Introduction

The Revised Authoritative Guide To Vaccine Legal Exemptions

Vaccines Have Serious Side Effects

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Gene therapy is an emerging field for treating diseases by using DNA as the remedy. It can be used to treat systemic diseases and various organ disorders. The goal of gene therapy is to treat a specific disease process with the protein product of the introduced gene. This protein can be either used locally or systemically (e.g., clotting factors and hormones) (1).

An essential component of the revolution in molecular biology has been the arrival of transfer methods by transfection or transduction. In principal, 3 different ways of introducing genetic material consist of (1) viral/bacterial vectors; (2) calcium phopshate, DEAE-dextran, or liposomes; and (3) physical methods such as direct injection, electroporation, or the gene gun (2). Each method has its own inherent strengths and weaknesses, and is especially suitable for a particular application.

In 1987, Sanford and coworkers invented a new addition to the armentarium of gene delivery vehicles, which was called ''biolistic method of gene transfer'' (3). Other terms include ''particle bombardment'' and ''gene gun.'' In principal, the technique implies that DNA is coated onto 1- to 5-^m heavy metal particles (usually gold or tungsten) that were accelerated in the early days using gun powder, today using helium, to sufficient velocity to penetrate the target cells. Historically, the first cells to be penetrated were plant cells (3,4). Its most notable application was the production of the first transgenic crop (i.e., the transfection of maize) (5,6).

To design potential treatment strategies for gene therapy, it is relevant to discuss 2 general approaches: the in vivo approach and the ex vivo approach of gene transfer (Fig. 1). In the in vivo approach, the desired genes are introduced directly into the target organ, whereas in the ex vivo approach, the target cells are cultured from biopsy specimens and the desired gene is inserted while these cells are being propagated in tissue culture. The genetically modified target cells are grown in culture and eventually grafted back onto the donor. Both the in vivo and the ex vivo approaches have their relative advantages and disadvantages, which reflect their potential applications. The biggest advantage of the in vivo approach is that it is simple and direct. In the case of skin, it takes advantage of its easy accessibility. The biggest disadvantage is that expression of the desired gene is usually transient because the gene is introduced locally and only into a limited number of target cells. Generally, stem cells have not been successfully targeted using in vivo approaches with the potential exception of hematopoiesis and liposomal gene transfer (7,8). However, it should be noted that transient expression of the desired gene may be adequate for a variety of applications such as genetic vaccination.

A plethora of different techniques has been developed in gene therapy to enhance the uptake of DNA (containing the gene of interest) (2). Initially, chemical DNA transfection has used calcium chloride and DEAE-dextran to transfer genetic material followed by the use of cationic lipids or liposomes. Because of the low transfection efficiency and the substantial in vivo toxicity of cationic lipids, other methods of gene transfer have increasingly been developed to insert DNA into cells. Viral gene transfer has exploited the capability of recombinant viruses such as retrovirus, adenovirus, or adeno-associated virus to infect cells and efficiently transport the genetic material containing the gene of interest into the cells. In addition, 3 physical techniques have been developed to introduce DNA into target tissues (2). These physical techniques are similar in that they can directly introduce DNA into the target organ such as skin. Consequently, these techniques will generally be used for in vivo approaches. Beside particle bombardment, direct injection using a syringe and a small needle has been

Figure 1 Schematic outline of in vivo and ex vivo gene transfer.

added as another alternative for gene transfer. Upon direct injection of DNA into skin or muscle, the DNA is taken up by the target cells and the desired genes are transiently expressed (9-12). In the case of injected muscle, the transgene can be detected for up to 1 year with some albeit low level of expression.

A variety of tissues has been successfully transfected using direct injection such as epidermis, muscle, thyroid, liver, lung, synovia, and melanoma (13-17). The exact mechanisms of how epithelial cells or muscle cells, the first tissue demonstrated to take up and express naked DNA, accomplish this is not yet clear, but there is evidence that a specialized transport process for small molecules, called potocytosis, may be involved (18,19). The existence of DNA-binding proteins on keratinocyte and muscle membranes is currently being investigated in the laboratory.

Feasibility studies have shown that plasmid DNA can also be expressed upon microinjection into the nucleus of mammalian cells (mouse fibroblast cell line LMTK~), but not when injected into the cytoplasm (20). Furthermore, ultrasound has been demonstrated to allow gene transfer into mammalian cells in culture (21).

Alternative ways of introducing genetic material into the skin consist of various techniques to overcome the epidermal barrier that limits the delivery of plasmid DNA. Such methods comprise applying a pulsed electric field on topically applied plasmid DNA (22). A simpler way for gene transfer to the skin is constant puncturing using a device with oscillating fine needles (23). Topical application of plasmid DNA complexed to various cationic lipids has been demonstrated to allow gene transfer to the skin (24), although this approach has not worked for others. Topical application of plasmid DNA coding for the murine interleukin-10 gene to the scarified cornea has been shown to suppress an ongoing ocular inflammation caused by herpetic keratitis (25). The corneal route also proved effective in generating an immune response when expression plasmids coding for the herpes simplex glycoprotein B were applied (25).

The focus of this chapter is to review the progress made on genetic vaccination procedures using DNA to induce humoral and cytotoxic T cell responses.

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