An in Vitro Human Platform to Efficiently Study Angelman Syndrome Class I/II Deletion Genes

Angelman Syndrome (AS) is a neurodevelopmental disorder characterized by mental disability, speech impairment, and ataxia. Deletions and loss-of-function mutations at the maternal allele of chr15q11-q13 can lead to AS. Most AS individuals have Class I/II deletions, large cytogenetic deletions spanning at least 5 Mb. Among the genes deleted within this region, UBE3A has been reported to be responsible for the core symptoms of AS. However, UBE3A and its surrounding genes constitutively contribute to the AS etiology. Hence, we need human-specific platforms that will enable us to investigate UBE3A and its understudied neighboring genes. The aim of this study is to establish an isogenic AS platform derived from human pluripotent stem cells which will help us investigate the roles of deleted or mutated genes in AS Class I/II deletion. Accordingly, we pursue: (i) Designing and integrating a recombinase-based landing pad into a safe harbor locus of AS patient-derived cell lines to rescue the expression of Class I/II deletion genes, (ii) Generating AS Class I deletion cell lines from human embryonic stem cells, (iii) Integrating a recombinase-based landing pad into the deletion region of an H9 maternal UBE3A deletion cell line to analyze UBE3A mutations. We successfully designed a landing pad and integrated it into the AAVS1 safe harbor locus of H9 cells and are in the process of integrating it into AS deletion cell lines. In parallel, we are using CRISPR/Cas9 to induce large cytogenetic deletions in H9 and H1 cells. Our work on deletion cell lines showed that there were differences in gene orders between genome assemblies for the AS deletion region. To shed light on this, we have obtained haplotype aware, phased genome sequences of H1 and H9 cells.