**2. Hydrodynamic naked DNA transfer**

#### **2.1. Hydrodynamic methodology**

The possibility of expressing heterologous genes with high efficacy after the delivery of naked DNA was firstly described during the mid-1990s [22, 23]. In 1999, Zhang et al. [24] and Liu et al. [25] introduced the hydrodynamic gene transfer procedure. This procedure consisted of the rapid injection of a large volume (2 ml in 5–7 s) of saline solution bearing the gene of interest through the tail vein in the mouse (20 g average weight). The possibility of transferring naked DNA efficiently aroused a great interest among researchers and clinicians since the hydrodynamic procedure permitted expressing high levels of a heterologous protein, employing a safer strategy than viral gene therapy. Different research groups focused their efforts on improving the technique in order to be safer, more efficient and reproducible [26].

#### **2.2. Hydrofection mechanism**

In **Figure 3**, the sinusoid circulation within the liver before and after the retrograde injection of a saline solution containing a plasmid is shown. The gene solution injected through the tail vein reaches the liver in a retrograde sense and increases the pressure inside the

**Figure 3.** Schematic representation of sinusoid organization after retrograde hydrodynamic injection of gene solution**.** When a plasmid is injected, vessel pressure increases inducing the separation of endothelium cells. This permits the access of gene constructs to the Disse space and to the hepatocytes through the massive formation of endocytic vesicles.

vessel. This distends the wall mediating the transient separation of endothelium cells. When this occurs, the DNA leaves the blood vessels through the sinusoid pores and intercellular spaces and reaches the Disse space. From this space, the DNA can access the hepatocyte, the massive formation of endocytic vesicles playing a relevant role in this process. This DNA must reach the nucleus of the hepatocyte in order that the gene information delivered can be decoded. When this process takes place efficiently, the DNA is transcribed to RNA and this is translated to the protein, which is released into the bloodstream (in case of plasma proteins).

Employing this procedure, therapeutic plasma levels of alpha-1-protein were achieved for periods of more than 6 months in mice [27]. Nowadays, many gene therapy experiments for different pathology treatments are being studied in mice [28–30]. The possibility of achieving therapeutic levels of heterologous proteins after hydrodynamic human gene transfer in the murine model boosted the efforts of research teams to develop and adapt the procedures in larger animals aiming to translate it into the clinics, since the hemodynamic changes induced by the hydrodynamic injection are not compatible with its use in humans.

The perfusion conditions that permitted achieving the most efficient results in the mouse implied doubling the animal's volemia in a very short period of time. The procedure had to be necessarily adapted since larger animals would have not tolerated these conditions. The modifications of the procedure were directed to diminish the systemic hydrodynamic pressure. This was performed by transferring the gene of interest to the one and only target organ by image-guided catheterization procedures. Studies were performed in rats [31, 32] and rabbit [33] models but the results obtained were much less efficient than that observed in the mouse. The most recent efforts focused on developing models of liver hydrodynamic perfusion in pigs given their anatomical proximity with humans [34].

In the swine model, different strategies for minimally invasive gene transfer were designed through liver catheterization [35, 36]. Although these procedures proved to be safe, the efficiency achieved was not remarkable. Some authors highlighted the possibility that higher intravascular pressure within the liver could be required. For this reason, different strategies to block the venous backflow and employ more demanding perfusion conditions were studied. Levels of heterologous protein expression were not close to therapeutics in any case. After several works carried out by research groups around the world, no significant result was achieved [37–41]. However, given the huge interest of this procedure and its potential to be translated to human clinical practice, different groups evaluated minutely the molecular process of the transferred gene decoding in order to confirm or refuse this possibility. Evaluating at molecular level the detailed delivery, transcription and translation of a transferred gene permitted in identifying the step of the decoding process that limited the final efficacy in liver tissue and comparing this process in different animal models: mouse, pig and human. The best conditions of efficacy and safety for liver hydrodynamic gene therapy have been established in pig liver 'in vivo' (by catheterization and surgery) and the human liver 'ex vivo' (by catheterization in watertight segments). The methodology permitted comparing quantitatively the efficiency of different procedures of liver gene transfer. These procedures included partial and complete vascular exclusion aiming to pressurize the organ without affecting the systemic hemodynamics.

**Figure 3.** Schematic representation of sinusoid organization after retrograde hydrodynamic injection of gene solution**.** When a plasmid is injected, vessel pressure increases inducing the separation of endothelium cells. This permits the access of gene constructs to the Disse space and to the hepatocytes through the massive formation of endocytic vesicles.

of hydrodynamic gene therapy and the wide range of application in clinics (especially in the liver), the translational process of the technique has been performed from the successful murine model to human liver segments. The swine model permitted adapting the procedure for 'in vivo' liver transfer. Different radiologic and surgical approaches performed to improve

