Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele UBE3A. UBE3A is biallelically expressed in nearly all cells of the body except in mature neurons, where the paternal allele is silenced by an extremely long noncoding RNA called UBE3A-ATS. In light of this biology, the most direct way to treat behavioral dysfunctions associated with AS is to unsilence the intact paternal UBE3A allele in neurons.

CRISPR/Cas9 technology can be used to target specific regions of the mammalian genome for mutagenesis or transcriptional repression. Mark Zylka’s laboratory has recently generated a library of S. pyogenes (Sp)Cas9 guide RNAs (gRNAs) that target regions throughout UBE3A-ATS. His team has evidence that several of these gRNAs, when transfected along with SpCas9, unsilence paternal Ube3a in cultured mouse cortical neurons.

In the current project, the group plans to test the hypothesis that central nervous system-directed delivery of Cas9 and a gRNA that targets UBE3A-ATS can unsilence paternal Ube3a and ameliorate behavioral phenotypes in a mouse model of AS. Zylka’s team will use adeno-associated virus (AAV) for delivery because it can drive gene expression for years in the brain. Pilot studies suggest that this gene-therapy approach can be used to enduringly unsilence paternal Ube3a in mice.

To advance this innovative gene therapy toward the clinic, the team will evaluate efficacy, on- and off-target effects and mechanism of action of gRNAs that target UBE3A-ATS. They will package a smaller Cas9 variant and an optimized gRNA into a single AAV vector, and then evaluate unsilencing efficacy and longevity for up to two years in mice. Lastly, the team will evaluate the extent to which AAV-mediated delivery of this CRISPR/Cas9-based gene therapy ameliorates behavioral phenotypes in AS model mice.

Findings from this study are expected to provide the first preclinical evidence that CRISPR/Cas9 can be used to unsilence paternal Ube3a in a mammalian model of AS. This new knowledge has the potential to advance a first-in-class treatment for an autism spectrum disorder.