AAV9/SLC6A1 gene therapy rescues abnormal EEG patterns and cognitive behavioral deficiencies in Slc6a1–/– mice
Weirui Guo, … , Chao Xing, Steven J. Gray
Weirui Guo, … , Chao Xing, Steven J. Gray
Published November 26, 2024
Citation Information: J Clin Invest. 2025;135(3):e182235. https://doi.org/10.1172/JCI182235.
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AAV9/SLC6A1 gene therapy rescues abnormal EEG patterns and cognitive behavioral deficiencies in Slc6a1–/– mice

Abstract

The solute carrier family 6 member 1 (SLC6A1) gene encodes the γ-aminobutyric acid (GABA) transporter GAT-1, the deficiency of which is associated with infantile encephalopathy with intellectual disability. We designed 2 AAV9 vectors, with either the JeT or MeP promoter, and conducted preclinical gene therapy studies using heterozygous and homozygous Slc6a1-KO mice at different developmental ages and various routes of administration. Neonatal intracerebroventricular administration of either vector resulted in significantly normalized EEG patterns in Slc6a1–/– or Slc6a1+/– mice as well as improvement in several behavioral phenotypes of Slc6a1–/– mice. However, some mortality and adverse effects were observed in neonatal-treated mice. Intrathecal administration of either vector at P5 normalized EEG patterns in Slc6a1+/– mice, but in Slc6a1–/– mice, the treatment only rescued nest building without impact on EEG. Both vectors were well tolerated in all mice treated at P5 or later (including WT mice), up to 1 year after injection. Overall, our data demonstrate compelling efficacy when mice are treated at an early development age. We also identified that outside of the neonatal treatment window, the severe homozygous KO model is more refractory to treatment, whereas our treatments in the heterozygous mice, which genotypically match human patients, have resulted in stronger benefits.

Authors

Weirui Guo, Matthew Rioux, Frances Shaffo, Yuhui Hu, Ze Yu, Chao Xing, Steven J. Gray

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Figure 3

i.t. administration of AAV9/hSLC6A1 to P5 mice rescues abnormal EEG patterns in Slc6a1+/– mice and some cognitive behaviors in Slc6a1–/– mice, but fails to rescue the abnormal EEG patterns in Slc6a1–/– mice.

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i.t. administration of AAV9/hSLC6A1 to P5 mice rescues abnormal EEG patt...
(A) Experimental design of AAV9/hSLC6A1 gene therapy via i.t. injection in P5 mice. Either vehicle, AAV9/MeP229-hSLC6A1, or AAV9/JeT-hSLC6A1 were administered at doses of 5.6 × 1011 vg or 1.12 × 1012 vg. Study readouts at specified ages are listed. (B) Representative EEG and EMG recording examples for treated Slc6a1+/– and Slc6a1–/–. A 10 Hz high pass filter was used to smooth the EEG signal. (C) Quantification of total spike train duration across a 48-hour EEG recording period (% recording time) for treated Slc6a1+/– mice (left to right: n = 7, 9, 12, 9, and 9). (D) Quantification of total spike train duration across a 48-hour EEG recording period (% recording time) for treated Slc6a1–/– mice (left to right: n = 6, 8, 5, 7, and 6). (E) Nest-building scores in treated Slc6a1–/– mice compared with vehicle-treated Slc6a1+/– mice (left to right: n = 10, 10, 10, 8, 10, and 9). For C–E, data are represented by the mean ± SEM. One-way ANOVA with Dunnett’s multiple-comparisons test was used for statistical analysis. (F) RNAscope multiplex fluorescent images showing mRNA expression of the hSLC6A1 transgene (green) and mouse endogenous mSlc6a1 (red) from the cortex in treated Slc6a1+/– mouse brain sections, counterstained with DAPI (blue). Scale bar: 100 μm. (G) Quantification of the percentage of transduced human SLC6A1-positive cells in the cortex of treated Slc6a1+/– mice. The percentage of hSLC6A1 transduced cells are expressed as a ratio of green hSLC6A1-positive cells to the total number of blue nuclear DAPI positive cells (counting at least 1,000 nuclei of each sample/brain area); (H) Quantification of the percentage of endogenous mSlc6a1-positive cells in hSLC6A1-transduced cells in each brain region (expressed as a ratio of the double SLC6A1-positive cells [red and green] to transduced hSLC6A1-positive cells [green]). Data are represented as mean ± SEM. n = 6 per group. One-way ANOVA with Dunnett’s multiple-comparisons test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.