**3.1 Viral methods for gene delivery in** *ex vivo* **gene therapy**

Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes into cells. Viral vectors have developed by taking advantage of the molecular mechanisms of the virus to deliver exogenous DNA into target cells. Currently, viral vectors are frequently used molecular biology tools for delivery of genetic material into cells of a living organism (*in vivo*) or in cell culture (*in vitro*). Viral vectors are tailored to their specific applications but share a key property. Because viral vectors are essentially created from pathogenic viruses, they are modified to minimize the pathogenic properties of the original viruses. This usually involves the deletion of a portion of the viral genome that is critical for viral replication. Such viral vectors can efficiently infect cells but once the infection has taken place, they cannot replicate. The viral vectors require helper genes to provide the missing proteins for production of new virions. Replication of viral vectors is usually conducted in packaging cells that were engineered with helper genes. Therefore, viral vectors can only replicate in packaging cells and exist solely to deliver exogenous DNA to target cells where the viral vector cannot replicate.

Table 1 summarizes the types of viral vectors currently developed. In general, viral vectors are very efficient in terms of gene delivery into target cells. However, viral vectors have common problems including the following: (i) a limited DNA capacity, (ii) expression of viral genes, (iii) initiation of the antiviral immune response, (iv) reversion to a replication competent state and (v) decreasing expression over time.

Viral vectors can be classified as DNA or RNA viral vectors. DNA viral vectors are derived from viruses such as adenovirus or herpes virus, which carry their genetic material in the form of DNA. Because these viral vectors persist as an extrachromosomal element after delivery into target cells, the viral vectors remain only temporarily. One advantage of the episomal presence of the vectors is that the gene expression level is high. Because exogenous genes cannot stay indefinitely, these vectors are not suitable for *ex vivo* gene therapy. Currently, these vectors are mostly used for *in vivo* gene therapy.

Retroviral and lentiviral vectors are examples of RNA viral vectors that are replicated in target cells via reverse transcriptase to produce DNA from their RNA genomes. The DNA is then incorporated into the host's genome by an integrase. Thereafter, the virus replicates as part of the host cell's DNA, permitting long-term expression of the exogenous gene and ensuring transmission of the exogenous gene to the progeny of transduced cells. Therefore, these vectors are suitable for *ex vivo* gene therapy in which permanent gene expression is required in *in vitro* cell culture. These vectors are also widely used for *in vivo* gene therapy.

#### **3.2 Non-viral methods for gene delivery in** *ex vivo* **gene therapy**

Gene therapy was originally devised for the treatment of inherited genetic diseases, such as hemophilia and cystic fibrosis. However, the realm of gene therapy has been expanding to develop strategies for cancer, infectious diseases like HIV, and various complex diseases,

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

Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes into cells. Viral vectors have developed by taking advantage of the molecular mechanisms of the virus to deliver exogenous DNA into target cells. Currently, viral vectors are frequently used molecular biology tools for delivery of genetic material into cells of a living organism (*in vivo*) or in cell culture (*in vitro*). Viral vectors are tailored to their specific applications but share a key property. Because viral vectors are essentially created from pathogenic viruses, they are modified to minimize the pathogenic properties of the original viruses. This usually involves the deletion of a portion of the viral genome that is critical for viral replication. Such viral vectors can efficiently infect cells but once the infection has taken place, they cannot replicate. The viral vectors require helper genes to provide the missing proteins for production of new virions. Replication of viral vectors is usually conducted in packaging cells that were engineered with helper genes. Therefore, viral vectors can only replicate in packaging cells and exist solely to deliver exogenous DNA to

Table 1 summarizes the types of viral vectors currently developed. In general, viral vectors are very efficient in terms of gene delivery into target cells. However, viral vectors have common problems including the following: (i) a limited DNA capacity, (ii) expression of viral genes, (iii) initiation of the antiviral immune response, (iv) reversion to a replication

Viral vectors can be classified as DNA or RNA viral vectors. DNA viral vectors are derived from viruses such as adenovirus or herpes virus, which carry their genetic material in the form of DNA. Because these viral vectors persist as an extrachromosomal element after delivery into target cells, the viral vectors remain only temporarily. One advantage of the episomal presence of the vectors is that the gene expression level is high. Because exogenous genes cannot stay indefinitely, these vectors are not suitable for *ex vivo* gene therapy.

Retroviral and lentiviral vectors are examples of RNA viral vectors that are replicated in target cells via reverse transcriptase to produce DNA from their RNA genomes. The DNA is then incorporated into the host's genome by an integrase. Thereafter, the virus replicates as part of the host cell's DNA, permitting long-term expression of the exogenous gene and ensuring transmission of the exogenous gene to the progeny of transduced cells. Therefore, these vectors are suitable for *ex vivo* gene therapy in which permanent gene expression is required in *in vitro* cell culture. These vectors are also widely used for *in vivo* gene therapy.

Gene therapy was originally devised for the treatment of inherited genetic diseases, such as hemophilia and cystic fibrosis. However, the realm of gene therapy has been expanding to develop strategies for cancer, infectious diseases like HIV, and various complex diseases,

**3.1 Viral methods for gene delivery in** *ex vivo* **gene therapy** 

target cells where the viral vector cannot replicate.

competent state and (v) decreasing expression over time.

Currently, these vectors are mostly used for *in vivo* gene therapy.

**3.2 Non-viral methods for gene delivery in** *ex vivo* **gene therapy** 

non-viral.


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

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 delivery methods and chemical gene delivery methods.

