2. Nonviral approaches for nucleic acid transfer

This section focuses on gene delivery methods using nonviral vector-based approach. Nucleic acids loaded in artificial or natural cargos or in naked condition are transferred to target cells. The characteristics of various gene deliveries are briefly described in Table 1.

glycol [7] and cell-specific targeting ligand on the surface of the liposome, have been extensively studied. Development of a promising linker also improves stability, biodegradability, and transfection efficiency and reduces cytotoxicity [8]. Lipofection has been utilized in 4.4% of clinical trials worldwide [2]. The results of human gene therapy for cystic fibrosis in clinical trials of phase I/IIa and IIb have been reported in the UK [9, 10]. Patients had cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and suffered from hypofunction of CFTR in multiple organs. Because secretory fluid becomes viscous, the patient may experience repeated respiratory infection and, finally, respiratory failure. CFTR gene was nebulized as lipoplex every 28 days for 1 year for significant stabilization of lung function [9, 10]. In 2016, other clinical trials for genitourinary cancers and solid tumors reportedly used the truncated forms of the RB gene

High efficiency Site specific

Method Functional component Advantages Disadvantages

Needle injection Mechanic force Simple Low efficiency

Electroporation Electric pulse High efficiency Tissue damage

Sonoporation Ultrasound Site specific Low efficiency

Magnetofection Magnetic field Site specific Low efficiency

Hydrodynamic pressure Simple

Gene gun Pressure Good efficiency Limited to target area

Ease to prepare

Ease to prepare

data)

Less toxic (Insufficient

Low efficiency in vivo Acute immune response

Acute immune response

Limited target area

Tissue damage

animals

Limited target area

Low efficiency? (Insufficient data)

http://dx.doi.org/10.5772/intechopen.80741

Expression limited to needle track

Need surgical procedure for internal organ

Need surgical procedure for internal organ

Need surgical procedure for internal organ

Need catheter insertion technique in large

Toxic to cells

Nucleic Acid-Based Therapy: Development of a Nonviral-Based Delivery Approach

Lipids Cationic lipids High efficiency in vitro

Polymers Cationic polymers Highly effective in vitro

Exosomes Natural or modified

Hydrodynamic delivery

exosomes

Cationic polymer is an artificially synthesized vehicle, and various types of polymer have been studied. DNA condensed in cationic polymer (polyplex) acquires tolerance to enzymatic degradation, which results in stability in the blood. Cellular uptake is via receptor-mediated endocytosis, which leads to a high level of transfection activity. Clinical trials using this approach for cystic fibrosis and ocular degenerative disease have been reported [13, 14]. Nevertheless, the stability of polyplex and persistent positive charge leads to high cytotoxicity.

and p53 gene with docetaxel, respectively [11, 12].

Table 1. Characteristics of nonviral gene delivery method.

2.2. Polymer-based approach

#### 2.1. Liposome-based approach

Lipofection, a cationic lipid-mediated approach, is widely used in numerous in vitro and in vivo studies. The first study reporting lipofection was published in 1987 [6]. Molecules comprising hydrophilic head, linker, and hydrophobic anchor form a spherical structure. The positively charged hydrophilic head plays a role in condensing the negatively charged DNAs. It also helps in establishing an electrostatic interaction with the negatively charged cell membrane. As a result, it promotes the cellular uptake of DNA-loaded liposome (lipoplex), endosomal escape, and subsequent release of the condensed DNAs into the cytoplasm. On the contrary, the hydrophobic anchor protects DNAs from degradation by nucleases. Liposome is a popular carrier to deliver even large-sized transgene; it is easy to prepare and modify and is utilized in numerous laboratories worldwide. Nevertheless, there are several drawbacks for its use in gene therapy. It has difficulty in achieving therapeutic level of transgene expression, shows no tropism to desired cells, and exhibits a short life span. Furthermore, the positively charged head has cell toxicity. An inflammatory response occurs when unmethylated CpG DNA is transported, which is one of the obstacles that need to be addressed. Various strategies to achieve high level of safety and efficiency, such as introduction and improvement of polyethylene


