Functions of the Heterologous Intron-Derived Fragments Intra and Extra Factor IX-cDNA Coding Region on the Human Factor IX Expression in HepG2 and Hek-293T Cells

Iranian Journal of Biotechnology

Haddad-Mashadrizeh, Aliakbar¹  Sam, Mohammad Reza²  Zomorodipour, Alireza³  Ghorbani, Mahdi⁴ 

[1]Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

[2]Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran

[3]Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

[4]Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran

关键词: Aldolase B intronic enhancer;    Beta-globin introns;    Hepatocyte;    Hemophilia B;    Human factor IX (hFIX);    Plasmid;   

DOI  :  10.21859/ijb.1753

学科分类：生物技术

来源: National Institute for Genetic Engineering and Biotechnology

【 摘 要 】

Background: Human FIX (hFIX) gene transfer into hepatocytes has provided a novel approach for treatment of hemophilia B. To obtain an improved expression of hFIX, the functional hFIX-expressing plasmids with appropriate intron-derived fragments which facilitate transcription and promote an efficient 3′-end formation of mRNAs are required.Objectives: We aim to evaluate the functions of the heterologous intron-derived fragments intra and extra hFIX-cDNA coding region with respect to the hFIX expression in the hepatocytes and kidney cells.Materials and Methods: HepG2 cells as differentiated hepatocytes and Hek-293T cells as embryonic kidney cells were transfected with the different hFIX-expressing plasmids containing various combinations of the two human beta-globin (hBG) introns within the hFIX-cDNA and Kozak sequence. In the next stage, as a hepatocyte-specific sequence, the rat aldolase B intronic enhancer sequence (rABE), was isolated from the first intron of the rat aldoase B gene and inserted within the upstream CMV promoter (CMVp) and efficacies of the engineered vectors were investigated in the stably-transfected HepG2 cells.Results: Our data indicate that the intron-less construct and hBG intron-I containing construct are more effective with regard to hFIX expression compared to other constructs in Hek-293 cells. In HepG2 cells, the rABE in combination with CMVp in context of intron-less plasmid induced an increase in total expression of hFIX protein dramatically; ranging from 2.3 to 40 folds increase compared to other constructs. The rABE in combination with CMVp in the hBG intron-I, hBG intron-II, and hBG intron-I,II containing plasmids induced 3.7, 2, and 1.6-fold increase in the total expression of hFIX protein, respectively. The presence of both hBG intronic sequences within the hFIX-cDNA induced a higher secretion level of hFIX than either intron-I or II alone and provided correctly spliced hFIX transcripts in HepG2 and kidney cell lines. The intron-less construct with or without rABE induced the highest hFIX mRNA levels in HepG2 and Hek-293T cells respectively compared to other constructs.Conclusions: The embryonic kidney cells in addition to the differentiated hepatic cell lines could be successfully targeted by plasmid vectors. The intron-less and hBG intron-I containing plasmids represent a particular interest in producing recombinant hFIX in Hek-293T cells. The synergistic function on the hFIX expression that was achieved by combining the CMVp with the liver-specific rABE would be a useful approach for future designing of the expression cassettes for hepatocyte-mediated gene expression in hemophilia B.

【 授权许可】

Unknown