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Keywords:

  • acute liverinjury;
  • CCL5 ;
  • CX3CL;
  • hepatocyte-specific;
  • interleukin 10;
  • multifunctional;
  • RNA interference

Abstract

Background & Aims

Liver disease, such as malignancy and hepatitis, often correlates with several genetic disorders. We aimed to construct a hepatocyte-specific vector that could manipulate multiple genes simultaneously.

Methods

We selected a highly efficient hepatocyte-specific α-foetoprotein (AFP) enhancer/albumin promoter (an RNA polymerase II promoter) to express our gene of interest and transcribe microRNA-based shRNAs (shRNAmir). Multiple shRNAmirs were assembled together in tandem to enhance the gene-silencing effect. By employing the AFP enhancer/albumin promoter and inserting an internal ribosome entry site (IRES), a hepatocyte-specific, multi-reporter vector that overexpressed both β-galactosidase (LacZ) and DsRed2 while simultaneously knocking down both EGFP and luciferase expression was successfully constructed and functionally tested in vitro.

Results

The reporter genes in the multireporter vector were easily replaced by immune-related genes to construct the Multi-Vector, which overexpressed human interleukin 10 and silenced both CCL5 and CX3CL1 (FKN) simultaneously in vivo; visualization of DsRed2 coexpressed to monitor vector function in vivo confirmed that the Multi-Vector was successfully introduced into the host. Simultaneous manipulation of these multiple genes by the Multi-Vector synergistically inhibited acute liver injury induced by Poly I:C/D-GalN injection in mice. The multifunctional cassette was also packaged in and successfully delivered by an adenoviral vector.

Conclusions

We successfully engineered a vector that can simultaneously regulate multiple genes from a single multigene-containing vector in a hepatocyte-specific manner, suggesting the possibility that this method could be extensively and practically utilized in liver gene therapy.