**2. Basic principles of current gene therapeutic approaches**

Gene therapy is classified as somatic gene therapy or germ line gene therapy. The application of current molecular genetic techniques used during the manipulation of transgenic or knock-out animals would definitely make gene therapy possible in virtually any type of germ line. However, all civilized societies in the world currently legally prohibit any attempts to genetically modify embryos. Thus far, gene therapy essentially implies somatic gene therapy. Compared to the easy genetic manipulation of embryonic stem cells, the genetic manipulation of somatic cells, including adult stem cells, is limited such that none of the somatic gene therapies are used practically thus far. Therefore, it is not surprising that the main quest of current gene therapy is to improve the efficiency of genetic manipulation in gene therapy, and the future success of gene therapy depends on the efficiency of genetic manipulation.

Genetic manipulation in gene therapy can be achieved by two different approaches: direct genetic manipulation of somatic cells in the body and genetic manipulation of autologous cells outside of the body. These two different strategies for gene delivery are termed *in vivo* and *ex vivo*, respectively. In the *in vivo* strategy, therapeutic genes are delivered into cells *in situ* using a variety of vectors to produce therapeutic proteins in specific sites in the body. In *ex vivo* gene therapy, genetically modified autologous cells are surgically implemented into the body. The *ex vivo* and *in vivo* gene therapies both have positive and negative aspects. Although gene therapy has been a very hot topic in biomedical science for several decades, it is still in its infancy, and a number of hurdles must be overcome to achieve the practical application of gene therapy to patients.

#### **2.1** *In vivo* **gene therapy**

*In vivo* gene therapy is a process in which a therapeutic gene is delivered through a vector directly into the target cells of patients to produce a therapeutic effect that prevents or treats diseases (Fig. 1). Theoretically, once an ideal gene delivery vehicle for a therapeutic gene transfer is developed, the *in vivo* gene therapy should involve a very simple procedure: the injection of a solution containing the gene delivery vehicle into the body. Because of this potentially easy treatment procedure in clinics, *in vivo* gene therapy is considered the preferred gene therapeutic method than *ex vivo* gene therapy. However, *in vivo* gene therapy has a basic and fundamental problem in the delivery of therapeutic genes to target cells: the low efficiency of gene transfer.

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

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

Gene therapy is classified as somatic gene therapy or germ line gene therapy. The application of current molecular genetic techniques used during the manipulation of transgenic or knock-out animals would definitely make gene therapy possible in virtually any type of germ line. However, all civilized societies in the world currently legally prohibit any attempts to genetically modify embryos. Thus far, gene therapy essentially implies somatic gene therapy. Compared to the easy genetic manipulation of embryonic stem cells, the genetic manipulation of somatic cells, including adult stem cells, is limited such that none of the somatic gene therapies are used practically thus far. Therefore, it is not surprising that the main quest of current gene therapy is to improve the efficiency of genetic manipulation in gene therapy, and the future success of gene therapy depends on the

Genetic manipulation in gene therapy can be achieved by two different approaches: direct genetic manipulation of somatic cells in the body and genetic manipulation of autologous cells outside of the body. These two different strategies for gene delivery are termed *in vivo* and *ex vivo*, respectively. In the *in vivo* strategy, therapeutic genes are delivered into cells *in situ* using a variety of vectors to produce therapeutic proteins in specific sites in the body. In *ex vivo* gene therapy, genetically modified autologous cells are surgically implemented into the body. The *ex vivo* and *in vivo* gene therapies both have positive and negative aspects. Although gene therapy has been a very hot topic in biomedical science for several decades, it is still in its infancy, and a number of hurdles must be overcome to achieve the practical

*In vivo* gene therapy is a process in which a therapeutic gene is delivered through a vector directly into the target cells of patients to produce a therapeutic effect that prevents or treats diseases (Fig. 1). Theoretically, once an ideal gene delivery vehicle for a therapeutic gene transfer is developed, the *in vivo* gene therapy should involve a very simple procedure: the injection of a solution containing the gene delivery vehicle into the body. Because of this potentially easy treatment procedure in clinics, *in vivo* gene therapy is considered the preferred gene therapeutic method than *ex vivo* gene therapy. However, *in vivo* gene therapy has a basic and fundamental problem in the delivery of therapeutic genes to target cells: the

**2. Basic principles of current gene therapeutic approaches** 

al., 2009).

transfer protocol.

efficiency of genetic manipulation.

application of gene therapy to patients.

**2.1** *In vivo* **gene therapy** 

low efficiency of gene transfer.

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 production of the needed protein.

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* gene therapy.

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 therapeutic methods have not a satisfactory result.

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

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

The Mechanical Agitation Method of Gene Transfer for *Ex-Vivo* Gene Therapy 95

Fig. 3. Strategies for *Ex Vivo* Gene Therapy. *Ex vivo* gene therapy is performed with the genetic alterations of patients target cells happening outside of the body in as culture. Target

current molecular biological techniques can solve the low efficiency of gene transfer. *Ex vivo* gene therapy has the potential to ultimately solve this major problem of gene therapy, and it could be practically used in clinics. However, normal somatic cells, including adult stem cells, do not only propagate well in a typical cell culture environment and are also vulnerable to epigenetic modification (Islam et al., 2007; Martinez-Climent et al., 2006), requiring that the transfer of therapeutic genes to the isolated cells be performed as soon as possible. Therefore, one of the key factors for the success of *ex vivo* gene therapy is to deliver therapeutic DNA molecules into isolated cells promptly with high efficiency. If these problems could be solved successfully, the *ex vivo* technique could be practically applied to

As the name implies, the success of gene therapy depends on introducing therapeutic genes into target cells with high efficiency. Since Friedmann and Roblin formulated the concept of gene therapy in 1972 (Friedmann & Roblin, 1972), the biggest challenge in gene therapy has been the development of a method to deliver therapeutic genes to target cells with high efficiency. Although gene delivery in *in vivo* gene therapy is much easier than in *ex vivo* gene therapy, gene delivery into primary cells of *in vitro* cell cultures is also quite difficult.

cells from the patient are infected with a recombinant virus containing the desired therapeutic gene. These modified cells are then reintroduced into the patient body, where

they produce the needed proteins that correspond to the inserted gene.

