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

Resumen

Pathological alterations in cardiac tissue structure and functioning lead to cardiovascular diseases, which are the leading cause of disease burden and mortality in the world. They are also a major cause of disability and health care costs. The main risk factor for the development of cardiovascular diseases is increasing age. Aging is a natural biological process in which the body’s homeostatic adaptive response is progressively altered, increasing the susceptibility to environmental stress and disease. Aging is particularly linked to an increase in the prevalence of heart-related pathologies such as heart failure and atrial fibrillation. With the average lifespan of the human population continuously increasing, it is expected that the problem of cardiovascular diseases will only continue to grow in the following years. In addition, despite current pharmacological treatments for heart failure and atrial fibrillation dramatically changed the prognosis of these patients in the last decades, they still present important efficacy and safety problems and are not regarded as definitive cures. In this context of limited therapeutic efficacy of the available medicines and unmet medical needs, the use of advanced therapy medicinal products (ATMPs), including stem cell therapy, extracellular vesicles (EVs) and biomaterials such as hydrogels has emerged as a potential breakthrough. ATMPs, according to the EMA, are medicines for human use based on genes, tissues or cells. In general, they are considered innovative therapies that target diseases with high unmet clinical needs. In the cardiovascular field, the discovery of stem cell niches in the heart drove significant attention towards ATMPs due to the limited regenerative capacity of the human heart. Stem cells, EVs and natural hydrogels present high therapeutic potential because of their ability to prevent and counteract different molecular, cellular and structural changes behind cardiac pathologies, as they are able to improve cell survival and protection, cell-cell communication, angiogenesis, cardiomyogenesis, reduce inflammation and molecularly regulate proliferation and cell cycle. However, despite the results of the preclinical studies using cell therapy for cardiac repair and regeneration seemed very promising, rapid implementation into clinical trials only led to modest outcomes. This made the scientific community to re-think and re-define the field of cell therapy in cardiac applications, stablishing which were the main limitations and the priorities that needed to be solved before further moving them into the cardiac clinical scenario. Some of the most highlighted limitations are the lack of deep understanding of their mechanism of action (MoA), their large variability and lack of standardization (including inadequate potency tests), and in particular for cells and EVs, their low in vivo retention at the target site. 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 provide a practical guide for the development of potency assays for cell and cell-based cardiac reparative and regenerative products. We then explore markers of anti-aging potency of cardiosphere-derived cell (CDCs) and their secreted EVs (CDC-EVs) and evaluate if the in vitro anti-senescent and pro-angiogenic activity of human CDC-EVs could be used as predictor of their in vivo potency in an animal model of cardiac aging. We continue developing and characterizing an optimized ATMP by combining solubilized cardiac extracellular matrix (cECM), polyethylene glycol (PEG), and EVs to yield an injectable hydrogel with short gelation time and improved retention of the EVs. We finally investigate and characterize the antiarrhythmic MoA of CDC-EVs in a suitable in vitro model of atrial fibrillation. Although, highly challenging, the potency tests for cell products are considered as a priority by the regulatory agencies and attempts should be made to develop robust and reproducible potency assays to predict therapeutic efficacy. In this work, we describe the main characteristics and challenges for a cell therapy potency test focusing on the cardiovascular field. Moreover, we discuss different steps and types of assays that should be taken into consideration for an eventual potency test development by tying together two fundamental concepts: target cardiovascular disease and the product’s expected MoA. We conclude that the use of in vivo and some in vitro assays, despite being closer to the clinical scenario and more representative of the MoA, commonly present high costs and are time consuming, making them unfeasible for mass production. The use of simple and easier to scale assays such as surrogates related to the specific MoA (i.e. as gene and protein expression or the secretion of specific factors) could be more practical. For cardiomyogenesis, potential to induce cardiomyocyte (CM) division or to differentiate into the CM lineage is proposed. Expression of ion channels (sodium, calcium and potassium channels) and connexin 43 could be indicative of electromechanical maturation and coupling, whereas secretion of pro- and anti-inflammatory factors (TNF-α, IL-6 and IL-10) could determine immunomodulation. The anti-senescent and antiapoptotic potential of the product can be determined in vitro on CM and stromal cells. VEGF and specific cytokine secretion can be indicative of the angiogenic potential, and TGF-β, MMP and TIMP secretion of the antifibrotic potential. However, to develop these, or other scalable and accurate potency assays, further work is needed to validate that the potential surrogate measurements correlate to the efficacy of the product in patients. CDCs in particular have demonstrated to induce rejuvenation of the heart and to improve cardiac structure and diastolic function in old animals. However, the extent of the reparative effects of human CDCs seem to variate among different CDC-donors: younger donors are thought to generate more potent stem cells. While it is highly desirable to predict the in vivo efficacy at the early stages of product development to discard unsuitable donors, there is controversy on the relevance of donor and cell characteristics in determining cell potency. In this study, we evaluate if the chronological age of the donor and/or cellular senescence of CDCs better predict their potency. We focus specifically on the rejuvenating and pro-angiogenic potency and use CDC-EVs as therapeutic product. CDCs from 34 human donors (3 months to 81 years old, both sexes) are obtained and characterized. After CDC-EVs obtaining, we determine their rejuvenating and pro-angiogenic in vitro efficacy on different cell types relevant in heart physiology and pathology, and donor and CDC characteristics are correlated to their CDC-EVs potency. We find that ability to form