371 General Discussion - hiPSC-CMs Disease Modelling and Future Perspectives 13 3). This finding indicates that GSK-3β inhibition alone is not sufficient to stimulate cardiac proliferation in the postnatal and adult phases. Previously, the administration of a constitutively active inhibitor-1 (I-1c) delivered by the cardiotropic AAV2/8 (BNP116) virus in ischaemic heart failure pigs improved cardiac function (LV EF +5.7%).117 I-1 decreases protein phosphatase 1 (PP1) activity, thereby upstream regulating the SERCA2a/phospholamban complex, and is also closely linked to the β-adrenoceptor system. Upon activation of I-1c, PP-1 is suppressed, thereby increasing SERCA2a activity and phospholamban phosphorylation and enhancing basal cardiac function.118 In heart failure patients, PP-1 levels are increased, resulting in a decreased PLN phosphorylation.119 Decreased PLN phosphorylation promotes binding to SERCA2a, leading to decreased SERCA2a activity.120 The reduced SERCA2a activity is associated with a defect in cardiomyocyte Ca2+ cycling and impaired cardiac contractility.121 As described in this discussion (Chapter 13), the PLN-R14del mutation causes a reduced sensitivity of Ser16 phosphorylation by PKA, causing the loss of SERCA2a activation. Thus, we hypothesized that PLN-R14del will be amenable to treatment by AAV-mediated overexpression of I-1c, by alleviating the detrimental effects of SERCA2a regulation through specific modulation of the PLN-coupled PP1 activity. In Chapter 12, we aimed to test this hypothesis, by first modeling the cellular phenotype of the PLN-R14del disease in 3D spheroids grown in 384-wells for 3 weeks. After 2-3 weeks, PLN-R14del spheroids recapitulated the cellular phenotype associated with the clinical features of the disease, including abnormal structure, decreased calcium handling activity, increased fibrosis, and decreased cardiac gene expression. Next, we treated the spheroids with a single addition of the AAV-based I-1c gene therapy for 1 week and used kinetic high-throughput screening analysis of intracellular calcium handling activity. AAV-mediated I-1c gene augmentation therapy restored normal calcium handling function, increased cardiac gene expression, and reduced the fibroblast activation in patient-derived and genome-edited PLN-R14del hiPSC lines. To the best of our knowledge, we showed in Chapter 12 the first proof of concept that the I-1c AAV2/8-based gene therapy has the potential to be used for the treatment of genetic cardiomyopathy modeled in hiPSC-CMs. Although the partial restoration of the calcium function and structure may be enough to preserve cardiac function in patients with PLN-R14del cardiomyopathy, the therapeutic targeting of the PLN-gene itself could hold even better promise for the genetic modification of the PLN-R14del disease. As previously described, the AAV6-mediated overexpression of PLN reduced the frequency of arrhythmogenic episodes in a PLN-R14del patient.109 The combination of AAV2/8, AAV9, or AAV2/9 with the overexpression of PLN could offer the perfect strategy for efficient delivery and optimal restoration of the PLN-R14del phenotype. In the far future, gene editing approaches such as CRISPR-CAS or prime editing will allow the editing of the specific gene causing the pathologic mutation122, although the limitations of the off-target effects, delivery, and immunogenic toxicity need to be overcome. Therefore,
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