Recapitulating fabry disease with human induced pluripotent stem cells and CRISPR interference reveals a signaling axis involved in cardiac phenotypes

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<p>Fabry disease is caused by mutations in GLA, an X-linked gene, which encodes lysosomal enzyme α-galactosidase (α-GAL). The mutations reduce the enzymatic activity, resulting in accumulation of enzyme substrates, sphingolipids, in lysosomes. However, how the accumulated sphingolipids lead to disease phenotypes remains unknown. Here we applied human induced pluripotent stem cells (hiPSCs) and Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) to model the disease. The CRISPRi is a technique that can repress genes of interest. Mechanistically, the repression is achieved by recruiting catalytically dead (d)Cas9 fused with KRAB, a transcription repressor domain, on a promoter region of the gene in a single guide (sg)RNA dependent manner. </p><p>By employing a disease-free CRISPRi hiPSC line in which dCas9-KRAB can be induced by doxycycline (Dox) treatment, we first established GLA CRISPRi hiPSC lines in which sgRNA against GLA is constitutively expressed. We found that, in the lines, the α-GAL expression is strongly suppressed by Dox treatments without obvious phenotypic changes, suggesting α-GAL is dispensable in hiPSCs. </p><p>Next, since a cardiac involvement (e.g. left ventricular hypertrophy) is common in Fabry disease patients, we differentiated the hiPSC lines into cardiomyocytes. Regardless of Dox treatments, we efficiently obtained beating cardiomyocytes from the hiPSC lines. RNA sequencing using the enriched cardiomyocytes revealed that only handful genes are affected by GLA suppression, suggesting that dysfunction of the enzyme did not substantially affect the cardiac differentiation. However, the Dox treatments significantly altered beating behavior; each beating is constituted by a contraction and relaxation cycle, and we observed that relaxation velocities were decreased and relaxation durations were prolonged by the Dox treatments. Additionally, the Dox treatments enlarged cardiomyocytes and altered sarcomere structures as are seen in hypertrophy. Thus, we conclude that the cardiac phenotypes of Fabry disease can be recaptured in vitro by employing the GLA CRISPRi.</p><p>To elucidate Fabry disease mechanisms, we are now querying protein expressions and modifications of molecules that play roles in regulating cardiac beating. Our preliminary data suggest that GLA suppression attenuates a signaling pathway. We will discuss Fabry disease mechanisms and drug candidates for the disease based on the mechanisms in this meeting.</p>

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