The possibility of expressing heterologous genes with high efficacy after the delivery of naked DNA was firstly described during the mid-1990s [22, 23]. In 1999, Zhang et al. [24] and Liu et al. [25] introduced the hydrodynamic gene transfer procedure. This procedure consisted of the rapid injection of a large volume (2 ml in 5–7 s) of saline solution bearing the gene of interest through the tail vein in the mouse (20 g average weight). The possibility of transferring naked DNA efficiently aroused a great interest among researchers and clinicians since the hydrodynamic procedure permitted expressing high levels of a heterologous protein, employing a safer strategy than viral gene therapy. Different research groups focused their efforts on improving the technique in order to be safer, more efficient and

In **Figure 3**, the sinusoid circulation within the liver before and after the retrograde injection of a saline solution containing a plasmid is shown. The gene solution injected through the tail vein reaches the liver in a retrograde sense and increases the pressure inside the

the liver hydrodynamic gene transfer 'in vivo' will be discussed in this chapter.

**2. Hydrodynamic naked DNA transfer**

**2.1. Hydrodynamic methodology**

188 Liver Research and Clinical Management

reproducible [26].

**2.2. Hydrofection mechanism**

#### **2.3. Therapeutic targets**

Since gene transfer can deliver a gene functionally complete to the cell, it presents a great interest for the treatment of inherited metabolic diseases [42–45], such as alpha-1-antitrypsin deficiency [46], in which the entire functional gene could be implemented.

catheter-mediated delivery strategies. These strategies included portal vein occlusion, left hepatic artery occlusion, portal vein and left hepatic artery occlusion and both vessels' occlusion with blood flow washout. Yoshino et al. injected the gene solution through the cava vein. The occlusion of portal vein and hepatic artery with the washout mediated the most efficient outcomes achieving disperse protein plasma levels for several weeks. For the first time, the procedure showed interesting results in pigs, for those proteins with low expression. In another work, hydrodynamic retrovenous gene transfer was performed in large and small areas of pigs' liver. Alino et al. [36] reported the presence of gene and protein expression in tissues, mainly within the perivenous area. Targeting smaller areas but employing same volumes of gene solution, higher plasma protein levels were achieved, much lower than those considered therapeutic. Fabre et al. [37] targeted the entire liver and isolated the hepatic segment of the inferior vena cava by clamping it suprahepatically and infrahepatically. Gene solution was transferred by a hydrodynamic procedure through two parallel syringes and, although the efficiency of gene delivery was much lower than the one observed in the mouse and rat, they confirmed the clinical feasibility of the technique as determined by systemic blood pressures, ECG, heart rate and so on.

Liver Gene Therapy: Employing Surgery and Radiology for Translational Research

http://dx.doi.org/10.5772/intechopen.72665

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Pressure reached within the liver during the hydrodynamic injection played an important role. For this reason, Fabre et al. [40] focused their work on pressurizing individual lobes of the liver by isolating them. Aiming to achieve localized high pressure without affecting the systemic circulation, they proposed individualizing the lobe by employing catheters with balloon and ligation. Although most of the authors suggested blood pressure to be the most important feature of hydrodynamic injection for efficient gene transfer, others have pointed other characteristics such as impulse [49] and flow rate [50, 51] to be relevant. However, nearly all authors agree to the need for isolating target areas or the entire liver to improve the

Firstly, the implication of the complete vascular exclusion in the final efficacy of the procedure should be evaluated in order to determine its relevance. As previously reported, the complete liver vascularization of the pig can be occluded up to 20 min without neither hepatic injury nor systemic damage [52]. Considering this fact, Carreño et al. [50] described in pigs a surgical procedure to completely exclude liver vascularization 'in vivo' and perform hydrodynamic gene delivery, targeting the entire organ.  The clamping sequence was as follows: first, the hepatic artery, then the portal vein and finally the infrahepatic vena cava, to interrupt hepatic inflow. The suprahepatic vena cava was clamped last, to secure total hepatic vascular exclusion. Depending on the flow sense of gene transfer, three different models were designed. In model 1 the portal vein was clamped, and only a longitudinal incision was made on the anterior surface of the cava vein to insert the perfusion cannula. In model 2, the process was the same as in model 1 but with the clamping of the vena cava and perfusion through the portal vein. In model 3 (**Figure 4**), the gene solution was injected simultaneously through suprahepatic IVC (Inferior Vena Cava) and the portal vein employing two catheters connected by a Y connector and a high-volume pump. After solution perfusion, the liver was kept under total vascular exclusion for no more than 5 min to allow gene penetration into the cell nuclei.

In all three models, when suprahepatic IVC was occluded and liver vasculature was completely excluded, the systemic pressure decreased rapidly. However, 1 min after revascularization this parameter was entirely normalized and animals recovered in few hours. Due to the invasiveness of the surgical procedure that included a laparotomy, same authors designed

procedure efficiency. This vascular isolation could be partial or complete.

Gene therapy can also play an important role in the treatment of different acquired pathologies. Its application for modulating the immune response in different proinflammatory conditions, such as liver transplantation, has been studied by implementing genes of antiinflammatory cytokines such as interleukin-10 (IL10).