a+/+ and Scn1a+/− mice (left panel) and from Ctrl-dCas9A- and Scn1a-dCasA-treated Scn1a+/+ and Scn1a+/− neurons at 22–25 DIV (center and right panels). (F) Densitometric quantification of immunoreactive bands in the western blots of adult mouse brains. Values corresponding to the Nav1.1 band were normalized to Calnexin levels (n = 3, p = 0.03, Student’s t test). (G) Densitometric quantification of immunoreactive bands in the western blots of Scn1a+/+ and Scn1a+/− neurons transduced with Ctrl-and Scn1a-dCas9A.Values corresponding to the Nav1.1 band were normalized to Calnexin levels (n = 4, one-way ANOVA followed by Turkey’s multiple comparisons test). Data are shown as mean ± SEM, with dots representing individual samples.
Figure 6
Figure 6
Scn1a-dCas9A Rescues Neuronal Excitability Defects in Cortical Mature Scn1a+/− Interneurons (A) Schematic drawing showing the experimental time frame for lentiviral transductions and functional analysis of Scn1a+/+;GAD67-GFP+ or Scn1a+/−;GAD67-GFP+ primary hippocampal neurons transduced with the two depicted lentiviruses. (B) Representative images of a patch-clamp-recorded Scn1a+/− interneuron expressing both GFP under the GAD67 promoter and tdTomato reflecting the active Scn1a-dCas9A system (Materials and Methods). (C and D) Representative current-clamp traces of APs induced by single current steps administered to Ctrl-dCas9A-transduced WT interneurons (black trace, C), Scn1a-dCas9A-transduced Scn1a+/+interneurons (blue trace, C), Ctrl-dCas9A-transduced Scn1a+/− interneurons (gray trace, D), and Scn1a-dCas9A-transduced Scn1a+/− interneurons (cyan trace, D). (E and F) Firing frequency versus injected current for Ctrl-dCas9A-transduced (E) and Scn1a-dCas9A-transduced (F) Scn1a+/+ and Scn1a+/− interneurons. Ctrl-dCas9A wild-type, n = 12; Ctrl-dCas9A Scn1a+/−, n = 10; Scn1a-dCas9A wild-type, n = 10; Scn1a-dCas9A Scn1a+/−, n = 1 (p < 0.05, 2-way ANOVA). (G and H), Histogram plots of the maximum frequency (G) and current threshold (H) reached by interneurons during the current step protocol (p = 0.02, p = 0.04, 2-way ANOVA/Bonferroni’s multiple comparisons tests). (I and J) Activity clamp analysis for the number of events during the full traces (I) and cumulative plot for AP frequency (J) (p = 0.03, 2-way ANOVA/Bonferroni’s multiple comparisons tests).
Figure 7
Figure 7
In Vivo Scn1a-dCas9A Delivery through Intracerebroventricular Brain Injections Attenuates Seizures in the Scn1a+/− Mice (A) Schematic illustration showing the experimental setting for in vivo delivery of the Scn1a-dCas9A system through intracerebroventricular injections into P0 pups of AAVs (2.9) carrying the Ctrl-dCas9A and Scn1a-dCas9A system. After 1 week, treated mice were genotyped, and then Scn1a+/− animals then selected for implantation of EEG electrodes and analysis of the epileptic phenotype. Wild-type (WT) litters were processed for molecular (2 weeks) and histological (5 weeks) characterization of in vivo AAV targeting. Doxycycline (dox) was administered in drinking water or food until final analysis. (B) Scheme of cerebral cortex dissection in treated mice for Scn1a expression at the mRNA level (Cx1, medial cortex; Cx2, lateral cortex; R, right; L, left). (C) qRT-PCRs performed on dissected areas of the brains in Ctrl-dCas9A- and Scn1a-dCas9A-treated wild-type mice (n = 6 for each group, p = 0.042 for Cx1_R, p = 0.03 for Cx1_L, Student’s t test). (D and E) Mean (± SEM) threshold temperatures (D) for the occurrence of myoclonic seizures (n = 6 for each group, p = 0.048, Student’s t test) and severity of the epileptic seizures, evaluated by a modified Racine score (E) in Ctrl-dCas9A-and Scn1a-dCas9A-treated Scn1a+/− mice (n = 6 for each group, p = 0.02, chi-square test). (F and G) Duration (F) and spike frequency (G) of temperature-induced seizures in Ctrl-dCas9A-or Scn1a-dCas9A-treated Scn1a+/− mice (n = 5 for Ctrl-dCas9A and n = 6 for Scn1a-dCas9A treated mice, p = 0.029 for duration and p = 0.2 for spike frequency, Student’s t test). (H) Representative EEG traces of hyperthermia-induced seizures in Ctrl-dCas9A-and Scn1a-dCas9A-treated Scn1a+/− mice.
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