Table 1. Characteristics of nonviral gene delivery method.

however, carcinogenesis and lethal immune reaction were reported [3–5]. Numerous researchers have been attempting to overcome these serious obstacles to enable safe and efficient therapy. For this purpose, the improvement of viral vector has been extensively studied in the last decade, and in addition, nonviral vector-based gene delivery method has developed with great promise. As expected, it resulted in less antigenicity and less chance of integration into the human genome than viral vector; therefore, it can be regarded as a biologically safer method than viral vector-based gene delivery method. However, the period

This chapter focuses on nonviral vector-based delivery method, which could be used for the nucleic acid-based therapy. In these methods, a transgene is not integrated into the host genome; hence, gene expression is transient. Because temporal transgene expression is applied to promising technologies, such as generation of iPS cells and gene editing by CRISPR/Cas9,

The last section of this chapter outlines the recent progress in the HGD, which enables the highest level of delivery efficiency among nonviral vector-based approaches and the clinical application utilizing the well-established method of catheter insertion into the vessels in the

This section focuses on gene delivery methods using nonviral vector-based approach. Nucleic acids loaded in artificial or natural cargos or in naked condition are transferred to target cells.

Lipofection, a cationic lipid-mediated approach, is widely used in numerous in vitro and in vivo studies. The first study reporting lipofection was published in 1987 [6]. Molecules comprising hydrophilic head, linker, and hydrophobic anchor form a spherical structure. The positively charged hydrophilic head plays a role in condensing the negatively charged DNAs. It also helps in establishing an electrostatic interaction with the negatively charged cell membrane. As a result, it promotes the cellular uptake of DNA-loaded liposome (lipoplex), endosomal escape, and subsequent release of the condensed DNAs into the cytoplasm. On the contrary, the hydrophobic anchor protects DNAs from degradation by nucleases. Liposome is a popular carrier to deliver even large-sized transgene; it is easy to prepare and modify and is utilized in numerous laboratories worldwide. Nevertheless, there are several drawbacks for its use in gene therapy. It has difficulty in achieving therapeutic level of transgene expression, shows no tropism to desired cells, and exhibits a short life span. Furthermore, the positively charged head has cell toxicity. An inflammatory response occurs when unmethylated CpG DNA is transported, which is one of the obstacles that need to be addressed. Various strategies to achieve high level of safety and efficiency, such as introduction and improvement of polyethylene

nonviral vector-based gene delivery may play a big role in future medicine.

The characteristics of various gene deliveries are briefly described in Table 1.

2. Nonviral approaches for nucleic acid transfer

of transgene expression tends to be limited.

4 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

multiple organs.

2.1. Liposome-based approach

glycol [7] and cell-specific targeting ligand on the surface of the liposome, have been extensively studied. Development of a promising linker also improves stability, biodegradability, and transfection efficiency and reduces cytotoxicity [8]. Lipofection has been utilized in 4.4% of clinical trials worldwide [2]. The results of human gene therapy for cystic fibrosis in clinical trials of phase I/IIa and IIb have been reported in the UK [9, 10]. Patients had cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and suffered from hypofunction of CFTR in multiple organs. Because secretory fluid becomes viscous, the patient may experience repeated respiratory infection and, finally, respiratory failure. CFTR gene was nebulized as lipoplex every 28 days for 1 year for significant stabilization of lung function [9, 10]. In 2016, other clinical trials for genitourinary cancers and solid tumors reportedly used the truncated forms of the RB gene and p53 gene with docetaxel, respectively [11, 12].

