The past decade has seen the development of exciting therapeutic candidates ranging from antisense oligonucleotides (ASOs) to gene replacement therapies, both aimed at replacing the UBE3A protein that is missing in the neurons of individuals living with Angelman syndrome (AS). However, approximately 70% of individuals with Angelman syndrome also have diminished levels of six to twelve other proteins encoded by genes near the UBE3A gene. This typically occurs through large deletions of genomic regions containing UBE3A and these nearby 6-12 genes. Importantly, many of these individuals are also found to have a more severe clinical presentation than those that only have a single gene of UBE3A impacted (e.g. point mutations, imprinting center defects, or uniparental disomy). Therefore, understanding how these other genes and proteins contribute to the clinical presentation of those living with Angelman syndrome could provide insight into therapeutic treatment strategies and targets that could alleviate significant clinical impairments outside of just UBE3A replacement or reactivation.

There are two challenges facing this goal. First, current research models of Angelman syndrome including rodents, pigs and human stem cells have focused on UBE3A itself.  An efficient and rapid approach is needed to create models that can capture the effects of the 6-12 other genes deleted in the majority of individuals with Angelman syndrome. Dr. Keung’s research will focus on the different types of deletions, and the most common genes impacted, outside of UBE3A.

Additionally, because it remains unclear how these genes can affect the diverse cell types present in the human brain, creating these lines, and then organoids of these lines, can support human translational research into the deletion genotype of Angelman syndrome, outside of the rodent.

FAST is funding Dr. Albert J. Keung and his lab at North Carolina State University to address these challenges directly through the engineering of human stem cells that contain a deletion of UBE3A and 10 neighboring genes (‘Class I deletion’).  

In these cell lines created in Dr. Keung’s lab, a synthetic ‘landing pad’ sequence will be inserted into the deleted region of the genome. This landing pad will enable efficient insertion and rescue of different combinations of the various deleted genes. These stem cells will then be coaxed to form human cerebral organoids, small 5mm diameter tissues that contain most cell types of the human brain. This system will be used to study the effects of rescuing each deleted gene, or combination of deleted genes, on neuronal function and cellular states.

To efficiently assess the effects of many different combinations of these genes, mosaic organoids will be created that are comprised of cells with different rescued gene combinations. This work is important in revealing the functions of these clinically relevant, yet understudied, genes. This will also provide a human experimental platform to efficiently test and screen putative therapeutics to address specific defects that can be discovered through this model.  As individuals with AS have highly diverse molecular and genetic profiles, including the loss of many other genes in addition to UBE3A in the case of a deletion genotype, Dr. Keung’s work becomes increasingly more important in allowing us to determine if additional therapeutics outside of UBE3A replacement/activation would be beneficial.

The grant runs through August 2022.