In vivo gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector

Abstract

Lipoproteins are emulsion particles that consist of lipids and apolipoproteins. Their natural function is to transport lipids and͞or cholesterol to different tissues. We have taken advantage of the hydrophobic interior of these natural emulsions to solubilize DNA. Negatively charged DNA was first complexed with cationic lipids containing a quaternary amine head group. The resulting hydrophobic complex was extracted by chloroform and then incorporated into reconstituted chylomicron remnant particles (Ϸ100 nm in diameter) with an efficiency Ϸ65%. When injected into the portal vein of mice, there were Ϸ5 ng of a transgene product (luciferase) produced per mg of liver protein per 100 g injected DNA. This level of transgene expression was Ϸ100-fold higher than that of mice injected with naked DNA. However, such a high expression was not found after tail vein injection. Histochemical examination revealed that a large number of parenchymal cells and other types of cells in the liver expressed the transgene. Gene expression in the liver increased with increasing injected dose, and was nearly saturated with 50 g DNA. At this dose, the expression was kept at high level in the liver for 2 days and then gradually reduced and almost disappeared by 7 days. However, by additional injection at day 7, gene expression in the liver was completely restored. By injection of plasmid DNA encoding human ␣1-antitrypsin, significant concentrations of hAAT were detected in the serum of injected animals. This is the first nonviral vector that resembles a natural lipoprotein carrier.

Successful expression of β-galactosidase and factor IX transgenes in fetal and neonatal sheep after ultrasound-guided percutaneous adenovirus vector administration into the umbilical vein

“…Approaches to achieve longer lasting and repeated gene expression could include the application at pre-immune stages of pregnancy 15 and/or the use of less immunogenic vectors such as the new generation of 'gutless' adenoviral vectors, 23 or of non-viral vector systems. [24][25][26] Alternatively, and probably even more suitable, the fetal organs may contain a sufficient number of dividing cells with stem cell character to achieve permanent transduction by retroviral/lentiviral 27,28 or adeno-associated virus vectors. 21,22,29 A related problem which may be overcome with the future refinement of the vector systems is the acute doserelated toxicity seen in our investigations.…”

Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes

“…Although some of the virus-mediated gene transfer systems have been found to be quite effective, their usefulness is limited, given that they induce an immune response, leading to the rapid rejection of transduced cells. To overcome this problem, artificial, non-viral gene delivery systems are being developed, including small liposomes, [6][7][8] particle bombardment, 9 gene gun, 10 electroporation, 11 chylomicron remnants 12 and cationic polymers. 13 Successful transfer and expression in the liver can also be achieved by systemic administration of naked plasmid using a hydrodynamic-based procedure.…”

Long-term expression of human alpha1-antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a hydrodynamics-based procedure

“…A variety of vectors have been used for introducing genes into the liver, including recombinant retrovirus, [1][2][3][4][5] adenovirus, 6-10 adeno-associated virus, [11][12][13] and nonviral vectors such as cationic polymers, [14][15][16] liposomes, 17,18 and reconstituted chylomicron remnants. 19 Although significant progress has been made in demonstrating successful gene delivery to the liver, a number of remaining difficulties and technical problems are associated with each of these methods. While retroviral vectors can generate a long-term expression of the introduced gene in the liver by using either ex vivo or direct injection, the procedure normally requires partial hepatectomy 20 or liver damage 2 in order to stimulate cell division.…”