327 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 of the specific Ca2+ regulation and PLN function is required, which is thought to be mainly due to the reduced PKA-dependent phosphorylation on Serine 16. Although the single-cell sequencing technique used in this study was effective in determining the pathological pathway patterns and gene expression profiles, the challenges that arose were severalfold. First, the dissociation of the spheroids before fixation could be optimized, thereby avoiding sequencing bias of non-CMs due to CM damage and loss during the dissociation. Secondly, the cell type identification was challenging since the cardiac spheroids were generated solely from hiPSC-CM differentiations. Further cell-type analyses of cardiac spheroids would establish a better understanding of cellular complexity. Lastly, by using specific primer labeling with this SPLiT-sequencing technique, allelic expression per cell could be studied. This experiment could provide insights into each cardiomyocyte’s allelic imbalance and genetic makeup harboring the PLN-R14del mutation. Additionally, RNA sequencing on multiple spheroid ages could identify the spatiotemporal processes to identify mechanistic insights in the ‘first to fail’ mechanism of the PLN-R14del cardiomyopathy. The AAV therapy described in this study is hopeful, but further analysis of the transduction efficiency of this AAV.I-1c in vitro and in vivo needs to be performed using immunofluorescent label detection. Additionally, future studies focussing on the effect in comparison to the modulation of PLN activity are required. For example, AAV-mediated Cas9-mediated exon excision in Duchenne muscular dystrophy can correct up to 7% of the genome, which was sufficient to avoid fibrosis of the left ventricle in a large animal model.84 Another study showed that the MYBPC3 gene replacement strategy circumvents haploinsufficiency of cMyBP-C (restoration of a correct amount of protein) and reduces CM hypertrophy.85 In this study, collagens (COL1A1 and COL3A1), and fibronectin (FN1) were significantly reduced and Ca2+ handling genes were upregulated after the MYBPC3 gene transfer, indicating that PLN gene transfer might efficiently eliminate the PLN disease phenotype. However, in one of the largest AAV clinical trials (CUPID2), the AAV-mediated upregulation of SERCA2A resulted in no safety issues or adverse effects, but the low-dose delivery of SERCA2A by AAV1 did not improve symptoms of heart failure in patients.86 In contrast, much higher levels of viral transgene DNA (range 8000–42,000 ssDNA copy number/µg DNA) were used in preclinical small and large animal models of HF where SERCA2A was tested. Therefore, the gene delivery, toxicity, and clinical impact need to be identified and addressed and aid the design of future trials in this field. AUTHOR CONTRIBUTIONS Manuscript writing; R.G.C.M. Collection of data; R.G.C.M, F.v.D and T.B. Analysis of data: R.G.C.M, T.B. Supervision and manuscript editing; J.P.G.S, P.A.D, J.C, F.S, R.H., and J.W.B. All authors have read and agreed to this thesis version of the manuscript.
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