1Medizinische Hochschule Hannover Institut für Molekulare und Translationale Therapiestrategien, OE-8886 Hannover, Deutschland
Background: Cardiovascular diseases (CVDs) is one of the leading causes of death worldwide. Unfortunately, current therapies for CVDs are not as effective as desired. Therefore, to explore new and innovative therapeutic approaches are still necessary. Notably, the power and potential of RNA technology have been underscored by the remarkable success in the application of mRNA vaccines during the COVID-19 pandamic, which has paved the way for a revolutionary shift towards broader clinical trails of RNA therapy. Circular RNAs (circRNAs) are a a unique type of noncoding RNA with RNA loop structure. This distinctive characteristic makes circRNAs more stable compared to linear RNAs, indicating the high value for development of RNA drug. In our perivious work, we observed that a CircRNA derived from INSR locus (Circ-INSR) can prevent doxorubicin mediated cardiomyocytes death. In this project，we aim to further understand the function of recombinant circ-INSR in caridmyocytes and in vivo.
Methods and Results: The overarching goal of this project is to evaluate the efficacy of recombinant Circ-INSR as a promising RNA-based therapeutic approach for preventing doxorubicin-induced cardiotoxicity. We generated recombinant Circ-INSR production system via using the in vitro transcribed and circularized RNA technology. Subsequently, 0.1 to 1µg of recombinant Circ-INSR was transfected into the hiPSC-CMs and further cardiomyocytes apoptosis is evaluated via LDH assay and γ-H2Ax staining. We observed that the recombinant Circ-INSR not the linearized RNA showing the protecting effect of doxorubicin induced cardiomyocytes damage.
Moreover, in order to assess the functionality of recombinant Circ-INSR in vivo, we implemented a large-scale production platform for generating recombinant Circ-INSR. Remarkably, we successfully achieved the production of minimum 100ug of RNA products within a 100ul production system. Next, 5 and 50µg of recombinant Circ-INSR are packaged into LNPs(GenVoy-ILM) via using NanoAssemblr system and delivered into mice by IV injection. To assess the expression and distribution of recombinant Circ-INSR in vivo, we performed qPCR analysis. Our results indicated that the recombinant Circ-INSR was expressed in the heart，liver， kidney and spleen 24 and 48h after recombinant Circ-INSR injection. Interestingly, the recombinant Circ-INSR expression is more stable in heart tissue compared to the other organs after 48h.
Conclusion: These findings provide valuable insights into the potential therapeutic effects of recombinant Circ-INSR and its ability to be delivered and expressed in heart. This knowledge will contribute to further investigations and the development of RNA-based therapies for preventing doxorubicin-induced cardiotoxicity.