Angelman Syndrome (AS) is a rare neurogenetic disorder characterized by developmental delay, intellectual disability, speech impairment, and ataxia. Currently, there are no effective therapies to cure AS; hence, there is a high unmet need for developing therapeutic approaches. Deletions and loss-of-function mutations at the maternal copy of chromosome 15q11-q13 is the common genetic lesion driving AS. The majority of AS individuals have Class I/II deletions, large interstitial deletions spanning approximately 5-7 million base pairs of DNA. Among the genes deleted within this region, the disruption in UBE3A has been reported to be responsible for the core symptoms of AS. However, AS individuals with large deletions show more severe symptoms indicating that not only UBE3A but its surrounding genes may collectively contribute to the disease pathology of AS. 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 construct an isogenic AS platform derived from human pluripotent stem cells which will help elucidate the roles of deleted or mutated genes in AS Class I/II deletion. Accordingly, we are pursuing three approaches: (i) Designing and integrating a synthetic 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/II deletion cell lines from human embryonic stem cell lines H1 and H9, (iii) Integrating a synthetic recombinase-based landing pad into the deletion region of an H9 maternal UBE3A deletion cell line to analyze UBE3A mutations and isoforms. 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 deletions in H9 and H1 cells to create isogenic AS Class I/II deletion cell lines. With our work on deletion cell lines, we noticed there were differences in gene orders between genome assemblies for the AS deletion region close to the centromeric region of chromosome 15. To shed light on this repetitive and complex genomic region, we have obtained haplotype aware, phased genome sequences of H1 and H9 cells, with broader utility in genome engineering of these widely used cell lines and in studying mechanisms of imprinting.

In vitro human platform to efficiently study Angelman syndrome class I/II deletion genes

ACS Spring 2022

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