**3. Clinical translation of hydrodynamic gene therapy**

Several animal models have been employed for hydrodynamic gene transfer. The murine model has resulted in the gold standard of the procedure since therapeutic levels of the protein encoded by the transferred gene have been achieved. The translational process has been carried out in rat, rabbit, guinea pig, dog, pig and human liver segments.

The murine model consisted of the rapid injection of gene saline solution in a volume equivalent to the animal volemia. This large volume facilitates the backflow of the gene solution and provokes its retrograde access to the liver. The high heart rate of the mouse permits the injection of such volumes with animal survival. Similar conditions to those employed for mice were carried out in rats, although different adaptations for diminishing the solution volume have been proposed in order to follow up the translation process. Other researchers [47] studied different strategies to improve hydrodynamic gene delivery efficiency by targeting the right lateral liver lobe of the rat through the portal vein branch. The need for outflow blockade in the target area was reported since the portal vein pressure was too low to avoid backflow. In another attempt to improve the efficiency of the procedure, the left liver lobe was targeted in the rat and outflow occlusion was performed to compare its effect to free-flow control rats [32]. It was reported that outflow blockade is demanding to obtain efficient outcomes in transgene expression. Larger animals do not have the ability to increase the heart rate as mice and doubling their volemia would be incompatible with survival. Thus, the hydrodynamic injection had to be adapted to reduce the final volume and minimize the systemic hemodynamic impact. These adaptations focused on targeting an organ. Regarding this fact, Eastman et al. injected a gene to a single liver lobe employing a balloon catheter and to the entire organ of the rabbit with hepatic venous occlusion and achieved protein plasma expression in 2 days. The safety of the liver hydrodynamic gene transfer was also assessed in dogs to prove its feasible application in large animals [48]. They performed four successive injections in four different main liver lobes. Authors observed no significant harmful effects and rapid recovery of animals. However, the results obtained were poor.

The following step for the clinical translation of the procedure was to test its potential use in anatomically more similar animals to human beings such as pigs and primates. The techniques for gene delivery that were employed should be applicable in human settings.

Yoshino et al. [35] and Aliño et al. [36] described the first attempts performed in a pig. The total volume employed was reduced by targeting an area of liver and compared different 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.

**2.3. Therapeutic targets**

190 Liver Research and Clinical Management

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

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 anti-

Several animal models have been employed for hydrodynamic gene transfer. The murine model has resulted in the gold standard of the procedure since therapeutic levels of the protein encoded by the transferred gene have been achieved. The translational process has been

The murine model consisted of the rapid injection of gene saline solution in a volume equivalent to the animal volemia. This large volume facilitates the backflow of the gene solution and provokes its retrograde access to the liver. The high heart rate of the mouse permits the injection of such volumes with animal survival. Similar conditions to those employed for mice were carried out in rats, although different adaptations for diminishing the solution volume have been proposed in order to follow up the translation process. Other researchers [47] studied different strategies to improve hydrodynamic gene delivery efficiency by targeting the right lateral liver lobe of the rat through the portal vein branch. The need for outflow blockade in the target area was reported since the portal vein pressure was too low to avoid backflow. In another attempt to improve the efficiency of the procedure, the left liver lobe was targeted in the rat and outflow occlusion was performed to compare its effect to free-flow control rats [32]. It was reported that outflow blockade is demanding to obtain efficient outcomes in transgene expression. Larger animals do not have the ability to increase the heart rate as mice and doubling their volemia would be incompatible with survival. Thus, the hydrodynamic injection had to be adapted to reduce the final volume and minimize the systemic hemodynamic impact. These adaptations focused on targeting an organ. Regarding this fact, Eastman et al. injected a gene to a single liver lobe employing a balloon catheter and to the entire organ of the rabbit with hepatic venous occlusion and achieved protein plasma expression in 2 days. The safety of the liver hydrodynamic gene transfer was also assessed in dogs to prove its feasible application in large animals [48]. They performed four successive injections in four different main liver lobes. Authors observed no significant harmful effects and rapid recovery of animals. However, the results obtained were poor.

