Poster R11 - Ultrasound-Mediated Gene Therapy: pre-sensitization to cure

Introduction: Recent clinical trials using immune checkpoint blockade therapy have shown immense promise against aggressive cancers. This is credited to its ability to reprogram the resident immune cells to have improved tumor recognition, a stronger cytotoxic response and long-term memory formation. Currently, the gold standard for immune checkpoint blockade is using antibody treatment. Such treatment is often limited by the high risk of cytokine storm, poor tumor targeting and high-dosing requirements. An alternate strategy to achieve a sustained potent outcome is to employ gene silencing with advanced lipid nanoparticle technology. However, the hyper populated environment with resident tolerogenic immune cells, limits their efficiency. To further increase the penetration of nanoparticles through the dense extracellular matrix of the tumor as well as the cytosolic delivery of their cargoes, we propose the ultrasound mediated delivery of siRNA-carrying lipid nanoparticles to genetically silence immune checkpoint proteins in metastatically aggressive murine breast cancer models.

Materials and

Methods: Our therapeutic strategy focuses on the pre-sensitization of the tumor microenvironment, to make it more readily susceptible to nanoparticle infiltration and delivery. We introduce lipid- shelled C3F8 core nanobubbles that are typically used as contrast agents in ultrasound imaging, and cavitate them using therapeutic ultrasound at their peak distribution in the tumor. The resulting local shear stresses and jet stream formation loosen the tumor microenvironment and temporarily sonoporate the resident cells. This cavitation also has an immunomodulatory effect on them, due to the sudden increase in ER stress in the tumor. This combination allows for lipid nanoparticles (LNPs) to be readily distributed and internalized , and direct delivery of the cargo straight into the cytosol for enhanced immune-checkpoint silencing. We tested the therapeutic efficacy of the strategy in mouse models of aggressive breast cancer. In addition to the macroscopic effects to the tumor growth, we assessed the biodistribution and immune cellular response using flow cytometry analysis.

Results, Conclusions, and Discussions: Our preliminary results have shown a pan-increase of the uptake of LNPs in all leukocytes in the tumor. The induced stressors have also allowed for uptake of LNPs by the T cell subset, which has no natural mechanism for this. The LNP-positive cells show higher transfection for silencing the immune checkpoint protein which in turn has activated and expanded the immune cells into their anti-tumor phenotype. We also observed a remarkable 2-fold decrease of the immunosuppressive cell subsets in as little as three hours after treatment. Also, antigen-presenting cells have expanded 10-fold in the tumor-draining lymph node which demonstrated improved tumor-recognition within 48 hours. Upon assessing the long-term impact, we observed a significant increase in overall survival and tumor regression in the US-mediated treatment group compared to the controls. This effect is sustained through multiple rechallenges, demonstrating relapse resistance due to systemic memory formation.

Our study shows that using ultrasound-induced nanobubble cavitation to help deliver siRNA-carrying lipid nanoparticles can effectively silence immune checkpoint proteins in aggressive breast cancer models. This approach resulted in increased activation and expansion of anti-tumor immune cells, reduced immunosuppressive cell subsets, and enhanced tumor recognition by antigen-presenting cells. The treatment also led to increased overall survival, tumor regression, and resistance to relapse. Our findings support the potential of ultrasound-mediated delivery as a promising strategy for enhancing immune checkpoint blockade therapy in aggressive cancers.

Immunotherapy remains vulnerable to multi-drug resistance and systemic toxicity. Most current solutions do not effectively engage the adaptive arm of the immune system and therefore generate a response that is generic with a time-limited therapeutic effect. This has become an increasingly important barrier to overcome, since the primary reason for breast cancer related deaths in the clinic are silent, metastatic relapse. Our solution allows for ultrasound-mediated direct priming of the adaptive immune system, as well as improved antigen recognition due to the release of tumor-associated antigens and reprogramming of the innate immune system to be pro-inflammatory. This combined with the remodeled tumor extracellular matrix has a synergistic effect on cancer therapy, with the flexibility to be augment the effects of clinically approved modalities.

Acknowledgements (Optional): This work was supported by grants from the National Cancer Institute R01CA253627 (E.K.), R01CA278633 (E.K. ), P30CA043703 (E.K.), and the Angie Fowler AYA Cancer Research Fund (E.K.). A.B. was supported by a fellowship from the NIH Interdisciplinary Biomedical Imaging Training Program (T32EB007509).