Development, characterization and evaluation of advanced therapies for the treatment of cardiac pathologies

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Cardiovascular diseases (CVDs) are the leading cause of disease burden and mortality in the world, as well as a major cause of disability and health care costs. With the average lifespan of the human population continuously increasing, it is expected that the problem of CVDs will only continue to grow in the following years. Current pharmacological treatments for age-associated cardiac pathologies such as heart failure and atrial fibrillation present severe clinical efficacy and safety problems and are not regarded as definitive cures. This makes it necessary to develop new treatment strategies that target the involved molecular pathways and trigger endogenous reparative responses. Contrary to current molecular treatments, advanced therapy medicinal products (ATMPs) such as stem cells, extracellular vesicles (EVs) and biomaterials such as hydrogels could have the potential to treat cardiac aging-associated pathologies from a more fundamental level. However, many problems and unknowns still need to be solved before they can reach the clinical scenario. Some of the most highlighted limitations we focus on in this work are: (i) the lack of deep understanding of their mechanism of action (MoA), (ii) their large variability and lack of standardization (including inadequate potency tests) and (iii) low in vivo retention at the site of interest. Therefore, the main objective of this thesis is to develop, characterize and evaluate advanced therapies for the treatment of cardiac pathologies solving some of their current limitations to enhance their therapeutic potential. To achieve this aim, we first focus on improving standardization and development of potency assays. We describe the main characteristics and challenges for a cell therapy based potency test in the cardiovascular field and we review and propose different types of assays that could be taken into consideration based on the product’s expected MoA and the target cardiovascular disease. Secondly, as cardiosphere-derived cells (CDCs) and their secreted EVs (CDC-EVs) have previously reported to have anti-senescent effects and this is considered important in aging-related cardiac diseases, we explore potential predictors of rejuvenating potency with a special focus on the chronological age of the CDC-donors and CDC-senescence, among others. Multiple in vitro tests allow us to conclude that more than cell particular biological markers or characteristics, the cell bioactivity relative to the expected MoA should be a better predictor for the ATMP potency. Thus, we evaluate if the in vitro anti-senescent and pro-angiogenic effect of the CDC-EVs, scored with a matrix assay, can be used to predict the in vivo potency of the CDC-EVs in an animal model of cardiac aging. Our results show that EVs classified in vitro as potent with the matrix assay have more cardiac reparative potential in vivo than EVs classified as non-potent. After further validation, the matrix assay proposed here could be a suitable in vitro potency test for discerning suitable allogenic biological products in the cardiac aging clinical scenario. Next, with the purpose of improving EV retention at the site of interest, we develop an optimized product combining hydrogels from cardiac extracellular matrix (cECM), polyethylene glycol and EVs to overcome some of their individual limitations: long gelation time of the cECM and poor retention of the EVs. We conclude that the combined product rapidly gels at physiological temperature and presents improved mechanical properties while maintaining the injectability, the biodegradability, and the bioactivity of its individual components. In addition, it serves to better retain the EVs on-site in vivo. Finally, we explore the electrophysiological modifications induced by CDC-EVs on arrhythmogenic tissue to better understand the mechanisms behind their antiarrhythmic effect. We found that CDC-EVs reduce spontaneous activation complexity and increase conduction velocity of cardiomyocytes leading to a less arrhythmogenic profile. If validated in other cellular models, CDC-EVs may be used specifically as antiarrhythmic agents in a wide range of cardiac pathologies. Although future work should aim to further validate these results both at preclinical and clinical level, these findings together partially overcome some of the main challenges for the therapeutic use of cellular therapies and open a new horizon for the treatment of cardiac-aging related pathologies, some still considered as unmet medical needs.
Cardiovascular diseases, Cardiac regenerative therapy, Cell therapy, Antiarrhythmic effects, Optical mapping, Extracellular matrix, Hydrogel
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