Scientists report on the use of nanoparticle-delivered siRNA targeting the chemokine receptor CCR2 to reduce damaging inflammatory responses in a range of disorders. The Massachusetts General Hospital and Harvard Medical School-led team found that systemic administration of a lipid nanoparticle encapsulated siRNA targeting CCR2 in mouse models led to reductions in inflammatory monocytes in atherosclerotic plaques and reduced infarct size after coronary artery occlusion.
The treatment also improved the outcome of pancreatic islet transplantation and reduced tumor volumes and the numbers of tumor-associated macrophages in mouse models of lymphoma and colorectal cancer. Matthias Nahrendorf, Ph.D., Florian Leuschner, Ph.D., and colleagues report on their approach in Nature Biotechnology in a paper titled “Therapeutic siRNA silencing in inflammatory monocytes in mice.”
Inflammatory monocytes - Ly-6Chigh in the mouse and CD14+CD16− in humans give rise to classical macrophages. Prolonged or excessive activity by these cells can propagate disease progression, the researchers explain. Targeting monocytes and their macrophage lineage descendents as a means of reducing prolonged inflammatory responses in disease is hindered because many such innate immune cells also have critical protective functions. Ideally, it would be possible to temporarily target only those cellular subsets involved in damaging inflammatory activity.
Mobilization of specifically inflammatory monocytes to sites of disease or damage is dependent on the chemokine MCP-1 and its receptor CCR2. The CCR2 receptor is also involved in recruiting monocytes from bone marrow. Unfortunately, the investigators note, small molecule or antibodies that target the MCP-1/CCR2 axis have often lacked specificity or efficacy in vivo.
An alternative approach to blocking CCR2 is provided by siRNAs: “We hypothesized that monocyte-targeting siRNA nanomaterials could silence CCR2 mRNA in the inflammatory monocyte subset to selectively inhibit migration (and consequently adverse function) of these cells and their progeny.”
The team thus developed a lipid-like siRNA carrier that had previously been described and a CCR2 siRNA identified using an in vitro screen. The nanoparticle was formulated using C12-200 lipid, disteroylphosphatidyl choline, cholesterol, PEG-DMG, and siRNA in a spontaneous vesicle formation procedure.
Tests to evaluate the distribution of fluorochrome-labeled siRNA nanoparticle after intravenous injection in mice showed that the siRNA accumulated in spleen red pulp, bone marrow, and liver. A particularly high uptake by splenic Ly-6Chigh monocytes was observed. This accumulation led to a significant reduction in CCR2 mRNA in Ly6Chigh monocytes, lower CCR2 protein levels, and a marked reduction in the ability of Ly-6Chigh monocytes to migrate toward MCP-1.
The investigators then evaluated systemic administration of the nanoparticle siRNA in murine models of disease. When administered to a mouse model of cardiac ischemia-reperfusion, the treatment led to a significant reduction in the number of monocytes and macrophages in the heart, and a 71% reduction in the number of Ly-6Chigh monocytes specifically, compared with control animals. The nanoparticle-siRNA-treated animals also demonstrated a 34% smaller infarct/area-at-risk ratio.
A three-week course of siRNA treatment was also given to apo-E knockout mice with established atherosclerosis. In comparison with control animals, the treated knockouts displayed an 82% reduction in the number of monocytes/macrophages in atherosclerotic plaques, including a marked reduction in inflammatory Ly6Chigh monocytes along with a 46% reduction in myeloid cells and 38% smaller lesion size in the aortic root.
The use of pancreatic islet transplantation as a feasible treatment option for patients with type 1 diabetes is hampered by the development of allograft rejection, an event which in mice is characterized by recruitment of Ly-6Chigh monocytes and monocyte differentiation into highly active antigen-presenting cells, the team notes.
They therefore investigated the effects of intravenous siCCR2 therapy on pancreatic islet transplantation in mice with streptozotocin-induced diabetes. The results showed that intravenous injection of nanoparticle-encapsulated siCCR2 significantly prolonged the normoglycemic period and thus islet graft function.
A fourth in vivo test investigated the effects of siCCR2 therapy in mice that had developed palpable tumors following transplantation with lymphoma EL4 cells. Compared with control animals, the treated mice showed marked reductions in tumor volume five days after siCCR2 therapy was initiated, a 54% reduction of in tumor-associated macrophages, and reduced VEGF expression and microvessel formation. Treatment of a separate mouse model of colorectal CT26 tumors demonstrated a 75% reduction in tumor-associated macrophages.
“To our knowledge, this is the first demonstration of siRNA delivery to the inflammatory monocyte subset,” the authors conclude. “Although we have concentrated on CCR2 in this study, it is logical to extend the approach to other targets in innate immune cells, for instance, proteins involved in proliferation, maturation, differentiation, and antigen presentation, such as transcription factors and cytokines. This study describes the merging of in vivo RNAi with recent insights into monocyte biology, opening a new translational avenue to approach the many diseases driven by recruitment of these cells.”