The following step for the clinical translation of the procedure was to test its potential use in anatomically more similar animals to human beings such as pigs and primates. The tech-

Yoshino et al. [35] and Aliño et al. [36] described the first attempts performed in a pig. The total volume employed was reduced by targeting an area of liver and compared different

niques for gene delivery that were employed should be applicable in human settings.

deficiency [46], in which the entire functional gene could be implemented.

**3. Clinical translation of hydrodynamic gene therapy**

carried out in rat, rabbit, guinea pig, dog, pig and human liver segments.

inflammatory cytokines such as interleukin-10 (IL10).

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

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. A complete midline laparotomy was carried out, exposing all the abdominal organs. 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 a technique for liver venous sealing mediated by image-guided catheterization [53]. Two strategies with different degrees of liver vasculature closure were proposed:


These three procedures, surgery and open and closed catheterization, proved to be safe. After gene transfer and animal awakening, their recovery was very fast and presented normal

**Figure 4.** Schematic figure of liver simultaneous cava and porta perfusion with laparotomy surgery. Suprahepatic inferior vena cava and hepatic artery are ligated, and gene solution is transferred simultaneously by portal vein and infrahepatic inferior vena cava. Modified from [50].

behavior few hours after the intervention. Furthermore, all of them mediated tissue expression of the protein encoded by the transferred gene. The rate of protein translation showed a direct relation with the degree of vasculature closure: surgery-mediated complete liver vasculature exclusion > catheterization-mediated venous vasculature closure > catheterization-mediated

**Figure 6.** Whole liver catheterization by balloon-catheters. Upper panel is a schematic figure of catheters localization. Infrahepatic IVC and portal vein are closed at liver access by inflated balloon-catheters. Lower left panel shows a radiologic image of supra and infrahepatic IVC catheters position. Lower mid panel shows an inflated balloon-catheter placed at portal vein blocking its exit. Lower right panel shows the iodinated contrast solution injection in the entire

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Transferring the human alpha-1-antitrypsin, the single liver lobe strategy mediated 20,000 copies of protein per cell in the liver. Targeting the entire organ with the closure of suprahepatic IVC, infrahepatic IVC and portal vein mediated a higher translation rate up to 100,000 copies per cell. The complete exclusion of liver vasculature by occlusion IVC, portal vein and hepatic artery with surgical procedure increased this rate up to 400,000 copies per cell in the liver tissue. The highest rate of tissue translation achieved was only 10-fold lower than the one obtained with the successful gold standard procedure performed in the mouse. This suggests that the hydrodynamic procedure of liver gene therapy with vascular exclusion mediated by radiological and surgical strategies mediated efficient delivery with efficacious

Once proved the efficiency of these procedures in pig and the confirmation of their safety for gene transfer 'in vivo,' the following step of translational process consisted of demonstrating

single lobe without organ vasculature closure.

liver. The gene solution is injected through suprahepatic inferior vena cava [54].

translation protein.

the efficacy in human liver tissue.

**Figure 5.** Single-lobe catheterization by balloon-catheter. Left panel is a schematic figure of catheter localization. Only the hepatic vein employed for gene transfer is occluded. Suprahepatic IVC, infrahepatic IVC and portal vein are not closed. Right panel shows two radiologic images of catheter position and iodinated contrast solution injection in single lobes defining the area affected by solution injected. The gene solution is injected through hepatic veins. The backflow is blocked by inflated balloon [54].

Liver Gene Therapy: Employing Surgery and Radiology for Translational Research http://dx.doi.org/10.5772/intechopen.72665 193

**Figure 6.** Whole liver catheterization by balloon-catheters. Upper panel is a schematic figure of catheters localization. Infrahepatic IVC and portal vein are closed at liver access by inflated balloon-catheters. Lower left panel shows a radiologic image of supra and infrahepatic IVC catheters position. Lower mid panel shows an inflated balloon-catheter placed at portal vein blocking its exit. Lower right panel shows the iodinated contrast solution injection in the entire liver. The gene solution is injected through suprahepatic inferior vena cava [54].

behavior few hours after the intervention. Furthermore, all of them mediated tissue expression of the protein encoded by the transferred gene. The rate of protein translation showed a direct relation with the degree of vasculature closure: surgery-mediated complete liver vasculature exclusion > catheterization-mediated venous vasculature closure > catheterization-mediated single lobe without organ vasculature closure.

