title: Construction and application of a novel hepatocyte-directed vector to simultaneous knockdown and overexpression of multiple genes.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China; Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui, China.
authors.forename: Jianlin
authors.initials: J
authors.lastname: Geng
authors.forename: Haiming
authors.initials: H
authors.lastname: Wei
authors.forename: Rui
authors.initials: R
authors.lastname: Sun
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Tian
mesh_terms: D000256:Adenoviridae; D000818:Animals; D056486:Chemical and Drug Induced Liver Injury / Q000235:genetics / Q000517:prevention & control*; D018946:Chemokine CCL5 / Q000235:genetics / Q000378:metabolism; D054428:Chemokine CX3CL1 / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D055785:Gene Knockdown Techniques / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics; D011070:Poly I-C; D011401:Promoter Regions, Genetic / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D000509:alpha-Fetoproteins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: C483024:CCL5 protein, human; C515538:CX3CL1 protein, human; D018946:Chemokine CCL5; D054428:Chemokine CX3CL1; D008164:Luminescent Proteins; D035683:MicroRNAs; D034741:RNA, Small Interfering; D000509:alpha-Fetoproteins; C413663:fluorescent protein 583; D016753:Interleukin-10; D012319:RNA Polymerase II; D001616:beta-Galactosidase; D011070:Poly I-C
keywords: CCL5; CX3CL; RNA interference; acute liverinjury; hepatocyte-specific; interleukin 10; multifunctional