Genome editor therapy for Angelman syndrome - PROJECT SUMMARY Angelman syndrome (AS) is a severe neurodevelopmental disorder that is caused by deletion or mutation of the maternally inherited UBE3A allele. The paternal UBE3A allele is silenced in cis by a long non-coding antisense RNA (UBE3A-ATS) exclusively in neurons. This biology explains why deletion of maternal UBE3A results in near-complete loss of UBE3A protein in the nervous system. We recently found that an adeno-associated virus (AAV)-Cas9 vector can be used to unsilence paternal UBE3A (patUBE3A) in mouse and human neurons when targeted to UBE3A-ATS. However, as a potential safety concern, the AAV vector integrated into Cas9-generated double-strand breaks at guide RNA target sites. In preliminary studies, we found that a Cas9 nickase (nCas9) AAV vector did not integrate at target sites and was more effective at unsilencing patUbe3a when compared to an equivalent Cas9 cleavase vector. Preclinical studies suggest that AS could be prevented or reduced in severity if patUBE3A is unsilenced in the early postnatal period. Thus, the overarching goal of this project is to de-risk the neonatal use of an AAV-nCas9 genome editor vector to enduringly treat AS. We will advance this somatic genome editor therapy as follows, Discovery (UG3) phase (2 years): 1) We generated a knock-in mouse harboring ~170 kb of human UBE3A-ATS sequence and validated its utility with antisense oligonucleotides and with AAV-Cas9 vectors that target human UBE3A-ATS. We will identify lead AAV-nCas9 vectors that target human UBE3A-ATS and perform biodistribution studies with this innovative humanized mouse line. 2) We will independently validate on-target activity of the lead vectors and evaluate potential off-target activities with human neurons containing the maternal 15q11-q13 deletion that typically causes AS. One lead vector will be selected as the clinical candidate using quantitative selection criteria and advanced to the Development (UH3) phase (3 years): 3) Complete development phase preparatory activities, including large-scale manufacturing of the clinical candidate vector with a NIH Blueprint Neurotherapeutics Network contractor and initiate the pre-investigational new drug (IND) meeting with the Food and Drug Administration (FDA). 4) Perform IND-enabling studies in rodents and rhesus monkeys. We will de-risk early postnatal use of the clinical candidate vector in rhesus monkeys, a species that closely models human neurodevelopment. All assays are in-place to quantify target engagement, biodistribution, and vector safety. Short and long-term IND-enabling safety studies are proposed, including noninvasive, longitudinal in vivo imaging in rhesus monkeys to assess the potential for adverse findings such as neuroinflammation. An IND application will be prepared and submitted to the FDA at the conclusion of the proposed studies.