Transferring the human alpha-1-antitrypsin, the single liver lobe strategy mediated 20,000 copies of protein per cell in the liver. Targeting the entire organ with the closure of suprahepatic IVC, infrahepatic IVC and portal vein mediated a higher translation rate up to 100,000 copies per cell. The complete exclusion of liver vasculature by occlusion IVC, portal vein and hepatic artery with surgical procedure increased this rate up to 400,000 copies per cell in the liver tissue. The highest rate of tissue translation achieved was only 10-fold lower than the one obtained with the successful gold standard procedure performed in the mouse. This suggests that the hydrodynamic procedure of liver gene therapy with vascular exclusion mediated by radiological and surgical strategies mediated efficient delivery with efficacious translation protein.

Once proved the efficiency of these procedures in pig and the confirmation of their safety for gene transfer 'in vivo,' the following step of translational process consisted of demonstrating the efficacy in human liver tissue.

**Figure 5.** Single-lobe catheterization by balloon-catheter. Left panel is a schematic figure of catheter localization. Only the hepatic vein employed for gene transfer is occluded. Suprahepatic IVC, infrahepatic IVC and portal vein are not closed. Right panel shows two radiologic images of catheter position and iodinated contrast solution injection in single lobes defining the area affected by solution injected. The gene solution is injected through hepatic veins. The backflow

**Figure 4.** Schematic figure of liver simultaneous cava and porta perfusion with laparotomy surgery. Suprahepatic inferior vena cava and hepatic artery are ligated, and gene solution is transferred simultaneously by portal vein and

a technique for liver venous sealing mediated by image-guided catheterization [53]. Two

**a.** Inject the gene solution through a balloon catheter placed in a single lobe (**Figure 5**), and

**b.** Place simultaneously three catheters with balloons within suprahepatic IVC, infrahepatic IVC and portal vein around the liver entry (**Figure 6**) in order to close its vasculature. The gene solution is injected through the catheter placed in suprahepatic IVC and the entire

These three procedures, surgery and open and closed catheterization, proved to be safe. After gene transfer and animal awakening, their recovery was very fast and presented normal

strategies with different degrees of liver vasculature closure were proposed:

only target this part of the liver and

organ is targeted.

192 Liver Research and Clinical Management

is blocked by inflated balloon [54].

infrahepatic inferior vena cava. Modified from [50].

In this sense, human liver segments proceeding from surgical resection in patients with cancer were injected with different genes to evaluate the potential transferability of this technique. Given their precedence, the vasculature of these human liver segments is entirely excluded so they are watertight and hence, pressurized. The gene is retrogradely transfected through a catheter placed in a hepatic vein (**Figure 7**) and the segment remains watertight for 5 min.

The molecular quantitative evaluation of decoding is demanding for a correct interpretation of the process. Quantitative determination of the molecular process provides real data of delivery, transcription and translation indexes. It would be important that researches achieved an agreement in data quantitation and expression to be able to objectively compare results and define the better conditions for gene transfer. The units should be expressed in molecular units (as number of copies or moles) or other units of mass. It is also very important that the data are referred to a common circumstance, such as a standard or 'normalized cell.' The normalized cell is defined as 'typical mammalian hepatocyte with defined content of total DNA' (genome weight of each specific animal, for instance, human: 6.6 pg), RNA (20 pg) and protein (500 pg). This strategy offers an objective analysis that permits expressing the data as the copy number of each molecular specie, considering the standard content of DNA, RNA and protein in a normalized cell. This offers a more comprehensive interpretation of the entire process and

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To sum up, the hydrodynamic procedure is an efficient strategy for gene delivery demonstrated by the levels of tissue protein that is observed. The more the vasculature is occluded, the better is the final protein expression. The surgical procedure permits, excluding liver, entire vasculature and mediates the higher expression rate. However, non-invasive image-guided catheterization permits good levels of protein production without the need of a laparotomy incision.

