Ben Philpot, Ph.D. UNC Chapel Hill Mapping UBE3A in Non‐Human Primates to Inform UBE3A Gene Therapy and Reinstatement Strategies to Treat Angelman Syndrome
Angelman syndrome is caused by deletions or mutations of the maternally‐inherited gene copy of UBE3A. The paternally‐inherited gene copy of UBE3A lies dormant, thus loss of the maternal UBE3A gene copy leads to a near complete loss of UBE3A protein in the brain. This biology suggests two major strategies to treat AS at its genetic core.
- First, one could treat AS by delivering UBE3A using a traditional gene therapy approach – that is, adding the UBE3A gene back to brain neurons through a safe viral‐like delivery.
- Second, one could treat AS by reactivating the dormant paternal gene copy of UBE3A.
Both of these approaches require knowing when, where, and in what cell types UBE3A protein is normally expressed in the developing brain, as this information is paramount to guide the desired biodistribution of delivered therapeutics, which likely changes over development. Currently we know a great deal about expression of UBE3A in the rodent brain, but almost nothing is known about the developmental expression pattern of UBE3A protein in the primate (human and non‐human primate; NHP) brain.
If the distribution of UBE3A in the primate brain is improperly inferred from rodent studies, this could lead to inappropriate delivery of AS treatments. To overcome this limitation, we propose to perform a complete study of UBE3A expression across development, from prenatal to adult ages, in the closest proxy we can get to the human brain – the brain of the rhesus macaque monkey. This non‐human primate (NHP) brain closely resembles that of the human brain, and has a high degree of evolutionary conservation. We will perform a complete study of the whole‐brain expression patterns of UBE3A in the primate brain, spanning early gestational periods to adulthood.
The temporal and regional expression patterns of UBE3A that we reveal in NHPs will critically guide emerging gene addition and reinstatement strategies to treat AS, including eventual prenatal treatments for AS.
Why This Study is Important
This work will generate a non-human primate model of AS for testing therapeutics. It is difficult to anticipate how many features of AS will be rescued by different therapeutics because mice are not as similar to humans as researchers would like. Furthermore, many of the therapeutics under development so far will not restore UBE3A in mice. This model would enable the testing of therapeutics directly in a model closest to humans. It would also help the understanding of how AS develops over time in a model more closely related to humans.
Study Updates
(May 2023) This study is working to produce a non-human primate (NHP) model of Angelman syndrome to develop safe and effective therapeutics. Work has focused on improving the oocyte and embryo culturing protocols and preparing for large-scale egg collections and embryo storage. A team at University of California at Davis has worked to optimize the collection of oocytes and culturing of oocytes and early embryos to maximize the percentage of early-stage embryos reaching blastocyst stage. There has been increased reliability with embryos reaching the blastocyst stage, which is when they would either be implanted or cryopreserved.
Additionally, significant effort has focused on expanding the pool of candidate egg donors to pair with existing sperm donors. Many rhesus macaque females were assessed to be candidate egg donors based on health and menstrual cycles measures. Blood from 20 candidate female donors was collected, shipped from UCD to UNC, and processed to extract DNA for SNP genotyping.
New female rhesus macaques for IVF experiments were identified and selected using SNP genotyping information to ensure ability to distinguish between paternal inheritance of UBE3A alleles in the subsequent embryos. Researchers are currently tracking cycles of several of these females to continue Cas9 editing experiments this breeding season.
(October 2021) The first step in this study was to demonstrate that this approach could restore UBE3A in AS mice.
(May 2024) The study shows that key features of UBE3A biology observed in rodents are mirrored in macaques; UBE3A expression is much higher in neurons compared to glia, and paternal UBE3A silencing occurs in neurons but not glial cells. Their detailed development time course also revealed that silencing of the paternal UBE3A allele begins in the second trimester of gestation and peaks during the third trimester in macaques, suggesting that the maternal loss of UBE3A could virtually eliminate UBE3A expression in neurons by the time of birth in humans.