317 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 significantly increased after AAV.I-1c treatment in the PLN-R14del hCSs (Supplementary Figure 7E). Interestingly, AAV.I-1c overexpression did not significantly affect gene expression patterns in healthy control hCSs compared to the non-transduced controls (Supplementary Figure 7A-E). Based on the log2 fold expression of these genes, a heat map was generated using Euclidean distance to study the hierarchical clustering of control and PLN-R14del hCSs with or without AAV.Ic1 treatment (Figure 6G). PLN-R14del hCSs treated with the AAV.I-1c clustered towards the CTRL and CTRL+AAV.I-1c, rather than with the PLN-R14del untreated group, indicating a gene expression profile shift towards the healthy control group after AAV.I-1c treatment. AAV-mediated overexpression of I-1c restores Ca2+ handling in PLN-R14del spheroids The Ca2+ intensity and peak amplitude showed improvements after 14 days by a single dose of the AAV. I-1c (MOI of 10.000) (Figure 7A-B), which was further quantified and reflected in improved decay time in both control and PLN-R14del hCSs (Figure 7D), increased peak amplitude (Figure 7E) while both rise time and CTD10 partially increased but not to healthy control levels (Figure 7F-G). In contrast, Interestingly, CTD90 did significantly improve in both transduced controls and PLN-R14del spheroids (Figure 7H). Lastly, the beating rate was reduced upon AAV.I-1c transduction and declined from 38±22 beats per minute (BPM) to 30±22 BPM, which is more similar to non-transduced (23±7 BPM) or transduced (22±7 BPM) controls (Figure 7I). To exclude beating-rate variability in Ca2+-handling, we normalized for beating rates and observed that PLN-R14del spheroids had similar reduced trends in Ca2+ parameters while these were normalized by AAV.I-1c supplementation (Supplementary Figure 8). Together, these results indicate that AAV.I-1c was able to restore Ca2+ handling in PLN-R14del cardiac spheroids, which makes the AAV.I-1c gene therapy a potential therapeutic option for PLN-R14del patients.
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