Cas9 gene therapy for Angelman syndrome trapsUbe3a-ATSlong non-coding RNA

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a mutation or deletion of the maternally inheritedUBE3Aallele. In neurons, the paternally inheritedUBE3Aallele is silenced incisby a long non-coding RNA calledUBE3A-ATS. Here, as part of a systematic screen, we found that Cas9 can be used to activate ('unsilence') paternalUbe3ain cultured mouse and human neurons when targeted toSnord115genes, which are small nucleolar RNAs that are clustered in the 3 ' region ofUbe3a-ATS. A short Cas9 variant and guide RNA that target about 75 Snord115genes were packaged into an adeno-associated virus and administered to a mouse model of AS during the embryonic and early postnatal stages, when the therapeutic benefit of restoringUbe3ais predicted to be greatest(1,2). This early treatment unsilenced paternalUbe3athroughout the brain for at least 17 months and rescued anatomical and behavioural phenotypes in AS mice. Genomic integration of the adeno-associated virus vector into Cas9 target sites caused premature termination ofUbe3a-ATSat the vector-derived polyA cassette, or when integrated in the reverse orientation, by transcriptional collision with the vector-derived Cas9 transcript. Our study shows that targeted genomic integration of a gene therapy vector can restore the function of paternally inherited UBE3A throughout life, providing a path towards a disease-modifying treatment for a syndromic neurodevelopmental disorder. Genomic integration of an adeno-associated virus vector in a mouse model of Angelman syndrome unsilences paternalUbe3aand rescues anatomical and behavioural phenotypes, suggesting a pathway towards the treatment of this neurodevelopmental disorder.