Modulation of the molecular conformation of a hepatocyte-targeting gene drug in order to improve its expression efficiency in vitro.
OBJECTIVE
To construct different conformations of a plasmid DNA/vector complex (pcDNA3.1/IFN-gamma-ASOR-PLL) and transfect cells of the hepatoma cell line BEL7402 to investigate the optimal conformation of the complex for improved expression efficiency in the target cell.


METHODS
Double-distilled water and adjuvant were added to the naked pcDNA3.1/IFN-gamma, target vector ASOR-PLL and the ASOR-PLL-pcDNA3.1/IFN-gamma complex to create different conformations; molecules that were transfected into BEL7402 cells and the expression efficiency was determined by measuring the IFN-g concentration in the culture supernatant by ELISA.


RESULTS
Naked pcDNA3.1/IFN-gamma DNA distributed linearly in double-distilled water and condensed into a mica configuration in adjuvant; ASOR-PLL had a net-like distribution without adjuvant and a spider-like form in the adjuvant-treated group; the ASOR-PLL-pcDNA3.1/IFN-g complex had a divaricate form without adjuvant, but a bead-like or granular conformation in 0.1 and 0.2 mol/L of adjuvant, a homogeneous bacilliform or chromatoid-shaped conformation in 0.3 mol/L adjuvant, and varied shapes in 0.4 and 0.5 mol/L adjuvant. The supernatant IFN-gamma expression in the bacilliform/chromatoid conformation complex group was the highest among the different conformation groups and controls. When chloroquine was added the supernatant IFN-gamma concentration increased in the liposome group and decreased in the bacilliform/chromatoid conformation group .


CONCLUSIONS
The two structural molecules and their complex, ASOR-PLL-pcDNA3.1/IFN-gamma, were adjustable in the liquid mode. The specific bacilliform/chromatoid conformation of complex was lysosome enzyme-resistant and could play an active role in improving the efficiency of gene expression. The hypothesis that a chromosome-like conformation of the target gene molecule is involved in enhancing exogenous gene expression is proposed.
Liu
Xiao Wei
XW
Department of Gastroenterology, the Second XiangYa Hospital, Central South University and Sheng Life Gene Drug Development Company, Changsha, Hunan Province, China.
Lu
Fang Gen
FG
Wu
Xiao Ping
XP
Ouyang
Chun Hui
CH
Yang
Dong Ye
DY
You
Yu
Y
Lian
Guang Hui
GH
D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology*; D001212:Asialoglycoproteins / Q000737:chemistry* / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology*; D004247:DNA; D004337:Drug Carriers; D004797:Enzyme-Linked Immunosorbent Assay; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry* / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology*; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology*; D010957:Plasmids; D011107:Polylysine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology*; D011487:Protein Conformation; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D001212:Asialoglycoproteins; D004337:Drug Carriers; D009961:Orosomucoid; C057326:asialoorosomucoid-polylysine conjugate; D011107:Polylysine; D007371:Interferon-gamma; D004247:DNA