Figure 3.

Administration of LNP-mRNA results in hepatic arginase 1 expression and resolution of arginase-deficient hepatocyte subcellular abnormalities

Images demonstrate murine arginase 1 expression (Arg1; red), glutamine synthetase (Glu1; green), and DAPI (blue) in (A) wild-type Arg1^+/+ hepatocytes; (B) lack of Arg1 expression in murine Arg1^−/− hepatocytes; (C) and restored hepatocyte expression of ARG1 when LNP B formulated with ARG1 is administered; this is markedly greater than the LNP A formulation (D). RNA in situ hybridization (E, LNP A; F, LNP B) demonstrates ARG1 mRNA both at 12 h (left images) after administration that is reduced by 24 h (right images). (G) Murine Arg1^+/+ liver control demonstrates specificity of human ARG1 probe while (H) demonstrates lack of ARG1 in murine Agr1^−/− liver. (I) Low-magnification representative image showing a hepatocyte with a spherical nucleus (N), several glycogen patches are pointed by arrow heads in wild-type Arg1^+/+ murine liver. Bar, 5 μm. (J) Low-power representative image showing a hepatocyte with an irregular shape, broken nucleus (N), and many enlarged mitochondria surrounding the nucleus in an untreated Arg1^−/− liver. Some mitochondria are seen but no prominent ER and glycogen granules are present. Bar, 2 μm. (K) Low-power representative image showing a hepatocyte having a spherical nucleus (N) that contains a prominent nucleolus; surrounding the nucleus are several patches of glycogen granules (arrowhead) when LNP A formulated mRNA was delivered. Bar, 2 μm. (L) High-power representative image of Figure 3K showing prominent rough ER and several mitochondria close to the nucleus. The ultrastructural features of the hepatocyte demonstrate a complete recovery in the subcellular morphology. ARG1, arginase 1; Arg 1, arginase 1; RNA, ribonucleic acid; LNP, lipid nanoparticle; N, nucleus; M, mitochondria. (A–H) Bar, 50 μm; (I) bar, 5 μm; (J and K) bar, 2 μm; (L) bar, 0.5 μm.