#### **3.2.1 Physical gene delivery methods**

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

The Mechanical Agitation Method of Gene Transfer for *Ex-Vivo* Gene Therapy 99

exogenous gene in the lipoplexes would not have a chance to be released into the cytoplasm for gene expression if the endosomes are stable. Therefore, helper lipids are added to form lipoplexes to facilitate the endosomal escape of the exogenous gene (Herringson et al., 2009a, 2009b; Savva et al., 2005). This approach is very successful because it increases the transfection efficiency dramatically. There are various combinations of cationic lipids and helper lipids available. More than 40 products are commercially available for cationic lipidbased gene delivery, including LipoTAXI (Agilent Technologies), LipofectaminTM (Invitrogen), NanoJuice® (Merck), Transfectam® (Promega), and LipoJet TM (SignaGen Laboratories). The cationic lipid-based gene delivery shows a very high transfection efficiency of up to 90 in *in vitro* cell culture. Because the cationic lipid-based lipoplexes are not stably maintained in the blood, these methods are best for *ex vivo* gene therapy. However, cationic lipid-based lipoplexes show a very poor transfection efficiency with primary cells, such as stem cells, indicating that new methodological developments are required for the practical application

Other than cationic lipids, several different positively charged materials are used as a base material for non-viral DNA delivery, such as cationic polymers (Segura & Shea, 2001), cationic peptides consisting of poly-L-Lysine (D'Haeze et al., 2007; Mullen et al., 2000; Niidome et al., 1997), or other types of cationic proteins (De Lima et al., 1999; Jean et al., 2009; Lam et al., 2004; Lee et al., 2003; Oliveira et al., 2009; Vighi et al., 2007). These approaches produce DNA carrying complexes that are more stable. However, the transfection efficiency of this method is not better than cationic lipid-based lipoplexes.

**4. Application of the mechanical agitation method to** *ex vivo* **gene therapy** 

One of the main obstacles for the application of adult stem cells in *ex vivo* gene therapy is the low efficiency of gene transfer to these cells. For example, electroporation or transfection in mesenchymal stem cells yields 5-10% gene delivery efficiency (Cai et al., 2002; Ding et al., 1999; Eiges et al., 2001; Lakshmipathy et al., 2004; Peister et al., 2004). Therefore, improved gene delivery methods would potentially be very beneficial for the practical application of *ex vivo* gene therapy in patient care. In current gene transfer protocols, virus particles or lipoplexes passively diffuse through the liquid culture medium to reach their target cells, which are layered on the bottom of a culture dish (Chuck & Palsson, 1996). Because the virus particles or lipoplexes contact the target cells by passive diffusion, increasing the chance of contact between virus particles or lipoplexes and their target cells would increase the chance of gene transfer and to promote higher transfer efficiencies. One simple way to increase contact between viruses or lipoplexes and target cells is through mechanical agitation. Based on this hypothesis, we developed a mechanical agitation method for retroviral transduction of primary cells or transfection by lipoplexes (Park et al., 2009). In this method, we simply implemented a step in which virus-containing or lipoplexescontaining cell suspensions are agitated to increase the movement of viruses or lipoplexes and target cells with the purpose of generating more frequent contact between them. Suspended target cells have a better chance of making physical contact with virus particles or lipoplexes than adherent target cells because of the possibility for three-dimensional contact between the cells and viruses or lipoplexes. The simple addition of the mechanical agitation step to the conventional transduction or transfection protocol increased gene transfer efficiency two-fold above the current rates these protocols (Fig. 4). In the following

Therefore, most of these methods are designed for *in vivo* gene therapy.

of *ex vivo* gene therapy.

physical gene delivery methods. The easiest method to deliver genes into cells is to draw naked DNA into a microneedle and then inject the microneedle into cells to transfer the naked DNA directly to the cells. Though gene transfer efficiency by this method is very efficient, the method is very slow and laborious. The main drawback of this method is that microinjection can be only performed on one cell at a time, which means that this approach cannot be used for typical gene therapeutic approaches. The approach is limited to use for gene delivery into germ-line cells to produce transgenic organisms.

Currently, the most popular physical methods for gene delivery into cells are electroporation and sonoporation. The cellular membrane is punctured by an electric pulse (electroporation) (Neumann et al., 1982) or ultrasonic wave (sonoporation) (Yizhi et al., 2007). The pores in the cellular membrane are only temporarily formed, and DNA molecules pass through during the short period of time when the pores open. These methods are generally efficient and work well across a broad range of cell types. However, a high rate of cell death limits their use, especially in gene therapy. These methods are widely used for gene delivery of immortal cells in which cell viability is not a critical issue during gene transfer.

Another popular method for physical gene delivery is the use of particle bombardment. In this method, gold particles (gene gun) (Gan et al., 2000) or magnetic particles (magnetofection) (Scherer et al., 2002) are coated with naked DNA. In the gene gun method, the DNA-coated gold particles are shot into the cell using high pressure gas, and the particles pass through the cellular membrane to introduce the particles inside the cells. In the magnetofection method, a magnet is placed underneath the tissue culture dish to attract DNA-coated magnetic particles. Then, the DNA-coated magnetic particles come into contact with a cell monolayer to introduce the particles inside the cells. These methods yield reasonably high efficiency gene transfers, but do not yield better efficiencies compared to other non-viral gene transfer methods, despite the requirement for expensive equipment. Also, it is quite difficult to control the DNA entry pathway, and the metal particles in the cells following gene transfer could negatively affect cells. Therefore, these methods are not widely used.
