Optimisation of intradermal DNA electrotransfer for immunisation

Abstract

The development of DNA vaccines requires appropriate delivery technologies. Electrotransfer is one of the most efficient methods of non-viral gene transfer. In the present study, intradermal DNA electrotransfer was first optimised. Strong effects of the injection method and the dose of DNA on luciferase expression were demonstrated. Pre-treatments were evaluated to enhance DNA diffusion in the skin but neither hyaluronidase injection nor iontophoresis improved efficiency of intradermal DNA electrotransfer. Then, DNA immunisation with a weakly immunogenic model antigen, luciferase, was investigated. After intradermal injection of the plasmid encoding luciferase, electrotransfer (HV 700 V/cm 100 μs, LV 200 V/cm 400 ms) was required to induce immune response. The response was Th1-shifted compared to immunisation with the luciferase recombinant protein. Finally, DNA electrotransfer in the skin, the muscle or the ear pinna was compared. Muscle DNA electrotransfer resulted in the highest luciferase expression and the best IgG response. Nevertheless electrotransfer into the skin, the muscle and the ear pinna all resulted in IFN-γ secretion by luciferase-stimulated splenocytes suggesting that an efficient Th1 response was induced in all case.

Introduction

The skin is an attractive target for antigen delivery and immunisation [1]. It is accessible, easy to assay and to remove if problems occur. After gene transfer, the encoded protein may exert local or systemic effect. As the half-life of skin cells is short, other organs and particularly the muscle are more appropriate for long term expression of proteins [2]. Nevertheless in the context of vaccination, long term expression is not required. The skin acts as an immunological barrier, containing a high density of immunocompetent cells. Although Langerhans cells represent only 1 to 4% of the total cells in the epidermis, it is believed that they cover over 25% of the skin area [3]. These antigen-presenting cells greatly contribute to develop immune responses after DNA delivery.

For gene therapy, the use of non-viral DNA offers several advantages: (i) lack of immunogenicity of the vector, (ii) absence of size limit for the therapeutic cassette, (iii) simpler GMP (Good Manufacturing Practice) production and (iv) improved safety and toxicity profiles. However, topical application or intradermal injection of naked DNA has so far resulted in low transgene expression [4], [5]. This is why different chemical, mechanical and physical methods have been developed to enhance non-viral DNA delivery to skin cells (for review [6], [7]).

Electrotransfer is one of the most efficient and promising methods of non-viral gene transfer. It involves plasmid injection into the tissue and application of electric pulses. It is hypothesised that the electric field plays a double role in DNA transfection. First, it transitorily disturbs membranes, and thus increases cells permeability. Second, it promotes electrophoresis of negatively charged DNA [8], [9]. However, the relation between these different effects of the electric field and transfection efficiency is controversial and still to be elucidated [10]. Volts, duration of pulses and the more appropriate type of electrodes must be evaluated for each tissue. A previous study has demonstrated that a combination of a short high-voltage pulse (HV) and a long duration low-voltage pulse (LV) was efficient for DNA electrotransfer in the skin [11]. For muscle transfection, we used a classical and validated procedure consisting in delivery of a series of identical electric pulses [12]. Widera et al. demonstrated that electroporation increased DNA vaccine delivery and immunogenicity in the muscle [13].

The aim of this research was to optimise intra-dermal DNA immunisation by electrotransfer. The effect of parameters such as the injection method or the dose of DNA was investigated. The effect of different pre-treatments to promote plasmid distribution before the electrotransfer was also studied. The first pre-treatment consisted in the application of an iontophoretic current to enhance DNA diffusion. Iontophoresis consists in using a low electric field to promote the movement of ions into tissues. This technique has been used for many years to deliver drugs or oligonucleotides into the eye or into the skin [14], [15], [16]. The second pre-treatment consisted in hyaluronidase injection to break down extra-cellular matrix components and facilitate plasmid distribution. Hyaluronic acid is an ubiquitous glycosaminoglycan of the extra-cellular matrix present around muscular fibres and in the skin, which contains approximately one-half of the hyaluronic acid of the body [17]. As a pre-treatment, bovine hyaluronidase has been shown efficient to enhance electrotransfer into the muscle [18], but its efficacy into the skin had not yet been investigated.

The immune response was evaluated using luciferase as a model antigen. Luciferase gene is widely used as reporter gene. Usually this protein, which is expressed intra-cellularly, induces no immune response. However, immune response occurs when high luciferase expression is reached in the muscle [19]. Luciferase was chosen because we considered that a protein with limited immunogenicity was a better model of the tumor antigens, which are often poorly immunogenic. Because the route and gene administration parameters influence the immune response [20], [21], [22], [23], electrotransfers into skin, muscle and ear pinna were performed.