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

Abstract

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...

Enhancement of calcium phosphate-mediated transfection by inclusion of adenovirus in coprecipitates

“…As indicated above, adenovirus has been incorporated into other gene transfer vectors, including cationic lipids and receptortargeted polylysine complexes. [5][6][7][8]10,12,14 In these cases adenovirus is also thought to facilitate escape of the plasmid from the endosome.…”

“…A number of methods have been developed to facilitate plasmid-mediated gene transfer, including direct injection, electroporation, and incorporation of plasmid in complexes with cationic lipids, polylysine, and other molecules. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] The use of calcium phosphate (CaPi) to coprecipitate DNA was one of the first methods used for DNA transfer to cells. 16 CaPi, as well as cationic lipids and other cationic polymers, condense the DNA and facilitate its uptake into the cell.…”

“…A similar strategy has been described in which adenovirus was included in complexes of DNA plus a cationic molecule (polylysine or cationic lipids), and in some cases a targeting moiety. [5][6][7][8]10,12,14 In such strategies, the adenovirus was either covalently linked to the cationic molecule or included in the complex via electrostatic interactions. Including adenovirus in the complex by either method markedly improved delivery and expression of plasmid DNA.…”

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

“…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. 14,15 One major concern for this technique is that it requires an injection volume of 8% of the body weight, which may alter the physiological conditions of the liver.…”

Gene therapy in the CNS

“…Coupling polylysine to a specific ligand, eg transferrin or insulin, induces endocytosis and targets DNA transfer to specific cell types. 108,109 Cationic lipids condense and encapsulate DNA in positively charged complexes (liposomes) that enter the cell by endocytosis. To avoid degradation by the endocytic pathway and to facilitate fusion with the cell membrane, fusion proteins derived from Sendai virus have been incorporated into liposomes.…”