mCherry mRNA (no modification)

mRNA-mediated protein replacement therapy represents a promising concept for the treatment of liver diseases. Children born with mutations in fumarylacetoacetate hydrolase (FAH) develop hepatorenal tyrosinemia type 1 (HT-1), which leads to renal dysfunction, liver failure, neurological impairment, and cancer. Protein replacement therapy using FAH mRNA holds promise for curing HT-1 but is currently hampered by the development of effective mRNA vectors that can function in the diseased liver. Structure-guided, rationally optimized 5A2-SC8 mRNA-loaded dendrimer lipid nanoparticles (mDLNPs) improve the delivery of FAH mRNA, resulting in functional FAH protein and sustained restoration of body weight and liver function in FAH-/- knockout mice. Optimization with luciferase mRNA enabled the preparation of DLNP vectors at in vivo doses as low as 0.05 mg kg−1. mDLNPs transfected more than 44% of hepatocytes in the liver, produced high levels of FAH protein (0.5 mg kg−1 mRNA), and were well tolerated in a knockout mouse model of impaired liver function. Genetically engineered FAH-/- mice treated with FAH mRNA mDLNPs had statistically comparable levels of TBIL, ALT, and AST to those of wild-type C57BL/6 mice and maintained normal body weight over a month-long treatment period.

To further understand the delivery of mDLNPs to the liver, the researchers quantified the time-dependent Luc activity after intravenous injection. Luc protein expression peaked at 6 h after injection and remained high for about two days at both the organ (Figure 1A) and systemic levels. Next, they investigated the in vivo dose-response behavior of delivery (Figure 1B). Luc expression increased with increasing doses from 0.05 mg kg−1 to 0.2 mg kg−1 mRNA. The average radiance of the entire liver was calculated and plotted for time-dependency and dose-response studies (Figure 1A, B). With expression levels above 106 photons/second at a dose of 0.05 mg kg−1 mRNA, mDLNPs may be the most efficient mRNA carrier reported to date. Next, the researchers quantified delivery to liver hepatocytes. mCherry mRNA was delivered at a dose of 0.5 mg kg−1, and mCherry expression was compared to that of a PBS-injected control group by fluorescence imaging of liver sections (Figure 1C). Strong red mCherry signals were observed in the liver. To further analyze the transfection efficiency, the researchers isolated hepatocytes from the injected mice and used flow cytometry to quantify the specific delivery of mCherry mRNA to hepatocytes. The researchers found that 44.2% of hepatocytes strongly expressed mCherry protein 6 hours after intravenous injection of mCherry mRNA mDLNPs.

Figure 1. mDLNPs deliver mRNA in a dose-dependent manner with high transfection efficiency of liver hepatocytes. (Cheng Q, et al., 2018)