cardiospheres and chronological age and sex of the donor are not related to most CDC properties and do not determine CDC-EV rejuvenating and pro-angiogenic effect in vitro. CDC senescence relates to other CDC bioactive properties, but this is insufficient to predict CDC-EV anti-senescence and pro-angiogenic in vitro potency. This study confirms that CDC-EVs have multiple rejuvenating and pro-angiogenic effects on different cell types involved in heart pathology and regeneration, but the extent is variable among donors and cannot be predicted by using chronological age of the donor or CDC senescence as surrogate markers. According to these results, we hypothesize that for determining cell potency, it is important to evaluate functionality relative to the expected MoA, since merely cell identity and specific donor or cell characteristics may not translate into boosted efficacy for the treatment of a particular pathology. Therefore, we continue evaluating if the in vitro anti-senescent and pro-angiogenic effect of the CDC-EVs can be used to predict their in vivo efficacy. CDC-EVs from 18 human donors are obtained and their anti-senescent and pro-angiogenic potency evaluated in vitro using a matrix assay with several markers. According to the performance in these tests, potency is scored and CDC-EVs with the highest and lower score are classified as potent (P-EVs) and non-potent (NP-EVs) respectively. The rejuvenating effect of both type of EVs are tested in an in vivo model of cardiac aging. In the heart of aged rats, P-EVs have the potential to significantly reduce the expression of the senescence-associated gene GLB1 and to non-significantly reduce cardiac hypertrophy and TGFB1 expression. On the contrary, NP- EVs do not significantly produce any effect and in fact significantly increase cardiac hypertrophy, cardiac fibrosis and reduce perfusion. At systemic level, while P-EVs significantly improve glucose metabolism and tend to drive total antioxidant capacity and hair growth to a healthier profile, NP-EVs do not significantly improve any of the explored parameters and even significantly increase total antioxidant capacity. After further validation, the matrix assay proposed here could be used as an in vitro potency test to evaluate EV suitability as an allogenic product before its use in the treatment of cardiac aging. Cell and EV efficacy may be limited by their poor retention at the target site. Cardiac extracellular matrix hydrogels (cECMH) seem promising as drug-delivery materials and could improve the retention of EVs, but may be limited by their long gelation time and soft mechanical properties. To contribute solving this limitation, in this work we develop and characterize an optimized product combining cECMH, polyethylene glycol (PEG), and EVs (EVs–PEG–cECMH) in an attempt to overcome their individual limitations: long gelation time of the cECMH and poor retention of the EVs. The new combined product presents improved physicochemical properties (60% reduction in half gelation time, and threefold increase in storage modulus vs. cECMH alone), while preserving injectability and biodegradability. It also maintains in vitro bioactivity of its individual components (55% reduction in cellular senescence vs. serum-free medium, similar to EVs and cECMH alone) and increased on-site retention in vivo (fourfold increase vs. EVs alone). The combination of EVs–PEG–cECMH is a potential multipronged product with improved gelation time and mechanical properties, increased on-site retention, and maintained bioactivity that, all together, may translate into boosted therapeutic efficacy. Finally, although cell-based therapies have shown potential antiarrhythmic effects that could be mediated by their paracrine action, the mechanisms and the extent of these effects have not been deeply explored. Therefore, we investigate the antiarrhythmic mechanisms of CDC-EVs on the electrophysiological properties and gene expression profile of HL-1 CMs. HL-1 cultures are primed with CDC-EVs or serum-free media alone for 48 hours, followed by optical mapping and gene expression analysis. In optical mapping recordings, CDC-EVs reduce the activation complexity of the CMs by 40%, increase rotor meandering and reduce rotor curvature, as well as induce an 80% increase in conduction velocity (CV). HL-1 cells primed with CDC-EVs present higher expression of SCN5A, CACNA1C and GJA1, coding for proteins involved in INa, ICaL and Cx43 respectively. Our results suggest that CDC-EVs reduce the activation complexity by increasing CV and modifying rotor dynamics, which could be driven by an increase in expression of SCN5A and CACNA1C genes, respectively. Our results provide new insights into the antiarrhythmic mechanisms of cell therapies, which should be further validated using other models. Altogether, the work in this thesis provides new insights into mechanisms to enhance the therapeutic potential of advanced therapies in the cardiac field. In particular, as CDC-EVs therapeutic translation may have been limited by their lack of standardization, their poor retention, and the lack of knowledge about their specific mechanisms of actions, the work done here proposes ways of partially overcoming these limitations. To ease standardization, successful potency assays based on the product’s expected MoA and the target cardiovascular disease should be developed. We provide a guide that suggests some potency assays based on the product’s expected MoA and the target cardiovascular disease. Here we show that the extent of the therapeutic potential is variable among donors and cannot be adequately predicted by using simple characteristics of the donor (e.g. age) or their parenteral cells (e.g. senescence, cardiosphere size). Accordingly, we propose as a potency test an in vitro matrix assay that classifies the CDC-EVs according to their anti-senescent and pro-angiogenic efficacy and illustrate its potential to predict their therapeutic benefit in cardiac aging. To solve the limitation of low EVs retention at the site of interest, we develop and optimize a new drug-delivery advanced therapeutic medicinal product. This product combines natural and synthetic biomaterials for the intramyocardial delivery of CDC-EVs. Finally, we look in vitro at the electrophysiological changes induced by CDC-EVs on CMs to shed light on the mechanisms by which they exert their poorly explored antiarrhythmic properties. Our findings indicate that EV increase the CV of the arrhythmogenic substrate and reduce its activation complexity, probably by inducing a higher expression of connexins and Na+ and Ca2+ ion channels. Despite future work is still needed, these findings together propose solutions to some of the limitations of CDC-EVs and new ways to understand and enhance their therapeutic efficacy in the field of cardiac-aging related pathologies.