# CRISPR/Cas9-based gene therapy for Angelman syndrome

> **NIH NIH R01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2020 · $569,876

## Abstract

PROJECT SUMMARY
Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by deletion or mutation of the
maternal allele of UBE3A. UBE3A is biallelically expressed in nearly all cells of the body except in mature
neurons, where the paternal allele is silenced by an extremely long non-coding RNA called UBE3A-ATS. In
light of this biology, the most direct way to treat behavioral dysfunctions associated with AS is to unsilence the
intact paternal UBE3A allele. CRISPR/Cas9 technology can be used to target specific regions of the
mammalian genome for mutagenesis or transcriptional repression. In unpublished studies, we generated
hundreds of S. pyogenes (Sp)Cas9 guide RNAs (gRNAs) that target regions throughout UBE3A-ATS. Several
of these gRNAs, when transfected along with SpCas9, potently unsilenced paternal Ube3a in cultured mouse
cortical neurons. Some of our most effective gRNAs targeted a region of Ube3a-ATS that is conserved
between mice and humans, making it possible to translate our findings to human neurons. Here, we will test
the central hypothesis that CNS-directed delivery of Cas9 and a gRNA that targets Ube3a-ATS can
enduringly unsilence paternal UBE3A and treat behavioral phenotypes associated with Angelman
syndrome. We will use adeno-associated virus (AAV) for delivery because it can drive gene expression for
years in the brain. Pilot studies with S. aureus (Sa)Cas9, a smaller Cas9 variant, suggest that our gene
therapy approach can be used to unsilence paternal Ube3a in mice for at least three months. To advance this
innovative gene therapy towards the clinic, we will evaluate efficacy, on- and off-target effects, and mechanism
of action of candidate therapeutic SaCas9 gRNAs that target Ube3a-ATS. We will use cultured neurons from
AS model mice and AS-derived human neurons. We will package SaCas9 and an optimized gRNA into a
single AAV vector, and then evaluate unsilencing efficacy and longevity for up to two years in mice, as well as
biodistribution and toxicity. Lastly, we will evaluate the extent to which AAV-mediated delivery of this
CRISPR/Cas9-based gene therapy treats behavioral phenotypes in AS model mice.

## Key facts

- **NIH application ID:** 10011898
- **Project number:** 5R01NS109304-02
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Mark J. Zylka
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $569,876
- **Award type:** 5
- **Project period:** 2019-09-15 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10011898

## Citation

> US National Institutes of Health, RePORTER application 10011898, CRISPR/Cas9-based gene therapy for Angelman syndrome (5R01NS109304-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10011898. Licensed CC0.

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