This work has been partially supported by Spanish Ministerio de Economía y Competitividad

1 Pharmacology Department, Medicine Faculty, University of Valencia, Valencia, Spain

3 Radiology Department and Biomedical Imaging Research Group (GIBI230), La Fe

4 Unit of Hepato-Biliary-Pancreatic Surgery and Transplantation, La Fe University and

5 Department of General Surgery, Digestive Surgery Unit, La Fe University and Polytechnic

2 Pharmacogenetics Unit, La Fe Health Research Institute, La Fe University and Polytechnic

, Eduardo García-Granero5

, Eva M. Montalvá4

,

permits comparing the results among different works and research groups.

(SAF2011-27002, SAF2007-64492) and Grifols ALTA Award 2017.

Luis Sendra1,2, María José Herrero1,2\*, Luis Martí-Bonmatí3

\*Address all correspondence to: maria.jose.herrero@uv.es

University and Polytechnic Hospital, Valencia, Spain

, Matteo Frasson<sup>5</sup>

**Acknowledgements**

**Author details**

Rafael López-Andújar<sup>4</sup>

Hospital, Valencia, Spain

Hospital, Valencia, Spain

Polytechnic Hospital, Valencia, Spain

and Salvador F. Aliño Pellicer1,2

The first studies of gene transfer with human liver segments [56] used the eGFP tracer gene in order to easily determine its expression efficacy, and it was demonstrated that the gene could be efficiently delivered and the protein was produced within the liver tissue as observed by fluorescence microscopy. After confirming the feasibility of the technique in this type of tissue, genes with clinical interest were employed to define the translational potential to clinical real settings.

Sendra Gisbert et al. [55] transferred a plasmid bearing the human interleukin-10 gene (IL10). Interleukin-10 is an immunomodulatory protein with pleotropic effects with potential interest for the treatment of inflammatory diseases or for inducing tolerance in organ transplantation. The rate of tissue protein translation achieved was around 1000 copies per cell, this meaning the potential therapeutic production of protein (IC50 of IL10 for TNFa = 124 pg) if compared with other results of the same group.

Our group also transferred in similar human liver segments a plasmid with the same human alpha-1-antitrypsin employed in mice and pigs but modified. In order to permit differing endogenous and exogenous genes and proteins, a sequence of nucleotides encoding the flag peptide was added. Preliminary experiments demonstrate that the procedure is efficient and the use of a human gene in human tissue favors the production of protein. First, results prove a rate of tissue protein translation of 104 –105 copies of hAAT-flag protein per cell, this accounting for up to 22% of all the hAAT proteins present in the liver tissue in 1 week.

The efficacy of gene transfer can be measured by different techniques and authors have studied many variables to present their results and evaluate how efficient a procedure is. This requires the use of a more detailed analysis that allows to identify the effectiveness of each of the stages of the process of delivery of the gene, its decoding of protein and its subsequent location.

**Figure 7.** Catheterization of human liver segment. Left panel shows a schematic figure of a human liver segment with a catheter placed in a hepatic vein. Right panel is a radiographic image of a human liver segment injected with iodinated contrast solution through hepatic vein. Modified from [55].

The molecular quantitative evaluation of decoding is demanding for a correct interpretation of the process. Quantitative determination of the molecular process provides real data of delivery, transcription and translation indexes. It would be important that researches achieved an agreement in data quantitation and expression to be able to objectively compare results and define the better conditions for gene transfer. The units should be expressed in molecular units (as number of copies or moles) or other units of mass. It is also very important that the data are referred to a common circumstance, such as a standard or 'normalized cell.' The normalized cell is defined as 'typical mammalian hepatocyte with defined content of total DNA' (genome weight of each specific animal, for instance, human: 6.6 pg), RNA (20 pg) and protein (500 pg).

This strategy offers an objective analysis that permits expressing the data as the copy number of each molecular specie, considering the standard content of DNA, RNA and protein in a normalized cell. This offers a more comprehensive interpretation of the entire process and permits comparing the results among different works and research groups.

To sum up, the hydrodynamic procedure is an efficient strategy for gene delivery demonstrated by the levels of tissue protein that is observed. The more the vasculature is occluded, the better is the final protein expression. The surgical procedure permits, excluding liver, entire vasculature and mediates the higher expression rate. However, non-invasive image-guided catheterization permits good levels of protein production without the need of a laparotomy incision.
