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<p>Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of maternal <i>UBE3A</i>. In neurons, the paternal (pat)<i>UBE3A</i> allele is silenced by a long non-coding antisense transcript called <i>Ube3a-ATS</i>. Previous genome-editing approaches used active nucleases to unsilence <i>patUbe3a</i> by disrupting <i>Ube3a-ATS.</i> However, these methods create DNA double-strand breaks (DSBs) and promote integration of adeno-associated virus (AAV) vector genomes, both of which raise potential safety concerns. Here, we found that a nickase <i>Neisseria meningitidis</i> Cas9 variant (nNmCas9-D15A) disrupted <i>Ube3a-ATS</i> transcription when targeted to the non-template strand and unsilenced <i>patUbe3a</i> in cultured mouse neurons without generating DSBs or causing AAV integration. Intracerebroventricular delivery of AAV9-nNmCas9-D15A in AS model mice potently and durably reduced <i>Ube3a-ATS</i> and elevated <i>Ube3a</i> throughout the cerebral cortex and hippocampus for at least 6 months. Further, this vector restored UBE3A expression in ~ 87% of cortical neurons, which compares favorably to previously reported efficiencies with active Cas9, dead Cas9, and zinc finger nuclease vectors. These results demonstrate that nNmCas9 is a highly effective and potentially safer genome editor for the treatment of AS.</p>

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Nickase NmCas9 unsilences paternal Ube3a in a mouse model of Angelman syndrome without causing AAV vector integration

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  • 发表日期：

    2026年5月14日

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  • Hannah O. Bazick,
  • Lucas M. James,
  • Mark J. Zylka

摘要

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of maternal UBE3A. In neurons, the paternal (pat)UBE3A allele is silenced by a long non-coding antisense transcript called Ube3a-ATS. Previous genome-editing approaches used active nucleases to unsilence patUbe3a by disrupting Ube3a-ATS. However, these methods create DNA double-strand breaks (DSBs) and promote integration of adeno-associated virus (AAV) vector genomes, both of which raise potential safety concerns. Here, we found that a nickase Neisseria meningitidis Cas9 variant (nNmCas9-D15A) disrupted Ube3a-ATS transcription when targeted to the non-template strand and unsilenced patUbe3a in cultured mouse neurons without generating DSBs or causing AAV integration. Intracerebroventricular delivery of AAV9-nNmCas9-D15A in AS model mice potently and durably reduced Ube3a-ATS and elevated Ube3a throughout the cerebral cortex and hippocampus for at least 6 months. Further, this vector restored UBE3A expression in ~ 87% of cortical neurons, which compares favorably to previously reported efficiencies with active Cas9, dead Cas9, and zinc finger nuclease vectors. These results demonstrate that nNmCas9 is a highly effective and potentially safer genome editor for the treatment of AS.