by Jim Daley, Ph.D., a FAST Advisory Council Member and Assistant Professor at University of Texas Health Science Center, San Antonio
The majority (about 70%) of individuals with Angelman syndrome (AS) have a large deletion on the maternal copy of chromosome 15, encompassing the UBE3A gene. Another ~11% have a point mutation in UBE3A that renders the gene nonfunctional or reduces its activity. Rarer forms of AS involve uniparental disomy (UPD; 3-5%), in which two copies of chromosome 15 are inherited from the father (instead of one from each parent), or an imprinting center defect (ICD; 3-5%), which prevents the normally active maternal copy of UBE3A from being turned on.
Many of the most promising Angelman syndrome therapeutics in the pre-clinical or clinical pipelines involve activation of the normally silent paternal copy of the UBE3A gene. One way to achieve this is by targeting an antisense transcript called UBE3A-AS, which itself blocks UBE3A on the paternal chromosome. Inhibiting UBE3A-AS thus removes this obstruction and allows UBE3A to be turned on – this is often referred to as “stopping the stop.” This strategy is well suited for all genotypes missing the UBE3A protein, but care needs to be taken as we consider those that have two silent copies of the gene (on both maternal and paternal alleles), as is the case with UPD and ICD. In these individuals, inhibition of UBE3A-AS could result in double activation of UBE3A. This paternal activation approach is being carefully investigated in many therapeutic platforms including antisense oligonucleotides (ASOs), CRISPR-based approaches, and gene therapy (shRNA, miRNA, etc). It is unclear whether UBE3A protein levels will need to be refined in neurons in order to compensate for the potential of “double activation” of the gene. There is promising data in cellular and animal models suggesting that if these neurons go to a 200% expression, which is what could happen if we get full biallelic expression, it could be safe, but further investigation is still needed.
A key bottleneck in resolving this question has been the lack of models to study the UPD and ICD genotypes, including those with mosaicism. This newly funded FAST grant to Dr. Albert Keung of North Carolina State University will address this question in a series of elegant cellular models. Dr. Keung’s lab will epigenetically modify human stem cell lines to mimic the UPD or ICD epigenotype of chromosome 15, in which methylation of the DNA reflects paternal patterns on both chromosomes. These cell lines will be a valuable, globally available tool for evaluating therapeutics for UBE3A activation.
A major part of this work involves Dr. Keung’s team creating unique “landing pad” cell lines for studying the various noncoding RNA genes present in the AS/PWS region on chromosome 15. In addition to the UBE3A gene, the AS/PWS region contains imprinted genes that generate a series of noncoding RNAs whose function is not well understood. Individuals with UPD/ICD subtypes of AS have altered expression of these RNAs, and also produce twice as much UBE3A-AS RNA as a neurotypical individual, but the impact of these changes on individuals with AS is unknown. These novel engineered cell lines will enable researchers to efficiently introduce diverse numbers and combinations of genes at tunable expression levels, without having to use inefficient genome editing technologies for each new gene one may want to study. FAST previously funded Dr. Keung to evaluate large deletion Angelman syndrome patient cell lines, which include the 10-12 genes situated next to UBE3A on the maternal allele. The goal of this isto better understand the impact of these genes on neuronal function and communication, and to determine which specific genes outside of UBE3A might need to be addressed in the future after UBE3A replacement is optimized. The new grant expands this study to provide cell lines that can be rapidly used by other investigators to evaluate ICD/UPD genotypesand their impact on neuronal function. In addition, these systems will also facilitate the study of mosaic forms of Angelman syndrome that necessarily require the ability to generate multiple cell lines that share the same overall genetic background.
Collectively, this project aims to provide the Angelman syndrome research community a set of cell lines that can be used to efficiently model the biology of ICD and UPD, as well as organoids that model the mosaic genotype, and understand the impact of gene overexpression in these different regions. This work will create valuable resources which will be shared with the AS research community and will help to drive AS research forward with the ultimate goal of accelerating drug development in Angelman syndrome for each and every genotype.