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 (Figure 2). Moreover, it was clarified that the pores naturally reduce and disappear in 24–48 h [70]. Although serum transaminase shows transient increase after a hydrodynamic injection, these values return to the background level within a short period. Considering the short life time of transaminase, an increase in serum transaminase is speculated to be caused by leakage from the transient pores. If the intravascular pressure is kept within an adequate range, this change in hepatocyte is reversible and does not result in apoptosis and necrosis; therefore, acute liver

Figure 2. Scheme of hydrodynamic gene delivery. The hepatocyte partly faces to the blood stream via the fenestra in the sinusoidal structure. A rapid stream of hydrodynamic injection has the blood in the sinusoid washed out transiently, and the nucleic acid can be delivered into hepatocytes without being degraded by nucleases. A high intravascular pressure makes dimples on the surface of hepatocyte, and finally generates transient pores. Nucleic acid is pushed into the

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

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

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) [71]. 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

failure is not a concern.

hepatocyte through the transient pores.

Figure 1. Technical details of the tail vein injection in a mouse. (a) When inserting a needle tip, the tail vein and needle shaft should be at the same angle. The puncture can be performed from the top of the tail curve. (b) If a needle tip successfully enters the tail vein, backflow of the blood is visible on the needle tip. Once the backflow is confirmed, a needle tip can be further inserted to the proximal side of the tail vein.

Nucleic Acid-Based Therapy: Development of a Nonviral-Based Delivery Approach http://dx.doi.org/10.5772/intechopen.80741 9

3. Principle and progress of hydrodynamic gene delivery toward human

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

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

Figure 1. Technical details of the tail vein injection in a mouse. (a) When inserting a needle tip, the tail vein and needle shaft should be at the same angle. The puncture can be performed from the top of the tail curve. (b) If a needle tip successfully enters the tail vein, backflow of the blood is visible on the needle tip. Once the backflow is confirmed, a

needle tip can be further inserted to the proximal side of the tail vein.

3.1. Principle, efficiency, and safety of hydrodynamic gene delivery

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

acid to pass through the cell membrane into the cytoplasm and nucleus.

gene therapy

Figure 2. Scheme of hydrodynamic gene delivery. The hepatocyte partly faces to the blood stream via the fenestra in the sinusoidal structure. A rapid stream of hydrodynamic injection has the blood in the sinusoid washed out transiently, and the nucleic acid can be delivered into hepatocytes without being degraded by nucleases. A high intravascular pressure makes dimples on the surface of hepatocyte, and finally generates transient pores. Nucleic acid is pushed into the hepatocyte through the transient pores.

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 (Figure 2). Moreover, it was clarified that the pores naturally reduce and disappear in 24–48 h [70]. Although serum transaminase shows transient increase after a hydrodynamic injection, these values return to the background level within a short period. Considering the short life time of transaminase, an increase in serum transaminase is speculated to be caused by leakage from the transient pores. If the intravascular pressure is kept within an adequate range, this change in hepatocyte is reversible and does not result in apoptosis and necrosis; therefore, acute liver failure is not a concern.

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) [71]. 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.

volume per second. Several studies have tried to resolve these problems using catheter technique. A balloon catheter is inserted from the jugular vein into the hepatic vein under X-ray guidance, which is often performed in clinic [56]. When the catheter is placed in the hepatic vein, the balloon on its tip is inflated, which causes venous occlusion to prevent leakage of DNA solution from the hepatic vein to the IVC. This technique targeting each lobe of the liver can reduce injection volume per one procedure to <1% BW, maintaining efficiency of gene

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

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

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 (Figure 4). Inconsistent intravascular pressure caused by leakage of DNA solution to the adjacent area, which results from physiological connections of intrahepatic vessels and tissue elasticity, is highly possible, and the leakage volume can be also associated with intravascular pressure during injection. To achieve precise control of intravascular pressure, a computer-controlled injector with feedback mechanism has been developed [54]. Although the initial version of the injector utilized CO2 as its driving force, the current version adopts electric motor for pursuit of more accurate control [58, 77] (Figure 5). This injection system leads to reproducible results of efficiency. Not only efficiency but also safety is confirmed in various aspects, such as blood test, electrocardiogram, hemodynamic CT study, laparoscopic observation, and

Figure 4. Relationship between time-pressure curve and transgene expression on site-specific delivery to a large animal. (a and b) HGD was performed to right and left lateral lobes of the pig liver. (c and d) Both injections achieved 75 mmHg of a peak intravascular pressure. (e and f) Gene expressions after the injections of (c) and (d) are shown in (e) and (f), respectively. This figure is partly reused and modified with updated information from Figures 3, 5, and 6 in [56] with their permission. RLL, right lateral lobe; RML, right medial lobe; LML, left medial lobe; LLL, left lateral lobe; CL,

delivery.

caudate lobe.

histologic assessment [56, 78, 79] (Figure 6).

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 [72]. In a rat model with bile duct ligation, hydrodynamic delivery of MMP13 gene indicated prophylactic effect on liver fibrosis [67]. 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 [75], muscle [61], and pancreas [76].

