AAV (Serotype 2)

Familial hypercholesterolemia (FH) is an inherited lipoprotein metabolism disorder caused by defects in the LDL receptor (LDLR), leading to severe hypercholesterolemia and associated with an increased risk of coronary heart disease and myocardial infarction. Here, researchers developed an FH gene therapy protocol using AAV2, AAV9, and lentiviral vectors and tested safety and efficacy in LDL receptor-deficient Watanabe hereditary hyperlipidemia rabbits. Studies have found that LV-LDLR can significantly reduce serum total cholesterol long-term, while AAV9-LDLR only causes a temporary reduction in serum cholesterol, and AAV2-LDLR cannot reduce serum cholesterol levels. A significant pathological side effect, namely bile duct hyperplasia, associated with increased Cyr61 stromal cell protein expression, was observed in the livers of AAV2-LDLR rabbits. Special attention should be paid to hepatic changes in gene therapy applications when using genes affecting cholesterol and lipoprotein metabolism for therapy.

Based on the histological features, bile duct hyperplasia was evaluated as reactive rather than malignant. Abnormal ductules stained with pancytokeratin antibody, confirming their epithelial origin (Figure 1f). In addition, histologically normal bile ducts in the AAV2 control and AAV9 control groups, as well as bile ducts in the AAV9-LDLR group, were pancytokeratin-positive (Figure 1e, g, h). This antibody also stained hepatocytes surrounding the ductules, but almost exclusively in animals with the most prominent bile duct hyperplasia (Figure 1f). Only a few single pancytokeratin-positive hepatocytes were seen in either AAV2 or AAV9 control animals, whereas in AAV2-LDLR animals, clusters of pancytokeratin-positive hepatocytes were seen in the bile duct lining surrounding the hyperplastic area as well as in other parts of the liver tissue (Figure 1e-h). Immunohistochemical staining with Ki-67 antibody showed a few proliferative cells in the hyperplastic areas of the bile duct at the 1-year time point. Their frequency was similar to the number of proliferative cells in normal liver tissue (Figure 1a-d) and did not differ between the groups. One month after gene transfer, β-galactosidase immunostaining of the livers of the AAV2 control group showed positive staining in inflammatory cells and biliary epithelium in addition to hepatocytes (Figure 1i, j).

Figure 1. Immunohistochemical characterization of representative rabbit liver samples after AAV mediated gene transfers. (Hytönen E, et al., 2019)