POSTER NO: 302

A novel DNA-peptide complex for efficient gene delivery in mammalian cells

¹I.A. Ignatovich, ¹E.B. Dizhe, ¹S.V. Orlov, ²A.V. Pavlotskaya, ²S.V. Burov, ¹A.P. Perevozchikov
¹Institute of Experimental Medicine, Academy of Medical Sciences, St.-Petersburg, Russia, ²aInstitute of Macromolecular Compounds, Academy of Sciences, St.-Petersburg, Russia

The major challenge in development of gene therapy strategies is design of carriers for gene delivery into cells. The most efficient means for delivering genes to target cells are based on viral-mediated gene transfer (mainly by recombinant adenovirus or retrovirus vectors). However, those methods have many disadvantages including immunogenicity, toxicity (for adenoviral vectors) and risk of side effects (for retroviral vectors). The alternative approach is using of non-viral gene delivery systems (molecular conjugates, cationic oligopeptides, cationic lipids, etc.). Although the ones are less effective than viral vectors, they are lack of mentioned above disadvantages and may be used repeatedly.

TAT is a transcription factor involved in HIV genome replication in virus-infected cells. It was shown recently, its basic region (47-57 a.a.) having a positive charge is responsable for non-receptor-mediated TAT transfer following nuclear translocation into cells. Corresponding TAT-peptide is used for effective transduction of fusion proteins into cells. The previous attempts to use TAT-peptide for plasmid DNA delivery were unsuccessful.

In present work we show direct complex formation between plasmid DNA and TAT-peptide through electrostatic interactions. The stehiometry of DNA/TAT complexes was studied. Plasmid DNA is capable of binding with TAT-peptide up to 1.7 fold excess of complex positive charge. Dependence of TAT/DNA complex formation on ionic strangth was investigated. Conditions for cooperative interactions between TAT-peptide and plasmid DNA were analyzed by DNaseI protection assay. Our previous studies of electrostatically formed DNA/cationic peptide complexes allowed us to suggest that they may be very efficient in delivery of foreign DNA into the cells if these complexes are positive charged. According to this suggestion the efficacy of beta-galactosidase expression vector delivery into human hepatoma (HepG2) and murine fibroblasts (NIH 3T3) cells was increased when charge of DNA/TAT-peptide complexes was changed to positive. Interestingly, the optimum charge ratio of peptide:DNA within complex was 3:1, that suggests about the stimulatory role of free TAT-peptide for DNA/TAT-complexes transfer into cells. To compare the mechanisms of intracellular transfer of TAT-peptide and DNA/TAT-complexes the role of temperature was investigated. It is well known, the transfer of TAT-peptide alone into cells doesn't depend on temperature. Surprisingly, the efficiency of DNA delivery was severely decreased when the experiments were performed under 4°C. Kinetics of transfer of DNA/TAT-complexes and TAT-peptide alone into cells was studied by using FITC-labeled TAT-peptide.

Obtained results allowed us to formulate the working hypothesis about mechanisms of DNA/TAT-peptide complex transfer into mammalian cells. Moreover, our data are the first evidence for perspective usage of TAT-peptide for delivery of foreign DNA into human cells.