#### 2.2. Polymer-based approach

Cationic polymer is an artificially synthesized vehicle, and various types of polymer have been studied. DNA condensed in cationic polymer (polyplex) acquires tolerance to enzymatic degradation, which results in stability in the blood. Cellular uptake is via receptor-mediated endocytosis, which leads to a high level of transfection activity. Clinical trials using this approach for cystic fibrosis and ocular degenerative disease have been reported [13, 14]. Nevertheless, the stability of polyplex and persistent positive charge leads to high cytotoxicity. Because cationic polymer is easy to prepare and improve, various constructs, such as polyethylenimine, polyamidoamine, polyallylamine, chitosan, dendrimers, cationic proteins, and peptides, have been studied to overcome the obstacles.

they are divided into solid, coated, and dissolving types [31]. In a mouse study, siRNA delivery is reported to be effective for skin conditions with aberrant gene expression, such as alopecia, allergic skin diseases, hyperpigmentation, psoriasis, skin cancer, and congenital

Nucleic Acid-Based Therapy: Development of a Nonviral-Based Delivery Approach

http://dx.doi.org/10.5772/intechopen.80741

Gene gun is known as microprojectile bombardment, and the first study reporting its use was published in 1987 [34]. At first, this method was developed for gene delivery into plant cells. A bullet with the microparticles containing DNA is shot to a target cell, and gene delivery is achieved. On the basis of the principle of obtaining a driving force, a gene gun is divided into three major groups: powder gene gun [34], high-voltage electric gene gun [35], and gas gene gun [36]. The driving force moves the microparticles containing DNA toward a target tissue and penetrates the cell membrane. Because delivery efficiency and cell damage are two sides of the same coin, appropriate operating pressure is required. A phase I clinical study was performed to treat melanoma using IL-12 gene [37]. Although an attempt of combining delivery with microneedles reportedly enhanced the penetration depths of microparticles [38], gene

Sonoporation, using ultrasound [39, 40], and electroporation, using electric pulse [41], increase the permeability of cell membrane for cellular uptake of nucleic acid. Magnetofection utilizes magnetic field to enable microparticles with nucleic acid to pass through the cell membrane [42]. These methods are used in combination with other methods, such as lipofection, to protect nucleic acid against degradation by nucleases. To increase gene delivery efficiency of sonoporation, microbubbles were shown to be effective [43] and applied for delivery to cancer cells [44, 45] and the central nervous system [46, 47]. Clinical trials in phases I and II have been

HGD is one of the simplest methods for gene transfer. The efficiency of HGD is the highest among nonviral vector-based delivery methods, and its physical force to deliver the gene into the cells relies on a high level of flow rate and volume of the injected solution. Since the first published reports in 1999 [52, 53], many researchers have utilized this methodology for gene transfer in animal experiments, particularly in rodent studies. For its application in human, safety and efficacy of this approach have been extensively studied and improved. To date, various types of nucleic acid have been delivered by this approach in rodents as well as pigs [54–57], dogs [58, 59], and rhesus monkeys [60, 61]. Functional analyses of therapeutic gene were reported in nonalcoholic steatohepatitis [62], hepatitis B and C [63], fulminant hepatitis [64, 65], liver fibrosis [66, 67], liver regeneration [68], Fabry's disease [64], and colon cancer

[69]. The next section describes its principle and progress in human gene therapy.

gun may be more appropriate for delivery to the skin, such as for vaccination.

reported for the treatment of melanoma [48–50] and solid tumors [51].

2.7. Sonoporation, electroporation, and magnetofection

2.8. Hydrodynamic gene delivery (HGD)

pachyonychia [33].

2.6. Gene gun

#### 2.3. Lipopolyplex-based approach

Lipopolyplex comprises polycation (cationic polymer or peptide) and condensed DNA with lipid shell and is divided into diverse categories according to the combination and ternary structure. Its advantages are of both lipoplex and polyplex, that is, more efficient transfection and less cytotoxicity. Previous study [15] and reviews [16, 17] have described the strategy, variety, and preparation of lipopolyplex.

