Parameters Affecting Image-guided, Hydrodynamic Gene Delivery to Swine Liver

Abstract

Development of a safe and effective method for gene delivery to hepatocytes is a critical step toward gene therapy for liver diseases. Here, we assessed the parameters for gene delivery to the livers of large animals (pigs, 40-65 kg) using an image-guided hydrodynamics-based procedure that involves image-guided catheter insertion into the lobular hepatic vein and hydrodynamic injection of reporter plasmids using a computer-controlled injector. We demonstrated that injection parameters (relative position of the catheter in the hepatic vasculature, intravascular pressure upon injection, and injection volume) are directly related to the safety and efficiency of the procedure. By optimizing these parameters, we explored for the first time, the advantage of the procedure for sequential injections to multiple lobes in human-sized pigs. The optimized procedure resulted in sustained expression of the human α-1 antitrypsin gene in livers for more than 2 months after gene delivery. In addition, repeated hydrodynamic gene delivery was safely conducted and no adverse events were seen in the entire period of the study. Our results support the clinical applicability of the image-guided hydrodynamic gene delivery method for the treatment of liver diseases.Molecular Therapy-Nucleic Acids (2013) 2, e128; doi:10.1038/mtna.2013.52; published online 15 October 2013.

Figure 1

Influence of insertion site on vascular distribution of injected phase contrast medium. (a) Schematic presentation of the relative location for catheter insertion at proximal (white rounded rectangle), intermediate (gray rounded rectangle), and distal (black rounded rectangle) site in a hepatic vein. (b–d) Venography showing the blockade of blood vessel by balloon catheter located at (b) proximal, (c) intermediate, or (d) distal site of the right lateral vein. The ball-like structure represents the location of the catheter balloon. (e–g) Venography images showing distribution of contrast medium in the targeted liver lobes upon injection of 20 ml of contrast medium in 10 seconds from (e) proximal, (f) intermediate, or (g) distal site. The white arrow in (e) points to the IVC. Black arrows point the area where the injected contrast medium accumulates.

Figure 2

Effect of injection site on intravascular pressure and level of reporter gene expression. Image-guided hydrodynamic injection of saline-containing pCMV-Luc plasmid (100 µg/ml) was performed from the injection sites shown in Figure 1 at an injection pressure of 300 psi. Intravascular pressure upon the injection from (a) proximal, (b) intermediate, and (c) distal site and average level of luciferase gene expression 24 hours after the hydrodynamic gene delivery in the targeted liver lobe (d). Two pigs were used for each measurement and the average luciferase activity was calculated from 15 liver samples collected from different parts of each targeted liver lobe (n = 30 for each injection site). The values represent mean ± SD. *P < 0.05, one-way ANOVA followed by Bonferroni's multiple comparison test.

Figure 3

Effect of injection volume on hydrodynamic gene delivery efficiency. (a) The balloon catheter was inserted into the right lateral hepatic vein (RLHV), (b) right medial hepatic vein (RMHV), or (c) left lateral hepatic vein (LLHV) followed by the venography. Each lobe was injected from the intermediate site with the volume of 1.25×, 2.5×, or 3.0× lobe volume. (d) Level of luciferase gene expression in right lateral lobe (RLL), (e) right medial lobe (RML), (f) and left lateral lobe (LLL). Two lobes from two different animals were used for each volume and 15 liver samples from different parts of each targeted lobe were used for luciferase assay (n = 30 for each data point). The values represent mean ± SD. *P < 0.05, one-way ANOVA followed by Bonferroni's multiple comparison test.

Figure 4

Effect of sequential injections to multiple liver lobes. A sequential injection of pCMV-Luc plasmids to each of the three hepatic veins in RLL, RML, and LLL in three pigs was performed. (a) Average luciferase activity for the entire liver after sequential injection to three lobes of each animal using injection volumes of 1.25×, 2.5×, or 3.0× lobe volume of the targeted lobe. Ten samples were collected from RLL, RML, LML, LLL, and the caudate lobe (CL) (n = 50 for each injection volume). The values represent mean ± SD. *P < 0.05, One-way ANOVA followed by Bonferroni's multiple comparison test. (b) Map of reporter gene expression level in the liver. Ten liver samples were collected from different parts of each liver lobe. (c–f) Immunohistochemical staining of liver sections was performed using an anti-luciferase antibody. Three samples were collected from each lobe and representative images from noninjected CL (c, e) and injected RML (d, f) are shown. Scale bars represent 100 µm. Black arrows indicate hepatocytes stained positive with anti-luciferase antibody. Black arrowheads indicate the central vein in the acinus.

Figure 5

Effect of hydrodynamic gene delivery on serum concentrations of marker enzymes. Blood samples were collected from the ear vein or peripheral vein in the limb of animals before (time = 0), 2 hours, 1, 4, 7, 14, 21, 28, 35, 42 (the day when the second injection was administered), 43, 49, 56, and 63 days after the first injection from pig 1 and 2 and as well as 70, 77, 84, 91, 98, and 105 days after the first injection from pig 2. Concentrations of (a) aspartate aminotransferase (AST), (b) alanine aminotransferase (ALT), (c) and lactate dehydrogenase (LDH). Arrows indicate first and second injection on day 0 and 42. The white square and black diamond-shape represent results of pig 1 and pig 2, respectively.

Figure 6

Persistent expression of human α-1 antitrypsin in the hepatocytes. Immunohistochemical staining of human α-1 antitrypsin was performed in the liver after the hydrodynamic gene delivery of pCAG-hAAT plasmid. (a, d) Noninjected CL in pig 1; (b, e) injected RML in pig 1; (c, f) injected RML in pig 2. Scale bar represents 100 µm (a, b, c, 200× d, e, f, 400×). Black arrows represent positively stained hepatocytes. (g) Quantitative analysis of positively stained cells. Ten liver tissue samples collected from each lobe (total of 50 samples in a liver) in pigs 1 and 2 and a quantitative analysis was performed on three fields (total of 150 fields in a liver) from each section. The values represent mean ± SD (n = 30 for each lobe). P < 0.05 between all injected lobes and CL. One-way ANOVA followed by Bonferroni's multiple comparison test.

Figure 7

Tissue distribution of plasmid DNA. Multiplex PCR analysis was performed on genomic DNA collected from pigs by 35 cycles of PCR amplification using primers for ampicillin resistance (Amp) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) genes, respectively. Lanes: 1, plasmid DNA; 2, normal liver without plasmid injection; 3, injected lobe in pig 2; 4, spleen; 5, kidney; 6, brain; 7, heart; 8, stomach; 9, colon; 10, ovary; 11, lung. The arrow represents the 660 bp Amp fragment. The arrowhead represents the 122 bp Gapdh fragment.

Figure 8

Assessment of tissue damage by histochemistry. Hematoxylin & eosin staining of liver samples from CL (a–c), and RML (d–f). Samples were collected immediately following the injection (a, d), 4 hours after the injection (b, e), and 24 hours after the injection (c, f). Scale bars represent 100 µm. White arrows represent central vein in the lobular structure.