Protein is expressed in all major organs after intravenous infusion of mRNA-lipid nanoparticles in swine

Abstract

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.

Keywords: bone marrow; brain; extrahepatic expression; gene therapy; large animal model; lipid nanoparticles; mRNA; megakaryocyte; nanomedicine; swine.

Graphical abstract

Figure 1

Hepatic and extrahepatic protein expression in swine following infusion of NanoLuciferase mRNA-LNPs (A) Swine were injected with a single dose of mNLuc (purple), scRNA (blue), or PBS (no RNA, red). Hepatic (B) and extrahepatic (C–F) NanoLuciferase protein signals measured ex vivo 24 h post-injection. N = 2–5. ∗p < 0.05, ns, not significant. Error bars represent mean $\pm$ SEM.

Figure 2

Hepatic and extrahepatic NanoLuciferase mRNA delivery without hepatotoxicity (A) mRNA NanoLuciferase levels measured by qPCR in mNLuc- (purple) or scRNA (blue)-treated swine. B.D., below detection limit; N.S., additional organs that were not significantly different. (B) Hepatotoxicity enzyme levels of alanine aminotransferase (ALT; i), aspartate aminotransferase (AST; ii), and alkaline phosphatase (ALP; iii) measured prior to injection (black), at 4 h (purple), and at 24 h post-injection (pink) of mRNA-LNPs or PBS. N = 3–5. ∗p < 0.05, ns, not significant. Error bars represent mean $\pm$ SEM.

Figure 3

Exogenous protein expression in circulating blood cells (A and B) NanoLuciferase protein signal in whole blood (A) or plasma (B) of swine treated with mNLuc (purple) or scRNA (blue). (C) NanoLuciferase protein signal in white blood cells (WBCs), red blood cells (RBCs), and platelets. N = 3–5. ∗p < 0.05, ns, not significant Error bars represent mean $\pm$ SEM.

Figure 4

Exogenous protein expression in the bone marrow (A) NanoLuciferase protein signal in unsorted bone marrow. (B) NanoLuciferase protein signal in sorted bone marrow cells: granulocytes, hematopoietic stem cells (HSCs), megakaryocytes, and platelets. N = 3–5. ∗p < 0.05, ns, not significant Error bars represent mean $\pm$ SEM.