Evidence for targeted gene transfer by receptor-mediated endocytosis

641

336

Complexes containing plasmid DNA, transferrin-polylysine conjugates, and polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or -galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with polylysine, (i) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (Mi) the release from endosomes by using membrane-disrupting influenza peptides. The presence of these influenza peptide conjugates in the DNA complexes renders the complexes active in membrane disruption in a liposome leakage assay and results in a substantial augmentation of the transferrin-polylysine-mediated gene transfer.

mentioning

confidence: 99%

Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle.

Wagner

Plank

Zatloukal

et al. 1992

641

336

“…The classical procedure makes use of retroviruses with which efficacious transfer and expression of genes is usually obtained. We and others (1,2) have explored the alternative method of transferring genes by receptor-mediated endocytosis (3)(4)(5). In particular, we have used the transferrin/ transferrin-receptor system to facilitate endocytotic transport of DNA.…”

mentioning

confidence: 99%

Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes.

Wagner

Zatloukal

Cotten

et al. 1992

450

215

We are developing efficient methods for gene transfer into tissue culture cells. We have previously shown that coupling of a chimeric adenovirus with polylysine allowed the construction of an adenovirus-polylysine-reporter-gene complex that transferred the transporter gene with great efficiency into HeLa cells. We have now explored simpler, biochemical means for coupling adenovirus to DNA/polylysine complexes and show that such complexes yield virtually 100% transfection in tissue culture cell lines. In these methods adenovirus is coupled to polylysine, either enzymatically through the action of transglutaminase or biochemically by biotinylating adenovirus and streptavidinylating the polylysine moiety. Combination complexes containing DNA, adenovirus-polylysine, and transferrin-polylysine have the capacity to transfer the reporter gene into adenovirus-receptor-and/or transferrinreceptor-rich cells.Gene therapeutic manipulations in vivo or in vitro require efficient methods of gene transfer in chosen target cells. The classical procedure makes use of retroviruses with which efficacious transfer and expression of genes is usually obtained. We and others (1, 2) have explored the alternative method of transferring genes by receptor-mediated endocytosis (3-5). In particular, we have used the transferrin/ transferrin-receptor system to facilitate endocytotic transport of DNA. In some tissue culture cells transferrinpolylysine/DNA conjugates provided a very efficient vector for gene transfer. Thus, after transferrinfection of the erythroleukemic cell line K-562 virtually 100%o of the cell population was found to express the transfected reporter gene for a protracted period of days (6).DNA delivered by receptor-mediated endocytosis suffers from the limitation that endocytosed DNA is trapped in intracellular vesicles and later largely destroyed by lysosomal action. Addition of chloroquine during transfection, preventing acidification of the endosomal and lysosomal compartment, is one measure to ensure better survival and transfer of DNA into the nuclear compartment (4). Simultaneous addition of (replication-deficient) adenoviruses during transfection is another (7). The added adenoviruses are thought to disrupt endosomes upon endocytosis and to admit DNA into the cytoplasm and eventually into the nucleus. The adenovirus-aided transferrinfection was found experimentally to augment gene transfer and expression of a reporter gene by as much as a factor of 1000 in HeLa (7,8) or BNL CL.2 (8), whereas without adenovirus, transfection efficiency, even in the presence of chloroquine, was moderate to poor for these cell types. For adenovirus-aided transfection (7, 8) the simultaneous presence of adenovirus receptor and transferrin receptor on target cells is a precondition for efficient gene transfer.We have recently demonstrated that coupling of adenovirus to polylysine/DNA complexes by means of an adenovirus-specific antibody (see Fig. 1 C) results in efficient transfer of DNA into cells with high levels of adenovirus r...

“…(i) Polymeric DNA-binding cations (such as polylysine, protamine, and cationized albumin) linked to cell targeting ligands [such as asialoorosomucoid (8), insulin (9), galactose…”

mentioning

confidence: 99%

Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artificial viruses.

Rémy

Kichler

Mordvinov

et al. 1995

240

117

Optimal in vitro gene delivery with cationic lipids requires an excess of cationic charges with respect to DNA phosphates. In these conditions, in vivo delivery will be hampered by interference from cationic lipid-binding macromolecules either circulating or in the extracellular matrix. To overcome this problem, we are developing a modular transfection system based on lipid-coated DNA particles reminiscent of enveloped viruses. The particle core consists of the lipopolyamine-condensed nucleic acid in an electrically neutral ratio to which other synthetic lipids with key viral properties are hydrophobically adsorbed.