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

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.

The Mechanical Agitation Method of Gene Transfer for *Ex-Vivo* Gene Therapy 97

capacity Tropism Inflammatory

Dividing and nondividing cells

Dividing and nondividing cells

Dividing and nondividing cells

Dividing and nondividing cells

such as diabetes, dementia and hypertension. Genetic manipulations for these diseases are more complicated than genetic manipulations for the treatment of inherited genetic diseases. This means that current gene therapies need to deliver DNA, RNA, siRNA, or antisense sequences that alter gene expression within a specific cell population to manipulate cellular processes and responses. Viral vector-mediated gene deliveries are by far the most effective means of DNA delivery. However, the recombinant vector containing the therapeutic gene has to be packaged with viral coat proteins to make gene delivery possible, meaning that viral vector-mediated gene deliveries are limited to a DNA molecule of a certain size because the viral coat proteins have a limited DNA carrying capacity. Other than the physical limitation of viral vector-mediated gene deliveries, there are more limitations, such as immunotoxicity caused by viral coat proteins, restricted targeting of specific cell types, and recombination. Therefore, non-viral gene deliveries have been a very popular research topic, and many interesting and creative methods have been developed. The efficiency of gene delivery (*i.e.* transfection efficiency) is crucial to the success of non-viral gene deliveries. Various non-viral gene delivery methods currently developed could be classified into two groups: physical gene

Physical gene delivery methods are methods for transferring DNA molecules from the surrounding medium into cells. Naked DNA (*i.e.,* an uncomplexed form of DNA) is used in

potential

cells Low Integrated

Low

High Non-

Low Integrated

High Non-

Vector genome forms

integrated

Nonintegrated (90%) Integrated (>10%)

integrated

Main Advantages

Persistent gene transfer in dividing cells

Extremely efficient transduction of most tissues

Noninflammatory; nonpathogenic

Persistent gene transfer in most tissues

Large packaging capacity; strong tropism for neurons

Packaging

Vector Genetic

material

**Adenovirus** dsDNA 7.5 kb

**virus** ssDNA < 5kb

**Lentivirus** RNA 8 kb

**Herpes virus** dsDNA > 30 kb

Table 1. A comparison of different viral vectors used for gene therapy

delivery methods and chemical gene delivery methods.

**3.2.1 Physical gene delivery methods** 

**Adeno-associated** 

**Retrovirus** RNA 8 kb Dividing

Typical efficiencies of gene delivery to primary cells are 5-10% in most current methods (Cai et al., 2002; Ding et al., 1999; Eiges et al., 2001; Lakshmipathy et al., 2004; Peister et al., 2004), which is not high enough for satisfactory *ex vivo* gene therapy. Because of this, many different methods of gene delivery have been developed using primary cells for *ex vivo* gene therapy. Generally, gene delivery methods can be divided into two categories, viral and non-viral.
