Assistant Professor of Engineering Brown University, United States
Introduction: Nucleic acid based therapeutics are a key component of modern medicine. Messenger RNA (mRNA)-encapsulated lipid nanoparticle (LNP)-based COVID-19 vaccines demonstrated the safety and efficacy of this platform technology, at a global scale. Nucleic acid, and RNA-based therapeutics in particular, provide numerous advantages over other therapeutic modalities, including: (i) the ability to target not only proteins but specific genetic sequences of interest, thereby allowing highly targeted therapeutics, and (ii) a relative ease in targeting extracellular as well as intracellular cytokines. Advancements in RNA chemical modifications and LNP delivery vectors both play critical roles in determining in vivo delivery, immunogenicity, and toxicity, and ultimately in determining the clinical applicability of RNA-LNP based therapeutics. In this work, we explored the use of modified siRNA-LNP constructs to silence checkpoints of innate immune cell (macrophage and natural killer (NK) cell) activation in vivo; and explored the therapeutic efficacy of siRNA-LNP ‘checkpoint inhibitors’ in murine models of metastatic cancer.
Materials and
Methods: Short interfering RNA (siRNA) was designed in silico to target highly conserved, and unique, genetic sequences within checkpoint-genes that inhibit macrophage or NK cell activation. siRNAs with various sequences and chemical modification patterns were screened in vitro to assess silencing efficiency, at the level of transcription, using RT-qPCR; and at the level of translation, using flow cytometry. Lipid nanoparticles (LNPs) were formulated with a novel, biodegradable, ionizable lipid (previously selected from a large library of ionizable lipids) and screened for in vivo delivery to macrophages or NK cells following intraperitoneal injection.
Therapeutic efficacy was assessed in murine models of peritoneal disseminated cancer: melanoma or ovarian cancer, generated via intraperitoneal injection of B16F10-melanoma or ID8-ovarian cancer cells into fully immunocompetent mice (C57BL/6). siRNA-LNPs were administered twice weekly, via intraperitoneal injection, and tumor burden tracked via IVIS imaging. Flow cytometry was also used to assess checkpoint silencing, immune cell recruitment, and phenotype.
Results, Conclusions, and Discussions: Optimized siRNA-LNPs allowed high levels of silencing in peritoneal macrophages (~90%) and NK cells (~70%) following low-dose (0.01 mg/kg), intraperitoneal, administration. LNPs formulated with our novel ionizable lipid demonstrated uptake mediated by clathrin-mediated endocytosis and macropinocytosis in macrophages; and ApoE/LDLR dependent uptake by NK cells. Macrophage checkpoint silencing siRNA-LNPs shifted macrophages towards tumoricidal phenotypes, increased phagocytosis, and tumor antigen presentation. Ultimately, in a murine model of peritoneal metastasized ID8 ovarian cancer, intraperitoneal administration of macrophage checkpoint silencing siRNA-LNPs + a cancer opsonizing-antibody significantly prolonged survival vs treatment with control siRNA-LNP + antibody. Administration of macrophage checkpoint silencing siRNA-LNPs with standard chemotherapy (cisplatin + paclitaxel), significantly slowed tumor growth, even following treatment cessation. The slowed growth was concomitant with increases in tumor-specific effector memory CD4 and CD8 T cells.
NK cell checkpoint silencing siRNA-LNPs increased NK cell sensitivity and activation driven by IL15. NK cell checkpoint silencing increased the number of IL15 receptors on NK cells, and when combined with intraperitoneal administration of recombinant IL15, yielded 4-fold expansion of NK cells. In murine models of peritoneal disseminated melanoma, NK checkpoint silencing siRNA-LNPs prolonged survival. Therapeutic efficacy was significantly abrogated following NK cell depletion, confirming the key role of NK cells in therapeutic efficacy.
Collectively, these data demonstrate that siRNA-LNPs allow for efficient checkpoint silencing on both macrophages and NK cells, in vivo, following intraperitoneal administration. Further, checkpoint silencing is a promising strategy for novel immune checkpoint inhibitor, cancer immunotherapy.
