B001 CAGinSTEM, a human embryonic stem cell platform to identify genetic factors implicated in Huntington’s disease

<h3>Background</h3> It is well known that HD patients with similar CAG length show a wide range of variability in motor onset that can account for up to two decades. One possible explanation resides in the fact that the inherited CAG repeats may expand in somatic tissues, especially in post-mitotic neurons, giving rise to a HTT mosaicism that results in longer than inherited CAG tracts in affected tissues, such as the striatum and cortex. This expansion may continue during the lifetime of the individual and contribute to exacerbate neuronal toxicity and selective neuronal degeneration. More recently, trans- and cis- modifiers of age of onset (AOO) have been identified. However, if and how they cause the progressive accumulation of CAG instability is still unclear. <h3>Aim</h3> To identify new cis and trans modifiers of CAG instability, we aimed to establish an isogenic human stem cell platform that, combined with third generation long-read sequencing, allows to monitor HTT CAG size over time, both during mitotic cell replication and in post-mitotic neurons. <h3>Methods</h3> Starting from H9 human embryonic stem cell (hES) line, we inserted a monoallelic Recombinant Mediated Exchange Cassette within HTT exon 1, which can be subsequently exchanged with any exon1 variant in an efficient way. We generated a wide variety of exon 1 modified cell lines, which we refer to as the CAGinSTEM platform. <h3>Results</h3> Our data show that the CAGinSTEM platform is technically robust as for each genotype we have multiple cell lines which have been quality checked. By exploiting the properties of the CAGinSTEM platform, we are testing how CAG length and composition impact on CAG instability in terminally differentiated medium spiny neurons and in active proliferating hES cells. <h3>Conclusions</h3> The CAGinSTEM platform offers a distinctive biological model system designed to explore genotype-phenotype correlations and investigate the mechanisms underlying CAG instability accumulation in postmitotic human neurons and other cell types. Funded by an ERC Advanced Grant from the European Commission.