#### 2.4. Exosome-based approach

Exosome is a kind of extracellular vesicle secreted by various cells. It comprises a lipid bilayer with several surface antigens derived from the parent cell. DNA, mRNA, miRNA, and protein can be included in the lipid bilayer. Moreover, exosome is known to have organ and cell tropism; however, the mechanism is not completely clarified. This indicates that exosome plays a role in intercellular communication. Cancer cells as well as healthy cells secrete exosome. Integrin included in exosome reportedly determines organ tropism for metastasis. Exosome from metastatic lung tumor of breast cancer induced lung metastasis of breast cancer, which originally had metastatic ability only to the bone [18]. An attempt to utilize cancer-derived exosome for cancer therapy was also reported, wherein the cancerderived exosome was used as a natural carrier of CRISPR/Cas9 plasmids. Compared to epithelial cell-derived exosome, cancer-derived exosome with CRISPR/Cas9 plasmids selectively accumulated in cancer cells, suppressed PARP-1 gene expression, and achieved induction of apoptosis [19]. Recently, many researchers have been studying exosome as delivery system for cancer therapy. Surface antigens of exosomes are known to be modified directly and genetically. The exosomes from leukemia cells, marrow stromal cells, adipose-derived mesenchymal stem cells, breast cancer cells, and kidney cells including siRNA and miRNA were reported to be used for colorectal tumor, glioma, hepatocellular carcinoma, breast cancer, and chronic myelogenous leukemia [20–24]. Although the exosome-based approach has been seen as a new and promising method of gene delivery, it is rather obvious that further understandings of the mechanisms and structures as well as improvement in exosomes' preparation are necessary to achieve the high level of efficiency and safety needed for clinical application.

#### 2.5. Needle injection

Direct injection to the tissue is the simplest approach for the physical delivery of nucleic acid. The first report for delivery to muscle was published in 1990 [25]. Needle injection was expanded to the skin [26], heart muscle [27], liver [28], and tumor [29]. Currently, microneedle is studied as a minimally invasive delivery for skin disease and vaccination [30, 31]. Microneedles are arrays of 25–2000-μm long needles [32]; on the basis of the delivery mechanism, they are divided into solid, coated, and dissolving types [31]. In a mouse study, siRNA delivery is reported to be effective for skin conditions with aberrant gene expression, such as alopecia, allergic skin diseases, hyperpigmentation, psoriasis, skin cancer, and congenital pachyonychia [33].

#### 2.6. Gene gun

Because cationic polymer is easy to prepare and improve, various constructs, such as polyethylenimine, polyamidoamine, polyallylamine, chitosan, dendrimers, cationic proteins, and pep-

Lipopolyplex comprises polycation (cationic polymer or peptide) and condensed DNA with lipid shell and is divided into diverse categories according to the combination and ternary structure. Its advantages are of both lipoplex and polyplex, that is, more efficient transfection and less cytotoxicity. Previous study [15] and reviews [16, 17] have described the strategy,

Exosome is a kind of extracellular vesicle secreted by various cells. It comprises a lipid bilayer with several surface antigens derived from the parent cell. DNA, mRNA, miRNA, and protein can be included in the lipid bilayer. Moreover, exosome is known to have organ and cell tropism; however, the mechanism is not completely clarified. This indicates that exosome plays a role in intercellular communication. Cancer cells as well as healthy cells secrete exosome. Integrin included in exosome reportedly determines organ tropism for metastasis. Exosome from metastatic lung tumor of breast cancer induced lung metastasis of breast cancer, which originally had metastatic ability only to the bone [18]. An attempt to utilize cancer-derived exosome for cancer therapy was also reported, wherein the cancerderived exosome was used as a natural carrier of CRISPR/Cas9 plasmids. Compared to epithelial cell-derived exosome, cancer-derived exosome with CRISPR/Cas9 plasmids selectively accumulated in cancer cells, suppressed PARP-1 gene expression, and achieved induction of apoptosis [19]. Recently, many researchers have been studying exosome as delivery system for cancer therapy. Surface antigens of exosomes are known to be modified directly and genetically. The exosomes from leukemia cells, marrow stromal cells, adipose-derived mesenchymal stem cells, breast cancer cells, and kidney cells including siRNA and miRNA were reported to be used for colorectal tumor, glioma, hepatocellular carcinoma, breast cancer, and chronic myelogenous leukemia [20–24]. Although the exosome-based approach has been seen as a new and promising method of gene delivery, it is rather obvious that further understandings of the mechanisms and structures as well as improvement in exosomes' preparation are necessary to achieve the high level of efficiency and safety needed

