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Doxorubicin is a commonly used anti‐cancer drug used in treating a variety of malignancies. However, a major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, the formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multidrug resistance‐associated protein 1 (MRP1) in cardiomyocytes derived from human iPS cells (hiPSC‐CM), to determine whether this will allow cells to effectively remove doxorubicin and confer cardioprotection. We generated a lentivirus vector encoding MRP1 (LV.MRP1) and validated its function in HEK293T cells and stem cell‐derived cardiomyocytes (hiPSC‐CM) by quantitative PCR and western blot analysis. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of fluorescent dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. Finally, we demonstrated that hiPSC‐CM transduced with LV.MRP1 were protected against doxorubicin injury. In conclusion, we have shown that we can successfully overexpress MRP1 protein in hiPSC‐CM, with functional transporter activity leading to protection against doxorubicin‐induced toxicity. [ABSTRACT FROM AUTHOR]

Sinoatrial node dysfunction can manifest as bradycardia, leading to symptoms of syncope and sudden cardiac death. Electronic pacemakers are the current standard of care but are limited due to a lack of biological chronotropic control, cost of revision surgeries, and risk of lead- and device-related complications. We therefore aimed to develop a biological alternative to electronic devices by using a clinically relevant gene therapy vector to demonstrate conversion of cardiomyocytes into sinoatrial node-like cells in an in vitro context. Neonatal rat ventricular myocytes were transduced with recombinant adeno-associated virus vector 6 encoding either hTBX18 or green fluorescent protein and maintained for 3 weeks. At the endpoint, qPCR, Western blot analysis and immunocytochemistry were used to assess for reprogramming into pacemaker cells. Cell morphology and Arclight action potentials were imaged via confocal microscopy. Compared to GFP, hTBX18-transduced cells showed that hTBX18, HCN4 and Cx45 were upregulated. Cx43 was significantly downregulated, while sarcomeric α-actinin remained unchanged. Cardiomyocytes transduced with hTBX18 acquired the tapering morphology of native pacemaker cells, as compared to the block-like, striated appearance of ventricular cardiomyocytes. Analysis of the action potentials showed phase 4 depolarization and a significant decrease in the APD50 of the hTBX18-transduced cells. We have demonstrated that rAAV-hTBX18 gene transfer to ventricular myocytes results in morphological, molecular, physiological, and functional changes, recapitulating the pacemaker phenotype in an in vitro setting. The generation of these induced pacemaker-like cells using a clinically relevant vector opens new prospects for biological pacemaker development. [ABSTRACT FROM AUTHOR]

Gene therapy is making significant impact on a modest, yet growing, number of human diseases. Justifiably, the preferred viral vector for clinical use is that based on recombinant adeno-associated virus (rAAV). There is a need to scale up rAAV vector production with the transition from pre-clinical models to human application. Standard production methods based on the adherent cell type (HEK293) are limited in scalability and other methods, such as those based on the baculovirus and non-adherent insect cell (Sf9) system, have been pursued as an alternative to increase rAAV production. In this study, we compare these two production methods for cardiotropic rAAVs. Transduction efficiency for both production methods was assessed in primary cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and in mice following systemic delivery. We found that the rAAV produced by the traditional HEK293 method out-performed vector produced using the baculovirus/Sf9 system in vitro and in vivo. This finding provides a potential caveat for vector function when using the baculovirus/Sf9 production system and underscores the need for thorough assessment of vector performance when using diverse rAAV production methods. [ABSTRACT FROM AUTHOR]