Bioengineering of differentiated hepatocytes with human factor IX-expressing plasmids in vitro.

For somatic gene therapy of hemophilia B, hepatocytes as the main cellular host for expression of hFIX are attractive targets. In gene therapy protocols, an efficient expression vector equipped with cis-regulatory elements such as introns is required. With this in mind, hFIX-expressing plasmids equipped with different combinations of 2 human ß-globin (hBG) introns inside the hFIX-cDNA and Kozak element were used for bioengineering of HepG2 cells as a model for differentiated hepatocytes and CHO cells a cell line generally used to produce recombinant hFIX (rhFIX). In HepG2 cells, the highest hFIX secretion level occurred for the intron-less plasmid with 8.5 to 53.8- fold increases, while in CHO cells, the hBG intron-I containing plasmid induced highest hFIX secretion level with 2.3 to 14.3-fold increases as compared to other plasmids. The first hBG intron appears to be more effective than the second one in both cell lines. The expression level was further increased upon the inclusion of the Kozak element. The highest hFIX activity was obtained from the cells that carrying the intron-less plasmids with 470 mU/ml and 25 mU/ml for HepG2 and CHO cells respectively. Secretion of active hFIX by all constructs was documented except for hBG intron-II containing construct in both cell lines. HepG2 cells were able to secret higher hFIX levels by 0.6 to 112.2-fold increases with activity by 5.3 to 16.4-fold increases compared to CHO cells transfected with the same constructs. Presence of both hBG intron-I and II inside the hFIX-cDNA provides properly spliced hFIX transcripts in both cell lines. In conclusion, the advantages of hBG introns as attractive cis-regulatory elements to obtain higher expression level of hFIX particularly in CHO cells were demonstrated. Hepatocytes could be effectively bioengineered with the use of plasmid vectors and this strategy may provide a potential in-vitro source of functional hepatocytes for ex-vivo gene therapy of hemophilias and production of rhFIX in vitro.

Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.

Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human ß-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias.