stm.sciencemag.org/cgi/content/full/12/558/eaaz6100/DC1

Supplementary Materials for

Antisense oligonucleotides increase Scn1a expression and reduce seizures and

SUDEP incidence in a mouse model of Dravet syndrome

Zhou Han, Chunling Chen, Anne Christiansen, Sophina Ji, Qian Lin, Charles Anumonwo, Chante Liu, Steven C. Leiser, Meena, Isabel Aznarez, Gene Liau, Lori L. Isom*

*Corresponding author. Email: lisom@umich.edu

Published 26 August 2020, Sci. Transl. Med. 12, eaaz6100 (2020)

DOI: 10.1126/scitranslmed.aaz6100

The PDF file includes:

Fig. S1. A control ASO showed dose-responsive effect on SMN2 transcript splicing in ReNcells. Fig. S2. Dose-dependent effects of ASO-22 on expression of Scn1a gene in ICV-injected neonatal mouse brains. Fig. S3. Dose-dependent effects of ASO-22 on expression of NaV1.1 in ICV-injected neonatal mouse brains. Fig. S4. A control ASO promoted exon 35 exclusion of Cep290 mRNA in neonatal mouse brain. Fig. S5. Expression of Scn1a mRNA in mouse brains at different post-injection days. Fig. S6. Expression of NaV1.1 in mouse brains at different post-injection days. Fig. S7. Numbers of Scn1a+/– mice that had 0, 1, or 2+ seizures after treatment. Fig. S8. Validation of the two anti-NaV1.1 antibodies. References (29, 30)

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/558/eaaz6100/DC1)

Movie S1 (.avi format). Terminal seizure event in a DS mouse injected with 60 μg of ASO at P14. Movie S2 (.avi format). Terminal seizure event in a DS mouse injected with 3 μl of PBS at P14. Data file S1 (Microsoft Excel format). Individual-level data for figures with n less than 20.

SUPPLEMENTARY MATERIALS

Supplementary Figures

A)

B)

Fig. S1. A control ASO showed dose-responsive effect on SMN2 transcript

splicing in ReNcells. ReNcells were treated with 20, 8 or 3 μM of a control ASO

(SMN2 ASO, 5’-ATTCACTTTCATAATGCTGG-3’) (29) for 72 h by gymnotic uptake.

ASO-22 and no ASO control (sham) were also included. Treatment of ReNcells with

control ASO increased inclusion of exon 7 of the SMN2 transcript in a dose-responsive

manor, as shown in panel A with the TBE PAGE of RT-PCR products and panel B with

probe-based qPCR. Treatment with ASO-22 had no effect on SMN2 exon 7 inclusion.

For qPCR, transcript expression was first normalized to the endogenous RPL32 signal

and then to the no ASO (sham) control. qPCR results are presented as mean ± SD (n=3

for each group). M: DNA ladder, NEB, N3231L.

Fig. S2. Dose-dependent effects of ASO-22 on expression of Scn1a gene in ICV-injected neonatal mouse brains.

C57BL/6J mice were ICV injected with PBS, a non-target control (NT, Cep290 ASO, 5’-CATGAAGGTCTTCCTCATGC-3’,

20μg), 0.3, 1, 3, 5, 10, 20 or 30 μg of ASO-22 at P2. Brains were harvested 5 days after injection and analyzed for Scn1a

mRNA expression. TBE PAGE of RT-PCR products shows Scn1a productive (bottom bands, 498bp, indicated by *) and

NMD-inducing transcript (upper bands, 562bp, indicated by §) in mouse brains. Quantification of the percentage of exon

21N inclusion is shown in Fig. 3B. Quantification of the Scn1a productive mRNA expression by qPCR is shown in Fig. 3C.

M: DNA ladder, NEB, N3231L.

