Multi-targeting zinc finger nuclease vector unsilences paternal UBE3A in a mouse model of Angelman syndrome.

Angelman syndrome (AS) is a severe neurodevelopmental disorder most often caused by deletion of the maternally inherited UBE3A allele (matUBE3A). In neurons, a long non-coding antisense RNA (Ube3a-ATS) silences the paternally-inherited UBE3A allele (patUBE3A). Here, we find that delivery of a zinc finger nuclease (ZFN) pair targeted to 86 Snord115 genes within Ube3a-ATS (ZFN17/18) using adeno-associated virus (AAV) can unsilence patUBE3A in primary neuron cultures and in the brain of a mouse model of AS for at least 9 weeks. The AAV vector genome integrated at ZFN17/18 on-target sites in cultured neurons and, as evidence of specificity, did not integrate at predicted off-target sites. AAV vectors carrying nickase and catalytically inactive ZFN17/18 variants failed to appreciably unsilence patUbe3a and did not integrate at on-target sites. In vivo, we observed significant knockdown of Ube3a-ATS in AS-model mice, resulting in some neurons reaching UBE3A levels like those of wild-type mice. ZFN17/18 did not downregulate Snrpn, Snord116, or IPW in vivo, genes that are associated with Prader-Willi syndrome. Overall, our findings demonstrate the potential use of multi-target ZFNs as therapeutics for AS.