#### 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 (Figure 3). Two major obstacles that should be overcome are poor site specificity and very large injection volume. HGD with adequate range of intravascular pressure, a key factor for efficient and safe delivery, is facile to achieve by a manual injection in mice. On the contrary, in larger animals, such as rabbits, pigs, dogs, and nonhuman primates, controlling intravascular pressure is difficult because of a large amount of injection

Figure 3. Improvements of hydrodynamic gene delivery toward human gene therapy.

volume per second. Several studies have tried to resolve these problems using catheter technique. A balloon catheter is inserted from the jugular vein into the hepatic vein under X-ray guidance, which is often performed in clinic [56]. When the catheter is placed in the hepatic vein, the balloon on its tip is inflated, which causes venous occlusion to prevent leakage of DNA solution from the hepatic vein to the IVC. This technique targeting each lobe of the liver can reduce injection volume per one procedure to <1% BW, maintaining efficiency of gene delivery.

solution and transfer of nucleic acid into the hepatocyte, the volume of the liver reportedly

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 [72]. In a rat model with bile duct ligation, hydrodynamic delivery of MMP13 gene indicated prophylactic effect on liver fibrosis [67]. 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

Based on efficiency and safety in rodents, HGD has been improved extensively and can be potentially applied in humans (Figure 3). Two major obstacles that should be overcome are poor site specificity and very large injection volume. HGD with adequate range of intravascular pressure, a key factor for efficient and safe delivery, is facile to achieve by a manual injection in mice. On the contrary, in larger animals, such as rabbits, pigs, dogs, and nonhuman primates, controlling intravascular pressure is difficult because of a large amount of injection

increased by 165% compared to the preinjected condition.

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

liver, such as the kidneys [75], muscle [61], and pancreas [76].

3.2. Improvement of a hydrodynamic injection for larger animals

Figure 3. Improvements of hydrodynamic gene delivery toward human gene therapy.

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 (Figure 4). Inconsistent intravascular pressure caused by leakage of DNA solution to the adjacent area, which results from physiological connections of intrahepatic vessels and tissue elasticity, is highly possible, and the leakage volume can be also associated with intravascular pressure during injection. To achieve precise control of intravascular pressure, a computer-controlled injector with feedback mechanism has been developed [54]. Although the initial version of the injector utilized CO2 as its driving force, the current version adopts electric motor for pursuit of more accurate control [58, 77] (Figure 5). This injection system leads to reproducible results of efficiency. Not only efficiency but also safety is confirmed in various aspects, such as blood test, electrocardiogram, hemodynamic CT study, laparoscopic observation, and histologic assessment [56, 78, 79] (Figure 6).

Figure 4. Relationship between time-pressure curve and transgene expression on site-specific delivery to a large animal. (a and b) HGD was performed to right and left lateral lobes of the pig liver. (c and d) Both injections achieved 75 mmHg of a peak intravascular pressure. (e and f) Gene expressions after the injections of (c) and (d) are shown in (e) and (f), respectively. This figure is partly reused and modified with updated information from Figures 3, 5, and 6 in [56] with their permission. RLL, right lateral lobe; RML, right medial lobe; LML, left medial lobe; LLL, left lateral lobe; CL, caudate lobe.

4. Conclusion

Acknowledgements

tuning of the system.

Conflict of interest

Author details

The authors declare no conflict of interest.

Takeshi Yokoo1,2, Kenya Kamimura2

\*Address all correspondence to: kenya-k@med.niigata-u.ac.jp

University School of Medicine, Chuo-ku, Niigata, Japan

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 in vivo gene editing based on CRISPR/Cas9, short-term transgene expression may be preferred, to prevent off-target effect. Therefore, the transient gene expression mediated by the nonviral vector-based delivery may have great advantages when it comes to gene editing. Among the methods, as described above, HGD may be a promising delivery approach as it is simpler and more efficient. Currently, we are modifying the original HGD method used in small animals in order to apply it into large animals to test its efficacy and safety. Metabolic and genetic diseases, which show lower level of normal functional protein, are so far good candidates for this type of procedure. Although there is evidence showing transgene expression and that the procedure was safely performed in pigs [54–57], dogs [58, 59], and baboons

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

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

[60, 61], further preclinical studies are necessary prior to human therapy application.

Society for the Promotion of Sciences, 16K19333 to Yokoo T, 17K09408 to Kamimura.

This work was supported in part by grant-in-aid for scientific research from the Japanese

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-

\*, Tsutomu Kanefuji<sup>3</sup>

1 Department of Preemptive Medicine for Digestive Diseases and Healthy Active Life, Niigata

, Takeshi Suda<sup>3</sup> and Shuji Terai<sup>2</sup>

Figure 5. Scheme of the computer-controlled hydrodynamic injection system. Prior to an injection, a user selects appropriate time-pressure pattern for delivery and preload the data to the command unit. The command unit transmits the data to the control unit, which modulates electric power based on the feedback information of an intravascular pressure during the injection from the pressure sensor placed at the peripheral vein of a target area.

Figure 6. Image-guided, computer-controlled HGD to the dog liver. The balloon catheter was placed at the appropriate position in the hepatic veins of right lateral lobe and the occlusion of the blood flow by the balloon was confirmed by injecting a small amount of contrast medium into the hepatic vein. Then the hydrodynamic injection of naked DNA solution was performed under the real time monitoring of liver structure by the laparoscope using the computercontrolled injection system (A). (B) Time-pressure curve and the volume of injected solution recorded in the injection system. Solid and dotted lines represent actual and preloaded time-pressure curves. The gray area shows cumulative volume of injected saline (ml). (C) Laparoscopic findings of the hydrodynamically injected right lateral lobe of the dog. The injected lobe was swollen, and the injected DNA solution transiently made the liver pale. Neither destruction nor bleeding was seen on the surface of the liver (arrowheads). (D) The effect of lobe-specific hydrodynamic gene delivery of luciferase expressing plasmid. (i) Liver samples were collected by needle biopsy under the ultrasound sonography 4 days after the injection. (ii) The immunohistochemical analyses showed positively stained cells in the injected right lateral lobe. No stained cells were found in noninjected left lateral lobe. This figure is partly reused and modified with updated information from Figure 1 in [58] with their permission.
