370 Chapter 13 approach the most interesting for the human heart. One phase IIb trial (CUPID2) explored the upregulation of SERCA2a, for the treatment of heart failure. Here, no safety issues or adverse effects were observed, but unfortunately, the delivery of SERCA2a by AAV1 did not improve symptoms of heart failure in patients 106, possibly due to the low intravenous AAV dosage. Other therapeutic strategies targeting SERCA activity, such as istraoxime and PST3093, have been tested to alleviate the PLN-R14del phenotype. Although Istraoxime in zebrafish ameliorates the in vivo Ca2+ dysregulation and improves cardiac relaxation107, PST3093 was not effective in PLN-R14del hiPSC-CMs76 and mice108 but affected Ca2+ dynamics parameters in the isogenic hiPSC-CMs. These therapeutic outcomes argue against SERCA2a super-inhibition as a mechanism of PLN-R14del. In an in vitro study, hiPSC-CMs harboring the PLN-R14del disease were treated with a combinatorial gene therapy approach by the overexpression of a miRNA-resistant PLN in an AAV6 vector to achieve the PLN gene correction. Interestingly, AAV6-mediated overexpression of PLN reduced the frequency of arrhythmogenic episodes when compared with non-infected cells.109 However, the hypertrophy markers, ANF, and BNP were significantly increased after AAV6 infection, suggesting a general stress response of AAV vectors in CM homeostasis. Therefore, the transgene expression of different serotypes could be used to improve the targeting of the organ of interest and thereby reduce the off-target effect and immune response. Interestingly, instead of AAV1 or AAV6, AAV9 has now become the method of choice for introducing genetic material into the heart, due to its ability to effectively transduce adult rodent hearts110 and hiPSC-CMs111. Recently, capsid re-engineering of adeno-associated vectors to a hybrid AAV2/8 and AAV2/9 virus has been reported to have up to 5-100 fold higher efficiency for transgene delivery as compared to AAV2 serotypes and are proven safe in rodent cardiomyocytes.112–114 Overall, the advanced AAV technology using suitable serotypes and hybrids, enables the generation of safe, effective viral delivery for any gene of interest. However, cardiac-specific delivery of therapeutic materials in vivo remains challenging, due to the invasive high systemic doses that potentially could lead to cardiomyocyte toxicity and systemic inflammation. For example, recently a young man with Duchenne Muscular Dystrophy died just days after receiving AAV9 due to an innate immune reaction that caused multi-organ failure including his heart, attributed to the high dose of the gene therapy.115Here, extracellular vesicles hold immense potential for the delivery of therapeutic agents, as this carrier is stable, biocompatible, and therefore non-mutagenic, less immunogenic, and non-cytotoxic.116 Moreover, lessons from the improved transfection efficiency in expanding hiPSC-CM (Chapter 5) could help to identify factors that regulate cardiac uptake of therapeutic materials in adult CMs. Likely, the molecular insights of this promising pro-proliferative factor CHIR99021 (CHIR) contribute to not only optimizing the generation of hiPSC-CM but also potential transfection, and transduction, which is essential to overcoming the current delivery inefficiency. Unfortunately, we showed that the in vivo administration of CHIR during late gestation resulted in a 1.8-fold increase in mitotic CMs, while we observed a progressive decline in the proliferative capacity of postnatal mouse cardiomyocytes (Chapter
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