Characteristics of nonviral gene delivery method.
Gene therapy returns to the center stage of medicine to treat patients with diseases that are unable to be cured with the conventional therapeutic strategies. This development is due to various reasons, including vector development and significant achievement in next-generation sequencing. Among the various methodologies of gene therapy, nucleic acid-based therapy has been considered to be promising in various diseases. The development of delivery methods to target cells in vivo, however, remains critical. These include viral vector-based and nonviral vector-based gene delivery methods as well as physical approaches such as hydrodynamic gene delivery (HGD). HGD is a simple and effective in vivo gene transfer method for the functional analysis of therapeutic genes and regulatory elements in small animals. Moreover, this chapter outlines the principle of HGD, gene expression studies in rodents, and recent advances in clinical application of HGD and provides future perspectives in developing a safe and efficient method for nucleic acid-based therapy.
- nucleic acid-based therapy
- nonviral delivery
- hydrodynamic gene delivery
- computer-controlled injection
- human gene therapy
In 1990, first human gene therapy was conducted, targeting adenosine deaminase deficiency
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, nonviral vector-based gene delivery may play a big role in future medicine.
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 multiple organs.
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.
|Lipids||Cationic lipids||High efficiency |
Ease to prepare
|Low efficiency |
Acute immune response
|Polymers||Cationic polymers||Highly effective |
Ease to prepare
|Toxic to cells|
Acute immune response
|Exosomes||Natural or modified exosomes||Less toxic (Insufficient data)||Low efficiency? (Insufficient data)|
|Needle injection||Mechanic force||Simple||Low efficiency|
Expression limited to needle track
|Gene gun||Pressure||Good efficiency||Limited to target area|
Need surgical procedure for internal organ
|Electroporation||Electric pulse||High efficiency||Tissue damage|
Limited target area
Need surgical procedure for internal organ
|Sonoporation||Ultrasound||Site specific||Low efficiency|
|Magnetofection||Magnetic field||Site specific||Low efficiency|
Limited target area
Need surgical procedure for internal organ
|Hydrodynamic delivery||Hydrodynamic pressure||Simple|
|Need catheter insertion technique in large animals|
2.1. Liposome-based approach
Lipofection, a cationic lipid-mediated approach, is widely used in numerous
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.
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  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 . An attempt to utilize cancer-derived exosome for cancer therapy was also reported, wherein the cancer-derived 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 . 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, 21, 22, 23, 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 . Needle injection was expanded to the skin , heart muscle , liver , and tumor . 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 ; on the basis of the delivery mechanism, they are divided into solid, coated, and dissolving types . 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 .
2.6. Gene gun
Gene gun is known as microprojectile bombardment, and the first study reporting its use was published in 1987 . 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 , high-voltage electric gene gun , and gas gene gun . 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
2.7. Sonoporation, electroporation, and magnetofection
Sonoporation, using ultrasound [39, 40], and electroporation, using electric pulse , 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 . 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  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, 49, 50] and solid tumors .
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, 55, 56, 57], dogs [58, 59], and rhesus monkeys [60, 61]. Functional analyses of therapeutic gene were reported in nonalcoholic steatohepatitis , hepatitis B and C , fulminant hepatitis [64, 65], liver fibrosis [66, 67], liver regeneration , Fabry’s disease , and colon cancer . The next section describes its principle and progress in human gene therapy.
3. Principle and progress of hydrodynamic gene delivery toward human gene therapy
3.1. Principle, efficiency, and safety of hydrodynamic gene delivery
HGD is achieved by the quick injection of a large amount of naked nucleic acid solution into the vein. In case of a rodent, the solution is injected from the tail vein. The most important step of successful gene delivery is a precise insertion of an injection needle into the tail vein. The details of technical tips are described in Figure 1. The quick injection can transiently increase an intravenous pressure. Mechanical force by rapid increase in venous pressure allows nucleic acid to pass through the cell membrane into the cytoplasm and nucleus.
Among various organs, the liver can achieve the highest level of gene expression because of the presence of the specific structure fenestra. Fenestra is a small window in the sinusoidal vessel, and hepatocytes are partly exposed to the blood stream. In other words, hepatocytes can be directly affected by intravascular pressure. A rapid stream of hydrodynamic injection can wash out the blood in the sinusoid vessel transiently and thoroughly, and nucleic acid can reach the hepatocytes without degeneration by nucleases. A high intravascular pressure creates dimples on the surface of the hepatocyte and finally generates transient small pores. The nucleic acid is pushed into the hepatocyte through the transient pores
To apply this method into the clinic, the modification of the original procedure is essential as in mouse studies, hydrodynamic injection is performed via the tail vein. Looking back to the original method, in detail, naked DNA solution equivalent to 10% of the body weight (BW) is injected for 5–7 s via the tail vein. The details of hydrodynamics during the injection have been reported using contrast medium under fluoroscopic imaging and cone-beam computed tomography (CT) . Briefly, the injected solution is led to the inferior vena cava (IVC) and then flowed back to the hepatic veins. The retrograde flow passes through the sinusoid vessel into the portal vein. Given that contrast medium transiently stayed in the liver after the injection, the flow generated transient pores on the surface of the hepatocyte while passing through the sinusoid vessel. Because of the filling of sinusoidal and interstitial space by the solution and transfer of nucleic acid into the hepatocyte, the volume of the liver reportedly increased by 165% compared to the preinjected condition.
The efficiency of transfer was indicated by microscopic images. Transgene expression was observed in approximately 20–40% of hepatocytes. Wide distribution of transgene expression in the liver can achieve therapeutic level of transgene expression . In a rat model with bile duct ligation, hydrodynamic delivery of MMP13 gene indicated prophylactic effect on liver fibrosis . Given its simplicity, safety, and efficiency, HGD has been utilized in numerous rodent studies [63, 65, 66, 73, 74]. HGD can be also applied to various organs other than the liver, such as the kidneys , muscle , and pancreas .
3.2. Improvement of a hydrodynamic injection for larger animals
Based on efficiency and safety in rodents, HGD has been improved extensively and can be potentially applied in humans
During the establishment of catheter technique, another important problem arises, that is, distinct response of injection pressure in a targeted area. Precise control of intravascular pressure is essential to achieve efficient and safe gene delivery
Currently, various approaches including both viral and nonviral vector-based delivery methods are studied for safe and efficient human gene therapy. They have their own properties, such as duration of gene expression, size of transgene to load, possible organs and their expected volumes in single procedure, and repeatability. Conditions to treat are also diverse. Congenital disease such as hemophilia possibly requires long-term transgene expression for decades. For
This work was supported in part by grant-in-aid for scientific research from the Japanese Society for the Promotion of Sciences, 16K19333 to Yokoo T, 17K09408 to Kamimura.
This work has finished due to Dexi Liu, and all members at Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University. The authors would like to appreciate all members at the Niigata city industrial promotion center and for their excellent assistance in producing the system, Yoshihiko Ohba for the supporting of fine-tuning of the system.
Conflict of interest
The authors declare no conflict of interest.