Novel approaches for cardiac reprogramming: Exosome delivery of reprogramming miRNAs and repressor targeting siRNAs

In situ reprogramming of scar fibroblasts into cardiomyocytes is therapeutically beneficial. However, functional improvements are relatively modest. The challenges for effective reprogramming are suboptimal timing of delivery, repressive barriers, and poor delivery to fibroblasts. Consequently, we hypothesize reprogramming efficacy will be improved by delivering reprogramming factors into the heart once the infarct has stabilized ablating the function of a repressor complex, and targeting reprogramming factors to cardiac fibroblasts. The hypothesis will be tested by three specific aims. Aim 1 will focus on the mechanism by which a repressor complex comprised of Cbx1, PurB and Sp3 inhibits cardiomyocyte genes. Studies will test the hypothesis that the repressor complex functions by inducing a DNA-loop and blocking access to enhancer elements. DNA-looping will be investigated by deleting or repositioning the repressor complex binding sites through the introduction of variable helical twists in a promoter reporter construct and then measuring luciferase reporter activity. Due to the apparent cooperativity between Cbx1, PurB and Sp3, we will also determine if knockdown of one repressor is sufficient to re-capitulate the effects of combined knockdown. Aim 2 will investigate the hypothesis that targeting the Cbx1-PurB-Sp3 repressor complex will improve miR combo efficacy in a mouse model of cardiac injury. The hypothesis will be tested by injecting fibroblast-selective C166-derived exosomes containing miR combo and a repressor-complex targeting siRNA into the hearts of fibroblast-lineage tracing mice immediately following MI or up to 4 weeks post infarct. The latter time-point will be used to determine if reprogramming can be employed as a salvage therapy once the infarct is completed with development of fibrosis. The effects on reprogramming (lineage tracing), cardiac structure (infarct size, fibrosis, etc.) and cardiac function (echocardiography) will be assessed over time. Aim 3 is tasked with moving reprogramming towards clinical applications via studies in a pre-clinical pig model of cardiac injury. Our preliminary data shows that pig cardiac fibroblasts internalize C166- derived exosomes and that miR combo improved cardiac function in infarcted Yorkshire mini-pigs. Following transient occlusion, C166-derived exosomes loaded with FITC-labelled miRNAs will be delivered into the hearts of Yorkshire mini-pigs and cardiac tissue analyzed for exosome uptake by measuring FITC incorporation in cardiac cells including fibroblasts, cardiomyocytes and endothelial cells. Additional studies will determine the function benefits arising from a therapy combining miR combo and a repressor targeting siRNA. In summary, successful completion of these studies is expected to delineate the role of transcription repressors as a newly discovered barrier to reprogramming. In addition, applying this knowledge in conjunction with fibroblast-selective delivery of miR combo via C166-derived exosomes is expected to enhance the efficacy and therapeutic outcomes of reprogramming fibroblasts to cardiomyocytes. Moreover, translational studies will move our MI- therapy closer to clinical applications. Project Number: 1R01HL178582-01 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Conrad Hodgkinson | Institution: DUKE UNIVERSITY, DURHAM, NC | Award Amount: $496,367 | Activity Code: R01 | Study Section: Therapeutic Development and Preclinical Studies Study Section[TDPS] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01HL17858201