Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.

Abstract

We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV Although extensive progress has been made, these techniques suffer from variable transfection efficiency, significant concern about possible recombination with endogenous virus, cellular toxicity, and immunologic host response reactions. These concerns have prompted the search for nonviral DNA transfection conditions for many cell types. Although nonviral techniques have overcome some of the problems of the viral systems, there remains a need for improved transfection efficiency in these newer nonviral systems (7, 13), although improved efficiency can be variably attained by the promoter enhancer elements utilized in the plasmid DNA constructs (19).As a nonviral system, liposomes have been used to encapsulate and deliver to cells a variety of materials, including nucleic acids (3-5) and viral particles (6,7,18,29). Recently, positively charged liposomes containing the membrane fusion- Of the viral vector systems, the recombinant adeno-associated virus (AAV) transduction system has proven to be one of the most efficient vector systems to stably carry the genes with high efficiency into a variety of mammalian cell types (17). AAV is a linear single-stranded DNA parvovirus which is dependent upon coinfection by a second unrelated virus to undergo productive infection. AAV carries two sets of functional genes: the rep genes, which are necessary for viral replication, and the structural capsid protein genes (10, 27). The rep and capsid genes of AAV can be replaced by a desired DNA fragment to generate AAV plasmid DNA. Transcomplementation of rep and capsid genes is required to create a recombinant virus stock. Upon infection, the recombinant virus uncoats in the nucleus and integrates into the host genome by its molecular ends (16,21). It has been well documented that AAV DNA integrates into cellular DNA as one to several tandem copies joined to cellular DNA through the termini (1, 14, 23).The AAV terminal repeats are an essential part of this transduction system, and recombinant virus is made primarily to transport the AAV genome into the cells. We examined whether AAV plasmid DNA transported into the cells by a nonviral system could be used to create systems that efficiently transfect primary mammalian cell types. The system developed here takes advantage of the simple carrier system of lipofection and the proficient expression capability of the AAV plasmid construct.

A novel N-acyl phosphatidylethanolamine-containing delivery vehicle for spermine-condensed plasmid DNA

“…The fact that N-C12-DOPE/DOPC (70:30) had much better transfection efficiency than EPC can be explained by both the better encapsulation efficiency and the cation-dependent fusogenic nature of the former composition. 33 Transfections of primary ovarian cancer cells have been previously reported using non-liposomal systems, such as retroviral 50 and cationic lipid vectors, 51 at efficiencies as high as 10-60% of the cells. This assessment depends of course on the plasmid and the sensitivity of the particular assay used and the amount of subliminal expression.…”

Lipid-mediated gene transfer of viral IL-10 prolongs vascularized cardiac allograft survival by inhibiting donor-specific cellular and humoral immune responses

“…46 Plasmid DNA was combined with the cationic lipid N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium bromide/dioleoyl phosphatidylethanolamine (␥AP DLRIE/DOPE), which was generously provided by VICAL (San Diego, CA, USA). DNAliposome complexes were diluted in lactated Ringer's solution and incubated for 10 min at room temperature before being perfused into donor hearts.…”

Efficient coexpression and secretion of anti-atherogenic human apolipoprotein AI and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors

“…In addition, the inverted terminal repeat sequences (ITRs) of AAV potentially facilitate entry and retention of the transfected plasmid DNA in target cell nuclei, and may allow vector integration into genomic DNA. Such nuclear accumulation of the transfected AAV plasmid DNA should enhance both the level and persistence of expression as demonstrated in vitro 38 and in vivo. 21 Our findings provide important support for use of this combination gene delivery system.…”