Overexpression of multidrug resistance-associated protein 1 protects against cardiotoxicity by augmenting the doxorubicin efflux from cardiomyocytes.

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.

Promoter Optimization Circumvents Bcl-2 Transgene-Mediated Suppression of Lentiviral Vector Production.

Lentiviral vectors are a robust gene delivery tool for inducing transgene expression in a variety of cells. They are well suited to facilitate the testing of therapeutic candidate genes in vitro, due to relative ease of packaging and ability to transduce dividing and non-dividing cells. Our goal was to identify a gene that could be delivered to the heart to protect against cancer-therapy-induced cardiotoxicity. We sought to generate a lentivirus construct with a ubiquitous CMV promoter driving expression of B-cell lymphocyte/leukemia 2 gene (Bcl-2), a potent anti-apoptotic gene. Contrary to our aim, overexpression of Bcl-2 induced cell death in the producer HEK293T cells, resulting in failure to produce usable vector titre. This was circumvented by exchanging the CMV promoter to the cardiac-specific NCX1 promoter, leading to the successful production of a lentiviral vector which could induce cardioprotective expression of Bcl-2. In conclusion, reduced expression of Bcl-2 driven by a weaker promoter improved vector yield, and led to the production of functional cardioprotective Bcl-2 in primary cardiomyocytes.

Potential Applications for Targeted Gene Therapy to Protect Against Anthracycline Cardiotoxicity: JACC: CardioOncology Primer.

Anthracyclines are associated with risk of significant dose-dependent cardiotoxicity. Conventional heart failure therapies have neither ameliorated declining cardiac function nor addressed the underlying cause. Gene therapy may confer long-term cardioprotection by rendering the heart resistant to anthracyclines after 1 treatment, although the optimal therapeutic target remains to be elucidated. Recombinant adeno-associated virus is now clinically approved for the treatment of lipoprotein lipase deficiency, spinal muscular atrophy, and hereditary transthyretin amyloidosis. High-throughput methods allow selection of recombinant adeno-associated virus capsids that facilitate efficient gene delivery to specific target cells. Vector safety is enhanced by incorporating cardiac-specific promoters into vector design and localizing delivery to reduce off-target risk. Any cardioprotective transgene may bear a degree of risk as they may play as yet unknown roles, which require careful assessment using clinically relevant models. The innovative technologies outlined here make gene therapy a promising proof of principle, with potential further application to nonanthracycline chemotherapeutics.