Molecular Therapy. 2023 Feb 18
Jinhui Li, Zhixin Shen, Yajing Liu, Zixiang Yan, Yuanhua Liu, Xiang Lin, Junjie Tang, Ruimin Lv, Guannan Geng, Zhi-Qi Xiong, Changyang Zhou, Hui Yang
Products used in the paper Details Operation
AAV vector packaging AAV-PHP.eb serotype was used in this study. The CAG-tdTomato,hSyn1-hfCas13x.1/U6-cr9, or hfCas13x.1/U6-NT plasmids were constructed and sequenced before packaging into AAV-PHP.eb vehicle; and the AAV vectors were packaged by PackGene Biotech. The vector titer was determined by qPCR specific for the inverted terminal repeat. Request Quote

Research Field: CNS

Keywords: Angelman syndrome, RNA editing, high-fidelity Cas13, paternal Ube3a, motor function

AAV Serotype: AAV-PHP.eb

Dose: For I.C.V. administration, neonatal mice were placed on ice for hypothermia anesthesia and injected with AAV virus into bilateral ventricles at four sites. A total of 5×10^10 AAV (containing hSyn1-hfCas13x.1/U6-cr9 or hSyn1-hfCas13x.1/U6-NT) particles plus 2.5×10^9 AAV (CAG-tdTomato) in PBS was injected into each mouse within 24 h of birth using Nanoject III (Warner Instruments). For immunofluorescence experiments, no AAV-containing CAG-tdTomato was injected. Tdtomato+ cortex or hippocampus tissues were dissected for RT-qPCR and western blotting. For I.V. injection, AAV solution was infused into the facial vein of neonatal mice as previously described.

Routes of Administration: I.C.V. injection and I.V. injection

Targeted organ: brain

Animal or cell line strain: Ube3am-/p+ mice and Ube3am+/pYFP mice were maintained on the C57Bl/6 background.


Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by loss of function mutations in maternally expressed UBE3A. No gene-specific treatment is available for patients so far. Although intact and transcriptionally active, paternally inherited UBE3A is silenced by elongation of antisense long noncoding RNA UBE3A-ATS in neurons. Here, we demonstrated that RNA targeting of paternal Ube3a-ATS with a high-fidelity CRISPR-Cas13 (hfCas13x.1) system could restore Ube3a expression to similar levels as that of maternal Ube3a in the cultured mouse neurons. Furthermore, injection into lateral ventricles with neuron-specific hSyn1 promoter-driven hfCas13x.1 packaged in adeno-associated virus (AAV-PHP.eb) could restore paternal Ube3a expression in cortex and hippocampus of neonatal AS mice for up to 4 months after treatment. Behavioral tests showed that expression of paternal Ube3a significantly alleviated AS-related symptoms, including obesity and motor function. Our results suggested that hfCas13x.1-mediated suppression of the Ube3a-ATS lncRNA potentially serves as a promising targeted intervention for AS.

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