CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice
Ralf S. Schmid, Xuefeng Deng, Priyalakshmi Panikker, Msema Msackyi, Camilo Breton, James M. Wilson
Ralf S. Schmid, Xuefeng Deng, Priyalakshmi Panikker, Msema Msackyi, Camilo Breton, James M. Wilson
View: Text | PDF
Concise Communication Genetics Neuroscience

CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice

Abstract

Gene editing holds the potential to correct mutations and cure devastating genetic disorders. The technology has not yet proven efficacious for therapeutic use in CNS diseases with ubiquitous neuronal defects. Angelman syndrome (AS), a severe neurodevelopmental disorder, is caused by a lack of maternal expression of the UBE3A gene. Because of genomic imprinting, only neurons are affected. One therapeutic approach focuses on the intact paternal UBE3A copy in patients with AS that is silenced by an antisense transcript (UBE3A-ATS). We show here that gene editing of Ube3a-ATS in the mouse brain resulted in the formation of base pair insertions/deletions (indels) in neurons and the subsequent unsilencing of the paternal Ube3a allele in neurons, which partially corrected the behavioral phenotype of a murine AS model. This study provides compelling evidence to further investigate editing of the homologous region of the human UBE3A-ATS because this may provide a lasting therapeutic effect for patients with AS.

Authors

Ralf S. Schmid, Xuefeng Deng, Priyalakshmi Panikker, Msema Msackyi, Camilo Breton, James M. Wilson

×

Figure 2

In vivo gene editing of Ube3a-ATS in a Ube3a-KO mouse model.

Options: View larger image (or click on image) Download as PowerPoint
In vivo gene editing of Ube3a-ATS in a Ube3a-KO mouse model. (A) Brains ...
(A) Brains of Ube3am–/p+ mice injected with ATS-GE vector were harvested 4 months later. We detected persistent paternal Ube3a expression in the cerebral cortex by Western blotting with Ube3a antibodies. Relative quantifications of the respective Ube3a band normalized to actin are shown below each lane. (B) IHC staining of the brains from A with Ube3a antibodies shows paternal Ubea3a expression throughout the brain. A representative cortical section is shown here (scale bar: 1 mm). Magnified cortical IHC images from Ube3am+/p+ (C), Ube3am–/p+ (D), and gene-edited Ube3am–/p+ (E) cortex (scale bar: 10 μm, E is a higher magnification of the section shown in B). (F) Amplicon sequencing analysis from the same cohort as shown in A revealed an average of 19.4% of cells with indels in injected pups. Injection of nontargeting CRISRP/Cas9 resulted in indel formation of 0.2% (n = 5 mice/group). (G) RNA extracted from cortices of the same cohort as shown in A was used to quantify Ube3a-ATS transcript levels at different locations between the site targeted by gene editing (E, about 35 kb distance to Ube3a 3′ UTR) and the imprinting center (IC). We observed a significant reduction of Ube3a-ATS starting approximately 5 kb away from E (1-way ANOVA with Tukey’s pairwise comparison, n = 5 mice/group). Means are shown with standard error; * P < 0.05, ** P < 0.01.