Development and evaluation of an auto-tunable CircR-miRNAs sponges as innovative anti-miRNA therapy for Idiopatic Pulmonary fibrosis (IPF).

IPF is a progressive fibrotic lung disease that leads to death within 3 to 5 years. Diseased alveolar type II (ATII) cells drive IPF by losing their regenerative ability and releasing pro-fibrotic factors. We found two miRNAs upregulated in IPF patients and Bleomycin-treated mice to prevent ATII-to-ATI transition and promote cell aging, worsening the disease. Blocking these miRNAs with anti-miRNA LNAs reduced lung fibrosis in Bleomycin-treated mice. Hence, we are developing an AAV vector encoding circular RNAs (circRNAs) designed to sponge the two selected microRNAs. These circRNAs are part of a negative feedback loop where the selected microRNAs levels regulate the circRNA sponge transcription rate, shutting off the system once disease is resolved. Specifically, we designed a self-regulating AAV6.2FF-CircR-miR-LacI platform, able to regulate the expression of our synthetic circRNAs according to the level of the sponged miRNAs while AAV6.2FF vectors specifically target ATII cells. First, constructs were validated by testing the sponging system in HEK293T and A549 cells with a luciferase reporter containing miRNA binding sites. Co-transfecting miRNA mimics showed that the circRNA sponge effectively sequestered miRNAs and restored reporter activity. Lastly, we are refining the CircR-miR-LacI negative feedback loop platform in HEK293T and A549 cells, using miRNA mimics and inhibitors to explore the platform switch's response. To sum up, our results suggest that engineered circRNAs targeting disease-associated miRNAs could be a promising therapeutic tool for developing innovative, safe AAV-based gene therapies for chronic endogenous miRNA inhibition.