Enhanced cationic liposome-mediated transfection using the DNA-binding peptide mu (mu) from the adenovirus core.

Promising advances in nonviral gene transfer have been made as a result of the production of cationic liposomes formulated with synthetic cationic lipids (cytofectins) that are able to transfect cells. However few cationic liposome systems have been examined for their ability to transfect CNS cells. Building upon our earlier use of cationic liposomes formulated from 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidyl-ethanolamine (DOPE), we describe studies using two cationic viral peptides, mu (mu) and Vp1, as potential enhancers for cationic liposome-mediated transfection. Mu is derived from the condensed core of the adenovirus and was selected to be a powerful nucleic acid charge neutralising and condensing agent. Vp1 derives from the polyomavirus and harbours a classical nuclear localisation signal (NLS). Vp1 proved disappointing but lipopolyplex mixtures formulated from pCMVbeta plasmid, mu peptide and DC-Chol/DOPE cationic liposomes were able to transfect an undifferentiated neuronal ND7 cell line with beta-galactosidase reporter gene five-fold more effectively than lipoplex mixtures prepared from pCMVbeta plasmid and DC-Chol/DOPE cationic liposomes. Mu was found to give an identical enhancement to cationic liposome-mediated transfection of ND7 cells as poly-L-lysine (pLL) or protamine sulfate (PA). The enhancing effects of mu were found to be even greater (six- to 10-fold) when differentiated ND7 cells were transfected with mu-containing lipopolyplex mixtures. Differentiated ND7 cells represent a simple ex vivo-like post-mitotic CNS cell system. Successful transfection of these cells bodes well for transfection of primary neurons and CNS cells in vivo. These findings have implications for experimental and therapeutic uses of cationic liposome-mediated delivery of nucleic acids to CNS cells.

Amelioration of established collagen induced arthritis by systemic IL-10 gene delivery.

A novel formulation of cationic liposomes containing the novel cytofectin ACHx was used for delivery of an anti-inflammatory cytokine gene, IL-10, to mice with established collagen induced arthritis. A single intraperitoneal injection of human IL-10 expression plasmid complexed with liposomes 2 to 4 days after the onset of arthritis was sufficient to give significant and prolonged amelioration of arthritis for 30 days. Preliminary experiments suggested that the therapeutic effect was IL-10 dose-dependent. The distribution of the human IL-10 DNA after injection was widespread, including the inflamed paws. Human IL-10 mRNA was also detected in the paws 24 h after injection. IL-10 protein was below the level of detection in paws and serum but was detected in some tissues up to 10 days after injection. The target cell of transfection was demonstrated to be the macrophage. These results suggest that systemic therapy with plasmid DNA complexed with cationic liposomes merits further development as an alternative method for anti-inflammatory treatment of arthritis.

Cationic liposome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon.

We have examined the potential of cationic liposomes as a tool for approaches to gene therapy in the CNS. Our previous work has shown that cationic liposomes formulated from 3 beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) could achieve high transfection levels in a neuronal cell line (McQuillin et al. Neuroreport 1997; 8: 1481-1484). We therefore wished to assess transfection efficiencies in organotypic cultures from the brain with a reporter plasmid expressing E. coli beta-galactosidase in order to mimic an in vivo model. Explant cultures were generated according to the method of Stoppini et al (J Neurosci Meth 1991; 37: 173-182) with slight modifications. Brain slices were maintained on transparent porous membranes and were observed to maintain their intrinsic connectivity and cytoarchitecture to a large degree over periods of up to 6 weeks in culture. CNS tissue was obtained from rats at birth or 5 days after birth. After transfection beta-galactosidase expression was detected in cells of both neuronal and non-neuronal morphology. Control cultures were exposed to liposome alone and a plasmid that had the beta-galactosidase gene insert removed. Only low levels of endogenous beta-galactosidase reactivity were seen in these control cultures. DC-Chol/DOPE-mediated transfection was confirmed using a RT-PCR protocol capable of differentiating between untranscribed plasmid DNA and RNA generated from the transfected vector. These results suggest that cationic liposomes, particularly DC-Chol/DOPE liposomes, will be useful as delivery agents for gene transfer to CNS cells in vitro and possibly in vivo.

Endothelial cell transfection with cationic liposomes and herpes simplex-thymidine kinase mediated killing.

Endothelial cells are a promising target for cancer gene therapy because neoangiogenesis is vital for the supply of oxygen and nutrients to solid tumours. However, endothelial cells have been reported to be difficult to transfect. We demonstrate high rates of transfection with the reporter gene pSV40 beta gal using DC-Chol/DOPE cationic liposomes and lower rates with the novel polyamine cationic liposomes ACHx/DC-Chol/DOPE and ACO/DC-Chol/DOPE. Endothelial cells transfected with HSV-thymidine kinase using DC-Chol/DOPE demonstrated 3 log10 increased cytotoxicity compared with controls when exposed to the prodrug ganciclovir, thereby demonstrating significant biological effect.

Enhanced in vitro and in vivo gene delivery using cationic agent complexed retrovirus vectors.

Retroviruses are, at present, the most efficient integrative vectors available for gene delivery. However, these viruses are still limited by relatively low titres. Although several protocols exist to improve virus titre most of them are time-consuming and unable to provide sufficient virus for in vivo applications. Virus titre can be enhanced by polybrene and other cationic agents. By investigating a broad range of cationic agents for their ability to enhance virus infectivity we found that both ecotropic and amphotropic retrovirus infection could be increased. In particular, the lipopolyamine dioctadecylamidoglycylspermine (DOGS) gave up to one order of magnitude enhancement above polybrene-mediated infection without cytotoxicity. To increase virus infectivity further we combined the enhancing effect of DOGS on virus infectivity with concentration of virus particles by ultrafiltration to reach titres of 1 x 10(9) IU/ml. The in vivo transduction of regenerating rat liver, by an amphotropic retrovirus was increased approximately five-fold by the addition of DOGS compared with virus alone. There was no animal toxicity observed following the administration of DOGS. The improved transduction efficiency seen both in vitro and in vivo following the co-administration of DOGS/virus complexes may be useful for future gene therapy applications.

Optimization of liposome mediated transfection of a neuronal cell line.

A cell line derived from sensory neurons was transfected with high efficiency using cationic liposomes, formulated from 3 beta [N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and dioleoyl L-alpha-phosphatidylethanolamine (DOPE). This is the first time that cationic liposomes of this type have been reported to transfect a neuronal cell line. We used a reporter gene construct expressing beta-galactosidase under the control of the cytomegalovirus immediate early promoter and routinely observed transfection efficiencies > 40%. Parameters affecting transfection efficiency were examined and the ratio of DNA to liposome proved to be crucial. Liposome formulation procedures and cell transfection protocols devised here will be used as a basis for further in vivo and in vitro work.