**1. Introduction**

90 Non-Viral Gene Therapy

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Gene therapy is a therapeutic method used to treat diseases by altering genes within a patient's cells. The concept of gene therapy emerged as molecular biology evolved from the mere discipline of studying DNA molecules to the scientific art of virtually manipulating the genes of cells. Explosive worldwide research was conducted after the first introduction of the concept of gene therapy into the scientific community. The original aim was to directly modify patient genes through *in vivo* gene therapeutic approaches. However, DNA molecules introduced into the body by *in vivo* gene therapy are delivered at a very low frequency into terminally differentiated tissue cells, which typically do not have the capability of self-renewal (Tenenbaum et al., 2003). Because of the short-lived nature of *in vivo* gene therapy, a defective gene in patients is only temporarily corrected by *in vivo* gene therapy (Kaloss et al., 1999). The development of gene therapeutic methods in which the corrected patient gene remains permanently has been actively pursued.

*Ex vivo* gene therapeutic methods have been considered as alternative options to gene therapy to overcome the short-lived nature of the corrected genes of *in vivo* gene therapy. In *ex vivo* gene therapy, the surgically removed adult stem cells, such as mesenchymal stem cells or hematopoietic stem cells, are typically cultured in a laboratory apparatus. The therapeutic DNA molecules are introduced into the isolated cells, and these transfected cells are then introduced into the patients. By using adult stem cells in *ex vivo* gene therapeutic methods, the corrected genes that are introduced are, in most cases, expressed permanently once they are corrected properly because the adult stem cells have the capability of selfrenewal (Dube & Denis, 1995; Muller-Sieburg & Sieburg, 2006; Tseng et al., 2006; Nehlin & Barington 2009). In *ex vivo* gene therapy, genetic manipulation is conducted in a lab outside of the body. However, normal somatic cells, including adult stem cells, do not propagate indefinitely and are vulnerable to epigenetic modification. Therefore, long-term cultures of somatic cells isolated from the body are very difficult to sustain (Beyer & Da sliva, 2006; Tonti & Mannello, 2008). This means that the long-term culture of adult stem cells in *ex vivo* gene therapy should be avoided as much as possible. Therefore, it is absolutely necessary to deliver therapeutic DNA molecules into isolated cells immediately with high efficiency.

The Mechanical Agitation Method of Gene Transfer for *Ex-Vivo* Gene Therapy 93

Fig. 1. Strategies for *in vivo* gene therapy. *In Vivo* gene therapy involves introduction of therapeutic DNA directly into the patient body. The DNA is introduced by cell–specific direct injection into tissue in need. Once inside the body and in contact with the specifically targeted cells, the inserted DNA is incorporated into the tissue cells where it encodes the

The bottleneck in development of *in vivo* gene therapeutic methods has been the development of an efficient method for delivery of a therapeutic gene into the target cells of the body. The main reason for poor gene delivery efficiency in *in vivo* gene therapy is rooted to the nature of the body. The cells in the body are typically surrounded by an extracellular matrix that usually provides structural support to the cells in addition to performing various other important functions (Fig. 2). The main constituent of the body is the extracellular matrix, not cells (Suki & Jason, 2008). For example, collagen proteins, which are one of the components of the extracellular matrix, constitute approximately 25-35% of the protein content of the entire body, implying that the extracellular matrix occupies the main volume of the body (Khan et al., 2009). The injected gene delivery vehicles must pass through the extracellular matrix to deliver therapeutic proteins into target cells in *in vivo* gene therapy. However, because the extracellular matrix spatially occupies such a large portion of the body, there is an unsolvable limitation for efficient gene transfer in *in vivo*

In addition to the low efficiency of gene delivery, *in vivo* gene therapy has another problem. The gene transfer vector is obligatorily exposed to the immune system of the body. This exposure causes an immune response that blocks gene delivery entirely. Overall, the potential immune response is another factor contributing to the low efficiency of gene delivery in *in vivo* gene therapy. Therefore, development of an ideal gene delivery vehicle for *in vivo* gene therapy is so extremely challenging that, until now, none of the *in vivo* gene

In *ex vivo* gene therapy, cells are removed from a patient, maintained in culture to introduce a therapeutic gene into the cells, and then transplanted into the patient (Fig. 3). The role of the transplanted cells, which are genetically modified, is to deliver a recombinant gene

production of the needed protein.

therapeutic methods have not a satisfactory result.

gene therapy.

**2.2** *Ex vivo* **gene therapy** 

In current gene transfer protocols, gene delivery vehicles containing therapeutic DNA molecules make only limited contacts with their target cells by passive diffusion, thereby limiting the chances of gene delivery. In our lab, we developed a very efficient method to deliver therapeutic genes to adult stem cells based on mechanical agitation (Park et al., 2009).

In this method, mechanical agitation of the gene delivery vehicles containing cell suspensions increases the movement of gene delivery vehicles and target cells, resulting in an increase in contact between them. The application of our mechanical agitation method to the gene delivery process of *ex vivo* gene therapy, both in transfection and transduction, has increased the gene transfer efficiency more than that of any other previously known gene transfer protocol.
