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<p>Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of maternally-inherited <i>UBE3A</i>. In neurons, paternally-inherited <i>UBE3A</i> is silenced in <i>cis</i> by a long non-coding RNA called <i>Ube3a-ATS</i>. Here, we found that <i>Neisseria meningitidis</i> Cas9 with two mutations (D15A and H587A) in the nuclease domains (dNmCas9) can unsilence the dormant paternal <i>Ube3a</i> allele in mouse and human neurons when targeted to <i>Snord115</i> snoRNA&#xa0;genes located in introns of <i>Ube3a-ATS</i>. Importantly, dNmCas9 disrupted <i>Ube3a-ATS</i> with a non-template bias and in the absence of a chromatin modifying domain, supporting a transcriptional interference mechanism. When packaged into an adeno-associated virus (AAV) vector, dNmCas9 exhibited dose-dependent <i>Ube3a-ATS</i> knock-down and paternal <i>Ube3a</i> unsilencing in vitro and in vivo. This vector also partially rescued the hind limb clasp phenotype when delivered to neonatal AS model mice. Collectively, our study underscores the potential of dCas9-based therapeutics without chromatin repression domains to mediate transcriptional downregulation.</p>

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AAV-dCas9 vector unsilences paternal Ube3a in neurons by impeding Ube3a-ATS transcription

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年9月2日

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  • Justin M. Wolter,
  • Lucas M. James,
  • Samantha L. Boeshore,
  • Hanqian Mao,
  • Eric S. McCoy,
  • Daniel F. Ryan,
  • Giulia Fragola,
  • Bonnie Taylor-Blake,
  • Jason L. Stein,
  • Mark J. Zylka

摘要

Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of maternally-inherited UBE3A. In neurons, paternally-inherited UBE3A is silenced in cis by a long non-coding RNA called Ube3a-ATS. Here, we found that Neisseria meningitidis Cas9 with two mutations (D15A and H587A) in the nuclease domains (dNmCas9) can unsilence the dormant paternal Ube3a allele in mouse and human neurons when targeted to Snord115 snoRNA genes located in introns of Ube3a-ATS. Importantly, dNmCas9 disrupted Ube3a-ATS with a non-template bias and in the absence of a chromatin modifying domain, supporting a transcriptional interference mechanism. When packaged into an adeno-associated virus (AAV) vector, dNmCas9 exhibited dose-dependent Ube3a-ATS knock-down and paternal Ube3a unsilencing in vitro and in vivo. This vector also partially rescued the hind limb clasp phenotype when delivered to neonatal AS model mice. Collectively, our study underscores the potential of dCas9-based therapeutics without chromatin repression domains to mediate transcriptional downregulation.