Sub‐organ Fractionation of Hepatic Cells after Antisense Oligonucleotide Treatment in Mice

Abstract

Nonalcoholic Steatohepatitis (NASH) causes chronic liver disease and leads to advanced fibrosis and cirrhosis. Current research is unclear which liver cell population is key for treatment of the disease. Antisense oligonucleotide (ASO) drugs are a likely therapy for NASH due to their preferential distribution to the liver. Moreover, conjugates can be attached to the ASO and enable targeted cell specific drug delivery to the liver. Conjugations to the ASO also facilitate enhanced potency in targeted cell types by utilizing different uptake mechanisms. Unconjugated ASOs preferentially accumulate in the non‐parenchymal (NP) cells of the liver including liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC) before reaching the hepatocytes. Conversely triantennary N‐acetyl galactosamine (GalNAc)‐conjugation of ASOs results in a shift of ASO to preferentially accumulate in the hepatocytes over the NP cells (Prakash et al. NAR, 42: 8796, 2014). A recent study was designed to investigate the ASO activity in hepatocytes, LSEC, KC, and HSC from mice fed a standard chow diet compared to mice fed a Western diet. The liver pathology of the mice fed a Western diet at the time of ASO treatment was indicative of a NASH diseased state (steatosis, steatohepatitis and fibrosis). Mice were injected subcutaneously, at the indicated doses, with unconjugated or GalNAc‐conjugated ASO for 6 weeks. Following treatment, mouse livers were perfused, the hepatocytes were isolated by Percoll gradient and the NP cells by Optiprep (Lodixanol) gradient. NP cells were further isolated into enriched populations by fluorescence‐activated cell sorting (FACS). Whole liver and isolated cell populations were evaluated for RNA reduction after treatment. Drug concentration in each cell type was also measured using an ELISA based assay. As expected, there was target mRNA reduction with unconjugated and conjugated ASOs in all hepatocyte cell fractions. There was also targeted mRNA reduction in all NP cell populations in both healthy and diseased mice. However, the HSC cell population contained the least amount of ASO resulting in the lowest level of targeted reduction. These results suggest that GalNAc‐conjugated ASO can decrease targeted RNA not only in hepatocyte cells, but also in LSEC, KC and HSC cells despite a lower dose and a sever diseased state. This finding illustrates the ability of GalNAc‐conjugated ASO to enhance drug potency yet still allow drug delivery to all liver cell types. Therefore, ASO treatment is an attractive therapy for liver diseases, such as NASH, that involve multiple cell types contributing to disease progression.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.