**3. Current methods for gene delivery in** *ex vivo* **gene therapy** 

patients in the near future.

Fig. 2. Typical Anatomical Structure of Connective Tissue. A) The Confocal Microscopic Image of a Mouse Connective Tissue. B) Schematic Illustration Depicting Extracellular Matrix. The extracellular matrix (ECM) is the extracellular part of animal tissue that usually provides structural support to the animal cells in addition to performing various other important functions. The extracellular matrix is the defining feature of connective tissue in animals.

product into the patient's body. The genetically modified cells are not required to reconstitute a particular organ or tissue for the purpose of reimplementation of the cells in a location where the cells were originally obtained. For example, genetically modified hepatocytes harvested from one liver lobe may be re-infused throughout any part of the liver of patients in *ex vivo* gene therapy.

The main disadvantage of *ex vivo* gene therapy is that it requires the surgical removal of cells from the body and transplantation of the cells back to the body. These surgical steps are very painful. However, *ex vivo* therapy has several advantages over *in vivo* gene therapy. First, the efficiency of gene transfer into the targeted cells is very high compared to *in vivo* gene therapy because gene delivery is performed under controlled, optimized conditions. Second, the transduced cells can be enriched if the vector has a selectable gene marker. Third, the immunological side effects that are caused by gene delivery vehicles in *in vivo* gene therapy are usually minimized in *ex vivo* gene therapy.

**2.3** *Ex-vivo* **gene therapy as a practical option to correct a defective gene permanently**  *In vivo* gene therapy introduces the therapeutic genes directly into the patient by intravascular injection. Because this approach is much simpler and less technically demanding than *ex vivo* gene therapy, which requires two surgical steps, the science of *in vivo* gene therapy has been preferentially developed. However, as discussed above, the nature of the mammalian body imposes innate, unsolved problems for *in vivo* gene therapy

to achieve gene expression at therapeutically effective levels. The limitation of *in vivo* gene therapy for practical application is mainly due to low efficiency gene transfer. Because genetic manipulations are conducted in a lab in *ex vivo* gene therapy, the application of

Fig. 2. Typical Anatomical Structure of Connective Tissue. A) The Confocal Microscopic Image of a Mouse Connective Tissue. B) Schematic Illustration Depicting Extracellular Matrix. The extracellular matrix (ECM) is the extracellular part of animal tissue that usually provides structural support to the animal cells in addition to performing various other important functions. The extracellular matrix is the defining feature of connective tissue in

product into the patient's body. The genetically modified cells are not required to reconstitute a particular organ or tissue for the purpose of reimplementation of the cells in a location where the cells were originally obtained. For example, genetically modified hepatocytes harvested from one liver lobe may be re-infused throughout any part of the

The main disadvantage of *ex vivo* gene therapy is that it requires the surgical removal of cells from the body and transplantation of the cells back to the body. These surgical steps are very painful. However, *ex vivo* therapy has several advantages over *in vivo* gene therapy. First, the efficiency of gene transfer into the targeted cells is very high compared to *in vivo* gene therapy because gene delivery is performed under controlled, optimized conditions. Second, the transduced cells can be enriched if the vector has a selectable gene marker. Third, the immunological side effects that are caused by gene delivery vehicles in *in vivo*

**2.3** *Ex-vivo* **gene therapy as a practical option to correct a defective gene permanently**  *In vivo* gene therapy introduces the therapeutic genes directly into the patient by intravascular injection. Because this approach is much simpler and less technically demanding than *ex vivo* gene therapy, which requires two surgical steps, the science of *in vivo* gene therapy has been preferentially developed. However, as discussed above, the nature of the mammalian body imposes innate, unsolved problems for *in vivo* gene therapy to achieve gene expression at therapeutically effective levels. The limitation of *in vivo* gene therapy for practical application is mainly due to low efficiency gene transfer. Because genetic manipulations are conducted in a lab in *ex vivo* gene therapy, the application of

animals.

liver of patients in *ex vivo* gene therapy.

gene therapy are usually minimized in *ex vivo* gene therapy.

Fig. 3. Strategies for *Ex Vivo* Gene Therapy. *Ex vivo* gene therapy is performed with the genetic alterations of patients target cells happening outside of the body in as culture. Target cells from the patient are infected with a recombinant virus containing the desired therapeutic gene. These modified cells are then reintroduced into the patient body, where they produce the needed proteins that correspond to the inserted gene.

current molecular biological techniques can solve the low efficiency of gene transfer. *Ex vivo* gene therapy has the potential to ultimately solve this major problem of gene therapy, and it could be practically used in clinics. However, normal somatic cells, including adult stem cells, do not only propagate well in a typical cell culture environment and are also vulnerable to epigenetic modification (Islam et al., 2007; Martinez-Climent et al., 2006), requiring that the transfer of therapeutic genes to the isolated cells be performed as soon as possible. Therefore, one of the key factors for the success of *ex vivo* gene therapy is to deliver therapeutic DNA molecules into isolated cells promptly with high efficiency. If these problems could be solved successfully, the *ex vivo* technique could be practically applied to patients in the near future.
