Efficient Transfection of Large Plasmids Encoding HIV-1 into Human Cells-A High Potential Transfection System Based on a Peptide Mimicking Cationic Lipid

Janich, C; Ivanusic, D; Giselbrecht, J; Janich, E; Pinnapireddy, SR; Hause, G; Bakowsky, U; Langner, A; Wolk, C

Abstract

One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

Keywords: gene therapy; cationic lipids; large plasmids; transfection; membrane fusion; HIV

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The HIV-1 Transfection System based on a cationic lipid utilizes positively charged lipid molecules to facilitate the delivery of genetic material into cells. This system enhances the efficiency of HIV-1 viral vector transfection by forming lipid-DNA complexes, which interact with the negatively charged cell membranes, promoting cellular uptake. The cationic lipids also protect the genetic material from degradation, increasing transfection success. This method is particularly advantageous for gene therapy and vaccine development, as it provides a non-viral alternative for introducing therapeutic genes or RNA into target cells. The use of cationic lipid-based systems is a crucial advancement in the field of HIV-1 research, offering a safer and more effective approach to gene delivery.