Acute and chronic liver diseases are often related to the disorder of liver regeneration. However, there is no drug specifically approved for promoting liver regeneration especially in acute-on-chronic liver failure (ACLF). According to recent studies, inhibition of mitogen-activated protein kinase kinase 4 (MKK4) protein is critical for promoting hepatocyte proliferation and liver regeneration. However, MKK4 is widely present in various parts of the body as well with the similar binding pocket as MKK7 for small molecular inhibitors, and non-specific inhibition of MKK4 expression may pose an off-target risk in clinical practice. Therefore, MKK4-siRNA (siMKK4) was designed and optimized as a therapeutic gene for specific knockdown of MKK4 and avoid the off-target binding to mitogen-activated protein kinase kinase 7 (MKK7). Moreover, N-acetylgalactosamine (GalNAc)-modified lipid nanoparticles (LNPs) were used as gene delivery carriers to construct a dual targeted gene therapy system GalNAc-LNP-siMKK4 with liver tropism and active targeting to hepatocyte. GalNAc-LNP-siMKK4 can be efficiently constructed by the reverse phase evaporation method, with uniform particle size, good stability, biocompatibility, hepatocyte targeting ability, and high gene silence effect on the expression of MKK4.andexperiments demonstrated that GalNAc-LNP-siMKK4 had good efficacy of gene therapy on promoting liver regeneration, reducing hepatocytes apoptosis, and promoting liver function recovery. The constructed hepatocyte-targeted gene therapy system GalNAc-LNP-siMKK4 could hold promise for treating ACLF based on reducing protein expression of MKK4 to promote hepatocytes proliferation specifically mediated by the targeting moiety of GalNAc. GalNAc-LNP-siMKK4 has targeting ability to deliver therapeutic genes to liver and hepatocytes, achieving a highly efficient gene therapy for promotion of liver regeneration and providing new therapeutic strategies for ACLF. In vitro in vivo