Direct injection to the tissue is the simplest approach for the physical delivery of nucleic acid. The first report for delivery to muscle was published in 1990 [25]. Needle injection was expanded to the skin [26], heart muscle [27], liver [28], and tumor [29]. Currently, microneedle is studied as a minimally invasive delivery for skin disease and vaccination [30, 31]. Microneedles are arrays of 25–2000-μm long needles [32]; on the basis of the delivery mechanism,

tides, have been studied to overcome the obstacles.

6 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

2.3. Lipopolyplex-based approach

variety, and preparation of lipopolyplex.

2.4. Exosome-based approach

for clinical application.

2.5. Needle injection

Gene gun is known as microprojectile bombardment, and the first study reporting its use was published in 1987 [34]. At first, this method was developed for gene delivery into plant cells. A bullet with the microparticles containing DNA is shot to a target cell, and gene delivery is achieved. On the basis of the principle of obtaining a driving force, a gene gun is divided into three major groups: powder gene gun [34], high-voltage electric gene gun [35], and gas gene gun [36]. The driving force moves the microparticles containing DNA toward a target tissue and penetrates the cell membrane. Because delivery efficiency and cell damage are two sides of the same coin, appropriate operating pressure is required. A phase I clinical study was performed to treat melanoma using IL-12 gene [37]. Although an attempt of combining delivery with microneedles reportedly enhanced the penetration depths of microparticles [38], gene gun may be more appropriate for delivery to the skin, such as for vaccination.

#### 2.7. Sonoporation, electroporation, and magnetofection

Sonoporation, using ultrasound [39, 40], and electroporation, using electric pulse [41], increase the permeability of cell membrane for cellular uptake of nucleic acid. Magnetofection utilizes magnetic field to enable microparticles with nucleic acid to pass through the cell membrane [42]. These methods are used in combination with other methods, such as lipofection, to protect nucleic acid against degradation by nucleases. To increase gene delivery efficiency of sonoporation, microbubbles were shown to be effective [43] and applied for delivery to cancer cells [44, 45] and the central nervous system [46, 47]. Clinical trials in phases I and II have been reported for the treatment of melanoma [48–50] and solid tumors [51].

#### 2.8. Hydrodynamic gene delivery (HGD)

HGD is one of the simplest methods for gene transfer. The efficiency of HGD is the highest among nonviral vector-based delivery methods, and its physical force to deliver the gene into the cells relies on a high level of flow rate and volume of the injected solution. Since the first published reports in 1999 [52, 53], many researchers have utilized this methodology for gene transfer in animal experiments, particularly in rodent studies. For its application in human, safety and efficacy of this approach have been extensively studied and improved. To date, various types of nucleic acid have been delivered by this approach in rodents as well as pigs [54–57], dogs [58, 59], and rhesus monkeys [60, 61]. Functional analyses of therapeutic gene were reported in nonalcoholic steatohepatitis [62], hepatitis B and C [63], fulminant hepatitis [64, 65], liver fibrosis [66, 67], liver regeneration [68], Fabry's disease [64], and colon cancer [69]. The next section describes its principle and progress in human gene therapy.
