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Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Proyecto MINECO-Ministerio de Ciencia e Innovación (Spain)- PID2019-105674RB-I00 Proyecto DGA- Diputación General de Aragón (Spain)- LMP128_21 Introduction Cardiac microRNAs (miRNAs) have been found to be dysregulated in cardiac disease and aging both in animal models and humans. Thanks to their pleiotropic effects, miRNAs are spotlighted as promising therapeutic targets to treat cardiac conditions. However, their low stability and potential off-target effects in vivo difficult the development of effective and safe miRNA therapies. Nanotechnology enables the fabrication of biocompatible carriers to perform targeted delivery of both conventional and advanced therapies. In particular, nanostructures built with DNA (DNS) have been successfully developed for miRNA therapies in cancer, but they have not been applied in cardiac therapy yet [1]. Purpose The main goal of this study is to demonstrate the capacity of DNS to deliver miRNA therapies in human cardiomyocytes. Methods Our group has identified MIR24-2 upregulated with age in the human left ventricle and miR24-2-5p to interact with genes related with the cardiac function, such as SERCA2 [2]. Different versions of DNS were designed and generated by self-assembly of DNA strands containing anti-miR24-2-5p capture segments. Physicochemical characterization of DNS was then conducted to determine their auto-assembling capacity, size, morphology and thermal stability. The characterization of the biological effect of the DNS was also conducted in vitro and in cell culture of HEK293 and human induced pluripotent stem cells (iPSC)-derived cardiomyocytes (iCM) to assess their cytotoxicity and internalization capacity. Results DNS were assembled correctly and reproducibly into carriers of around 30-50 nm. In vitro, the DNS showed thermal stability at physiological temperature, proper stability in serum and they disassembled specifically in the presence of the target miR24-2-5p sequence. In HEK293 and iCM cultures, the DNS showed lack of cytotoxicity demonstrating in vitro biocompatibility. DNS were efficiently internalized by HEK293, but the uptake capacity of DNS by iCM was remarkably lower. Conclusions We created and characterized biocompatible anti-miR-loaded DNS that effectively captured the target miR-24-2-5p in vitro. DNS internalized in iCM, but with lower efficiency than in HEK293 cells. Our results indicate that DNS are suitable candidates to carry miRNA therapies to human cardiac cells in vitro, but the observed cell-type specific differences suggest that future efforts need to be directed towards developing DNS functionalization capable of promoting efficient and specific carrier uptake in vivo by primary cardiac cells in vivo.
Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Minsterio de Ciencia e Innovación (MICNN)INECO PID2019-105674RB-I00 ERC starting grant ERC-StG-638284 One of the main causes of drug withdrawal from the market is their unexpected toxic effect on the heart, such as arrhythmia generation. The Comprehensive In Vitro Proarrhythmia Assay (CiPA) from the Food and Drug Administration (FDA) promotes the use of in silico human models into the safety assessment workflow for early prediction of drug-induced cardiotoxicity. Despite being age a primary risk factor for cardiac arrhythmia and the elderly a primary object population of pharmacology, age-dependent effects are not considered in the CIPA initiative. The goal of our study is to create in silico human ventricular models of cellular electrophysiology that enable risk stratification of drugs according to age to optimize drug safety assessment. Two human adult ventricular electrophysiological models ORd 1 and Jae 2 were used to generate experimentally-calibrated populations of action potentials (AP) and calcium transients (CaT). Using the results of our previous age-related transcriptomic data analysis3, the effects of aging on ionic current properties were applied to such populations, with aging evaluated using two parameters of cardiac biological aging (BA): CDKN2A expression (CDKN2A-age), a single indicator of cell senescence, and apparent age (AppAge), a complex index computed from nearly 3000 age-related genes. For each of the three populations, namely adult, aged-CDKN2A and aged-AppAge, drug effect simulations were carried out for a list of CiPA high-risk compounds. The two aged populations were significantly different (p-value < 0.001) to their adult homologue in all the evaluated AP- and CaT-related parameters studied, both for ORd and Jae basis models. In particular, prolongation of AP duration (APD), increased AP triangular morphology and decreased membrane voltage peak were observed with aging. When analyzing drug effects, a higher number of individuals presenting arrhythmogenic events were found in the elderly populations than in the adult ones for all tested drugs, i.e. bepridil, dofetilide, quinidine and sotalol. Of those individuals with normal AP repolarization, drug effects on APD were significantly more accentuated on aged models compared to adult ones. As an example, treatment with dofetidile 50 nM lead to 16.67 % of arrhythmic events on adults, while it lead to 26.7% and 33.3% on aged individuals according to AppAge and CDKN2A-age, respectively. Dofetidile increased mean APD to 94.3% (CDKN2A-age) and 96.9% (AppAge) on elder over untreated individuals as compared to 78.59% in adults. To conclude, we created and tested a battery of in silico cellular models of cardiac electrophysiology that integrate age as pro-arrhythmic risk factor. Results show important differences between elder and adult populations both at baseline and after the administration of four different drugs. Our human in silico models may lead to improved early detection of potential cardiotoxic events in preclinical drug studies.
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