AAV-mediated AP-1 decoy oligonucleotide expression inhibits aortic elastolysis in a mouse model of Marfan syndrome

Aims Marfan syndrome is one of the most common inherited disorders of connective tissue caused by fibrillin-1 mutations, characterized by enhanced transcription factor AP-1 DNA binding activity and subsequently abnormally increased expression and activity of matrix-metalloproteinases (MMPs). We aimed to establish a novel adeno-associated virus (AAV)-based strategy for long-term expression of an AP-1 neutralizing RNA hairpin (hp) decoy oligonucleotide (dON) in the aorta to prevent aortic elastolysis in a murine model of Marfan syndrome. Methods and results Using fibrillin-1 hypomorphic mice (mgR/mgR), aortic grafts from young (9 weeks old) donor mgR/mgR mice were transduced ex vivo with AAV vectors and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Grafts were explanted after 30 days. For in vitro studies, isolated primary aortic smooth muscle cells (SMCs) from mgR/mgR mice were used. Elastica-van-Giesson staining visualized elastolysis, reactive oxygen species (ROS) production was assessed using dihydroethidine staining. RNA F.I.S.H. verified AP-1 hp dON generation in the ex vivo transduced aortic tissue. MMP expression and activity were assessed by western blotting and immunoprecipitation combined with zymography. Transduction resulted in stable therapeutic dON expression in endothelial and SMCs. MMP expression and activity, ROS formation as well as expression of monocyte chemoattractant protein-1 were significantly reduced. Monocyte graft infiltration declined and the integrity of the elastin architecture was maintained. RNAseq analysis confirmed the beneficial effect of AP-1 neutralization on the pro-inflammatory environment in SMCs. Conclusion This novel approach protects from deterioration of aortic stability by sustained delivery of nucleic acids-based therapeutics and further elucidated how to interfere with the mechanism of elastolysis.

STAT3 decoy oligonucleotide-carrying microbubbles with pulsed ultrasound for enhanced therapeutic effect in head and neck tumors

Signal transducer and activator of transcription-3 (STAT3) is an oncogenic transcription factor implicated in carcinogenesis, tumor progression, and drug resistance in head and neck squamous cell carcinoma (HNSCC). A decoy oligonucleotide targeting STAT3 offers a promising anti-tumor strategy, but achieving targeted tumor delivery of the decoy with systemic administration poses a significant challenge. We previously showed the potential for STAT3 decoy-loaded microbubbles, in conjunction with ultrasound targeted microbubble cavitation (UTMC), to decrease tumor growth in murine squamous cell carcinoma. As a next step towards clinical translation, we sought to determine the anti-tumor efficacy of our STAT3 decoy delivery platform against human HNSCC and the effect of higher STAT3 decoy microbubble loading on tumor cell inhibition. STAT3 decoy was loaded on cationic lipid microbubbles (STAT3-MB) or loaded on liposome-conjugated lipid microbubbles to form STAT3-loaded liposome-microbubble complexes (STAT3-LPX). UTMC treatment efficacy with these two formulations was evaluated in vitro using viability and apoptosis assays in CAL33 (human HNSCC) cells. Anti-cancer efficacy in vivo was performed in a CAL33 tumor murine xenograft model. UTMC with STAT3-MB caused significantly lower CAL33 cell viability compared to UTMC with STAT3-LPX (56.8±8.4% vs 84.5±8.8%, respectively, p<0.05). In vivo, UTMC with STAT3-MB had strong anti-tumor effects, with significantly less tumor burden and greater survival compared to that of UTMC with microbubbles loaded with a mutant control decoy and untreated control groups (p<0.05). UTMC with STAT3 decoy-loaded microbubbles significantly decreases human HNSSC tumor progression. These data set the stage for clinical translation of our microbubble platform as an imaged-guided, targeted delivery strategy for STAT3 decoy, or other nucleotide-based therapeutics, in human cancer treatment.