Fig. S3. Dose-dependent effects of ASO-22 on expression of NaV1.1 in ICV-

injected neonatal mouse brains. C57BL/6J mice were ICV injected with 0.3, 1, 3 or 10

μg of ASO-22 or PBS at P2. Brains were harvested 5 days after injection and analyzed

for NaV1.1 expression. NaV1.1 expression (~223 kDa, indicated by *) was assessed by

immunoblotting of two randomly selected brain samples from each dosing group. 50 g

of protein was loaded per lane. Immunoblotting was performed with anti-NaV1.1

antibody Alomone ASC-001 (panel A) or NeuroMab 75-023 (panel B). Ponceau S

stained blots (panel C and D as controls for panels A and B, respectively) are included

to show equal loading. ½ input of protein from a brain injected with 10 µg of ASO was

included for testing signal saturation of the immunoblotting. Brain samples from

untreated Scn1a+/- and WT littermate mice were included as controls. Quantification of

NaV1.1 expression by Meso Scale Discovery (MSD) method is shown in Fig. 3D.

Fig. S4. A control ASO promoted exon 35 exclusion of Cep290 mRNA in neonatal

mouse brain. C57BL/6J mice were ICV injected at P2 with PBS, ASO-22 (20 μg) or a

control ASO (5’-CATGAAGGTCTTCCTCATGC-3’, 20 μg) targeting the Cep290

transcript (30). Injected brains were harvested at P7 and analyzed for exon 35 exclusion

of Cep290 with RT-PCR. TBE PAGE of RT-PCR products showed that the control ASO

promoted exon 35 exclusion of the Cep290 mRNA. ASO-22 had no effect on Cep290

mRNA splicing. M: DNA ladder, NEB, N3231L.

Fig. S5. Expression of Scn1a mRNA in mouse brains at different post-injection

days. C57BL/6J mice were ICV injected with 3 or 10 g (panel A and B, respectively) of

ASO-22 or PBS at P2. Brains were harvested 1, 3, 5, 10, 20 or 30 days after injection

and analyzed for Scn1a mRNA expression. TBE PAGE of RT-PCR products shows

Scn1a productive (bottom bands, 498bp, indicated by *) and NMD-inducing transcript

(upper bands, 562bp, indicated by §) in mouse brains. Gapdh was used as loading

control. Quantification of the Scn1a productive mRNA expression by qPCR is shown in

Fig. 3G. M: DNA ladder, NEB, N3231L.

Fig. S6. Expression of NaV1.1 in mouse brains at different post-injection days.

C57BL/6J mice were ICV injected with 10 μg of ASO-22 or PBS at P2. NaV1.1

expression (~223 kDa, indicated by *) was assessed by immunoblotting of two randomly

selected brain samples from the 10-, 20-, and 30-day post-injection mice. 50 μg of total

protein was loaded per lane. Immunoblotting was performed with anti-NaV1.1 antibody

Alomone ASC-001 (panel A) or NeuroMab 75-023 (panel B). Ponceau S stained blots

(panel C and D) are included to show equal loading for panels A and B, respectively.

Quantification of changes in NaV1.1 expression by Meso Scale Discovery (MSD)

method is shown in Fig. 3H.

Fig. S7. Numbers of Scn1a+/– mice that had 0, 1, or 2+ seizures after treatment.

Scn1a+/- mice were ICV injected with 20 µg of ASO-22 or PBS at P2 and seizures were

monitored between P22 to P46. The numbers of mice that had 0, 1 or 2+ seizures, of 21

total mice, were plotted for each treatment group.

Fig. S8. Validation of the two anti-NaV1.1 antibodies. Specificity of the two anti-

NaV1.1 antibodies, Alomone ASC-001 (panel A) and NeuroMab 75-023 (panel B), was

tested using total protein prepared from a Scn1a-/- mouse brain (middle lane) and brains

of two WT littermates (left and right lanes). Both antibodies detected the NaV1.1 protein

at ~ 223 kDa in brain lysate from the WT mice, as expected. In contrast, NaV1.1 protein

was not detected in brain lysate from the Scn1a-/- mouse. The strong signals (smears)

seen with the NeuroMab 75-023 staining may have resulted from binding of the

secondary goat anti mouse antibody to the IgG presented in the mouse brain lysate.

Ponceau S stained blots (panel C and D for panels A and B, respectively) are included

to show equal loading.

Movie S1. Terminal seizure event in a DS mouse injected with 60 μg of ASO at P14.

Movie S2. Terminal seizure event in a DS mouse injected with 3 μl of PBS at P14.

Data file S1. Individual-level data for figures with n less than 20.