RESUMEN
In vivo delivery of mRNA is promising for the study of gene expression and the treatment of diseases. Lipid nanoparticles (LNPs) enable efficient delivery of mRNA constructs, but protein expression has been assumed to be limited to the liver. With specialized LNPs, delivery to extrahepatic tissue occurs in small animal models; however, it is unclear if global delivery of mRNA to all major organs is possible in humans because delivery may be affected by differences in innate immune response and relative organ size. Furthermore, limited studies with LNPs have been performed in large animal models, such as swine, due to their sensitivity to complement activation-related pseudoallergy (CARPA). In this study, we found that exogenous protein expression occurred in all major organs when swine were injected intravenously with a relatively low dose of mRNA encapsulated in a clinically relevant LNP formulation. Exogenous protein was detected in the liver, spleen, lung, heart, uterus, colon, stomach, kidney, small intestine, and brain of the swine without inducing CARPA. Furthermore, protein expression was detected in the bone marrow, including megakaryocytes, hematopoietic stem cells, and granulocytes, and in circulating white blood cells and platelets. These results show that nearly all major organs contain exogenous protein expression and are viable targets for mRNA therapies.
RESUMEN
Platelets contribute to a variety of physiological processes including inflammation, sepsis and cancer. However, due to their primary role in hemostasis, platelet transfusions are largely restricted to managing thrombocytopenia and bleeding. One way to expand the utility of platelet transfusions would be to genetically engineer donor platelets with new or enhanced functions. We have previously shown that lipid nanoparticles containing mRNA (mRNA-LNP) can be used to genetically modify authentic platelets in a non-clinical crystalloid solution. Currently, platelets collected for transfusion are stored in plasma or in plasma supplemented with platelet additive solution (PAS) at supraphysiological concentrations at room temperature, or at 4 ºC if intended for use in acute hemorrhage. Here we describe a new plasma-optimized mRNA-LNP for transfecting platelets directly in plasma and plasma supplemented with PAS that is scalable to physiological and supraphysiological platelet concentrations. Transfecting platelets in clinical solutions with mRNA-LNP does not affect aspects of in vitro physiology, and transfected platelets are storable. The compatibility of this transfection system with current clinical practices could enable future mRNA-LNP based platelet products and cell therapies.
RESUMEN
Genetic manipulation of animal models is a fundamental research tool in biology and medicine but is challenging in large animals. In rodents, models can be readily developed by knocking out genes in embryonic stem cells or by knocking down genes through in vivo delivery of nucleic acids. Swine are a preferred animal model for studying the cardiovascular and immune systems, but there are limited strategies for genetic manipulation. Lipid nanoparticles (LNPs) efficiently deliver small interfering RNA (siRNA) to knock down circulating proteins, but swine are sensitive to LNP-induced complement activation-related pseudoallergy (CARPA). We hypothesized that appropriately administering optimized siRNA-LNPs could knock down circulating levels of plasminogen, a blood protein synthesized in the liver. siRNA-LNPs against plasminogen (siPLG) reduced plasma plasminogen protein and hepatic plasminogen mRNA levels to below 5% of baseline values. Functional assays showed that reducing plasminogen levels modulated systemic blood coagulation. Clinical signs of CARPA were not observed, and occasional mild and transient hepatotoxicity was present in siPLG-treated animals at 5 h post-infusion, which returned to baseline by 7 days. These findings advance siRNA-LNPs in swine models, enabling genetic engineering of blood and hepatic proteins, which can likely expand to proteins in other tissues in the future.
