Highly Efficient Retrovirus-Mediated Gene Transfer into Rat Hepatocytes<i>In Vivo</i>

Kitten, Olivier; Cosset, François‐Loïc; Ferry, Nicolas

doi:10.1089/hum.1997.8.12-1491

Cited by 60 publications

(51 citation statements)

References 19 publications

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“…Many different gene delivery methods are available including cationic lipid-based DNA transfection [44][45][46][47][48][49] and retroviral and adenoviral gene transfer vectors; [50][51][52][53][54][55] however, the use of these methods for hepatocytes has not been effective because hepatocytes do not divide, are highly susceptible to toxicity, and transfection efficiencies in hepatocytes are extremely low (HC Isom, unpublished data). 11 Previous studies have reported baculovirus-mediated gene delivery to primary hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

et al. 2003

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Gene delivery to differentiated hepatocytes is notoriously difficult. Hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented medium acquire paracellular junctions, arrange themselves in multicellular islands and are an excellent in vitro model for studying liver function. Baculovirus-mediated gene delivery to hepatocytes in this culture system is restricted to peripheral cells of the islands. However, this limitation can be overcome by transient calcium depletion of the cells prior to and during baculovirus infection. Examination of the mechanism underlying this process revealed that calcium depletion was accompanied by a transient loss of intercellular contacts and paracellular junction complex integrity, increased distance between adjoining cells, and internalization of the tight junction protein, zona occludens ZO-1.Internalization of ZO-1 was accompanied by baculovirus infection of internal cells of hepatocyte islands. When calcium levels were restored, paracellular junction complex integrity returned to normal by 12 h. No permanent alterations in hepatocyte ultrastructure and albumin mRNA, and protein expression were caused by this gene transfer method. Loss in paracellular junction complex integrity exposes the basolateral (sinusoidal) surface of hepatocytes resulting in homogeneous baculovirus-mediated gene delivery to approximately 75% of the cells in long-term DMSO culture. We conclude that the use of recombinant baculovirus as a vector in combination with transient calcium depletion is a highly efficient method for delivering exogenous genes to hepatocytes without loss of hepatic differentiation.

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Section: Discussionmentioning

confidence: 99%

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

et al. 2003

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“…1 Asialofetuin (AF), a glycoprotein having triantennary galactose terminal sugar chains, is known as an excellent ligand molecule selectively recognized by ASGPr. 2 For liver gene delivery, both virus-mediated [3][4][5] and non-viral systems have been considered. 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.…”

Section: Introductionmentioning

confidence: 99%

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

2003

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A novel lipidic vector composed of DOTAP/Chol liposomes, asialofetuin (AF), protamine sulfate and DNA has been developed. The resulting protamine-AF-lipoplexes improved significantly the levels of gene expression in cultured cells and in the liver upon i.v. administration. Lipoplexes containing the optimal amount of AF (1 mg/mg DNA) showed a 16-fold higher transfection activity in HepG2 cells than nontargeted (plain) complexes. The uptake by cells having asialoglycoprotein receptors (ASGPr) on their plasma membrane was decreased by the addition of free AF, indicating that AF-lipoplexes were taken up specifically by cells via ASGPr-mediated endocytosis. Results from transfections performed in cells defective in ASGPr, ie HeLa cells, confirmed this mechanism. By addition of the condensing peptide, protamine sulfate, smaller complexes were obtained, which enhanced even more the uptake of AFcomplexes in HepG2 cells and in the liver. The optimal amount of protamine was 0.4 mg/mg DNA, and gene expression was about 5-fold over that obtained with AF-lipoplexes in the absence of the peptide, and 75-fold higher than that with plain conventional lipoplexes. Protamine-AF-lipoplexes increased by a factor of 12 luciferase gene expression in the liver of mice administered systemically via the tail vein, compared to plain complexes. In summary, our findings extend the scope of previous studies where AF-lipoplexes were used to introduce DNA into hepatocytes. The combination of targeting and protamine condensation obviated the need for partial hepatectomy, commonly required to obtain efficient gene delivery in this organ. Since protamine sulfate has been proven to be non-toxic in humans, the novel liverspecific vector described here may be useful for the delivery of clinically important genes to this organ.

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“…Liver is an important target organ for gene delivery due to its function in synthesizing many serum proteins and its involvement in numerous genetic and acquired diseases. 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.…”

Section: Introductionmentioning

confidence: 99%

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

2000

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The liver is an important target organ for gene transfer due to its large capacity for synthesizing serum proteins and its involvement in numerous genetic and acquired diseases. Previously, we and others have shown that an efficient gene transfer to liver cells in vivo can be achieved by an intravenous injection of plasmid DNA using a hydrodynamics-based procedure. In this study, we systematically characterized the expression of transgene encoding a secretory protein in mouse. Using human ␣1-antitrypsin (hAAT) gene as a reporter, we demonstrate that the serum level of hAAT can reach as high as 0.5 mg/ml by a simple

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Highly Efficient Retrovirus-Mediated Gene Transfer into Rat HepatocytesIn Vivo

Cited by 60 publications

References 19 publications

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

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

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

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